A DISEASE PREDICTIVE MODEL FOR CHEMOTHERAPY OF KARNAL BUNT OF WHEAT By Muhammad Abdul Shakoor M.Sc. (Hons) Agri. A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN Plant Pathology FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE, FAISALABAD 2009

To The Controller of Examinations, University of Agriculture, Faisalabad

We, the supervisory committee, certify that the contents and the form of the thesis

submitted by Mr. Muhammad Abdul Shakoor Regd. No 85-ag-1234 have been found

satisfactory and recommend that it be processed for evaluation by the External

Examiner(s) for the award of degree.

CHAIRMAN ___________________ Dr. MUHAMMAD ASLAM KHAN MEMBER ___________________ Dr. NAZIR JAVID MEMBER ___________________ Dr. MUHAMMAD JALAL ARIF

Contents

CHAPTER Title Page 1 2 2.1 2.2 2.3 2.4 2.5 2.5.1 2.5.2 2.5.3 2.5.4 2.5.5 2.5.6 2.6 2.6.1 2.7 2.8 2.9 3.0 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.2 3.2.1 3.2.2 3.3 3.3.1 3.3.2 3.4 3.4.1 3.4.2

INTRODUCTION 1 REVIEW OF LITERATURE 5 History and geographical distribution 5 Survey and economic losses 8 Taxonomic status 15 Symptoms of the disease 15 Host range 16 Biology of Tilletia indica 16 Dispersal of teliospores 16 Teliospore dormancy 17 Nature of teliospores and their survival in the soil 18 Teliospores germination 20 Dispersal of sporadia from soil level to ear head 21 Infection process 22 Epidemiology and disease prediction models 24 The Humid Thermal Index (HTI) model as a tool for 28 determination of potential disease distribution Sources of genetics resistance against the disease 35 Chemical control of the disease 40 MATERIALS AND METHODS 45 Survey of Karnal bunt disease of wheat 45 Field survey 45 Survey of grain markets 47 Sampling 47 Survey of agricultural farms 48 Screening of wheat germplasm for the source 48 of resistance Isolation and preparation of mass culture of 48 Tilletia indica Field evaluation of wheat germplasm lines/ varieties 50 for the source of resistance against Karnal bunt disease under artificial inoculation conditions Epidemiological studies 55 Cultivation of susceptible germplasm lines/ varieties 55 for epidemiological studies Development of disease predictive model 56 Chemotherapy of Karnal bunt disease of wheat 56 In vitro evaluation of fungicides at four dosages 56 rates In vivo control of Karnal bunt disease of wheat 57 by protective and eradicative spray of fungicides

3.4.3 3.4.4 4.0 4.1 4.2 4.3 4.3.1 4.3.2 4.4 4.4.1 4.4.2 5.0 5.1 5.2 5.3 5.4 6.0 7.0 8.0 9.0 10.0

Protective spray 57 Curative spray 59 RESULTS 60 Survey of Karnal bunt disease of wheat 60 Screening of wheat germplasm for the source of 67 resistance Epidemiological studies 67 Correlation of environmental conditions with 67 Karnal bunt disease development Disease predictive models 73 Chemotherapy of Karnal bunt disease of wheat 75 In vitro evaluation of fungicides against the mycelial 75 growth of Tilletia indica In vivo control of Karnal bunt disease of wheat by 77 protective and curative (eradicative) spray of fungicides DISCUSSION 80 Survey of Karnal bunt disease of wheat 80 Screening of advanced lines / cultivars of wheat 82 against the disease Weather conditions and disease predictive models 84 Evaluation of fungicides against the Karnal bunt 86 disease of wheat SUMMARY 89 CONCLUSIONS 90 RECOMMENDATIONS 91 FUTURE STUDIES 92 REFERENCES 93

LIST OF FIGURES

FIG# TITLE PAGE

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7

8

9

10

After centrifugation teliospores sedimented into pellet 49

in conical centrifuge test tubes

Disinfested teliospores by centrifugation with 5 % 49

chlorex in capped test tubes

Germination of teliospores after 10 days on plain agar 51

petri-plates

Germination of teliosporse after 20 days in petri-plates 51

containing plain agar

Germination status of teliospores after 30 days

Germinating teliospores with long slendrical primary 51

sporadia growing from tip of promycelium (basidium)

under power microscope 100x 60)

Pattern of spore shedding (secondary sporadia) 52

on PDA slants in conical flasks after second day

of inoculation

(A and B) Pattern of sporadia shedding on PDA 52

slants after third day of inoculation

(A & B) production of secondary sporidia on 53

PDA slants after 6 days of inoculation.

Screening of wheat germplsm lines/ cultivars’for 54

the source of resistance against Karnal bunt disease

under artificial inoculation condition and field

evaluation of fungicides.

FIG # TITLE PAGE 11 Mean inhibition zones of Tilleta indica by the 76

action of various fungicides at various ppm 76

(A) Control (water) 76

(B) Alert plus at 100 ppm 76

(C) Antracal at 100 ppm 76

(D) Dolomite at 100 pp 76

(E) Shelter at 100 ppm 76

(F) Shelter at 80 ppm 76

LIST OF TABLES

TABLE # TITLE PAGE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Rating scale used to calculate the severity of Karnal 46 bunt of wheat An example of the calculation of coefficient of 46 infection Rating scale used to determine level of 55 resistance/ susceptibility Fungicides evaluated against in vitro colony growth 58 of Tilletia indica and in vivo control of wheat grains by foliar spray Analysis of variance for the assessment of bunted seeds 60 in grain markets of various districts and locations of the Punjab All pairwise comparisons tests for Karnal bunt disease of 61 wheat for districts ingrain markets of the Punjab Percent wheat sample infection, range of infection 62 and average infection in grain markets and storage godowns of 17 districts of the Punjab by Karnal bunt disease during 2006. Analysis of variance for the incidence of Karnal bunt disease 63 of wheat under natural condition in various hot spot areas of the Punjab during field survey 2007 All pairwise comparisons test for Karnal bunt disease of 64 wheat for districts recorded in hot spot areas (field). Survey of Karnal bunt disease of wheat under natural 65 conditions in different hot spot areas of the Punjab during the crop year 2007. Range of infection and percent wheat grains infection 66 in hot spots of various research stations/ research institutes, model farms and seed corporation farms during wheat crop of 2007 Level of resistance/ susceptibility of advanced wheat 68 lines and commercial cultivars against Karnal bunt disease of wheat Analysis of variance of Karnal bunt disease incidences 69 recorded on wheat varieties during 2006 and 2007 Correlation of environmental factors with Karnal bunt 69 disease of wheat on different lines/ varieties. Comparison of monthly environmental conditions 72 and karnal bunt incidence recorded on wheat varieties during 2006 and 2007.

TABLE # TITLE PAGE 16 17 18 19 20

Summary of stepwise procedure for independent variables 74 influencing Karnal bunt disease of wheat during growing season 2005-07 Summary of stepwise procedure for independent variables 74 influencing Karnal bunt disease during growing season 2005-06. Summary of stepwise procedure for independent variables 75 influencing Karnal bunt disease during growing season 2006-07 Mean inhibition zones of colony of Tilletia indica by various 77 fungicides at 4 dosages rates amended on PDA medium Percent decrease in wheat kernels infection by Tilletia indica 78 by protective and eradicative spray of fungicides

Abstracts Karnal bunt disease caused by (Tilletia indica) is sporadic in nature. The fungus attacks

the wheat crop in Feb. and March which are the susceptible stages (booting stage) of the

host. During the survey it was estimated the disease is extending from north to south

Punjab that are hot and dry areas of the province. Epidemiological studies revealed that

range of minimum air temperature between (11-16 oC), maximum air temperature (27-31

oC) and rainfall more than (1.15 mm) during the vulnerable growth stages produced the

critical circumstances for the invasion of the fungus on the wheat crop. Based upon the

environmental conditions of two years data combined stepwise regression indicated that

all the employed environmental factors explained 84 % of the in disease development,

Out of 200 germplasm lines/ varieties only 16 lines were highly resistant and 9 were

resistant. Protective spray of fungicides (Dolomite and Shelter) reduced the disease 62 to

63 % respectively.

ACKNOWLEDGEMENT All appreciations and acclamations are for Almighty Allah, the Compassionate, the ever

Merciful, the only Creator of the universe and bestowed the mankind with knowledge and

wisdom. The humblest thanks to the Holy prophet Hazrat Muhammad (peace be upon

him), for the directive “Acquisition of knowledge is obligatory upon every Muslim man

and women”.

The author feels great pleasure and honour to express profound gratitude

and appreciation by heart and soul to Dr. M. A. Khan, Professor , Department of Plant

Pathology, University of Agriculture, Faisalabad, whose affectionate supervision,

scholastic guidance, encouragement and constructive criticism enable the author to plan,

execute and complete this task.

Heartful thanks are extended to Dr. Nazir Javed, Associate Professor,

Department of Plant Pathology, University of Agriculture, and Faisalabad for his

valuable suggestions to improve this dissertation.

Special thanks are also due to Dr. Muhammad Jalal Arif, Associate

professor, Department of Entomology, University of Agriculture, Faisalabad, who spared

the time without any hesitation for kind guidance, encourageous suggestions to improve

the dissertation.

Sincerest thanks to S. T. Sahi, Associate Professor and Principal;

Agricultural College Depalpur who contributed a lot of moral support during this study

and experiments.

Lastly the author is grateful and lacks words to express his heartful thanks

to parents who prayed for my success, along with economic supports and inspired from

time to time.

(M. SHAKOOR)

DEDICATIONS

Dedicated to Holy Prophet MUHAMMAD (Peace be upon him)

the greatest social reformers of human beings.

To my mother and father, who always prayed for my success in

this life and hereafter.

Chapter 1

INTRODUCTION Bread wheat (Triticum aestivum L.) is the main staple food crop and major

source of nutrition for the people of Pakistan. It is grown in winter months of

November to April on an area of 27.69 m. ha. (Anonymous, 2007a). Average

wheat yield of Pakistan is 2.38 mt/ha, which is extremely low as compared to

other wheat producing countries of the world such as Ireland, Denmark, United

Kingdom, France, Egypt and Saudi Arabia having 7.86, 7.83, 7.78, 6.23, 6.15

and 4.48 mt/ha, respectively (Anonymous, 2007b). Many factors contribute to

low yield in Pakistan, diseases being one of them. Karnal bunt (partial bunt) of

wheat caused by Tilletia indica (Mitra) Mundkur causes considerable losses in

the yield of wheat. The disease not only reduces the weight of grains, but also

deteriorates its quality and makes it unacceptable for human and animal

consumption (Gopal and Sekhon, 1988). According to Pamela et al., (2004) it

fulfills the following criteria of its inclusion in the list of emerging infectious

diseases (EIDs) of plants, (i) it has increased incidences, geographical

distribution and host range (ii) it has changed its pathogenesis (iii) it has been

newly evolved or newly recognized. The emergence of plant EIDs is similar to

those of humans, wildlife and domestic animals (Pamela et al., 2004). Modern

changes in the agricultural pattern, implementation of modern farming

techniques, ecosystem, intensification and globalization stimulate the attacking

mechanisms of the pathogen, which pose threats to agriculture or biodiversity

conservation. According to Dobson and Foufopoulos, (2001) understanding of factors

driving the plant EIDs require knowledge of host-parasite biology, use of multiple-host

system models parameterized for a large number of environmental, ecological and

biological factors.

Karnal bunt was first detected in 1931 at Karnal in Haryana, India and hence it is

called Karnal bunt (Mitra, 1931). It bears many names such as Karnal bunt, new bunt,

partial bunt, incomplete bunt, Indian bunt and stinking smut. Singh et al., (1989)

reported that Karnal bunt was a disease of wheat, durum, rye and triticale (a

hybrid of wheat and rye). Though the disease is native to South Asia but subsequently

it has been reported from Iran, Syria, Afghanistan, Iraq, Mexico (Joshi et al., 1983),

Nepal (Singh and Dhaliwal, 1989) and United States (Ykema et al., 1996). The disease

remained less damaging till late 1970 but subsequently severe epidemics started

occurring coinciding with the change over to high yielding, irrigated, semi dwarf and

high fertilizer input farming.

In early 50s, Karnal bunt disease was considered to be a disease of minor

importance and it was confined to hills of Pakistan. In 1971, the disease was reported

from Sialkot, Gujranwala and Mardan districts during the crop years of 1966-71

(Hassan, 1971) and the frequency of disease ranged from traces to 2.0 percent. The

disease remained endemic for considerable period of time in the Northern area of

Pakistan and later it spread to south and was reported as far as Jhang, Khanewal and

Muzaffargarh district of the Punjab (Bhatti and Ilyas, 1986). A little later the disease

became wide spread throughout the Punjab and was prevalent in 23 districts with a

frequency range of 0.32 to 3.50 per cent (Ilyas et al., 1989a). At present almost all

commercial varieties of wheat under cultivation are susceptible to Karnal bunt and the

disease incidence is aggravating with the passage of time. The disease reduces wheat

yields and can cause a fishy, unpalatable odor and taste in wheat flour, reducing flour

quality (Bonde et al., 1997; Sekhon et al., 1980; Singh and Bedi, 1985). Since karnal

bunt affects the international trade of commercial wheat grain and movement of

germplasm, the presence of the disease can cause economic loss to wheat exporting

countries (Bonde et al., 1997). The yield and quality losses are generally minor; the

most economic loss can be attributed to the quarantine status of the pathogen

(Babadoost, 2000; Butler, 1990).

The pathogen infects the ovaries in the emerging wheat heads and converts the

grains partially or completely into dark colored powdery masses of teliospores. The

diseased fields emit a foul smell like that of rotten fish due to production of

trimethylamine. Karnal bunt can reduce wheat yields. There is no estimate of losses, due

to this disease, occurring in Pakistan; however, survey in India conducted during the

years of heavy disease revealed a total loss of 0.5 percent, but in some fields where 89

percent of the kernels were infected, the yield losses ranged from 20-40 percent in highly

susceptible varieties (Anonymous, 2004). Brennan et al., (1990) estimated the economic

losses from Karnal bunt of wheat in Mexico to be US $ 7.02 million per year. Besides

yields losses, Karnal bunt can reduce wheat flour quality due to fishy, unpalatable odour

and taste, if a grain lot contains 1-4 percent infected seed (Bonde et al., 1997; Hussain et

al., 1988; Mehdi et al., 1973; Sekhon et al., 1980). If in a grain lot 5 percent of the grain

is infested, the quality of the flour recovery and chemical changes in composition of flour

and gluten contents cause poor dough strength (Sekhon et al., 1980; Gopal & Sekhon,

1988). Karnal bunt is also a disease of quarantine interest and it affects the international

trade of commercial wheat grain and movement of wheat germplasm through out the

world. Thus presence of diseased grain in wheat lots can cause economic loss to wheat

exporting countries (Bonde et al., 1997, Babadoost, 2000; Butler, 1990).

Karnal bunt differs from other diseases of wheat in that the pathogen infects

plants during anthesis and it sporulates on the same generation of the host which it

infects. Neither all spikes of plant nor all grains in spike are affected by the disease and

usually a few irregularly distributed kernels are bunted (Mitra, 1935; Bedi et al., 1949;

Dhaliwal et al., 1983). Infection of individual kernels varies from small points of

infection to completely bunted kernels but completely infected ones are rare (Chona et

al., 1961). The embryo of the infected kernels usually remains undamaged except when

infection is severe (Cashion and Luttrel, 1988) but the endosperms of the kernels get

shrunken to varying degrees. At maturity severely infected kernels are filled with

teliospores of the pathogen which serve as primary source of inoculum. The spikes of

infected plants are generally reduced in length with less number of spikelets (Mitra,

1937). During harvest, infection sori are broken resulting in contamination of healthy

seeds, soils, equipment, machinery and the vehicles with the liberated spores. The spores

may be blown by wind for long distances.

The teliospores of the Karnal bunt fungus can survive in soil for more than 5

years (Krishna and Singh, 1983b; Babadoost et al., 2004; Bonde et al., 2004). Although

many control strategies have been suggested for the management of Karnal bunt disease

and the strategies include seed treatment with hot water and solar energy, seed treatment

with fungicides and soil drenching with fungicides (Anonymous, 2005), however, the

results were not convincing. The cheapest and the most feasible method of Karnal bunt

control is the use of host resistance and breeding for varieties resistant to Karnal bunt

disease. Control of Karnal bunt has now become a major concern in Pakistan due to

scarcity or non-availability of resistance in commercial wheat varieties under

cultivation. The gravity of the situation of the disease calls for evaluation of

fungicitoxicants against the disease for its management. As the pathogen is soil, seed

and air-borne, it can penetrate locally into host plant, so application of spray fungicides

is very critical. Epidemiological factors have great influence on the epidemic

development of karnal bunt disease. Wheat is vulnerable to Karnal bunt fungus only

during a 2-3 week windows of its’ physiological development stages if the environmental

conditions happen to be conducive during this short period for successful infection and

the weather favourable for the disease development does not exist every year (Workneh

et al., 2008). Therefore spraying for the disease every year would be unnecessary waste

of time and resources. Characterization of environmental conditions conducive to partial

bunt disease is required to decide the timing of fungicide application. Development of a

disease predictive model for the chemotherapeutic control of this disease was the main

objective of the studies which was accomplished with following line of work: 1. Survey for the incidence of Karnal bunt disease of wheat in major

wheat growing areas of the Punjab.

2. Screening of advanced lines/varieties received from Wheat

Research Institutes, Faisalabad by artificial inoculation.

3. Determination of correlation of environmental factors with Karnal

bunt disease of wheat.

4. Development of a disease predictive model to determine the

relationship of environmental factors with disease incidence.

5. In vitro and in vivo evaluation of spray fungicide for the

management of Karnal bunt disease of wheat

Chapter 2

REVIEW OF LITERATURE

History and geographical distribution Karnal bunt was formally recorded in 1930 at the Botanical Research Station, Indian

Agricultural Research Institute (IARI), Karnal, Haryana in northwest India (Mitra, 1931),

now in the Punjab region of India.

The first report of Karnal Bunt from a non-Asian country was received from

Mexico in 1972, but the disease has been confined to localized areas within the state of

Sonora (Anon. 1991a). The Karnal bunt pathogen (Tilletia indica) was not identified

outside Asia until 1972, when it was reported from the state of Sonora in northern

Mexico (Fuentes-Davila, 1998; Sansford, 1998). At that time, the disease was restricted

to the Yaqui and Mayo valleys in Sonora and was found only in traces in farmers’ field.

However, in the early 1980s, during surveys in these valleys, Karnal bunt disease was

found in 64 % of the farms (Charles et al., 2005).

According to Boratynski et al., (1985) and Marshal et al., 2003, Karnal bunt

entered in the United States and Canada from Mexico, because in October 1983 the

pathogen was intercepted, as bunted kernels, at the Calexico, CA, and Port of entry in

Mexican boxcars. Subsequently, Mexican boxcars were found frequently contaminated

with bunted wheat grains, and viable teliospors of T. indica were found in dust and debris

in 9 of 20 randomly selected boxcars. It was concluded that Mexican boxcars destined for

United States and Canada were the best potential path way for the artificial movement of

Karnal bunt pathogen into the United States. On 8 March 1996, the first report of disease

in the United States was documented on durum wheat in Arizona, and a few weeks later

it was confirmed on bread wheat also in California (Ykema et al., 1996). Samples of

wheat, maintained by the Arizona Department of Agriculture, were tested for the

presence of Karnal bunt pathogen, and teliospores of T. indica were found in grains

harvested in 1993, indicating that the disease had been in Arizona in 1992. It was soon

discovered that bunted wheat grains also had been shipped to, and planted in, New

Mexico and Texas (Ykema et al., 1996). The disease has later been established in the

New World in northwest Mexico (Warham, 1986) and has been reported in southern

California, Arizona and Texas in the US (Rush et al., 2005). Da Luz et al., 1993 reported

the presence of T. indica in a seed lot produced in Rio Grande do Sul state, in 1989 but

there have been no reports since and establishment has not been proven.

T. indica was first noted from Brazil in 1993 in the southern part of the Rio

Grande do Sul and some efforts were made practically to get rid of it (Da Luz et al.,

1993). However, no further publications of its existing status in Brazil have been made. It

entered into the Brazil through imported wheat germplasm for research purposes during

accessions between 1990 and 1992 (Mendes and Ferreira, 1994, Oliveira et al., (2002)

. Karnal bunt also occurs in an area of Northern Cape Province in South Africa

(Crous et al., 2001). However, between 2002 and 2004 it was found to have spread to a

number of new areas (Naudé, 2002, Anon., 2004a). All locations where pathogen

(Tilletia indica) has been found to lie on or close to latitude 30o north and 30o south and

declared as having arid or semi-arid climates with mild/cool winters and hot summers. In

the main, they have been recognized as dry environments ranging from desert with little

or no rain to ‘arid steppe’ or ‘semi-desert’ with a short rainy season. In the plains of

northwest India, the climate is termed sub-tropical summer rain; Karnal bunt also has

been recorded in some fringing environments, but incidence is very low and outbreaks

can be sporadic. The climates of these areas are termed ‘subtropical rain’ and ‘tropical

summer rain’ (Pearce and Smith, 1990; Stefanski et al., 1994).

Regional distribution and gradual dispersal of the pathogen within a country

indicated that the disease has a close relationship to the environmental condition specific

to that area. The disease is not common in the Indian subcontinent out side the North

West region. Disease incidence is very low in central and eastern India. Partial bunt or

Karnal bunt disease has never been found in the south or northeast of the Indian

subcontinent (Anon. 1996; Joshi et al., 1983; Singh, 2005).

It has never been reported in the field in ‘oceanic temperate’, ‘temperate

continental’ or ‘sub-tropical winter rain’ environments prevailing in Europe. During

domestic survey and investigations research workers from UK had collected 25 samples

of wheat grain during the harvesting years 2002, and 2003 and 50 samples during 2004,

tested by using the EU/EPPO-recommended T. indica Diagnostic Protocol (Inman et al.,

2003, Anon., 2004c), all of which also proved negative for T. indica. The situation

regarding specific surveys for the presence of T. indica in other European Union

countries is unknown. However, Hungary and Lithuania are preceding inspection of

wheat grain and triticale originating in countries affected by T. indica (Talevi et al.,

2004).

The pathogen has not been reported in southern areas of India and Mexico due

to different environments to areas in these countries where the disease is established,

although there have been many chances for internal spread (Fuentes-Davila, 1996a;

Singh, 2005). In the northwest of India, where incidence of Karnal bunt is relatively high,

rain-fed wheat is sown in late October and irrigated wheat is sown in November.

Harvesting generally starts in mid-May. Four to six irrigations are recommended with

one coinciding with flowering (Anon., 2006).

In Pakistan, it was known to occur in Sialkot district of the Punjab in 1950

(Saleem and Akhtar, 1988). Mundkur, (1943) and Bedi et al., (1949) reported this

disease from Punjab and NWFP. They also reported that the disease was airborne

and increased with excessive irrigation. In the year 1986-87, it appeared in

epidemic form in the majority of wheat growing areas of the Punjab and upto

30% disease was recorded in wheat grain samples of various commercial

cultivars collected from the 17 districts of the Punjab. (Anon., 1986; Ilyas et al.,

1989). During the epidemic years, the disease also caused substantial losses to

crop (Bhatti and llyas, 1986). Teliospores of Tilletia indica were also found in

23.8% (5 of 21 samples) of germplasm lots from Pakistan in 1984 (Diekmann,

1987). Mirza (2005), during a survey collected 254 Karnal bunt samples from

Punjab and 96 from North West Frontier Province (NWFP) during growing

season 2003-04. Samples of the varieties MH-97, Inqilab-91 and Wattan which

were mostly grown in the wheat growing areas of the Punjab were infected with

Karnal bunt disease. Almost 83.33% from Kasur and Okara, 66.66% from

Lodhran, 60% from Pakpattan, 50% from Sialkot, Rawalpindi and Khushab,

47.6% from Gujranwala, 44.44% from Faisalabad, 32.14% from Bahawalpur and

15% from Multan were infected with Karnal bunt disease of wheat.

With the passage of time, the spread and geographical distribution of

Karnal bunt disease of wheat increased. Mustafa, (1965) reported the disease

from Iraq. The disease had also been recorded in southern Nepal (Singh, et al.,

1989), and reported from southern Iran (Torarbi et al., 1996). Wheat from

Afghanistan (Locke & Watson, 1955), Lebanon, Syria and Turkey (Lambat et al.,

1983) has been found contaminated with teliospores of T. indica, although the

pathogen has never been in the field in these countries (Warham, 1986, 1992).

Later wheat germplasm consignments from Lebanon, Syria and Turkey were

found free of teliospore contamination (Diekmann, 1987). The status of the

disease in these countries was unclear. However, an international survey using a

standardized protocol has been suggested as the only way of determining the

exact distribution of the Karnal bunt disease (Babadoost, 2000).

Survey and economic losses Karnal bunt disease caused Tilletia indica (Mitra) Mundkur) was reported

from Karnal, India for the first time; hence it was named as Karnal bunt. In India and

Pakistan, variations in losses have been noted during unusual and uneven surveys carried

out in different years. However, there is trend of increasing incidences with passage of

time. Royer and Rytter, (1985) stated that semi-dwarf cultivars of wheat imported from

Mexico were widely-grown in India during 1965. These cultivars were more susceptible

than those of used earlier and were the major cause of the increase in disease incidence

between 1969–1975. Indian farmers apparently replanted harvested seed irrespective of

whether conditions, thus perpetuating T. indica in the soil.

Agarwal et al., (1976) reported that Karnal bunt disease was considered

endemic in India until the 1969–70 wheat growing season when most of the imported

Mexican cultivars grown were affected by the disease with a maximum of 7.5% seed

infection in the cultivar S-331 in the foothills of the Himalayas of Uttar Pradesh. Joshi et

al., (1983), highlighted the occurrence of Karnal bunt in the ‘plains of undivided India’ as

being at ‘trace levels’ in the early years. During 1969–70 it was widespread in Delhi,

Punjab, Haryana, Rajasthan and western Uttar Pradesh. By 1974–75 it was reported as

severe in many areas of northern India, especially in the foothills of the Himalayas and

the Tarai region of Uttar Pradesh, Punjab and Himachal Pradesh; disease severity was

described as especially high in Hempur in Uttar Pradesh (15–23%). Percentage of lots of

8 wheat cultivars falling into different ranges of seed infection; the maximum being 8.3%

of the lots of cultivar HD-2009 falling into the 20–50% seed infection category.

Aujla et al., (1977) described that the ‘infection % of the bunt’ during 1975–

76 on different cultivars ranged between 2.2 and 52.9% in Jammu and Kashmir and 1.4

and 25.1% in the Punjab. They found that the incidence of disease was highest on late

flowering cultivars which coincided with temperatures of 18–22°C and >70% relative

humidity. A survey conducted in 1981 revealed that the disease was present in areas

where it had previously been unreported including new locations in central India (Zhang

et al., 1984).

Singh, (1994) summarized the distribution, incidence, and severity of Karnal

bunt disease in Uttar Pradesh containing 36% of the area of land under wheat production

at that time in India. A Karnal bunt distribution map was arranged for surveys. It was

noted that Karnal bunt was commonly distributed in various western and eastern districts

of Uttar Pradesh with northern hill and southern dry regions being generally free. It was

severe at many places in northern India especially in the foothills of the Himalayas, Uttar

Pradesh, Punjab and Himachal Pradesh. Disease severity was as high as 15–50% at

Hempur and Pantnagar in Uttar Pradesh. During 1975–76 maximum disease incidences in

India were found in the Punjab where 39% of grain samples were infected as compared to

9% in Haryana. During 1977–78, 38% of field samples from Haryana were infected as

compared to 25% in Himachel Pradesh and Kashmir. The disease increased in 1978–79

when the incidence of field samples infected in Punjab, Himachel Pradesh and Kashmir

were observed 78%, 71% and 89% respectively. Karnal bunt was also reported from

West Bengal, Bihar, Madhya Pradesh and Gujarat covering eastern, central and western

parts of India.

Singh et al., (1996) conducted post-harvest surveys from 1986–1994 in north-

western India and pointed out a high level of infection in the climatically favourable

years of 1986, 1987, 1990 and 1991 (34, 25, 21 and 16% of samples infected). Beniwal et

al., (2000) reported that in the state of Haryana (south of the Punjab) Karnal bunt

infection ranged from 0.05–9.90% and 0.05–0.30% during the 1995–96 and 1996–97

cropping seasons, respectively.

Indu-Sharma et al., (2004) highlighted the position of different grain markets of

Punjab in India with regards to status of Karnal bunt disease of wheat. Less affected areas

during the last 11 years (1994-2004) from where wheat may be procured for foreign

exchange at the grain market level or by contract farming were identified. Every year

variations were noted in the disease as an evident from the percent disease free samples

recorded in different years. Amongst different districts, the Karnal bunt disease incidence

was least in Patiala district where 85% samples were free from Karnal bunt disease

followed by samples from Amritsar (81.12%). Average Karnal bunt free samples were as

high as 90% and above in seven grain markets of the Punjab (Amritsar, Bagharian,

Bhadson, Muktsar, Kotshmeer, Delhon, and Bhasor). Varshney et al., (2004) during

monitoring the incidence of the disease rejected the certified seeds due to infection

caused by Tilletia indica in 1025 seed samples of 15 wheat cultivars from main wheat

producing areas in India and the number of discarded grains varied from seed lot to seed

lot of the same cultivar. The rejection for cultivar HD-2009 was 0.25% in 1995-96, but

was 100% in cultivar HS-240 in the same year.

Singh et al., 2002, collected the wheat grain samples from Aligarh, Faizabad,

Varanasi, Gorakhpur, Uttaranchal, Meerut, Dehradun, Udam, Singh Nagar, and Uttar

Pradesh, India during 1997-98 and 2000-01 to assess the scope of the disease incidence in

wheat caused by Tilletia indica in the two provinces of India. In an associated

experiment, seed grains of 15 bread wheat varieties cultivated by farmers as certified

seeds were evaluated for the confirmation of disease in Uttaranchal and Uttar Pradesh

India, during 1998-2001. Udam Singh Nagar indicated the highest number of diseased

seed grains in 1998 (57.4%) and 1999 (53.3%), whereas Dehradun recorded the highest

number of infected seed grains in 2000 (35.7%). The Maximum number of wheat seeds

plots discarded due to karnal bunt was recorded during 1999; however, wheat seed plots

from Gorakhpurh revealed the highest karnal bunt incidence during 1998. Seeds of

cultivar Sangam recorded the highest karnal bunt infection (36.4%), whereas HP1633

recorded the lowest (10.5%). Only the seeds of cultivar HP1633 were not rejected due to

karnal bunt.

In India Gill et al., (1993a), reported that the percentage of bunted wheat

kernels increased with passage of time. Infected kernels that were noted 25 % in 1997-78,

increased up to 86 % in 1982-83 cropping season. The percentage of wheat seed samples

with bunted seeds were 9, 34, and 17 in Haryana during the 1874-75, 1977-78, and 1978-

79 cropping seasons, respectively, and 60% of samples had infected kernels in 1982

(Singh et al., 1986). In 1989-90, 1990-91 and 1991-92, 62, 63, 94 and 62 % of wheat

samples respectively (Gill et al., 1993). However, data recorded by survey teams in

northwest India revealed that even during the worst epidemic years, the loss was only 0.2

to 0.5 of total yields (Joshi et al., 1980). According to Singh, (1994), the state of Utter

Pradesh in northeast India exhibited less than 1% losses even in the worst years.

A detailed analysis of wheat samples collected from 15 districts of eastern

Uttar Pardesh in 1987, a year of particularly high epidemics in India, revealed that an

average of 3.79 % of seeds per samples were infected (Rai and Singh, 1989; Rai et al.,

1988). In 1978-79 one of the wheat fields in Madhya Pradesh had 23.5 % of the seeds

infected. (Singh and Prasad, 1980). Singh et al., 2001, conducted a survey to collect the

samples from different wheat grain markets in Punjab, India during the period of bumper

crop yield with maximum wheat production, i.e. from April to mid-May in 1999 to

Karnal bunt disease. Analysis of the samples revealed that the Karnal bunt disease was

well-spread in Punjab. From a total of 552 samples collected, 42.7% samples were

infected by T. indica. The maximum bunted samples were found in Faridkot district

where the prevalence of the disease was 92.8%, whereas the minimum prevalence of bunt

was 3.4% in Amritsar district. The overall disease incidence in Punjab was 0.24%. The

disease incidence ranged from 0.002% in Kapurthala district to 1.34% in Ropar district.

The samples from Ropar (2.53% incidence) and Sangrur (2.13% incidence) revealed the

endemic occurrence of the disease in certain areas of Punjab. Wheat cv. HD 2329 showed

higher susceptibility to Karnal bunt compared to cv. PBW 343, which occupies the

maximum area in Punjab. The prevalence and incidence of the disease were 53.6 and

0.36%, respectively, on HD 2329, and 35.8 and 0.20%, respectively, on PBW 343. The

survey also indicated the existence of low bunt areas in Punjab, which need to be more

specified by continuous extensive surveys in these areas. Singh et al., 1999, reported that

during 1989-90 to 1996-97 wheat grain samples were collected from markets in different

districts of the Indian Punjab. No completely Karnal bunt free area was found in the state

but some areas of low disease severity were observed in Bathinda.

In Pakistan, Ilyas et al., (1989b) conducted a survey of 29 districts of Punjab

during the crop season of 1987-88 to determine the Karnal bunt infection. Grains samples

were collected randomly from grain markets and farmer’s fields. A composite sample of

25g was drawn from each main sample to work out the percentage incidence and range of

infection of karnal bunt in different districts of the Punjab. The disease was found to be

widespread in 23 districts of Punjab. The disease was; however, completely absent in six

districts namely Multan, Muzaffargarh, Layyah, R.Y. Khan, D.G. Khan and Rajanpur.

Ehsan et al., (2002), conducted a survey in 78 wheat growing localities of

Pakistan to assess the prevalence of Karnal bunt disease. The highest disease incidence

was observed on cv. WL-711 (46.0%) grown in Hathion. High disease incidence was also

recorded for Pak-81 (0.47-6.32%) and Pirsabak-85 (0.41-5.57%) and for the districts of

Mangora (2.53-23.42%), Mardan (0.41-46.00%), Malakand (6.32%), and Swabi (0.54-

5.85%). Wheat-growing areas in Manshera (0.42-1.04%), Peshawar (0.64-2.36%),

Abbottabad (0.00-1.37%), and Attock (0.00-1.25%) had low to medium disease

incidence. Bhutta et al., 1999, conducted a survey in Punjab. A total of 730 wheat seed

samples were tested to assess the incidence of karnal bunt using the dry inspection

method from 1993/94 to 1996/97. High infection percentage (3%) of karnal bunt in

various seed lots was noted. Seventy three % and twenty five % of the samples from

Central Punjab and northwest areas of Pakistan respectively were found contaminated

with bunted grains. Southern parts of the country were found free from 1994/95 to

1996/97.

Mirza, (2005) conducted a comprehensive survey and on farm disease

assessment in Punjab and North West Frontier Province (NWFP). During his survey he

collected three hundred and fifty Karnal bunt samples from both the provinces during

2003-04, of which 254 were from the Punjab and 96 from NWFP. Samples from the

varieties MH-97, Inqilab-91 and Wattan which were mostly grown in wheat growing

areas of Punjab were found infected with Karnal bunt disease. Almost 83.33 % samples

from Kasur and Okara 66.66 % from Lodhran, 60.00 % from Pakpattan, 50.00 % from

Sialkot, Rawalpindi and Khushab, 47.6 % from Gujranwala, 44.44 % from Faisalabad,

34.48 % from Sahiwal, 33.33 % from Lahore and 32.14 % from Bahawalpur were

affected.

Singh, (1998) assessed the yield losses upto 0.16 % in an epidemic year in the

foothills of the Himalayas. Brennan et al., (1992) divided the losses from Karnal bunt

into two types: direct losses and indirect losses. Direct economic costs caused by disease

affecting quality included :- (a) value of the yield loss; (b) value of quality loss (c) the

cost of handling and marketing infected and infested product and (d) economic cost of the

loss of markets through quarantine or marketing restrictions imposed following the

presence of the disease (loss of seed and grain exports). Indirect loss or control costs

aimed at preventing the spread of disease or reducing its’ severity includes: (a) cost of in-

crop control measures. (b) cost of quarantine or regulatory restrictions imposed on the

production and marketing of crops (c) regulatory cost associated with monitoring the

disease and (d) costs associated with extra processing or fumigation of the output from

infested area.

Karnal bunt normally has nominal impact on wheat yield. However,

comparatively small numbers of infected grain have a major effect on germinability and

quality of wheat products (Warham, 1986). The pathogen was not identified outside of

Asia until 1972, when it was reported from the state of Sonora in northern Mexico

(Fuentes-Davila 1998; Fuentes-Davila and Duran, 1986). The yield losses reported in the

literature vary from <1 % to 20 % and average losses due to Karnal bunt disease are less

than 1 % in effected areas of India, Pakistan and Mexico (Gordan and Brennan, 1998).

Brennan and Warham (1990) estimated the yield loss in north-western

Mexico to be 0.12 % per year and quality loss at 0.69 in the value of crop in infested area.

Fuentes-Dávila, (1996a) during a survey reported that levels of Karnal bunt were

‘noticeable’ in 1981–82 in the Yaqui and Mayo valleys in the state of Sonora,

Mexico.The disease remained limited to north-western Mexico reaching around 400 km

north, south and west with incidence varying annually depending upon meteriological

conditions. Disease dominated in years when high relative humidity and rainfall

coincided with anthesis of the crop. Low disease incidence developed in the Yaqui valley

in 1982, 1984, 1987, 1988, 1990, 1994 and 1996 with surveys showing 85.0 to 99.7% of

samples tested to be disease-free. The highest levels of infection established in 1983 and

1985 with 7.5 and 11.2% of samples with >1% infected grain.

Due to the favorable environmental conditions at the vulnerable growth stages

of wheat Karnal bunt disease has a considerable potential to establish in Australian wheat

belt. Sixty seven suitable sites were found at risk from Karnal bunt disease during survey

and modeling. They also expected a loss of $A 491 million per year due to the Karnal

bunt disease of wheat. These sites were through the southern wheat belt from Western

Australia to central to New South Wales. (Gordon and Brennan, 1998) Murray et al.,

1996, estimated the losses due to karnal bunt in Australia including direct and indirect as

54.46 $A/ ton of grain.

A national Karnal bunt survey programme was arranged in 1996 for the

confirmation of Karnal bunt disease only in localized areas and not widespread in the U.

S. wheat crops. The fundamental motto of the survey was to provide U. S. certifying

official the data to issue phytosanitary certificates demanded by the countries which

import U.S. wheat. Approximately 650 counties in 39 states participate in the national

survey each year, with samples ending by the middle of September. Bread wheat, durum

wheat and triticale were incorporated in the survey. Samples were collected from

countries where the vulnerable cultivars were grown (Charles et al., 2005). When

national survey was initiated in 1996 a considerable numbers of samples were

compulsory to confirm the limited distribution of Karnal bunt disease (caused by Tilletia

indica) in the United States. During the first year of the national survey, a field had been

quarantined at the discovery of a single teliospore of T. indica, but in 1997, United State

Department of Agriculture (USDA) and Animal and Plant Health Inspection Services

(APHIS) adopted the bunted kernel standard.

Primarily the monitoring of wheat grain samples for bunted kernels was done

by visual observation either on a plastic tray or in a vibrating grain inspection machine

(Anon. 2002). Since the samples typically consist of several thousand kernels, analysis

was tedious. In the beginning of 2002, a high-speed optical sorter was used that

selectively removed, based on seed discoloration, suspected bunted kernels from the

samples (Dowell et al., 2002). This technology to a large extent reduced the amount of

material to be examined manually and is now used for national Karnal bunt survey

samples.

In May 2001 USDA confirmed that wheat in an elevator in Young County,

TX. tested positive for Karnal bunt samples.. Consequently, during survey collection of

grain samples by APHIS, in and around the Young County, confirmed the presence of T.

indica in the adjoining Texas counties of Archer, Throckmorton and Baylor. In 2002,

because of convenience of a Kb positive field to the county line, the regulated area was

further extended into neighboring Knox County, but no Kb-positive field had been

identified in that county. Vocke et al., (2002) reported that the Karnal bunt regulatory

programme allows the US Department of Agriculture to issue phytosanitary export

certificates stating that wheat in a given shipment is from an area where Karnal bunt is

not known to occur.

Charle et al., (2005) during a comprehensive survey found that over 85 % of the 399

fields that have tested positive for Karnal bunt disease. The maximum number of bunted

kernels collected from individual field has 478 in Arizona in 2004, 209 in California, in

2004 and 224 in Texas in 2001 with 1.0, 0.46, and 0.5 % respectively.

Taxonomic status Tilletia indica (Mitra) Mundkur (synonym Neovossia indica Mundkur)

belongs to class Ustilaginomycetes, phylum Basidiomycota, order Ustilaginales, family

Tilletiaceae and genus Tilletia specie indica. Black, dusty-appearing teliospors of the

fungus gave it the name “smut” (Boned et al., 1997). The class name is derived from

“ustulatus”, meaning burned, in suggestion to the blackened appearance of the infected

plants (Carris, et al., 2006). Cereal-infecting species of Tilletia that produce teliospores

within the ovaries of their hosts plants are generally called bunt fungi, also considered to

be derivation of word burned (Duran and Fischer, 1961). Mundkur (1938, 1940) stated

that T. indica “probably belongs to the genus Neovossia” based on the large number of

non-conjugating basidiospores produced by the fungus. Based on a detailed taxonomic

study Krishna and Singh, (1982) justified its placement in Neovossia indica. However,

western scientific literature prefers to designate the name of causal agent of Karnal bunt

as Tilletia indica (Duran and Fischer, 1961; Duran, 1972). Symptoms of the disease

The Partial bunt is too much difficult to detect under field conditions, and

generally careful examination revealed evidence of disease (Morris et al., 1997, and

Bonde et al., 1997). Only a few kernels of some wheat heads become partially infected,

except, in severe cases in which whole of the kernels in an ear are replaced with fungal

sorus. There may be a slight swelling or darkening of infected florets. Therefore, Karnal

bunt is easiest to detect by observing the harvested grains carefully. The pathogen

converts the infected ovary into a sorus where a mass of dark brown coloured teliospores

are produced. Small sori are generally formed in longitudinal furrow, leaving the rest of

seed unaffected. However, in severe cases the major part of the endosperm along the

longitudinal furrow may get spoiled. Partially infected grains occur in clusters of

spikelets and many such aggregate clusters may be present in a spike (Nagarajan et al.,

1997).

In a standing wheat crop, infected spike can be detected by the shiny silvery

black spikelets with glumes spread apart and swollen ovaries. The infected grains emit a

fishy odour due to trimethylamine and wheat products from severely infected grains are

unpalatable. Cashion and Luttrell, (1988) reported that the pathogen (Tilletia indica) did

not invade the embryo and the mycelium growth was limited to the pericarp.

Transmission electron microscopes (TEM) study revealed that the mycelium proliferated

in the pericarp by disintegrating the middle layers of parenchymatous cells and prevents

fusion of the outer and inner layers of pericarps, with the seed coat. The mycelial mat

formes a compact hymenium-like structure which gives rise to short, septate stalks with

single teliospores (Roberson and Luttrell, 1987).

Host range Tilletia indica infects bread wheat (Triticum aestivum L.), durum wheat

(Triticum turgidum Desf.), and Triticale ( x Trititicosecale Wittm.) under natural

conditions (Fuentes-Davila et al., 1996). The wild wheat species Aegilops geniculata

Roch (as T. ovatum), Aegilops sbaronensis Eig., A. pereggrina (Hack.) Maire and Weiller

var. peregrine (as T. variabilis), and “Triticum scerrit” are reported as hosts for Tilletia

indica, (Aujla et al., 1985). With artificial inoculation, T. indica infects triticale, 3 species

of Triticum L., 11 species of Aegilops L., 2 species of Bromus L., 3 species of Lolium L.,

and Oryzopsis miliacea (L.) (Royer and Rytter, 1988). Host range lists for Tilletia species

contained naturally occurring hosts and artificially inoculated hosts (Warham et al.,

1986) and hosts resulting from synonymy of similar species (Duran, 1987; Duran and

Fischer, 1961). Biology of the Tilletia indica

Dispersal of teliospores: Dispersal of Karnal bunt pathogen is documented by means of its’ teliospores.

The long distance dissemination of teliospores contaminating wheat seeds or grains is

believed to be in a manner similar to other bunt disease of wheat. (Wilcoxson & Saari,

1996). These bunted seeds are known as reservoir of teliospores. Besides being seed-

borne the spores can be carried to new areas through machineries and hand tools.

Teliospores cling to plant parts, clothes, farm equipments, vehicles, threshers, and

combine harvesters. They may also be dispersed by rain water and animals, including

insects and birds, both as surface contaminants and through faeces. Teliospores may be

transferred to new areas in the form of wind-blown inoculums (Bonde et al., 1987).

Teliospores also germinate successfully after ingestion by livestock and insects like

grasshoppers, providing another mean of dissemination (Smilanick et al., 1985b).

Teliospore dormancy:

Most freshly collected teliospores are dormant and unable to germinate

(Mitra, 1931; Banasal et al., 1983; Smilanick et al., 1985a). The highest rate of

germination occurres with one year old teliospores (Mathur and Ram, 1963; Kiryukhina

and Shcherbakova, 1976). Three types of teliospore dormancy have been noted in T.

indica. Teliospores taken from freshly harvested grains germinate poorly compared with

several months to a year or more old bunted grains. (Bansal et al., 1983; Mitra, 1935; and

Rattan and Aujla, 1990). This type of dormancy is known as postharvest dormancy that

occurs in T. horrida also (Chahal, et al., 1993). The second type of dormancy is long-

term dormancy, in which germination rarely exceeded 50% under optimal laboratory

conditions and it remained between 15%–30% in most reports, although teliospores were

stored for more than one year. This type of dormancy, contributes to teliospore survival

under field conditions. The third type of dormancy can be induced by cold temperature.

Dry teliospores of T. indica kept at −18◦C progressively lost the capability to germinate

over a period of 12 weeks of treatment (Zhang et al., 1984). The teliospores in these

experiments were plated immediately after treatment and non-germinability was thought

to indicate lethality. However, the inhibition of germination was minimized if teliospores

were plated after a 20-day “thawing” period at 22◦C rather than immediately after the

cold treatment (Chahal and Mathur, 1992). Cold-induced dormancy had also been

observed in experiments performed over 4 to 8 days of freezing at 0◦C (Sidhartha et al.,

1995). Bedi et al., 1990 noted that with the passage of time germination of teliospores is

affected, 4-14 month old teliospores had maximum germination which declined with

further aging. Similar dormancy also occurs in Tilletia horrida (Chahal et al., 1993).

Cold-induced dormancy of the dwarf bunt pathogen T. controversa Kuhn also occurs

annually in natural field environments of temperate climates (Hoffmann, 1982; Hoffmann

and Goates 1981).

Nature of teliospores and their survival in the soil. Due to the hardy nature of teliospores of T. indica it remains viable in the soil

for a long period of time. Teliospores of T. indica, T. horrida, and other bunt species that

have been examined have relatively thick cell walls with four distinct layers, an

endosporium, a thin middle layer, an exosporium from which the ornamentation

develops, and an outer sheath (Hess and Gardner, 1983; Nawaz and Hess, 1987;

Roberson and Luttrell, 1987). The teliospore wall resists toxic gases and liquids including

methyl bromide and chloropicrin (Smilanick et al., 1988), hydrogen peroxide (Smilanick

et al., 1994), propionic acid, ozone (Rush et al., 2004), and degradation in the digestive

tract of animals (Smilanick et al., 1986). This enables teliospores of T. indica to survive

for several years in hot desert to cold temperate field environments (Babadoost et al

2004; Bonde et al., 2004; and Chib et al., 1990). Under typical laboratory conditions, T.

indica teliospores can survive for at least 16 years (Bonde et al., 1997)..

Teliospores are globose to subglobose in shape, 22-49µm in size, reticulate

with spines and are surrounded by a thick cover sheath of 2-4 microns (Nagarajan et al.,

1997). The peridium or sheath is fragile, fractured structure and is easily identifiable

under the electron microscope. The second sheath episporium is reticulate and has

numerous curved spines while the third layer is endosporium. Mature teliospores are dark

brown and immature spores are light brown Bonde et al., 1996.) Some hyphal fragments

on mature teliospores may be present. Pale orange to mainly dark, reddish brown to

opaque-black and densely ornamented with sharply pointed to truncate spines,

occasionally with curved tips make the distinguishable morphological characteristics of

T. indica compared with other two similar species T. horrida and T. walkri (Anon. 2004).

The teliospors of Tilletia indica are very resistant to adverse environmental

conditions and have been reported to survive in the contaminated soil for 2 to 5 years as

reported by various research workers (Chib et al., 1990; Krishna and Singh, 1983; Singh

et al., 1990). Teliospore, being the only source of survival in the soil provides the basis of

pathogen spread and play a vital role in the perpetuation of disease. Bonde et al., 2004,

conducted an experiment to study the survival of teliospores of Tilletia india in different

types of soils. A teliospore longevity study was initiated in Kansas, Maryland, Georgia,

and Arizona. Soil from each location was with T. indica teliospores and placed in

polyester mesh bags. The bags were placed within soil from same location with in

polyvinyl chloride pipes. Pipes were buried in the respective plots such that the bags were

at 5, 10 and 25-cm depths. Each pipe was open at the both ends to allow interaction with

the outside environment; however, it was fitted with screens preventing possibility of

teliospore escape. In the Karnal bunt–quarantine area of Arizona, bags of infested soil

also were placed outside the pipes. Teliospore-infested soil from each location was well

furnished with dry conditions in a laboratory. During the first 2 years, viability of the

teliospores of Karnal bunt declined more rapidly in pipes than outside pipes, and more

rapidly in fields in Kansas and Maryland than in Georgia or Arizona. After 2 years,

viability declined equally. In the laboratory over 3 years, viability decreased significantly

more rapidly in dry soil from Kansas or Maryland than in dry soil from Georgia or

Arizona, while pure teliospores remained unchanged. Consequentley Bonde et al., (2004)

concluded that soils, irrespective of weather, affect teliospore longevity.

Teliospores of T. indica are introduced into the soil at harvest and may persist

there for 45 months (Krishna and Singh 1983; Bonde et al., 1997; Singh, 1994 and

Warham, 1986). Teliospores on the soil surface germinate and produce primary and

secondary sporidia which, under favourable environmental conditions, infect plants at

flowering (Bonde et al., 1997; Singh 1994 and Warham 1986). Chib et al., and 1990, and

Gill et al., 1993a, reoorted that in India, T. indica teliospores survived for 3 years on soil

surface in the field, but spores buried 20 cm deep decreased to low viability after 2 years.

In another experiment Krishna and Singh 1983, investigated that in India, survival of

teliospores on the soil surface and at depths of 7.5 and 15 cm was 45, 39, and 27 months,

respectively. It was reported that viability of the teliospores of T. indica decreased with

increasing burial depth from 5 to 20 cm (Rattan and Aujla, 1990; Sidharta et., al 1995).

Teliospores survived better in loamy sand soil than in clay and sandy loam soil (Rattan

and Aujla, 1990). Singh, 1994, and Smilanick et al., 1989, demonstrated that the survival

of pathogen is longer in dry soil than in wet soil in the form of teliospores. In laboratory

conditions storage teliospores retained their viability for 5 to 7 years (Kiryukhina and

Scherbakova, 1976; Mathur and Ram, 1963 and Zhang et al., 1984.).

Study was conducted by Babadoost et al., (2004) for the assessment of

survival of T. indica teliospores in a location in the northern United States. Soils differing

in texture and other characteristics were collected from four locations, equilibrated to –

0.3 mega pascal (MPa), and infested with teliospores of T. indica to give a density of 103

teliospores per gram of dry soil. Samples (22 g) of the infested soil were placed in 20-μm

mesh polyester bags, which were sealed and placed at 2, 10, and 25cm depths in

polyvinyl chloride tubes containing the same field soil as the infested bags. Tubes were

buried vertically in the ground at Bozeman, in October, 1997. Soil samples were assayed

for recovery and germination of T. indica teliospores 1 day and 8, 20, and 32 months

after merging of teliospores into soil. Teliospores recovered from soil samples were 90.2,

18.7, 16.1, and 13.3% after 1 day and 8, 20, and 32 months after assimilation of

teliospores into soil, respectively, and was significantly (P < 0.01) affected by soil

source. The percentage of teliospore recovery from soil was the greatest in loam soil and

lowest from a silt loam soil. The rate of teliospores recovered from soil was not

significantly affected by depth of burial and the soil source–depth interaction during the

32-month period. The percentage of germination of teliospores was significantly (P <

0.01) affected by soil source and depth of burial over the 32-month period. The mean

percentage of teliospore germination at 1 day, and 8, 20, and 32 months after

incorporation into soils was 51.3, 15.1, 16.4, and 16.5%, respectively. In another

experiment, samples of silty clay loam soil with 5 × 103 teliospores of T. indica per gram

of soil were stored at different temperatures in the laboratory. After 37 months of

incubation at 22, 4, –5, and –18°C, the rates of teliospore recovered from soil were 1.6,

2.0, 5.7, and 11.3%, respectively. The percentage of spore germination from soil samples

was highest at –5°C. Microscopy studies revealed that disintegration of teliospores began

after breakdown of the sheath-covering teliospore. The results of this study showed that

teliospores of T. indica could survive in Montana for more than 32 months and remained

viable.

Teliospore germination Most locally infecting bunt fungi have echinulate, verrucose, or tuberculate

teliospores that germinate at approximately 200C–250C (Castlebury et al., 2005). Though

each teliospore generally produces one promycelium (Mitra 1931), several promycelia

may arise from a single teliospore (Krishna and Singh, 1981; Mitra, 1931; Warham

1988a; Rivera-Sanchez and Fuentes-Davila, 1997). Promycelia vary in length 500 µm

and bear at the apex a whorl of 32 to 128 or more primary sporidia (Mitra 1931).

Variation in the length of promycelial tips may occur (Peterson et al., 1984).

The effects of physical factors that influence teliospore germination in T.

indica and T. horrida have been studied extensively (Chahal et al., 1993; Dupler et al.,

1987; Krishna and Singh, 1983). Dupler et al., 1987, and Smilanick et al., (1985a)

reported that germinating teliospores were remarkably durable, strong and resilient

during the process even with wide swings in pH, temperature, and soil moisture including

freezing and desiccation (Dupler et al., 1987, Biswas, 2003). The basidium (also called a

“promycelium”) emerges through the ruptured wall of the teliospore and either produces

basidiospores instantly, or elongates to over 500 μm in length, depending on the species

and prevailing environmental conditions. Elongating basidia form retraction septa,

confining the cytoplasm to the apical region. A terminal whorl of 10 to 150 primary

basidiospores, also called primary sporidia, is formed. Castelebury et al., (2005) reported

that the number of basidiospores formed per basidium varied considerably among

different taxa and not all locally infecting species produce a large number of

basidiospores, and the number of basidiospores also varied considerably within a species

(Castlebury and Carris, 1999; Mitra, 1931; and Teng, 1931).

Basidiospores may elongate slightly after detachment, and become curved or

undulate before germinating to form either hyphae or a sterigma-like structure on which a

haploid, uninucleate, allantoids sporidium also called “ballistospore” ( Ingold, 1996) was

asymmetrically formed ( Singh and Pavgi, 1972). The allantoids sporidia formed by T.

indica, T. horrida, and other locally infecting Tilletia species studied had the same

morphology and forcible discharge (Ingold, 1996, 1997). Allantoids sporidia may

germinate directly via germ tubes or repetitively to produce additional sporidia. In

culture, passively dispersed, filiform sporidia were formed from short, lateral

sporogenous cells on the hyphae. Allantoids sporidia were the primary infective agents of

T. indica but had not been studied in other locally infecting bunts. The role of the

passively dispersed, filiform sporidia in the infection process is unclear.

Dispersal of sporidia from soil level to ear head.

Prescott (1986) reported that the microconidia or crescent shaped allantoids

spores are the product of macroconidia produced by the chlamydospors present in the

soil. The microconidia get deposited on the lowermost leaves by current of air and splash.

The maximum trapping of the microconidia occurres in air samples during early morning

hours when 100 % relatively humidity (RH) prevails. Nagarajan, (1991) explained that

microconidia in the presence of leaf wetness produce a secondary crop of spores and as

the leaf surface dries, these spores get dispersed to higher/upper leaves. Having climbed

up the leaves through monkey jumps, they reach the flag leaf, some also get wind

deposited and if at the boot emergence stage coincides with a mild drizzle or rain, and the

microconidia get washed down into the sheath. Bedi et al., (1949) revealed that with

presence of free water, once again crops of the microsporidia are produced. Before

complete ear emergence, if more number of rains and favourable weather occurs causing

run down, then more severe Karnal bunt develops. Occasionally, microconidia get lodged

on the floret parts at the time of anthesis and if conditions are favourable they infect to

produce Karnal bunt sorus on individual grains.

According to Bains and Dhaliwal, (1988) spikelets of whear crop inoculated

with sporidial suspension of Karnal bunt pathogen (N. indica) at the booting stage,

produced secondary sporidia after incubation (intact/detached) under favourable moist

conditions in the laboratory. Sporidia were also released from inoculated spikes in the

field where sporidial release exhibited diurnal periodicity. With the help of electron

microscope it was confirmed that viable and infective sporidia trapped in different

experiments were invariabily of the allantoid type. Maximum sporidia developed on the

outer glumes of florets. More sporidia were captured between 5 to 6 o'clock than later

parts of the day but no sporidia were trapped between 14 to 18 o'clock. However, they

could be trapped at any time of the day from the detached spikes incubated in the

laboratory under favourable and moist conditions. Sporidia developed at 15 and 20°C but

not at 30°C. These findings indicated that repeated cycles of sporidial production in

spikes provided more inoculum than expected from soil-borne teliospores of N. indica.

Infection process The Karnal bunt disease cycle is initiated with the germination of the

teliospores at or near the soil surface, producing basidiospores that form hyphae,

allantoids sporidia, and successive generations of sporidia. According to Prescott, (1986)

and Sidhartha et al., (1995) the diurnal release of Tilletia indica sporidia occurs in the

presence of high relative humidity mainly between 1800 and 0800 h but the most

favorable at approximately 0200 to 0300 h . Some sort of “defense mechanism” has been

suggested that insures the existence of sufficient basidiospores at the time of heading,

such as a factor that triggers teliospores to germinate when the plant is heading (Warham

1988; Whitney and Rrederiksen, 1975). Otherwise, teliospores would undergo a

hypothesized “suicidal germination”, if they germinate when the host is not in a

susceptible condition (Rush et al., 2005, and Stein et al., 2005).

Preliminary infection by T. indica occurs when sporidia accumulate on spikes

and germinate to produce hyphae that enter stomata (Goates, 1988). Hyphae then

penetrate intercellularly to the base of the floret and enter into the periderm of nascent

kernels passing through the funiculus. Scanning electron microscopy studies revealed that

the stomata of the rachis could also be essential path way for primary infection

(Dhaliwal, 1989). However, solid information collected during the movement of hyphae

within spikes indicates the rachis was not a regular site for initial infection (Rattan and

Aujla, 1991). Nagarajan et al., (1997) stated that basidiospores or hyphae of T. indica

frequently fuse to form a dikaryotic mycelium prior to attack on the host plants, and

basidiospore fusion during culture has been reported (Krishna and Singh, 1983).

During the study of infection process of T. indica visible hyphal anastomosis

was only vary infrequently observed prior to penetration into the wheat plant, was not

considered a normal means of dikaryon formation (Goates, 1988). Most probably,

haploid hyphae are capable of infecting the host plant but the dikaryotic state must be

attained for the development of teliosporogenesis (Duran and Cromarty, 1977). The stage

at which the fusion of two nucleuses started in the T. indica was not identified. The

method of penetration and timing of dikaryon formation in T. horrida are unknown,

although Singh & Pavgi, (1973) suspected that sporidia lodge on the feathery stigma and

enter into the style to reach at the chalazal end of ovary.

Frequently the embryo is not colonized except in sever infection when the

embryo is killed, and infected seed grains are often able to germinate (Singh & Pavgi

1973; Fuentes-Davila, et al., 1996). Teliospores of T. indica arise from sporogenous cells

that generate a thin hymenial stratum on the surfaces of the cavities formed by the

partition of the inner and outer layers of the pericarp and by the separation of the inner

pericarp from the seed coat (Roberson and Luttrell, 1987). Teliospores develop at the

terminal portion of sporogenous hyphae, where the dikaryotic cytoplasm becomes

surrounded by a septum and karyogamy occurs during enlargement of teliospore of T.

indica primary cells (Fuentes-Davila and Duran 1986). This was basically the same

procedure observed in Tilletia species infecting systemically (Trione et al., 1989). The

process of teliosporogenesis in Karnal bunt pathogen was influenced by temperature.

Even after flourishing introduction of infection, utmost diurnal temperatures of 35o–40oC

after the grain-filling stage decrease teliosporogenesis significantly. Goates and Hoffmann, (1987) reported that during initial stages of teliospore

germination, the diploid nucleus undergoes meiosis followed by several rounds of

mitosis, producing abundant haploid nuclei prior to the development of the basidium. In

T. indica, about 64 to 128 nuclei were observed migrating into the basidium. Nuclei

migrate into developing basidiospores, split synchronously, and one daughter nucleus

returns to the basidium. These nuclei in the basidium then either entered empty

basidiospores or degenerated. The sorting of 64 or more nuclei that were tightly grouped

at the tip of the basidium followed by orderly migration of 1 nucleus into each of

approximately 100 basidiospores is a remarkable feat of cellular mechanics. Each haploid

cell of the basidiospore can produce hyphae, or form allantoid or filiform sporidia.

According to Fuentes-Davila, 1984; Duran and Cromarty, 1977, T. indica is heterothallic

and bipolar with four alleles controlling mating and pathogenicity. Variable numbers of

chromosomes can be found among monospore isolates from single teliospores of T.

indica, indicating that chromosomal alteration or differential segregation occurs during

meiosis. Epidemiology and disease prediction models Karnal bunt disease spreads from season to season and various stages of

disease development begin when teliospores of the disease become dislodged from

infected kernels during harvest, become airborne, and settle in the field or spread to

adjacent and remote areas with wind currents or through other resources. The classic

work of Mundkur, (1943), and Bedi et al., (1949), revealed that natural infection by T.

indica occurs through airborne sporadic (inoculum) during heading, but there is

considerable disagreement on plant stages that are susceptible, and on the most

susceptible stage. Results from a lot of studies provide the justification of susceptibility

only during specific periods within boot swelling to anthesis stage (Aujla et al., 1986;

Bains, 1994; Nagarajan 2001; Rush et al., 2004). However, Goates, (1988), reported that

under natural conditions, the spikes were within the boot and airborne sporidia could not

reach the glumes, where hyphal penetration was initiated. According to the recent studies

of Goates, (2006) with Karnal bunt disease of wheat revealed that infection from airborne

inoculum can occur when florets begin to emerge from the boot up to the soft dough

stage of wheat kernel, and infection peak up after spikes had completely emerged, but

before the on set of anthesis. These results are contradictory to studies claiming

susceptibility prior to spike emergence (Kumar and Nagarajan, 1998; Nagarajan, 2001;

Sharma et al., 1998 and Bains 1994) and it was the first report of susceptibility much

beyond anthesis.

According to Indu-Sharma and Nanda, (2003) teliospores of Karnal bunt when

suspended over water, started germinating from 15 October onwards to March under field

conditions. Environmental conditions were condusive frequently for the disease at

vulnerable stage (ear emergence). Prolonged tenure of mist, fog or rain for 20 days in

December postponed the teliospore germination and the sporidia lost the potential to

cause the disease after a lapse of 45-50 days. Nutritional component of the medium in

which sporidia were cultured, affected the disease inducing potential.

Under specific climatic conditions of heavy dew or light rain, it appears that

flag leaf and the boot sheath may be essential for normal infection (Aujla et al., 1986 and

Kumar and Nagarajan, 1998). Dhaliwal et al., (1983), reported that after the primary

infection, spread to adjacent florets had been occurred as late as the dough stage. The

results of various studies on teliospore germination representing the wide range of

environmental conditions conducive for teliospore germination indicate that it is not a

limiting factor for disease development. The production of allantoids sporidia, which are

the infective agents of the disease, seemed to be the primary factor in epidemiology. Bedi

et al., (1990) reported that the optimum temperature and pH for germination and

production of sporidia were 20oC and 8.0 respectively. He further described that pre-

incubation storage of teliospores at 4oC for one week significantly enhanced their

capacity to produce sporidia where as storage at 40oC or more were not conducive. White

fluorescent light enhanced germination and no sporidia were formed under complete

darkness.

Disease-prediction models have been developed in the Karnal bunt disease

affected countries that integrate the climatic factors that influence significantly

production of sporidia including temperature, humidity, solar radiation, and rainfall

(Jhorar et al., 1993; Jhorar et al., 1992 and Mavi et al., 1992). Germinating teliospores

produces sporidia possessing thin cell walls that are considered to be short-lived and

transitory structures sensitive to drought (Aujla et al., 1985; Nagarajan et al., 1997 and

Smilanick et al., 1989). According to Smilanick et al., 1989, sporadia of T. indica at 95%

relative humidity, survived no longer than 14 h. However, according to the experiments

conducted by Goates (2006), T. indica, T. horrida, T. walkeri, and T. caries sporidia were

remarkably durable. Sporadia collected from natural discharge in air dried conditions

were viable for 30 days at 10–20% RH at 20◦–22◦C and after 60 days at 40%–50% RH at

18◦C, and newly formed sporidia were commonly observed germinating within 18 h of

rehydration. It was observed that sporadia could survive in common field even, after

several diurnal periods containing temperature above 38 oC associated with relative

humidity below 10% and then rapidly produced hyphae under humid rainy conditions to

infect host plants.

Similar results were obtained in field experiments over 46 days at

temperatures often exceeding 40oC and relative humidity as low as 10% (Lori et al.,

2006). It was concluded by these results that teliospore germination prior to a vulnerable

stage of host plant. The inoculum remains viable in dry field conditions, which can

regenerate rapidly during humid conditions normally associated with disease.

According to Sansford, (1998) weather extremes (extremely hot, extremely

dry, or very humid and cold) conditions are not favorable for development of Karnal bunt

disease and are considered to be limited to moderately cool climates (Jhorar et al., 1992;

Sansford, 1998; and Diekmann1998). However, T. indica had never been reported

outside of its’ allocation in the southwestern US and Mexico in spite of the historic

movement of wheat grains throughout the North America. This relatively limited

distribution of Karnal bunt could be attributed to specific weather conditions affecting on

the life cycle of the fungus (Diekmann, 1998).

Krishna and Singh, (1982) reported the optimum environmental conditions

for germination of teliospores of T. indica and concluded that 15-200C as adequate

temperature under alternate light and dark regime. Singh, (1994) suggested that

successful infection was dependent upon the suitable weather conditions during flowering

stage (inflorescence) of wheat plants, which is considered the most vulnerable stage to

infection, and optimum temperature range for teliospors germination is 15 to 250C.

Smilanick et al., 1985a, reported that the optimum temperature after 3 week incubation in

continuous light was 15 to 20oC, over a pH range of 6.0 to 9.5. Moisture was a critical

factor in determining weather in disease development (Singh, 1994; Smilanick et al.,

1985a and Gill et al., 1993a). Singh, (1994) reported that 82 % relative humidity and

preferably free water was required for the teliospores germination. He further added that

with high humidity and rainy weather during 2-3 week window at flowering, infection of

wheat kernels and individual seed grains increases. In the laboratory tests, survival of

teliospores was noted after freezing over several months, but with delayed or reduced

germination, (Chahal and Mathur, 1992; Zhang et al., 1984). According to Diekmann,

(1993) moisture at the time of flowering may be the most critical element for the

establishment of Karnal bunt disease in U.S.A. Prediction made from environmental data

and prerequisite of infection by pathogen suggest that, under current climatic conditions,

Karnal bunt would never cause major crop losses in the U.S.A.

Generally, teliospores at or very near the soil surface germinate in optimum

conditions, and consequently release secondary sporidia that become air-borne and infect

the host plant at flowering. However, the exact details of many steps of the disease

development are still poorly understood and are considered a matter of opinion or

conjecture. Krishna and Singh, (1983) reported that teliospores of T. indica survived

longer on the soil surface than in soil, and teliospore survival decreased with increased

soil depth. No teliospores survived after 27 months at a depth of 15 cm in Pantnagar,

India. But in the experimental studies of Babadoost, et al., (2004) it was indicated that

teliospores survived for more than 32 months at all three depths (2, 10, and 25 cm) in

Montana. There was non significant influence of soil depth on teliospore recovery from

soil. Even after a period of twenty months, germination of teliospores recovered from

various soil samples buried at 25 cm was significantly higher than those of samples

buried in the soil at depth of 2 and 10 cm. The differences between the report by Krishna

and Singh, (1983) and the results from studies of Babadoost et al., (2004) may be

consequently attributed to the effects of soil moisture and temperature on survival,

viability and germination of teliospores, as reported by Rattan and Aujla, 1992. When

Babadoost et al., (2004) stored infested soil samples with the teliospores of Karnal bunt

fungus in the laboratory in Bozeman, teliospore revival decreased as long as the soil

samples were wet. It was also pointed out that temperatures at depths of 2 and 10 cm

were higher than those at 25 cm during summer.

The Pest Risk Analysis Panel of the North American Plant Protection

Organization (NAPPO) estimated the risk of Karnal bunt disease, establishment in

commercial wheat growing regions of North America during October 2001. Critical

factor of environments conducive to induction of disease development were

acknowledged in coincidence with time of occurrence, in phonological terms, with

potential disease severity to categorize areas at risk of disease establishment. Data were

stratified by the phonological window matching to the critical stage of wheat anthesis

which was considered the vulnerable period for each country across North America. Data

were analyzed, characterized, and related to disease triangle including to production

features, like yield and cropping constituency of wheat, climatology, known disease

distribution (survey results), relevant wheat production technology, cultivation practices,

chronological disease dynamics and environmental conditions leading to epidemics of

disease. The risk model confirmed that majority of the wheat growing areas in North

America were not highly susceptible to the establishment of the disease most of the time.

Limited area had been acknowledged where risk may be occurred medium or high. By

the analysis of existing climatic conditions and planting patterns during winter and spring

wheat it was concluded that vulnerable period did not generally coincide with

environmental factors condusive to Karnal bunt disease at the greater part of North

America. The majority of wheat production territories in Canada and the United States

keep up a correspondence to the lowest risk category for the disease. This was true for

both winter and spring cultivated regions of both countries. (Anon. 2001).

Various models to estimate risk of establishment of Karnal bunt in different

countries have been developed (Kehlenbeck et al., 1997; Diekmann 1998; Murray and

Brennan 1998; Sansford 1998; Baker et al., 2000).

The Humid Thermal Index (HTI) model as a tool for determining

potential disease distribution On the basis of prevailing climatic conditions, there are examples for

determination, prediction and mapping the potential distribution of diseases. An example

of mapping the potential distribution of disease based on relevant environmental indices

is provided by studies of Karnal bunt disease of wheat. The disease has been established

in regions of southern USA. A number of pest risk areas (PRAs) had been selected to

determine whether the disease could be established other areas through grain shipments.

The HTI is a disease forecasting model developed by (Jhorar et al., 1992). In this model

he found a high correlation between a humid thermal index (HTI) and Karnal bunt

disease development over a period of 19 years in the central areas of Punjab, in India.

The HTI can be defined as the relative humidity (RH) in mid-afternoon divided by the

maximum daily temperature, calculated at the time between first awn visible to half

inflorescence emergence.

This model was used in the European Union (EU) sponsored to Karnal bunt

programme as a device to determine potential distribution within Europe (Sansford et al.,

2006). The HTI model has also been as a source to predict potential induction of Karnal

bunt in Australia (Murray & Brennan, 1998; Stansbury & McKirdy, 2002) and South

Africa (Stansbury & Pretorius, 2001). The model was devised when it was considered

that there was a strong positive relationship between disease intensity and both average

daily temperature and relative humidity during the month of anthesis of wheat crops

(Jhorar et al., 1992; Mavi et al., 1992). An HTI between 2.2 and 3.3 was shown to be

particularely favourable for the disease.

The HTI has been used to determine prevailing environmental conditions prior

to anthesis of European wheat crops for infection and this finding has been extrapolated

to predict that disease could establish on susceptible wheat varieties in many areas of

Europe (Baker et al., 2005; Sansford et al., 2006). After the analysis of long-term,

average data, the HTI model predicts that a number of places in Pakistan, India, Iran,

Arizona and South Africa had unfavorable climatic conditions for Karnal bunt despite the

disease being present. This contradiction is justified by the reasons that local temperature

and relative humidity levels are modified by irrigation (Stansbury & McKirday, 2002).

Secondly in some parts of the Punjab wet and humid conditions favor teliospores

germination, consequently inoculum is depleted at the stage of crop development when

the wheat is not susceptible to infection (Sharma & Nanda, 2003).

Teliospores at or near the soil surface possess a level of hypersensitivity to

environmental conditions and soil moisture and temperatures of around 5–20oC can

stimulate germination (Sansford, 1998). According to Smilanick et al., (1985b) only the

teliospores that germinate within the 2 mm of the soil surface will release sporidia that

are capable of spreading to canopy of host plants. However if favourable conditions are

present for the germination of teliospores other than at the vulnerable period of crops for

infection, teliospores undergo ‘suicidal germination’ and the teliospore population will

decrease without inducing disease. In contrast, if suboptimal climatic conditions for

teliospores germination remain dominant for most of the growing season of crop,

teliospores are more likely to survive longer with the potential to become an inoculums’

source for next crop infection (Bonde et al., 2004).

A long dry season in hot arid and semiarid climate may be an important

suboptimal weather element or device to inhibit ‘suicidal germination’. The dark-

coloured, hardy nature, thick-walled teliospores endure harsh, dry summer conditions

during the post-harvest period (Singh, 2005). Dormancy accompanied by dry period

ensured that large numbers of teliospores remained ungerminated until water stimulates

germination during the next growing season. Seasonal rains or irrigation water are

required for wheat production in dry regions and, because teliospores act as a reservoir of

inoculum by releasing sporidia in areas where the disease is prevalent.

However, the situation is different in environments in Europe where rain is

more evenly distributed throughout the year than in warmer regions. Consequently,

persistent moisture may also stimulate microbial antagonism and degradation that may

affect viability and reduce the longevity of teliospores. Therefore, in spite of favourable

weather circumstances, prior to anthesis of wheat crops there may be no great reservoir of

inoculum capable of sustaining the disease because of a gradual depletion of teliospore

reserves. Even if infection did occur under favourable low teliospore population levels

would result in reduced disease incidence and increase the possibility of pathogen

extinction (Bonde et al., 2004; Garrett & Bowden, 2002).

Diekmann, (1993) reported three temperature related parameters as sufficient

for discriminating environmental indices where the disease had established and those

where it had not. They were (1) the difference between mean daily maximum and

minimum temperature in the month of planting, (2) the mean daily maximum temperature

in the month of flowering and (3) the mean daily minimum temperature in the coldest

month of the year. Due to some drawback this model it was criticized by (Sansford et al.,

2006) as it was not based on temperature parameters only, neither rainfall nor soil

moisture was included as an essential parameter.

Jones, 2007 concluded that the climate throughout the year is important and

not just climatic conditions prior to antheis. There is a range of sometimes conflicting

information available on how abiotic factors during rest of the year influence the survival

of pathogen in establishment of Karnal bunt disease of wheat as summarized by Warham,

(1986). With respect to the suitability of the climate in the Pest Risk Analysis (PRA)

area, Warham, (1986) came to conclusion that low temperature and high humidity were

essential at wheat anthesis for the infection to occur, while dry weather, high temperature

and bright sunlight were not favorable for infection process. Rainfall is necessary but rain

on its’ own at flowering was not sufficient to cause infection, suggesting that a specific

combination of environmental factors was required. Crop irrigation is an additional factor

favouring disease.

Jhorare et al., (1992) devised a model for the prediction of Karnal bunt disease

in the central Punjab, India, by an empirical method. A study of the associations between

incidence of Karnal bunt disease and meteorological factors, using chronological

meteorological data and data pertaining to disease intensity for Ludhiana district, in the

central plains of the Punjab, was made for the reproductive stage of wheat crop. The

period studied corresponded to flag leaf emergence (starting 12 February in Ludhiana)

and subsequent stages of host plant ending on 18 March. This corresponded to the most

important period for the pathogen, when teliospors that germinated at the time could lead

to the production of infective sporidia that survived to infect the host and for the Karnal

bunt to develop.

The first meteorological model used to assess the risk of Karnal bunt

infection was developed by Jhorar et al., (1992). They showed that there were non-

significant relationships between the Karnal bunt disease development and maximum

daily temperature and sunshine duration during the vulnerable stages leading up to

anthesis for wheat (for India this was the 9th-11th standard meteorological weeks (SMW).

“The two most important factors were determined to be mean maximum daily

temperature (r =0.88) and 3 pm RH (r =0.93). The sunshine duration (r = -0.73) was

negatively related, while number of rainy days (r = 0.71) were positively related.

Regression analysis showed that evening relative humidity and maximum temperature

can be put in a disease model as independent variables in simple regression equations.

The data were then used to develop a HTI forecasting suitability for disease

establishment and spread. The HTI was the monthly average 3 pm RH, divided by the

average monthly maximum temperature, for the month leading up to anthesis of wheat

crop.

The most significant relationship was found between disease intensity and

evening relative humidity (A), maximum temperature (B), and the “Humid Thermal

Index” (A/B). A best-fit model was developed for forecasting the severity of Karnal bunt

in the central Punjab thus:

(1) DI = -0.8 + 1.5 HTI

(2) HTI = ERH ÷ TMX

Where DI = Disease index, HTI = Humid Thermal Index, ERH = evening relative

humidity recorded at 14.30 hrs (average of the third, fourth, and fifth reproductive

weeks), TMX = maximum temperature (average of third, fourth and fifth reproductive

weeks). Jhorar et al., (1992) concluded that the HTI during this part of growing period

varied between 1 and 5; the lowest values occurring in extremely dry and warm

environment, and highest values representing extremely humid and cold weather, neither

of which favoured the disease. An HTI of 2.2-3.3 throughout the third and fourth week of

the study period favoured the disease. These conditions resulted from frequent cloudiness

and irregular showers which could be predicted, thus permitting a disease forecast to be

made.

The second meteorological model for assessment of optimum conditions for

the Karnal bunt disease was developed for the Pacific Northwest, USA by Smiley, (1997)

who noted that temperature and relative humidity were essential for the region, but

stressed the significance of specific weather factors such as suitable rainfall and related

humidity levels that were essential for teliospore germination, secondary sporidial

reproduction, its penetration and infection. Smiley, (1997) reported that utility of these

events being synchronized with three to four week vulnerable period leading upto wheat

anthesis. Suitable rain and humidity events were defined as: “1. Measurable rain (>3 mm)

occurring on each of two or more successive days; 2. At least 10 mm being collected

within 2-day interval; 3. Average daily RH must also exceed 70% during both rainy

days”.

In order to predict the likely risk the pathogen establishing Pest Risk Analysis

(PRA) area, the 1996 UK PRA applied the HTI used climatic data from individual

meteorological stations in UK and showed that conditions during the “heading” period

(broadly speaking May and June) were favourable for infection and disease development,

i. e. the majority of the calculated HTI values fell within 2.2 and 3.3. Kehlenbeck et al.,

(1997) also calculated the HTI for the wheat growing areas of Germany and found that

some of the southern areas had HTI values which fell within optimum range for disease

development.

Murray and Brennan, (1998) also used this methology for Australia. They

assessed the locations for potential development of Karnal bunt in Australia. Data for

average monthly maximum and minimum temperatures and 3 pm relative humidity were

obtained from the Bureau of Meteorology (http://www.bom.gov.au) for 122 wheat

growing regions in Australia. The data were compiled up to the end of 1996. Times of

sowing and stage for anthesis development were collected from agronomists and wheat

breeders of concerned region. The humid thermal index (HTI) was computed for each

month coinciding anthesis occurrence at each location, as the ratio of the monthly

average 3 pm relative humidity to the monthly average maximum temperature. It was

noted that out of 122 sites within Australian wheat belt, 67 had HTIs condusive for the

Karnal bunt disease establishment.

The location was estimated for potential development of disease as follows:

“(i) if HTI < 2.2, locality is too hot or too dry (ii) if 2.2 £ HTI £ 3.3, site is appropriate for

Karnal bunt disease (iii) if HTI > 3.3, site is too cold or too wet of the 122 localities, 46

were of category (i), 67 were of category (ii) and 9 were of category (iii). The 67

“reasonable” sites were through the southern wheat growing regions from Western

Australia to central New South Wales. Category (i) sites or localities were in Queensland,

northern New South Wales, the Victorian and South Australian Mallee, and the northern

inland wheat belt of Western Australia. The cool and wet localities, category (iii), were

outside the main wheat producing areas, on the south coast of Western Australia,

southern Victoria and the north coast of Tasmania”.

As the sowing time and growth development stages of wheat crop vary from

country to country, region to region and even locality to locality with in a country

therefore, life-cycle of the pathogen differ with time between Asian and European

countries. The Australian, United Kingdom, and German studies were conducted using

individual data. For the improvement of this work further, Baker et al., (2000) undertook

provisional meteorological mapping using interpolated environmental data (1961-90)

with adjustments in the calculation to allow for the lack of mid-afternoon relative

humidity measurements. The result showed that for June, an area covering much of

central and southern England had HTIs falling between 2.2 and 3.3 which was therefore,

considered using this factor alone, favourable for Karnal bunt development in the crops

phenologicially susceptible at that time.

There is an existence of strong relationships between environmental

conditions at anthesis and establishment of Karnal bunt disease of wheat for sites in

India. Mavi et al. (1992) developed a model based on the average maximum temperature

during mid to late anthesis (-ve correlation), the “evening relative humidity” (presumably

2:30 pm, +ve corr.) and sunshine duration (-ve) during early to late anthesis, and the

number of rainy days in early anthesis (+ve). Although this model has R2 of 0.89, it is

likely to be location specific due to the inclusion of sunshine hours and therefore not

directly applicable to Australian conditions.

Diekmann, (1993) launched “geophytopathology” methology to develop a

correlation between Karnal bunt likelihood and (i) the distinction between the mean

maximum and minimum temperature in the month of crop sowing; (ii) the average daily

minimum temperature in the coldest month of the year; and (iii) the average daily

maximum temperature at anthesis of wheat crop. Diekmann compared localities in the

world where T. indica did and did not occur for the development of the model.

Stansbury & McKirdy, (2002) compiled two previously established meteorological

modelling techniques while determining areas in Western Australia (WA) where

circumstances were condusive for infection of wheat crop by Karnal bunt pathogen. A

strong correlation (r = 0.83) was found between the rainfall model, and the Humid

Thermal Index (HTI) model, which used average-monthly data. Rain fall model was

developed and based on the per cent opportunity of at least three Suitable Rain Events

(SRE) during the vulnerable stages (August to October) of wheat crop. It was concluded

that northern wheat growing belts are too hot and dry (HTI < 2.2, chance of SRE 15–

27%), southern regions are unimportant due to too cold and/or wet (HTI > 3, chance of

SRE 68–97%), eastern regions are marginal to too hot and dry (HTI around 2.2, chance

of SRE 24–50%), and western wheat growing sites are appropriate (HTI between 2.2 and

3.3, chance of SRE 41–78%). Analysis indicated that between and within year infection

due to T. indica was more likely to establish if anthesis occurred in northern areas in

August, in October in southern regions, in September in eastern belts, and in August,

September, and October in western and south-eastern wheat growing localities.

Stansbury and Pretorius, 2001, used the meteorological modeling techniques to

determine suitable condition for the development of infection, and influence of irrigation

schedules in South African wheat growing belt for the favourable environment for Karnal

bunt pathogen. Only rainfed spring wheat in the western and southern wheat production

areas of the Western Cape experienced climatic suitability to Tilletia indica development.

Humid Thermal Indexes (HTI) experienced an appropriate range of 2.2 to 3.3 (HTI range

2.09-3.20, mean 2.58) and most regions indicated at least one Suitable Rain Event (SRE)

(range 0.78-2.44) during the vulnerable period of crop. In distinction to central and

eastern wheat production belts in South Africa were dry and warm under natural

circumstances (HTI range 0.61-1.67, mean 1.06) possessing less than one SRE (range 0-

0.63) for early and mid-sowing wheat crop in these regions. It was concluded that the late

sowing of wheat in irrigated areas experienced less condusive climatic conditions for the

prevalence of Karnal bunt pathogen. This may not be applied to wheat growing regions

where crops are irrigated very 24 h, as minimum critical relative humidity (RH) for the

security and the chances of survival of T. indica spores would potentially be increased.

A linear disease prediction model was devised by Nagarajan, (1991) for the

climatic suitability of Tilletia indica infection by using average weekly weather had a R2

value of 0.89 indicating an appropriate degree of fitness. For the condition of North West

India, Y= 0.4381+2.97a-2.77b-0.09c=0.13d; where Y= is the predicted Karnal bunt

severity on bread wheat and the variable a to d, represent rainfall duration between 15-20

February, rainfall during 2-28 February, amount of rain days between 5-20 February,

amount of rain between 22-28 February, respectively. When an analogous exercise was

carried out for Mexico where Karnal bunt disease developed in Sinaloa and Sonora states,

the R2 value was 0.91 and the model when validated was found to indicate a reliable

forecasting Y= -0.71 +1.6A + 0.95B + 1.07C + 0.20D; where A to D stand for the

number of rainy days between 1-7 February, 8-14 February, and 22-28 February; and

total magnitude of rainfall between 21-28 February, respectively. Though there are minor

differences between both the models for fractional coefficient values, the common

denominator is that the number of rainy days during a particular period of February when

the ear head emerges. Sources of genetic resistance against the disease. The most successful, efficient and economic method of disease control is

through host plant resistance. Screening is carried out by creating artificial epiphytotic

circumstances at boot leaf stage. Studies with Tilletia indica aimed at developing

trustworthy and practical methods of inoculation for screening germplasm have

demonstrated the highest rate of infection occurred by hypodermically injecting a

suspension of sporidia into the wheat boot at awns-emerging stage, or slightly before

(Aujla et al., 1982, Aujla et al., 1986; Royer and Rytter, 1988; Bains, 1994;). Krishna and

Singh, (1982a) adopted injection technique at several phonologic stages of wheat growth

from panicle initiation to early milky stage using sporidial suspension and concluded that

the most susceptible stage for inoculation was when awns were just emerging. During the

artificial inoculations in wheat crop in the field, use of more precise propagules of the

allantoids secondary sporadia had required, in order to attain reliable high level of

infection (Singh et al., 1988; Fuentes-Davila et al., 1993). The susceptibility of bread wheat to Karnal bunt fungus had been well

acknowledged by the work of Fuentes-Davila, et al., (1992) achieving infection levels

greater than 50 % under artificial conditions; therefore it is significant to continue

evaluating new advanced lines and cultivars, as a measure to overcome the problems, and

guidelines for the release of commercial use. However, bread wheat cultivars that had

shown constantly low levels of infection were identified to occur (Fuentes-Davila and

Rajaram, 1994). According to Bedi et al., (1949) and Fuentes-Davila et al., (1992)

sources of resistance against the Karnal bunt disease were also present in durum wheat

and triticale germplasm under natural and artificial inoculated conditions.

Villareal et al., (1994) during an evaluation with three crop cycle under

artificial conditions, concluded that 49 % synthetic hexapolids (SH) were immune to the

disease. Villareal et al., 1996) identified 4 SHs as immune sources during a work to

evaluate elite bread wheat lines, synthetic hexaploid wheat derivatives, commercial

varieties and candidates to release for resistance against Karnal bunt disease of wheat

under artificial inoculation. Mujeeb, et al., (2006) evolved immune synthetic hexaploid

wheat by crossing (hybridizing) randomly high yielding durum wheat cultivars with

several Aegilops tauschii accessions. The F1 combinations were advanced by

conventional breeding protocols to F8. Crosses between Triticum aestivam and Aegilops

tauschii accessions resulted in F1 hybrids with 2n=3X=21, ABD plants. These hybrid

seedlings after colchicines treatment led to hexaploid C-0 (2n=6X=42, AABBDD) seed

formation. Stable plants with 42 chromosomes called synthetic hexaploids were screened

against biotic stress. An elite SH group of 95 advanced lines was prepared from the initial

420 SH wheats. These 95 germplasm lines were distributed by CIMMYTs’ germplasm

bank. Supplementary repositories were at the Kansas, State University Wheat Genetic

Resource Faculty, Manhattan, Kansas, USA and at National Agricultural Research

Center, Islamabad, Pakistan. High level of resistance in the elite SH wheats were

maintained during the Karnal bunt stress screening. The durum cultivars involved in

these SH combinations were generally susceptible under the severe greenhouse

inoculation tests. The corresponding SH wheats showed immunity under similar

conditions.

Singh et al., (2003) in his studies for mapping a resistance gene effective against

Karnal bunt of wheat reported that most sources of genetic resistance to KB were traced

in China, India and Brazil, Gill et al., 1993, Fuentes-Davila et al., (1995) and Singh et

al., (1995b) also had same conclusions.

In India Indu-Sharma et al., 2005, reported the genetics of Karnal bunt (KB)

resistance in populations derived from crosses of four resistant stocks (HD 29, W 485,

ALDAN 'S'/IAS 58, H 567.71/3*PAR) and a highly susceptible cultivar, WH 542. The

plant materials screened for KB response consisted of F2, BC1 and RILs from all

'Resistant' x 'Susceptible' crosses and RILs from the six possible 'Resistant' x 'Resistant'

crosses as well as the parents and F1s. The screening was performed under optimal

conditions for disease development with a mixture of isolates from North Western Plains

of India using the widely followed syringe method of inoculation. The KB scores of the

F1 from the four 'Resistant' x 'Susceptible' crosses indicated partial dominance of

resistance. Genetic analysis revealed that HD 29, W485 and ALDAN 'S'/IAS 58 each

carried two resistance genes whereas 3 genes were indicated in H 567.71/3*PAR. The six

'Resistant' x 'Resistant' RIL sets showed that the genes in the four resistant stocks were

different and that there may be as many as nine genes governing KB resistance in the four

parents.

Aujla et al., (1989) devised rating scale for identifying wheat cultivars

resistant to Karnal bunt disease of wheat. They categorized wheat varieties on the basis of

severity and response to T. indica into five classes ranging from highly resistant to highly

susceptible and expained the procedure for calculating the coefficient of infection with

the help of grades of infection and numerical values of infected grains. Dhaliwal et al.,

(1986) concluded that germplasm of wild wheat, and Aegiolops agropyron possess the

resistance to various diseases including Karnal bunt disease of wheat. Triticum urartua

was resistant to T. indica and some Aegiolops.

Warham, (1988b) conducted screening against Karnal bunt for resistance in

wheat, triticale, rye and barley. None of the bread wheat lines were immune to Karnal

bunt disease.

Gartan, et al., (2004) during detection of multiple resistance sources concluded

that among 100 advanced breeding lines of wheat (T. aestivum) and triticale (Secale

cereale), the genotypes HS450, HS455, HPW232, VL861, PW731, PW733, PW738 and

PW739 had multiple resistance against a number of wheat diseases including yellow

(Puccinia striiformis var. striiformis), brown rusts (Puccinia recondita), powdery mildew

(Erysiphe graminis) and Karnal bunt (Tilletia indica). The evaluation of these genotypes

may be used in crossing programme with other agronomically superior varieties to

incorporate the genes of desirable characters to obtain high yielding, disease resistant

varieties. Souza et al., (2005) reported a resistant germplasm line IDO602 (Reg. no. GP-

776, PI 620628), resulting from a first backcross of the hard white spring wheat Borloaug

M95 onto the hard red spring wheat Westbred 926, in Idaho, USA, and delivered it in

February 2003 for use in research and crop improvement programmes. It is semi-dwarf

wheat with resistance to Karnal bunt (T. indica) and stripe rust (Puccinia striiformis).

Sharma et al., (2004) obtained a Karnal bunt resistant wheat stock

('KBRL22') germplasm from a cross of two resistant lines ('HD29' and 'W485'). They

used it as a donor for introgression in KB-free trait into 'PBW343' (an 'Attila' sib), the

most widely grown wheat variety in India. The numbers of KB-free and KB-affected

plants in BC1, BC2, BC3 and BC4 as well as the F2 were obtained after artificial

inoculations. The segregation pattern in these generations clearly indicated two

independently segregating, dominant genes which jointly confer the KB-free attribute. Kaur and Nanda, (2002) evaluated wheat genotypes WL 711, WL 1562, HD

2329, PBW 343, PBW 396 and WH 542 along with resistant genotype HD 29 for

susceptibility to Karnal bunt disease (T. indica) during 1995-2002 by artificial

inoculation. Results showed that WL-711 was the most susceptible to disease. Singh et

al., (2003) obtained resistant sources of wheat from Advanced Varietals Trials and other

sources like CIMMYT and were tested at hot spot multilocations under artificially

inoculated conditions for Karnal bunt (T. indica) establishment during 1990/91-1993/94

in Indian Punjab, Himachal Pradesh, Haryana, New Delhi and Uttar Pradesh, India. Of

the 66 entries, 18 were resistant lines, namely HD 29, HD 30, HD 2385, RAJ 2296, WL

1786, WL 6975, WL 7247, HW 502, PBW 34, PBW 225, W 285, W 382, W 388, W 485,

DWL 5010, ND 589, ND 602 and HP 1531. PBW 34 and PBW 225 were released

varieties. Nineteen lines received from CIMMYT were also resistant to Karnal bunt. HD

29 and HD 30 had already been registered by the National Bureau of Plant Genetic

Resources, New Delhi, as INGR 99012 and 99011, respectively. Indu-Sharma, (2001) demonstrated that out of 43, 680 germplasm lines of

bread wheat (Triticum aestivum L. emend. Fiore & Paol.) tested against Karnal bunt

disease of wheat, 744 lines expressed stability for resistance. Out of 188 strains possessed

multiple resistances to Karnal bunt, brown (Puccinia recondita Rox.ex. Desm.) and

yellow (P. striiformis) rusts and were agronomically superior. Six Karnal bunt-free

('KBRL 10', 'KBRL 13', 'KBRL 15', 'KBRL 18', 'KBRL 22', 'KBRL 24') and 3 high-

yielding Karnal bunt-resistant wheats ('W 7952', 'W 8086', 'W 8618') were developed by

pyramiding of Karnal bunt-resistant genes and pedigree method of breeding respectively. Jafari et al., (2000) inoculated spikes of 26 wheat advanced cultivars/lines by

injection of a suspension of secondary sporidia of T. indica at booting stage. Coefficient

of infection and percentage of infected grains for each entry were estimated, they were

ranked into four groups for their responses to the disease. None of the entries was

completely resistant the Karnal bunt disease and most of them were vulnerable. Cultivars

Pastour, N-75-3 and N-75-5 were partially resistant. Darab-2 with 68.1% of infected

grains and a coefficient of infection of 44.1, was the most susceptible cultivar, Atrak and

Niknejad very susceptible and Falat susceptible. A significant correlation was found

between coefficient of infection and percentage of infected grain for each entry and the

regression model was calculated.

Beniwal et al., (1999) studied the effect of three different sowing dates on

Karnal bunt development was studied in ten commercially grown wheat varieties in India

during 1995-96. The coefficient of infection was greater for crops sown on 16 November

than for crops sown on 16 and 31 December. The varieties C.06 (31.96%), WH 147

(31.16%), HD 2009 (26.93%) and HD 2329 (20.93%) had a greater mean coefficient of

infection compared to WH 283, HD 2285 and WH 896. Weather parameters including

mean temperature (19.5 oC), relative humidity (62.6%) and rainfall (66.9 mm) spread

over 6 rainy days from the date of inoculation to harvesting for crops sown on 16

November made them more susceptible to infection than those sown on 31 December,

which had a mean temperature of 23oC, relative humidity of 53.7%, and 31.6 of mm rain

(3 rainy days only). In Pakistani wheat germplasm generally there is scarcity of resistance against

Karnal bunt disease of wheat. Out of 141 wheat varieties/lines evaluated by Ahmad et al,

(1999) at the Wheat Research Institute, Ayub Agricultural Research Institute, Faisalabad,

Pakistan, during 1993-94, four lines, T-91731, T-911734, T-91736 and T-91740,

remained free from karnal bunt infection caused by N. indica (T. indica). T-92733, T-

91729 and D-91690 were moderately resistant while 51 showed a moderately susceptible

response. Thirty nine varieties/lines were susceptible and 44 highly susceptible. Iftikhar

et al., (1988) during screening of wheat germplasm against T. indica concluded that out

of eighty cultivars evaluated only three entries T.C. L.83740, V-86354 and V-86257 were

totally free from every kind of infection and thus they were declared to be immune.

However, none of the entries was found to fall in resistant class. Eight germplasm lines, i,

e. V-85003, V-85028,V-85255, V-85409, V-86231, V-86326, V-86369 and V-86371

exhibited moderately resistant, 10 moderately susceptible (V-83134, V-83156-3, V-

83035, V-84021, V-85195, V-85165, V-85060, V-83152-1,V-85283 and V-86357), 21

susceptible (Dirk,Mexi-Pak-65, SA-75, B. Silver, Lyp.-73,Pb.85, C-591, C-518, C-271,

C-217, C-273, Yaccura, Suitleg-86, Sandal, V-85096, V-84658, V-86115, V-86061, V-

852992,V-86184,V-87240) and 38 were highly susceptible (Arz, Pavan, LU.26, Lu.26 S,

LU.26 S-1, WL-711, Indus-79, Pak-81, Pb.81, BWp-79, K.Noor-83, Morrocco, Potowar,

Barani-70,Chenab-79, SA-42, Pb.76, C-228, Fsd-85, Wandanak, Pari-75, Chakwal-86,

Barani-83, V-83171, V-84140, V- 85078, V-85205, V-85072-1, V-84133, V-85054, V-

86215, V-86299, V-85405, V-86303, V-86124, V-86124, V-86240 and V-87239. Chemical control of the disease Karnal bunt is very difficult to control when it is present in wheat area. Seed

treatments that are used to control other bunt and smut diseases of wheat are generally

ineffective because these only protect the wheat crop at seedling stage. The fungus T.

indica, rather infecting the host plant or seedling as in the case with other smuts, infects

the seed in the head as it emerges. Several attributes of the etiology of Karnal bunt and

the teliospores of T. indica make control a very complex problem. Cultural practices that

reduce the Karnal bunt incidence, such as delay in sowing date, reduced nitrogen

fertilization or reduced planting density, only affect modest reduction in karnal bunt

incidences and may themselves reduce yields (Gill et al., 1993a; Singh, 1994 and

Warham and Flores, 1988 Rivera-Castaneda et al., 2001). Due to the hardy nature

teliospors, are sturdy, long-lived and very resistant to chemical and physical treatments

(Smilaninick et al., 1988 and Warham 1988a). They are seed borne and protected by the

sorus and remainder of the partially bunted seeds typical of disease. According to

Smilanick et al., (1989) teliospores masked in the soil persist longer than those of present

on soil surface and are more sheltered from harsh climatic conditions and chemical

treatments. Seed and soil treatments applied for the control of Karnal bunt have been only

partially successful. Seeds treatments with fungicides do not kill the teliospores but

inhibit their germination (Hoffmann 1986 and Warham et al., 1989). They have reported

that fungicides applied to soil at seedling had not reduced the disease, probably because

the infection originated from airborne infectious sporidia from the teliospores that

germinated outside the test plots. According to Agarwal et al., (1993) numerous chemical

compounds have actively against this pathogen by reducing the germination of

teliospores. However, Anonymous (1997) proposed that the chemical seed treatments

have proved ineffective in killing teliospores with the exception of mercurial compounds

which are banned in most countries. Seed treatment does little to eliminate soil-borne

inoculum. So in case of chemical control the only valid and the most effective option is

the application of foliar fungicides at anthesis stage.

According to Smilanick et al., (1987) seed treatment fungicides do not protect

wheat plants from infection when seed are planted in teliospore-infested soil, and do not

persist long enough within the plant to inhibit the infection of florets. During the

evaluation of fungicides they estimated that out of eight seed-applied and 16 foliar-

applied fungicides against the Karnal bunt of wheat more than 80 % control was obtained

with two applications of either Propiconazole or etaconazole or four application of

mancozeb or copper hydroxide when these fungicides were applied to wheat crop when

the spikes were still enclosed (feeks’ growth stage 9), with the awns emerged about 1cm.

Best control of propiconazole and etaconazole was obtained when they were applied 72

hours after inoculation rather than before inoculation.

Foliar application of the fungicide has given control of Karnal bunt disease of

wheat to some extent. Two or more application of propiconazol at or after spike

emergence reduced the incidence of disease by 95 % (Aujla et al., 1989; Singh; 1994).

Sharma et al., (2005) evaluated new fungicides in greenhouse experiments to determine

efficacy against the karnal bunt disease of wheat and durum wheat, caused by T. indica.

The treatments comprised: 0.05, 0.10, 0.20, 0.40 and 0.80% Folicur (tebuconazole); 0.05,

0.10 and 0.20% Contaf (hexaconazole); 0.05, 0.10 and 0.20% Tilt (propiconazole); 50,

100 and 200 g a.i. thifluzamide/ha; and the control, applied at 48 h after inoculation of

sporidial suspension. Infected and healthy grains were counted in the inoculated ear

heads and the percent infection was calculated. Folicur at 0.20%, Contaf at 0.10%, Tilt at

0.10% and 100 g a.i. thifluzamide/ha resulted in more than 90% Karnal bunt control,

while Folicur at 0.40% and 0.80%, and Contaf at 0.20% resulted in 100% bunt control.. Rivera-Castaneda et al., (2001) during in vitro studies have reported the

effectiveness of few commercial fungicides in inhibiting teliospore germination of T.

indica. A number of native plants extracts from Sonora, Mexico, were tested to determine

their antifungal activity against T. indica. Dichloromethane (DCM) and methanol

(MeOH) extracts were incubated with the fungus to measure inhibition of mycelial

growth. DCM extracts from Chenopodium ambrosiodes and Encelia farinosa reduced

mycelial colony growth of the fungus, but total inhibition was obtained with 500 mg/ml

of the DCM extract from Larrea tridentata. Teliospores subjected to this treatment

showed no viability when transferred to fresh culture media. None of the evaluated

extracts stimulated fungal growth. The extract from Larrea tridentata illustrated potential

as control agent for T. indica. A study was carried out by Singh et al., (2000) from 1996 to 1999 wheat

cropping season to evaluate fungicides for the control of Karnal bunt (T. indica) through

foliar spray under field conditions to ensure healthy wheat seed production. Using a

knapsack sprayer the highly susceptible cultivar HD 2329 was sprayed with following

fungicides: propiconazole, hexaconazole, tricyclazole, flusilazole, thiophanate-methyl,

cymoxanil and carbendazim at 0.05 and 0.1% dosage level. Among the fungicides, the

maximum disease control (99.8%) was achieved by two sprays of propiconazole (0.1%)

whereas a single spray controlled (97.68%) disease, followed by hexaconazole (94.40%)

in the post-inoculation treatment. In the pre-inoculated spray treatments, maximum

disease control (99.78%) was achieved with two sprays of propiconazole, while a single

spray provided 96.46% control followed by hexaconazole (92.87%). None of the

fungicides assured in a complete control of the disease. Flusilazole controlled the disease

by 70.25-83.76%, but was phytotoxic to the wheat crop. Tricyclazole, cyamoxanil,

carbendazim and thiophanate-methyl resulted non-significant in disease control. Two

sprays of propiconazole (0.1%) at 15 days interval reduced the disease from 19.83 to 0.02

and 18.66 to 0.04% in post-inoculation and pre-inoculation treatments, respectively,

which is below the level of international seed certification standard for foundation seed

(0.05%).

Goel et al., (2000) reported that a foliar spray of propiconazole (Tilt 25 EC)

at 250 and 500 ml/ha, applied at the boot leaf stage, decreased Karnal bunt disease in

wheat up to 78 and 87% respectively, in multilocation trials conducted during 1988-93 at

New Delhi, Ludhiana and Gurdaspur (Punjab). Consequently it increased grain yield and

was therefore, efficient for the management of this disease in the field. According to

Salazar et al., (1997) out of 11 fungicides applied in varying combinations and

concentrations to control T. indica infection of wheat in Mexico, propiconazole resulted

the best disease control. However, based on costs and quarantine regulations in northwest

Mexico, the use of propiconazole would not be economically profitable. Agarwal et al.,

(1993) found that a range of fungicides that gave significant control of sporidia on wheat

crop if applied at the early heading stage. These included mancozeb, carbendazim, fentin

hydroxide, bitertanol and propiconazole. Findings from current EU (European Union)

research project (Anon. 2004b) reported that some active ingredients, particularly

azoxystrobin as well as propiconazole were effective against both pre and post-infection

by T. indica when applied as a single spray treatment at flag leaf ligule just visible (first

awn visible) or caryopsis watery ripe stage. Krishna and Singh, (1982) evaluated the fungicides for the contol of Karnal

bunt of wheat. In a glass-house test against T. indica Plantavax (oxycarboxin), Vitavax

(Carboxin), Bayletan (triadimefon) and Bavistan (carbendazim) gave 82-87.55 % control

when sprayed on the ear heads two days before inoculation of a sporidial suspension into

the boot leaf. Krishna and Singh (1983) reported the chemical that enhanced the

germination of teliospores of T. indica. Out of 16 treatments tested for obtaining

abundant and early germination of teliospores from the diseased grains of wheat, the best

were citrus and tomato juice, ether and hexan. Krishna and Singh, (1986) studied the

effect of some organic compounds on teliospores germination and screening of

fungicides against the Karnal bunt pathogen. Butyric acid and Lactic acid at 500 ppm.

increased percent germination. Among the alcohols and solvents, methanol, ethanol n-

butanol, ether and toluence induced early germination and xylene, hexane andchloroform

also increased germination. Phenol 2, 4-dihydroxy cinnamic acid, Chlorogenic acid had

stimulatory affect on germination.

Mancozeb and Metalaxyl are considered broad spectrum fungicides, and

exhibited a strong antisporolant activity against three major divisions of the fungi,

Oomycota, Ascomycota and basidiomycota when applied before inoculation rather than

post infection (curative applicative application). Metalaxyl (260 µg/ml) also provide

complete control of disease when used in protectant mode. (Ammerman et al., 1992.,

Baldwin et al., 1996., Gold et al., 1996 and Mitsuhiro et al., 1999).

Chapter 3

MATERIALS AND METHODS

Survey of Karnal bunt disease of wheat

Three types of surveys were conducted in various wheat growing areas of the Punjab.

1). Survey of hot spot areas.

2). Survey of grain markets /storage in various Districts and Towns.

3). Survey of various Research Stations / Research Institutes Seed Corporation and

Model

Farms etc.

(i) Field survey:

With the collaboration of agricultural extension workers, farmer’s fields were

visited in main wheat growing areas of the Punjab. Survey was conducted in the month of

April-June, (2005-2006), which are the harvesting and threshing months of wheat in the

Punjab, Pakistan.

Farmer’s report about complaints of wheat disease in the fields proved to be

the most helpful method in collecting the data about Karnal bunt disease in small towns’

villages and cities. On complaint report, farmer’s fields were visited on the spots during

harvesting, threshing and transportation of wheat before putting the grains into storage

godowns. A random sample from the edges of each infected field was taken in a

replicated manner in the form of at least 20 heads from one acre rendering a fair

representative of the entire field. The samples were packed in brown envelops before

placing them into a collection bag. They were labeled with following conventions:

1) Unique field number, sampler’s name, growers name, date and place.

2) Identification code, accompanying sample worksheet, name of the district, name of

extension worker and other necessary information required for fungus identification and

data analysis in the laboratory.

3) Name of wheat cultivars/ line with its pedigree.

The fields were located through cooperation of extension workers and close

coordination with wheat growers and farmers. Special protocol for sampling was taken

into consideration prior to carry over in disease diagnostic laboratory, to maintain

sanitation without cross-contamination. To determine the variation in incidence of the

disease in the various hot spot areas of the Punjab, 12 main wheat growing districrts were

selected. The samples were taken from five various localities in each of the districts, and

submitted to disease diagnostic laboratory of plant pathology section, Ayub Agricultural

Research

Table: 1. Rating scale used to calculate the severity of Karnal bunt of wheat.

Infection

category

Symptoms Coefficient

of infection

0 Healthy

0

1* Well developed point infection

c*. 25 % seed bunted.

0.25

2 Infection spreading along the groove

c. 50 % seed bunted.

0.50

3 Three-quarters of the seed converted to sorus. c.

75 % seed bunted.

0.75

4 Seed completely converted to sorus.

c. 100 % seed bunted.

1.00

*categories combined for use in calculations of coefficients of infection

Table: 2. An example of the calculation of coefficient of infection (from Aujla et al.,

1989)

Grade of infection 0 1 2 3 4

Numerical values 0 0.25 0.50 0.75 1.0

Number of grains 200 75 50 25 10

Multiplication with

numerical values

0x200 0.25x75 0.50x50 0.75x25 1.0x10

Value after

multiplication

0 18.75 25 18.75 10

Gross total 0.00 + 18.75 + 25 + 18.75 + 10 = 72.25

Total grains 200 + 75 + 50 + 25 + 10 = 360

Coefficient of infection 72.25x 100 ÷ 360 = 20.14

Institute, Faisalabad for confirmatory test through diagnostic scheme recommended by

European and Mediterranean plant protection organization (EPPO) Anonnymous

(2004c). To determine the severity of the disease a rating scale was used for Karnal bunt

(T. indica), based on Aujla et al., (1989) and Bonde et al., (1996). (ii) Survey of grain markets Survey of grain markets in towns and big cities was conducted to assess the

Karnal bunt disease in the territory. After counseling with dealers about the source (local

or abroad) and history of the wheat grains, only the local sources of wheat of the territory

were taken into consideration. Mostly the small and medium type dealers were selected

for sample collection. Big dealers were neglected due to the apprehension of abroad

source of wheat grains and mixture of varieties. To determine the precise estimate and

analysis of variance for the incidence of disease in grain markets of the Punjab a second

survey was conducted after an interval of ten days. During the survey 5 localities were

selected with four replications (samples) in each of 24 districts.

Sampling Seed lots were sampled according to International Seed Testing Association

(ISTA) rules (EPPO, 2004). As the grains for feed or processing were typically more

difficult to separate because consignments were usually very large, and transported or

stored as large loose bulks. Therefore, for monitoring purposes, grains were sampled in

an appropriate fashion to produce a 1–2 Kg thoroughly mixed sample representative of

the consignment. The samples were taken from one year old and new lots. The most

efficient and rapid wash test method for detecting teliospores in a sample, (size-selective

sieving and centrifugation technique of Peterson et al., (2000) was adopted. Direct visual

examinations for bunted kernels or teliospores contaminating seed surfaces were not

considered reliable methods. However, Karnal bunt teliospores were also detected by

visual examination with the naked eye and for the confirmatory test low power

microscopy (10– 70X magnifications) was used. To help visualize symptoms, seeds were

soaked in 0.2% NaOH for 24 h at 20 °C. This was especially useful for chemically

treated seed lots where coloured dyes obscure symptoms (Mathur & Cunfer, 1993;

Agarwal & Mathur, 1992). With severe contamination, teliospores had been seen on the

surface of seeds (Mathur & Cunfer, 1993). Teliospores were soaked in water; quickly

surface sterilized and then germinated on water agar plates. After 20-26 days they were

shifted on PDA (potato dextrose agar) media.

Identification was mainly based on morphological characteristics to avoid the possible

confusion with similar species like Tilletia wlakri (ryegrass bunt) and Tilletia horrida (rice

smut) (Durain and Ficher, 1961; Duran, 1987). After completing the entire possible

requirement in diagnostic scheme i.e. isolation, detection, germination and confirmation of

the culture, a number of replicated 50 g sub-samples were taken to evaluate % infection

from all the positive samples.

(iii) Survey of Agricultural Farms For the collection of entries and for the assessment of resistance/ susceptibility

in various germplasm lines and varieties against the disease, experimental areas of various

research institutes, agricultural research station, model farms, and seed corporations were

visited. Survey was continued at an interval of 10 days from sowing to threshing of wheat

during two consecutive growing seasons (2005-2006) and (2006-2007).

With the collaboration and support of research workers from Plant Pathology

section Ayub Agricultural Research Institute Faisalabad, germplasm lines/ varieties with all

possible desirable characteristics (short duration, early maturing, high yielding, drought

resistant, and cultivars possessing a degree of resistance to highly susceptibility to Karnal

bunt disease) were selected for screening and further epidemiological studies. Most of the

germplsm advanced lines/ cultivars were obtained from Wheat Research Institute

Faisalabad, Tareen Model Farm Lodhran, Punjab Seed Corporation Khanewal, Jalindher

Seed Corporation Sahiwal and Department of Plant Breeding and Genetics University of

Agriculture Faisalabad.

Screening of wheat germplasm for the source of resistance

(i) Isolation and preparation of mass culturing of Tilletia indica During the survey one year old bunted grains of wheat infected with T. indica,

were obtained from storage godowns of Tareen model farm at Lodhran, where wheat

cultivar AS 2002 was badly affected by Karnal bunt disease, along with some other bunted

old varieties, during the crop years 2004-2005 and 2005-2006. The disease had been

appearing here for the last few years on old varieties. Free teliospores were obtained by

gently crushing severely affected seeds, classified to category 3 to 5 (Warham, et al., 1986)

with a mortar and

Fig. 1: After centrifugation teliospores sedimented into pellet in conical centrifuge test tubes.

Fig. 2: Disinfested teliospores by centrifugation with 5 % chlorox in capped test tubes.

pestle. For each infected samples a suspension of suitable magnitude of teliospores in 200

ml of sterilized water was made. Teliospores suspended in water were filtered through 120

mesh sieve, which retained the larger debris on the sieve. The filtrate was centrifuged at

3000 rmp for one minute which sedimented the spores into a pellet in 40 ml of the test

solution in capped pre-sterilized, 50-ml-capacity conical centrifuges test tubes (Fig 1).

Excess of water was decanted out. The teliospores were disinfected with commercial

bleach (5% chlorx) in capped test tubes by centrifugation. The teliospores were again

collected in the sort of pellet (Fig. 2), and chlorox was decanted out. The teliospores were

rinsed twice in sterile water, each time recovering after centrifugation. Whole of the

process was repeated three times to avoid the chances of contamination. A spore

suspension was prepared by adding sterile water in the test tubes containing disinfected

teliospores. One ml of the spore suspension with the help of macro-pipette was distributed

to each of several Petri- plates containing plain agar medium (Fig. 3). The composition of

plain agar medium was 20 gm agar dissolved in one litter of water. Inoculated Petri-plates

were incubated at 15-20 0C with a regime of 14 hours light and 10 hours darkness. After

25-30 days of incubation, teliospores germinated and primary sporidia were visible on the

surface of plain agar medium in the Petri-plates in the form of thread like or star like

structures (Fig 4 & 5). Using this primary sporidial culture (Fig. 6), secondary sporidia

were produced on PDA. For this purpose about 100 ml of autoclaved PDA was taken into

each 250 ml flasks, and the media were solidified in slanting position in the flasks. The

slants were then inoculated with a primary sporidial culture by gently placing an inverted

culture block size of about 3 x 3 mm (making angle) on the upper edges of the slants. The

inverted block of the culture facilitated the sporidia, frequently shedding on the medium

(Fig.7, 8A & 8B). The culture slants in flasks were incubated at 18-20 0C. Within 5 to 6

days secondary sporidia started to discharge forcibly from primary sporidial culture in the

form of brittle, crustaceous, unibonate colonies with dendric margins (Fig. 9, A & B).

Thousand of the sickle shaped and allantoids types secondary sporidia were observed under

the microscope.

Field evaluation of wheat germplasm lines/ varieties for the source of resistance against Karnal bunt disease under artificial inoculation. Two hundred one advanced lines and forty three commercial cultivars or local

varieties received from Wheat Research Institute Faisalabad, were sown in a single row

sub.

Fig.3. Germinating teliospores after 10 days on plain agar petri-plates.

Fig.4 (Germination of teliospores after 20 days) in petri-plates containing plain agar.

Fig.5 (Germination status of tetiospores after 30 days) Germinating teliospores having star like structures of primary sporidia on plain agar.

Fig. 6 Germinating teliospores with long slenderical primary sporadia growing from the tip of promycelium (basidium) under power microscope. (100x 60)

Fig :7 Pattern of spore shedding (secondary sporadia) on PDA slants in conical flasks after second day of inoculation.

Fig. 8: (A and B) Pattern of sporadia shedding on PDA slants after third day of inoculation.

A B

Fig. 9 (A & B): production of secondary sporidia on PDA slants after 6 days of inoculation.

A

B

Fig.10: Screening of wheat germplsm lines/ cultivars for the source of resistance against Karnal bunt disease under artificial inoculation condition and field evaluation of fungicides.

plots of 3 meter each (Fig. 10). There was 30 cm row to row spacing and 15 cm plant to

plant distance. The test entries were boot inoculated by injecting 4 ml of the sporidial

suspension of Tilletia indica with a hypodermic syringe starting from second week of

February to mid March. Boot inoculation was carried following the procedure of Singh and

Krishna, (1983a) and Aujla et al., (1983). Ten heads of each test line were boot inoculated.

The inoculated plants of each line/ variety were tagged and labeled. To maintain the vigor

of plants, nitrogenous fertilizers were used in a well balanced quantity, with a suitable

interval of irrigation to lower the temperature and increase the relative humidity. All the

agronomic practices were homogenously applied for all the entries. Inoculated heads were

harvested at maturity between 20 to 30th of April. Heads of each line/ variety were hand

threshed and the total numbers of healthy grains as well as bunted grains of the inoculated

heads were counted and disease incidences were thus calculated. The levels of resistance/

susceptibility of the test cultivars were assessed by using the following modified disease

rating scale of Aujla et al., (1989). Table: 3. Rating scale used to determine level of resistance/ susceptibility.

Disease rating scale % Grain infection Level of resistance or susceptiblity

0 No infection on (panical) Highly resistant

1 1 % or less grains bunted Resistant

3 1.1-2 % of grains bunted Moderately resistant

5 2.1-5 % of grains bunted Moderately susceptible

7 5.1-10 % of grains bunted Susceptible

9 More than 10 % of the grains bunted Highly susceptible

Epidemiological studies

(i) Cultivation of susceptible germplasm lines/ varieties for epidemiological

studies During the survey for the assessment of Karnal bunt disease, about 66

susceptible germplasm lines/ varieties indicating various degree of infection (from

minimum to maximum) were collected from various agricultural research stations for

epidemiological studies. These were sown in experimental area of Department of Plant

Pathology University of Agriculture Faisalabad during consecutive growing season 2005-

2006 and 2006-2007. The agronomic practices were adopted homogenously in both

years, to keep the crop in good condition, producing conducive environment for the

development of disease. No fungicide was sprayed so that the entries could get maximum

chances for the development of Karnal bunt disease under natural conditions. The entries

were early sown in the second week of November in randomized complete block design

with four replications. There was 30 cm row spacing and 15 cm plant to plant distance.

The data were recorded in both years from February to end of April. At maturity the

kernels were picked from each entry and they were hand threshed. The incidence of the

disease for each entry was calculated by using the following formula.

Number of bunted grains in 10 kernels Disease incidence = ___________________________________ X 100 Total number of grain in 10 spikelets (ii) Development of disease predictive model Monthly environmental data comprising of maximum and minimum air

temperature, relative humidity, average rainfall and wind speed were collected from

meteorological station, situated at a distance of 100 meter away from the experimental

area of Department of Plant Pathology University of Agriculture Faisalabad. All the

above mentioned parameters of environment were collected from February to end of the

April during 2006 and 2007, respectively. The data of 66 entries and environmental

variables were subjected to analysis of variance. Differences in the environmental

parameters and disease incidence were determined by Least Significant Difference test

(LSD) at (p<0.05). The influence of these environmental parameters on the development

of disease was determined by correlation analysis (Steel et al., 1996).

All the data were subjected to stepwise regression analysis and the most

favourable environmental parameters were determined by significance level. The

coefficient (R2), Mallows Cp and Mean Square Error were used to select the best model

(Myers, 1990). Chemotherapy of Karnal bunt disease of wheat

(i) In vitro evaluation of fungicides at four dosage rates In vitro evaluations of ten fungicides at 40 ppm, 60 ppm, 80 ppm, and 100

ppm against the colony growth of T. indica, were carried out by inhibition zone

technique (Khan and Ilyas, 2007). A weighed quantity of each of ten fungicides (Table 4)

was amended into the sterilized water to obtain each of the 40, 60, 80 and 100 µg/mg

(ppm) aqueous concentration. Twenty ml of sterilized potato dextrose agar medium

(dextrose 20 g, starch 20 g, and 20 g agar agar dissolved in water to make the volume

1000 ml) was poured in equal quantity into replicated sets of 90 mm diameter petri-plates

and allowed to solidify. A secondary sporidial culture of T. indica (grown on PDA slants

in 250 ml conical flasks) was scratched with a sterilized scalpel, removed and stirred

thoroughly in 200ml sterilized water taken in a 1000 ml sterilized beaker. This sporidial

suspension was filtered aseptically through sterilized muslin cloth and the suspension was

diluted further to get about 10,000 sporidia/ ml of water. One ml of this sporidial

suspension was transferred to each of 90 mm diameter PDA plates and spread uniformly

on the surface of PDA with a sterilized bended (L-shaped) glass rod. With the help of a

sterilized 6mm diameter cork borer, a well was made in the center of each PDA Petri-

plate with sporidial suspension spread on its’ surface. The wells of the four PDA Petri-

plates were filled with aqueous solution of each of four concentrations of the each test

fungicide. The four PDA Petri-plates with wells were filled with sterilized water which

served as control. The Petri-plates were labeled with the fungicides and its’ dosage rates.

All the Petri-plates with wells either with fungicide solution or sterilized water were put

at 50C in a refrigerator for 24 hours to allow the diffusion of fungicide solution into the

medium of the Petri-plates. These Plates were then incubated at 200C. After 6 days of

incubation the diameter of inhibition zone of T. indica colony around the well for each of

the concentration of the test fungicides was measured (Fig.11, A-F).

(ii) In vivo control of Karnal bunt disease of wheat by protective and

eradicative spray of fungicides. A. Protective spray

Wheat grains of highly susceptible cultivar AS-2002 were sown in sub-plots

of 1.53 x 0.92 meter size, with three replications, with sub-plot to sub plot distance 60

cm, repeat to repeat distance 90 cm, row to row distance in sub-plot 30 cm and plant to

plant distance in a row 15 cm. At boot stage, the sub plots were sprayed with each of

ten fungicides at their recommended dosage rates (Table 4). The sub-plots sprayed with

tap water served as control. Each treatment was replicated four times. The layout of the

experiment was in RCBD. After 48 hours of fungicidal spray, each of the twenty heads

in each sub-plot was boot inoculated with 3 ml of sporidial suspension of Tilletia indica

(10,000 spores/ml). The sub-plots inoculated with sterilized water served as control.

Inoculated plants were tagged and labeled. The field was irrigated to lower the

temperature. Agronomic practices were homogenously applied in all the replications.

At maturity plants

Table: 4. Fungicides evaluated against in vitro colony growth of Tilletia indica and in vivo control of Karnal bunt disease of wheat grains by foliar spray

S.

No.

Common

name

Chemical name

& % composition Formulation

Dose

Rate Manufacturer

1 Protocol

Precombi

Thiophanate Methyl

(52.50%)

+

Diethofen carb

(12.5%)

65 W.P. 500g/ 100 lit

water Agrolet Co.

2 Agrohit

Diamethomorph

(6%)

+

Mancozeb (44%)

50 W.P. 350g/ acre Agrolet Co.

3 Dolomite

Metalaxyl (15%)

+

Mancozeb (65%)

58 W.P. 250g/ acre M/S Pak. China

Chemicals

4 Alert plus

Fosetyl aluminium

(40%)

+

Mancozeb (30%)

70 W.P. 350g/ acre Agrolet Co.

5 Crest Carbendazim 50 W.P. 100g/ acre M/S Pak. China

Chemicals

6 Anthacal

Propineb (61.25%)

+

Provali Carb (5.5%)

75 W.P. 250g/ 100 lit

water Agrolet Co.

7 Shelter Mancozeb (80%) 80 W.P. 600g/ acre M/S Pak. China

Chemicals

8 Thiomil Thiophaneti methyle

(70%) 70 W.P. 200g/ acre

M/S Pak. China

Chemicals

9 Reconil-M

Chlorothalonil

(30%) w/w

+

Mancozeb

(40%) w/w

+

70 W.P.

330g/ acre

or

150g/100 lit

water

M/S Pak. China

Chemicals

other ingredients

(30%) w/w

10 Aliette Aluninium ethyl

phosphate 80 W.P. 2.5g/lit water

Rhoune-poulenc-

Agrochimie

were harvested and hand threshed. The data of inoculated heads were recorded on

percent grain infection The data were analyzed statistically by subjecting it to analysis

of variance and Least Significance Difference Test (LSD) Steel and Torrie, (1996) to

visualize the difference between the effects of various fungicidal spray treatments.

B. Curative spray evaluation

In another experiment the variety (AS-2002) was sown in various sub-plots

and the experiment was designed similar to that of protective application of fungicides.

When crop reached to boot leaf stage each of the twenty heads in each replication were

boot inoculated with 3 ml of sporidial suspension of T. indica (10,000 spores/ml).

Sterilized water sprayed in sub-plots served as control. After 48 hours of artificial

inoculation, each of the test fungicides was sprayed at their recommended dosage rates

(Table 4) following RCBD. Inoculated plants were tagged and labeled and field was

irrigated. At maturity, the inoculated heads were harvested, hand threshed and percent

seed infections for each spray treatment was determined. The data were analyzed

statistically by subjecting it to ANOVA and LSD test (Steel and Torrie, 1996) to

visualize the difference between various fungicidal spray treatments.

Chapter 4

RESULTS Survey of Karnal bunt disease of wheat Analysis of variance (Table 5) revealed that there was a distinction in the

percentage of bunted seeds among the grain markets of 24 districts (Table 6). Significant

difference was obtained even in the samples drawn from various locations of a district.

Similar results were exhibited by the evaluation of samples collected from hot spot areas

(fields) and localities of 12 districts (Table 8). However, results of varieties were non-

significant indicating that most of the cultivars in the disease affected regions were

susceptible. Except varieties and the interaction of varieties with districts, all the possible

two way and three way interactions were significant (Table 8).

Table 5. Analysis of variance for the assessment of bunted seeds in grain markets of various districts and locations of the Punjab.

S. O. V. DF SS MS F P

District 23 90102 3917.48 86.67 0.00*

Location 4 3028 756.94 16.75 0.004*

Replication 3 592 197.48

Dist.x Locat. 92 79380 862.83 19.09 0.00*

Error 357 16137 45.20

Total 479 189240

Grand mean 28.41, CV 23.89 *Significant at 0.05

A comprehensive survey for the incidence of Karnal bunt disease of wheat in

17 districts (Table7) of the Punjab assessed in the crop of 2006 revealed that out of 624

samples collected from grain markets and storage godown of various cities and towns,

208 samples were found infected by Karnal bunt fungus. In other words, 33.3 percent of

the total collected samples harbored infection by T. indica. The number of infected

samples ranged from 6.97 percent in Muzaffargarh district to 60.97 percent in Toba Tek

Singh district. Among 17 districts surveyed in the Punjab, three districts such as

Muzaffargarh, Dera Ghazi Khan and Lodhran had 6.97, 8.00 and 17.85 percent infected

samples respectively (below 20 percent). Seven districts such as Sheikhupura,

Bahawalpur, Chakwal, Jhang, Sargodha,

Table: 6 All pairwise comparisons test for Karnal bunt disease of wheat for districts in grain markets of the Punjab. Alpha 0.05, Standard Error for Comparison 2.0692, Critical T Value 1.969, Critical Value for Comparison 4.0733, Error term used: Distt*location*sample 276, DF. There are 15 groups (A, B, etc.) in which the means are not significantly different from one another.

Nankana Sahib and Multan had 22.58, 23.68, 23.07, 23.68, 24.32, 25.00 and 32.60 %

infected samples respectively (i.e. below 40 percent), while the remaining seven districts

such as Sialkot, Gujranwala, Faisalabad, Sahiwal, Khanewal, Vehari and Toba Tek Singh

had 40.54, 48.57, 40.57, 42.85, 45.16, 49.05 and 60.97 percent Karnal bunt infected

samples respectively (i.e. above 40 percent). The low number (below 20%) of wheat

samples infected by Karnal bunt fungus (T. indica) in districts of Muzaffargarh, Dera

Ghazi Khan and Lodhran (Southern Punjab) can be attributed to relatively dry

environmental and soil conditions of these districts as compared to the districts of the

Name of district Mean Homogenous group 1-Sialkot 57.74 A 2-R. Y. Khan 49.15 B 3-Gujranwala 44.78 C 4-Faisalabad 42.87 CD 5-D. G. Khan 40.63 CD 6-Bahawalpur 39.37 DE 7-Multan 36.05 EF 8-Khanewal 35.73 EF 9-Sahiwal 34.65 F 10-Bhakkar 33.22 F 11-Nankana Sahib 31.96 FG 12-Chakwal 28.29 GH 13-Kasur 25.29 HI 14-Sargodha 24.93 HI 15-Jhang 24.92 HI 16-Shekhupura 23.03 IJ 17-Layyah 22.21 IJ 18-Ludhran 19.64 JK 19-Muzaffargarh 17.44 K 20-Gujrat 12.94 L 21-Rawalpindi 10.64 LM 22-Mianwali 7.25 MN 23-Vehari 7.14 MN 24-Rajanpur 5.41 N

Eastern Punjab where above 40 percent of the samples were found infected due to

existing of relatively moist environmental and soil conditions which probably favour the

germination of teliospores of Table 7: Percent wheat sample infection, range of infection and average infection in grain markets and storage godowns of 17 districts of the Punjab by Karnal bunt disease during 2006.

Sr no.

Districts

Total no. of samples collected

-ve sample

+ve sample

percent samples infected

Grade/Range of infection

Average infection percentage

1 Bahawal pur 38 29 9 23.68 0.57-3.51 1.75

2 Multan 46 31 15 32.60 0.75-4.59 1.75

3 Muzaffargar

h 43 40 3 6.976 0.50-2.15 1.46

4 D.G.khan 25 23 2 8.00 0.65-2.91 1.78

5 Khanewal 31 17 14 45.16 1.50-3.65 1.95

6 Vehari 53 27 26 49.05 1.57-3.74 1.85

7 Gujranwala 37 22 15 48.57 1.85-4.43 2.97

8 Sialkot 35 18 17 40.54 1.75-4.95 2.75

9 Sheikupura 31 24 7 22.55 0.75-2.75 1.05

10 Ludhran 28 23 5 17.85 0.55-3.38 0.95

11 Sahiwal 35 20 15 42.85 1.05-3.68 1.57

12 Faisalabad 69 41 28 40.57 0.95-4.75 2.45

13 Jhang 38 29 9 23.68 0.75-2.24 1.25

14 Toba-Tak Singh 41 16 25 60.97 0.85-4.96 02.45

15 Sarghodha 37 28 9 24.32 0.45-2.48 0.78

16 Chakwal 13 10 3 23.07 0.39-2.67 1.27

17 Nankana-

sahib 24 18 6 25.00 0.75-4.54 2.35

the Karnal bunt fungus and multiplication of its sporidia which could infect developing

grains in the heads of wheat crop.

The range of wheat grain infection and average grain infection varied greatly

among infected samples of the 17 districts (Table 7). The maximum range of grain

infection of 0.75 to 4.59, 1.85 to 4.43, 1.75 to 4.95, 0.95 to 4.75, 0.85 to 4.96 and 0.75 to

4.54 was found in the infected samples of Multan, Gujranwala, Sialkot, Faisalabad, Toba

Tek Singh and Nankana Sahib district, respectively with average grain infection of 1.75,

2.97, 2.75, 2.45, 2.45 and 2.35 percent, respectively. The minimum range of grain

infection of 0.50-2.15, 0.65-2.91, 0.75-2.75, 0.75-2.24, 0.45-2.48, and 0.39-2.67 was

found in infected samples of Muzaffargah, D.G. Khan, Sheikhpura, Jhang, Sargodha and

Chakwal districts, respectively with average grain infection of 1.46, 1.78, 1.05, 1.25, 0.78

and 1.27 percent, respectively. The intermediate range of wheat grain infection of 0.57-

3.51, 1.50-3.65, 1.57-3.74, 0.55-3.38, 1.05-3.68 was found in the infected samples of

Bahawalpur, Khanewal, Vehari, Lodhran and Sahiwal, respectively with average wheat

grain infection of 1.57, 1.95, 1.85, 0.95, 1.57 percent, respectively. Again the maximum

and minimum range of grain infection in various districts can be attributed to relatively

wet or dry conditions of the atmosphere and soil of the districts surveyed.

Table: 8. Analysis of variance for the incidence of Karnal bunt disease of wheat under natural condition in various hot spot areas of the Punjab during field survey 2007.

S.O.V. DF SS MS F P

Replication

(Samples)

2 9.62 4.81

Varieties 2 2.91 1.45 2.72 0.61ns

Locations 4 97.17 24.29 45.33 0.00*

Districts 11 313832.3 28530.21 53241.03 0.00*

Dist.x Loc. 44 2034.243 46.23 86.27 0.00*

Dist.x Var. 22 73.23 3.32 6.21 0.34ns

Loc.x Var 8 106.25 13.28 24.78 0.00*

Dist.x Loc.x Var. 88 1156.35 13.14 24.52 0.00*

Error 358 191.84 0.53

Total 539 317503.9

Grand mean 30.68, CV 5.67 *Significant at 0.05 ns= non-significant

The survey of kanral bunt disease of wheat crop of 2007 in various hot spots

of farmer’s field falling in 13 districts of the Punjab (Table 10) revealed that the hot spots

of Bahawalpur, Vehari, Toba Tek Singh, Sahiwal, Multan and Bhakkar had maximum

range of wheat grain infection of 0.97-6.5, 1.05-6.5, 1.5-6.5, 0.75-5.9, 0.57-5.5 and 0.75-

5.5, percent respectively with average grain infection of 4.9, 4.50, 4.25, 2.25, 2.25 and

3.00 percent, respectively. The hot spots of districts D.G. Khan and Muzaffargarh had

minimum range of grain infection of 0.75-2.0 and 0.87-2.7 percent, respectively with

average grain infection of 1.25 and 1.60 percent respectively while the hot spots of

district Rawalpindi, Chakwal, Layyah, Jhang and Faisalabad had intermediate range of

wheat grain infection of 0.5-3.7, 0.96-3.10, 0.55-3.50, 0.95-3.5 and 0.95-3.50 percent

respectively with average grain infection of 1.50, 1.50, 2.00, 2.50 and 1.25 percent,

respectively (Table 10).

Table: 9. All pairwise comparisons test for Karnal bunt disease of wheat for districts recorded in hot spot areas (field).

Name of district Mean Homogenous group 1-Bahawalpur 80.09 A

2-Vehari 65.19 B

3-T. T. Singh 51.95 C

4-Sialkot 43.15 D

5-Sahiwal 33.16 E

6-Chakwal 32.82 E

7-Khanewal 20.44 F

8-Faisalabad 14.30 G

9-Multan 11.61 H

10-Muzaffargarh 9.16 I

11-D. G. Khan 5.19 J

12-R. Y. Khan 1.13 K

Alpha 0.05, Standard Error for Comparison 0.3665, Critical T Value 1.965. Critical Value for Comparison 0.7204, Error term used: Samples*Varieties*Locations*Distt, 446 DF. There are 11 groups (A, B, etc.) in which the means are not significantly different from one another.

While conducting the survey of hot spot areas to observe variation in disease

incidences of 12 districts (Table 9) of the Punjab, there was a non significant difference

in varieties indicating that cultivars were either susceptible or resistant to Karnal bunt

disease of wheat (Table 8). Regarding the severity of the disease in the hot spots of areas,

variation was found among the districts and locations even in the same district, as

indicated by the significant interactions of districts, locations and varieties (Table 8).

Table: 10. Survey of Karnal bunt disease of wheat under natural condition in different hot spot areas of the Punjab during the crop year 2007. Sr no.

Districts

Location

Varieties

Percent infection

Range Average

1 Rawalpindi Pindi Gujran

Parwaz-94, Pasban-90 and MH-97

0.5—3.7 1.50

2 Chakwal Karyala Chakwal 97,Pb-85

and Yecora 0.96—3.10 1.50

3 Sahiwal Arifwala

Parwaz-94, MH-97 and unknown

0.75—5.9 2.25

4 Muzaffar –garh Seetpur, Ali pur AS-2002 and

unknown 0.87—2.7 1.60

5 Mutltan Sher shah, Bun bosan

SH-2003 and unknown 0.57—5.5 2.25

6 Bahawal-pur

Ahmad pur, Khan pur

Inqalab-91 and Unknown 0.97—6.5 4.90

7 D. G. khan Shadan Unknown 0.75—2.0 1.25

8 Layyah Karor lalesan AS2002 and

Faisalabad-85 0.55—3.5 2.00

9 Bhakkar Sarai muhajir,

Jahan khan AS2002 and unknown 0.75—5.5 3.00

10 Jhang

Mulwana morh,Hassu balail

Pasban-90 and MH-97 0.95—3.5 2.50

11 Vehari Burewala, Luden Unknown and

Kohinoor-83 1.05—6.5 4.50

12 Toba Tak- Sing

Chak 387 J.B, Chak 384,J.B.

AS-2002 and Inqabal-91 1.5—6.5 4.25

13 Faisalabad Amin pur Bangla Watan, and

Unknown 0.95—3.50 1.25

The survey/ assessment of Karnal bunt disease of various hot spots naturally developed at

research stations/institutes, model farms and seed corporation farms (Table 11) revealed

that hot spots of Jalindhar Seed Corporation Farm Arifwala (Sahiwal) and Barani Agri.

Research Institute, Chakwal developed 2.5 and 3.5 percent grain infection with a range of

infection of 1-5 and 1-7 percent, respectively while the hot spots at Punjab Seed

Corporation (Peeruwal) Khanewal and at Agronomic Research Institute (Karor Laleason)

Layyah developed 4.5 and 6.5 percent grain infection with a range of 1-15 and 5-10

percent infection, respectively, while the hot spots of Tareen Model Farm Lodhran and

Experimental Farm of the Department of Plant Breeding & Genetics, University of

Agriculture, Faisalabad, developed 7.5 and 10.5 percent grain infection with a range of

infection 5-15 and 5-45 percent, respectively.

Table: 11. Rang of infection and percent wheat grains infection in hot spots of various research institutes/ research stations, model farms and seed corporation farms during wheat crop of 2007.

Name of place/ Venue Lines/ varieties affected

Percent Range of infection

Percent Average Infection

Coefficient of infection

Tareen Model Farm Lodhran

AS-2002, Shafaq-06 and KC050

5-15

7.5

11.87

Punjab seed corporation (Peeruwal) Khanewal

AS-2002 and KC050

1-15

4.5

10.43

Jalindher seed corporation (Arifwala) Sahiwal

AS2002, KC050, Iqbal-91 and Pasban -90

1-5

2.5

7.98

Agronomic Research Station (Karore lalesan) Layyah

AS-2002 5-10

6.5

13.24

Experimental area of

Department of Plant Breeding and Genetics University of Agriculture Faisalabad

75 germplasm lines

5-45

10.5

15.56

Barani Agricultural Research Instt. Chakwal

35 germplasm lines

1-7

3.5

6.24

Screening of wheat germplasm for the sources of resistance Regarding the approaches towards breeding for disease resistance, the

screening of 201 advanced lines of wheat revealed that 16, 9, 12, 33, 44, and 87 lines

were highly resistant (immune), resistant, moderately resistant, moderately susceptible,

susceptible and highly susceptible, respectively (Table 12). Sixteen highly resistant lines

were V-02192, V-03079, V-04022, V-04178, V-05011, V-05020, V-05023, V-05025, V-

05039, V-05136, V-05144, V-05150, V-06122, V-06126, V-06127, V-06128 while the

nine resistant lines were V-01078, V-03138, V-04048, V-04076, V-04171, V-04183, V-

05132, V-05152 and V-08794. The twelve moderately resistant lines were V-02156, V-

04157, V-04189, V-04611, V-5010, V-05097, V-5610, V-06121, V-06123, V-06125, V-

06131 and V-06133. The remaining advanced lines were moderately to highly

susceptible.

Out of 44 commercial cultivars screened against Karnal bunt disease, none was

found to be highly resistant or resistant. However, 07, 07, 14 and 16 cultivars were found

to be moderately resistant, moderately susceptible, susceptible and highly susceptible

(Table 10). The seven moderately resistant cultivars were BWP-97, SA-42, SH-2002,

Farid-2006, Blue Silver, Shafaq-06 and Manthar. The seven moderately susceptible

cultivars were Chris, Crow, Fontana, Pavon, Moracco, Dirk and Daman-98. The fourteen

susceptible cultivars were Pak-81, Sahar, SA-75, Pb-76, Pb-81, Pb-96, Faisalabad-85,

Parwaz-94, Uqab-2002, GA-2002, Inqlab-91, Kohsar-95, Chakwal-96 and Chakwal-97

while the sixteen highly susceptible cultivars were C-271, C-273, C-518, C-591, WL-

711, Chenab-2000, Faisalabad-83, Kohinoor-83, Local White, MH-97, Pb-85, Pasban-90,

LH-26, Shalimar-88 and Yecora and Mexi-Pak-65.

Epidemiological studies

Correlation of environmental conditions with karnal bunt disease: Karnal bunt disease incidences recorded on 66 gremplasm lines/ varieties during

two consecutive growing season 2005-06 and 2006-07 revealed significant results (Table

13). The individual effect of years and varieties and their interactive effects were

significant. Variation in the disease incidence among the 66 germplasm lines/ varieties

during two years was observed. Each of the germplasm line/ variety indicated a different

degree of Table: 12. Level of resistance/susceptibility of advanced wheat lines and commercial

cultivars against Karnal bunt disease of wheat Grade in the disease rating scale and percent grain infection

Response of test line or cultivar against the disease

Advanced wheat lines

Commercial wheat cultivars

0 = No infection at all

Highly resistant or immune

V-02192, V-03079, V-04022, V-04178, V-05011, V-05020, V-05023, V-05025, V-05039, V-05136, V-05144, V-05150, V-06122, V-06126, V-06127, V-06128

-

1 = Less than 1% grain bunted

Resistant V-01078, V-03138, V-04048, V-04076, V-04171, V-04183, V-05132, V-05152, V-08794

-

3 = 1.1-2% grain bunted

Moderately resistant

V-02156, V-04157, V-04189, V-04611, V-05010, V-05097, V-05610, V-06121, V-06123, V-06125, V-06131, V-06133

BWP-97, SA-42, SH-2002, Blue Silver, Farid-2006, Shafaq-06 and Manthar

5 = 2.1-5% grain bunted

Moderately susceptible

V-03094, V-03285, V-04040, V-04122, V-04179, V-04188, V-04306, V-05003, V-05004, V-05009, V-05041, V-05042, V-05043, V-05044, V-05048, V-05049, V-05050, V-05053, V-05054, v-05055, V-05058, V-05060, V-05064, V-05065, V-05066, V-05067, V-05071, V-05072, V-05082, V-06069, V-09247, V-00BT08, V-00BT015

Chris, Crow, Fontana, Pavon, Moracco, Dirk, Daman-98

7=5.1-10% bunted grain

Susceptible V-02156, V-03007, V-03079, V-03094, V-03138, V-04009, V-04022, V-04040, V-04048, V-04068, V-04112, V-04157, V-04171, V-04178, V-04179, V-04181, V-04188, V-04189, V-04309, V-04611, V-05006, V-05008, V-05056, V-05062, V-05070, V-05093, V-05132, V-05136, V-05144, V-05150, V-05152, V-05620, V-06124, YR9-SEERI-E45, YR10-E30, YR15-E27, YR18-E23, YRA-E27, YRA-E40ANZA, LR-2C, LR-10, LR-13, LR-26, V2KC-050

Pak-81, Sahar, SA-75, Pb-76, Pb-81, Pb-96, Faisalabad-85, Parwas-94, Uqab-2002, GA-2002, Inqlab-91, Kohsar-95, Chakwal-96, Chakwal-97

9=More than 10 percent grain bunted

Highly susceptible

D-05603, D-05604, D-05607, D-05608, D-05609, D-05612, D-05623, D-05625, D-05627, D-05630, V-04148, V-05085, V-05086, V-05087, V-05088, V-05090, V-05094, V-05096,

C-271, C-273, C-518, C-591, WL-711, Chenab-2000, Faisalabad-83,

V-05100, V-05101, V-05104, V-05105, V-05107, V-05113, V-05115, V-05119, V-05121, V-05122, V-06129, V-06130, V-06134, V-06135, V-06136,V-06137, V-06138, V-06139, V-06140, V-085205, V-087049, V-087094, LR-1, LR-2, LR-2B, LR-3, LR-3KA, LR-3BG, LR-9, LR-11, LR-12, LR-14A, LR-14B, LR-15, LR-16, LR-17, LR-18, LR-19, LR-20, LR-21, LR-22A, LR-22B, LR-23, LR-24, LR-25, LR-27, LR-28, LR-29, LR-30, LR-32, LR-33, LR-34, LR-35, LR-36, LR-37, LR-38, YR1-E1, YR1-E18, YR2-E35, YR5-E19, YR5-E25, YR6-APR-E38, RR6-YR7, E37, YR7-E36, YR7-E10, YR8-E20, YR8-E26, YR9-E39

Kohinoor-83, Local White, MH-97, Pb-85, Pasban-90, LU-26, Shalimar-88, Yecora, MexiPak-65

Table: 13. Analysis of variance of Karnal bunt disease incidences recorded on wheat varieties during 2006 and 20007. Source of

variance

DF Sum of

squares

Mean square F Values Prob>F

Replication 3 12.2 4.06

Varieties 65 11749.3 180.76 105.82 0.000*

Year 1 4970.5 4970.45 2909.78 0.000*

Varieties vs Year 65 2341.00 36.02 21.08 0.000*

Error 393 671.30 1.71

Total 527 19744.30 Grand Mean 14.083 CV 9.28 **Significant at 0.05

infection during its growth stages under natural conditions, although similar production

technology, cultural and agronomic practices were adopted during both years. The data

were analyzed by correlation to determine the influence of environmental factors.

There was a significant correlation of maximum air temperature with 62

entries and minimum air temperature with 58 entries. (Table: 14). After the (min./max.)

air temperature rainfall had significant correlation in the development of Karnal bunt

disease as

Table: 14. Correlation of environmental factors with Karnal bunt disease of wheat on different lines/varieties.

Lines/ varieties

Temperature C Relative humidity

mm)

Rainfall (mm)

Wind velocity

Max. Min.

9013 0.89663* 0.0001

0.91809* 0.0098

- 0.6946 0.1256

-0.7885 0.0623

0.2184 0.9672

9092-2 0.89663* 0.0155

0.91809* 0.0098

0.6946 0.1256

0.7885 0.0623

0.0218 0.9672

9141 0.90476* 0.0132

0.92584* 0.0080

-0.7106 0.1135

-0.7954 0.0585

0.4957 0.9257

9148 0.85395* 0.0304

0.87621* 0.0220

-0.6249 0.1847

-0.7516 0.0849

-0.0832 0.8754

9181 0.92428* 0.0084

0.79822 0.0570

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7186

9189 0.91521* 0.0105

0.93581* 0.0060

-0.73527 0.0958

0.8044 0.0536

0.0965 0.8556

9191 0.92428* 0.0084

0.94345* 0.0047

-0.77558 0.0699

-0.8118* 0.0498

0.1911 0.7168

9233 0.91647* 0.0102

0.93385* 0.0064

-0.80172 0.0551

-0.8044 0.0536

0.2822 0.5879

9233-5 0.85395* 0.0304

0.87621* 0.0220

-0.6249 0.1847

0.7516 0.0948

-0.0832 0.8754

9292 0.90964* 0.0119

0.93059* 0.0071

-0.72150 0.1055

-0.7997 0.0561

0.0696 0.8958

9296 0.92259* 0.0088

0.94240* 0.0049

-0.76068 0.0791

-0.8106* 0.0504

0.1525 0.7729

9316-1 0.91521* 0.0105

0.93581* 0.0060

- 0.7352 0.0958

-0.8044* 0.0506

0.0965 0.8556

9316-2 0.92374* 0.0085

0.94243* 0.0049

-0.7847 0.0645

-0.8112 0.0536

0.2181 0.6780

9316-3 0.92207* 0.0089

0.94196* 0.0050

-0.7581 0.0807

-0.8102* 0.0506

0.1464 0.7819

9327 0.91799* 0.0098

0.93557* 0.0061

-0.7997 0.0561

-0.8058 0.0529

0.2733 0.6001

9332-1 0.92267* 0.0087

0.94103* 0.0051

-0.7898 0.0616

-0.8102* 0.0506

0.2350 0.6540

9372 0.92428* 0.0084

0.79822 0.0570

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9381 0.92160* 0.0090

0.93973* 0.0053

-0.7930 0.0598

-0.8091 0.0511

0.2465 0.6377

9428 0.83627* 0.0380

0.85869 0.0285

-0.5995 0.2084

-0.7363 0.0951

-0.1167 0.8257

9435-2 0.89663* 0.0155

0.91809* 0.0098

-0.6946 0.1256

-0.7885* 0.0503

0.0218 0.9672

9436 0.58094 0.2266

0.60232 0.2058

-0.2996 0.5640

-0.5134 0.2975

-0.4124 0.4165

Contd…

9437 0.86444* 0.0263

0.88657* 0.0186

-0.6406 0.1705

-0.7607 0.0790

-0.0613 0.9081

9451 0.92298* 0.0087

0.9414* 0.0050

-0.7886 0.0623

-0.81050 0.05305

0.2308 0.6598

9459 0.92428* 0.0084

0.9434* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9459-1 0.92428* 0.0084

0.94345* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9466 0.92428* 0.0084

0.94345* 0.0047

-0.7755 0.0699

-0.8118 0.498

0.1911 0.7168

9475 0.88515* 0.0190

0.89998* 0.0145

-0.8137* 0.0488

-0.7763 0.0695

0.3843 0.4519

9475-1 0.88241* 0.0199

0.90424* 0.0133

-0.6694 0.1458

-0.7763 0.0695

-0.0188 0.9718

9476 0.85395* 0.0304

0.87621* 0.0220

-0.6249 0.1847

-0.7516 0.0849

-0.0832 0.8754

9479 0.89341* 0.0164

0.91497* 0.0105

-0.6887 0.1303

-0.7857 0.0639

0.0118 0.9822

9480 0.88241* 0.0199

0.90424* 0.0133

-0.6694 0.1458

-0.7763 0.0695

-0.0188 0.9718

9481 0.91521* 0.0105

0.93581* 0.0060

-0.7352 0.0958

-0.8044 0.0536

0.0965 0.8556

9489 0.92428* 0.0084

0.94345* 0.0047

-0.7755 0.0699

-0.8118 0.05980

0.1911 0.7168

9492 0.90743* 0.0125

0.92848* 0.0075

-0.7165 0.1091

-0.7978 0.0572

0.0603 0.9096

9493-1 0.91293* 0.0110

0.93368* 0.0065

-0.7293 0.1000

-0.8025 0.0546

0.0847 0.8732

9496 0.87086* 0.0239

0.89290* 0..0166

-0.6506 0.1617

-0.7663* 0.05045

-0.0469 0.9296

9507 0.92428* 0.0084

0.94345* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9508 0.88241* 0.0199

0.90424* 0.0133

-0.6694 0.1458

-0.7763 0.0695

-0.0188 0.9718

9517 0.83627* 0.0380

0.85869* 0.0285

-0.5995 0.2084

-0.7363 0.0951

-0.1167 0.8257

9519 0.91126* 0.0115

0.92808* 0.0076

-0.8064 0.0526

-0.7997 0.0561

0.3069 0.5541

9520 0.86930* 0.0245

0.89136* 0.0171

-0.6482 0.1639

-0.7649 0.0764

-0.0505 0.9242

9520-2 0.85395* 0.0304

.87621* 0.0220

-0.6249 0.1847

-0.7516 0.0849

-0.0832 0.8754

9521 0.58094 0.2266

0.60232 0.2058

-0.2996 0.5640

-0.5134 0.2975

-0.4124 0.4165

9522-1 0.81144* 0.0500

0.83398* 0.0391

-0.5658 0.2418

-0.7147 0.1105

-0.1583 0.7645

Contd…

9527-1 0.92428* 0.0084

0.94345* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9529-2 0.82797* 0.0418

0.71872 0.1075

-0.5880 0.2196

-0.7290 0.1001

-0.1312 0.8043

9529-3 0.83148* 0.0402

0.85393* 0.0304

-0.5929 0.2149

-0.7321 0.0980

-0.1251 0.8132

9536 0.92207* 0.0089

0.9419* 0.0050

-0.7581 0.0807

-0.8102* 0.0506

0.1464 0.7819

9541 0.91378* 0.0108

0.9344* 0.0063

-0.7315 0.0985

-0.8032 0.0543

0.0889 0.8669

9542-3 0.85395* 0.0304

0.87621* 0.0220

-0.6249 0.1847

-0.7516 0.0849

-0.0832 0.8754

9546 0.92428* 0.0084

0.94345* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9554 0.84494* 0.0342

0.73305 0.0974

-0.6118 0.1968

-0.7438 0.0900

-0.1007 0.8493

9595 0.92207* 0.0089

0.94196* 0.0050

-0.7581 0.0807

-0.8102* 0.0506

0.1464 0.7819

9602 0.80795 0.0518

0.83050* 0.0407

-0.5612 0.2465

-0.7116* 0.01127

-0.1637 0.7565

9610 0.91809* 0.0098

0.93846* 0.0056

-0.7435 0.0902

-0.8068 0.0523

0.1137 0.8301

9618 0.92401* 0.0084

0.94281* 0.0048

-0.7825 0.0658

-0.8115* 0.0499

0.2115 0.6874

9622 0.92428* 0.0084

0.9434* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9629 0.92428* 0.0084

0.9434* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

9677 0.89282* 0.0166

0.9143* 0.0107

-0.6876 0.1311

-0.7852 0.0642

0.0101 0.9849

Iqbal-2000 0.58094 0.2266

0.6023 0.2058

-0.2996 0.5640

-0.5134 0.2975

-0.4124 0.4165

Barani-70 0.92428* 0.0084

0.9434* 0.0047

-0.7755 0.0699

-0.8118* 0.0498

0.1911 0.7168

Barani-83 0.91144* 0.0114

0.9322* 0.0067

-0.7257 0.1025

-0.8012 0.0553

0.0776 0.8837

Anmol-91 0.91903* 0.0096

0.93931* 0.0054

-0.7465 0.0882

-0.8076 0.0519

0.1202 0.8205

Faisalabad-85

0.91686* 0.0101

0.93734* 0.0058

-0.7398 0.0927

-0.8058 0.0529

0.1060 0.8416

Pasban -90 0.85395* 0.0304

0.87621* 0.0220

-0.6249 0.1847

-0.7516 0.0849

-0.0832 0.8754

Parwaz-94 0.88817* 0.0181

0.90986* 0.0118

-0.6793 0.1378

-0.7812 0.0665

-0.0033 0.9950

*Correlation significant at P =0.05 Upper values in a column indicate Pearsons correlation coefficient. Lower values indicate

Contd…

level of significance at 5 % level of probablity. Table: 15. Comparison of monthly environmental conditions and karnal bunt incidence recorded on wheat varieties during 2006 and 2007.

Environmental parameters

2006 2007 LSD

Maximum temperature

(oC)

31.1 a 27.8 a 16.53

Minimum temperature

(oC)

16.1 a 14.8 a 10.87

Relative humidity (%) 38.8 a 49.71 a 34.91

Rainfall (mm) 0.57 a 1.10 a 1.88

Wind speed (km/hr) 4.03 a 2.50 a 1.98

Disease incidence 11.16 b 17.15 a 0.223

Mean values sharing the same letters do not differ significantly as determined by the LSD test (P = 0.05) compared to relative humidity which significantly influenced only one line i. e. 9475.

Rainfall exerted significant influence on 21 lines/ cultivars. There was absolutely no

correlation between the Karnal bunt disease incidences and wind velocity. Eighteen lines

i.e. 9191, 9296, 9316-1 ,9316-3, 9332-1, 9435-2, 9459, 9459-1, 9475, 9496, 9507, 9527-

1, 9536, 9546, 9595, 9618, 9622, 9629 and one variety Barani-70 were influenced

significantly by more than 50 % environmental factors in the disease development (Table

14).

By the comparision of weather data during the vulnerable stages for the disease

incidence recorded on wheat varieties/ lines, (Table 15) it was concluded that during

2006, average maximum temperature was 31.1 oC while the average minimum

temperature was 27.8 oC. Relative humidity and rainfall were recorded 38.8 % and 0.57

mm respectively. High rainfall (1.10 mm), relative humidity (49.1 %), low maximum

temperature (27.8 oC) and low minimum temperature (14.8 oC) during the year 2007

caused the more disease incidences than year 2006.

Disease predictive models Two years combined environmental conditions and Karnal bunt disease

incidence data subjected to stepwise regression analysis revealed a model consisting of

air temperature (max./ min.) rainfall wind speed and relative humidity explained 84 % of

the variability in disease development (Table 16). Thus all the environmental variables

were exerting significant influence on Karnal bunt disease development during two years

period.

When these data were split by year, a model consisting of minimum and

maximum air temperature, rainfall, relative humidity and wind speed explained 81 % of

the variability in Karnal bunt disease development during 2005-06 (Table 17). However,

in the second year relative humidity and rain fall were eliminated by stepwise regression

and a model consisting of only three environmental variables (i.e. maximum air

temperature, minimum air temperature and wind speed) explained 90 % of the variability

in Karnal bunt disease development (Table 18).

Table.16. Summery of stepwise procedure for independent variables influencing Karnal bunt disease of wheat during growing season 2005-07

* Significant at P = 0.05

Regression equation Y = -53.78+1.81x1* + 0.19x2*+0.08x3 +5.69x4* -1.97x5. Where x1 indicate maximum temperature, x2 minimum temperature, x3 relative humidity,

x4

Step Variables entered Number In Model R2 C (p) F Prob>F

1 Min. temp 1 0.48 976.20 373.96 0.0001*

2 Rain fall 2 0.73 326.05 357.84 0.0001*

3 Wind speed 3 0.79 163.04 117.26 0.0001*

4 Relative

humidity 4 0.81 101.36 51.00 0.0001*

5 Max. temp. 5 0.84 30.41 66.74 0.0001*

indicates rain fall and x5 indicates wind speed respectively.

*Indicates significant regression at 5 % level of probably

Table: 17. Summary of stepwise procedure for independent variable influencing Karnal bunt disease during growing season 2005-06

*Significant at P = 0.05 Regression equation Y = -38.18 + 1.81x1 *+ 0.26x2 + 0.18x3* + 5.69x4* -1.97x5. Where x1 indicate minimum temperature, x2 maximum temperature, x3 relative humidity, x4 rainfall and x5 wind speed respectively. *Indicates significant regression at 5 % level of probably Table: 18. Summery of stepwise procedure for independent variables influencing karnal bunt disease during growing 2006-07.

Step Variables

entered Number In Model R2 C (p) F Prob>F

1 Min. temp 1 0.55 702.79 237.61 0.0001*

2 Wind

speed 2 0.85 99.31 400.40 0.0001*

3 Max.

temp. 3 0.90 3.00 98.31 0.0001*

*Significant at P = 0.05

Regression equation Y = 24.64 + 0.87x1* + 0.18x2 * - 1.08x3.

Step Variable entered

Number In Model R2 C (p) F Prob>F

1 Min temp. 1 0.43 410.84 155.07 0.0001*

2 Relative humidity 2 0.72 93.51 219.75 0.0001*

3 Rainfall 3 0.75 71.27 18.09 0.0001*

4 Max. temp. 4 0.76 56.19 13.54 0.0003*

5 Wind speed 5 0.81 3.00 55.66 0.0001*

Where x1 indicate minimum temperature, x2 maximum temperature, x3 wind speed respectively. *Indicates significant regression at 5 % level of probably Chemotherapy of Karnal bunt disease of wheat

(i) In vitro evaluation of fungicides against the mycelial growth of T. indica An in vitro evaluation of ten fungicides (Table 4), amended into PDA, against

colony growth of T. indica revealed that the effectiveness of the fungicides depended

on kind of fungicide and its dosage rate evaluated. However, the efficacy of fungicides

for inhibiting colony growth of T. indica increased with an increase in their dosage

rates (Table 19). Dolomite and Shelter at their 40µg/ ml and 100µg/ ml dosage rates

were the most and statistically equally effective fungicide in inhibiting the colony

growth of Tilletia indica as these fungicides produced 3.50 and 3.35 mm diameter at

40µg/ ml and 7.77 and 7.75 mm diameter inhibition zones of the fungus at 100µg/ml

(Fig. 11). Shelter however at 100 µg/ml dosage rate displayed the same and statistically

equal effectiveness as Dolomite at 80 µg/ml. Crest, Antracol Alert plus at 100 µg/ml

dosage rate were less effective than Dolomite and Shelter as these fungicides produced

3.77, 3.50, 3.10 mm diameter inhibition zones respectively. However, there was no

statistically difference between the effectiveness of Crest and Antracol and between

that of Antracol and Alert plus. Alert plus and Antracol at 100 µg/ml dosage rate

displayed the same effectiveness as Dolomite and Shelter displayed at 40 µg/ml.

Reconil, Agrohit and Thiomil were the least effective fungicides

A B

C

EF

D

Fig. 11 : Mean inhibition zones of Tilletia indica by action of various fungicides. A = control (water), B = Alert plus at100 ppm, C = Shelter at 80 ppm, D = Antracal at100 ppm, E = Dolomite at100 ppm F = Shelter at100 ppm

against T. indica, while Aliette and Protocol pre-combi were ineffective in inhibiting

the growth of the fungus (Table 19).

(ii) In vivo control of Karnal bunt disease of wheat by protective and

curative (eradicative) spray of fungicides.

The in vivo evaluation of effectiveness of the 10 test fungicides, as protective

spray (i.e. spray before inoculation) and as eradicative spray (i.e. spray after

inoculation) revealed that the protective sprays were more effective than eradicative

sprays in controlling Karnal bunt disease of field grown wheat plants (Table 20).

The protective spray of Dolomite and Shelter, though statistically equally effective,

were the most effective and caused 62 and 64 percent reduction in occurrence of Karnal

bunt disease respectively, while the eradicative spray of Dolomite and Shelter reduced

39 and 41 percent disease, respectively on wheat grain and there was no statistical

difference between the effectiveness of both fungicides.

Table: 19. Mean inhibition zones of colony of Tilletia indica by various fungicides at 4 dosages rates amended in PDA medium. Fungicides

Mean Inhibition zone (mm) at 4 dosage rates40 ppm 60 ppm 80 ppm 100 ppm

Crest 2.52 h* 3.40 ef 3.75 e 3.77 e

Dolomite 3.50 ef 5.70 c 7.75 a 7.77 a

Agrohit 0.25 l 0.50 l 0.5 0 l 0.5 l

Alert plus 0.00 l 0.00 l 2.00 j 3.1 fg

Shelter 3.35 ef 4.30 d 6.45 b 7.75 a

Reconil 0.00 l 1.00 k 1.00 k 1.00 k

Aliette 0.00 l 0.00 l 0.00 l 0.00 l

Antracol 2.25 ij 2.8 0 gh 3.50 ef 3.5 ef

Protocol pre-

combi 0.00 l 0.00 l 0.00 l 0.00 l

Thiomil 0.25 l 0.25 l 0.5 l 0.5 l

Water

(control) 0.00 l 0.00 l 0.00 l 0.00 l

* Values having the same letters do not differ significantly at 5% level of

significance as determined by LSD = 0.44 and EMS = 0.10

The protective sprays of Crest, Agrohit, Alert plus and Antracol were

intermediate in their effectiveness and respectively caused 33.79, 34.65, 18.93 and

41.19 percent reduction in Karnal bunt infection of wheat grains. However, there was

not significant different between the effectiveness of Crest, Agrohit and Antracol.

Table: 20. Percent decrease in wheat kernals infections by Tilletia indica by protective and

Fungicides

Spray of fungicides before

Inoculation

Spray of fungicides after

Inoculation

Mean percent

kernel infection

Percent

decrease in

kernel infection

over control

Mean percent

kernel infection

Percent

decrease in

kernel

infection over

control

Crest 22.45 efg* 33.79 28.81 bc 7.30

Dolomite 12.73 h 62.45 19.01 fg 38.84

Agrohit 22.16 efg 34.65 28.64 bc 7.82

Alert plus 27.49 cd 18.93 28.89 bc 7.04

Shelter 12.24 h 63.90 18.50 g 40.48

Reconil 33.01 ab 2.71 26.41 cde 14.98

Aliette 30.86 abc 8.99 30.97 abc 0.35

Antracol 19.94 fg 41.19 23.59 def 24.07

eradicative spray of various fungicides. *Values having the same letters do not differ significantly at 5% level of significance as

determined by LSD value = 4.91 at alpha = 0.05, EMS = 8.87

.

Antracol being comparatively less affective caused 24.07 percent decrease in

kernel infection by its curative spray. The eradicative sprays of Crest, Agrohit and Alert

plus were ineffective in controlling wheat kernel infection. The protective as well as

curative sprays of Reconil M, Alliette, Protcol Pre-combi and Thiomil were ineffective

in the control of wheat kernel infection by T. indica.

From the analysis of environmental paramaters for the development of Karnal

bunt disease it was concluded that during the vulnerable growth stages of wheat

minimum temperature, maximum temperature and rainfall played a vital role. If the

average minimum temperature ranges between 11 to 16 oC and max. temperature 27.5 to

31.1 oC in the month of Feb. and March that are commonly considered booting stage

(depending upon varietal character and date of sowing) then the spray of fungicides is

necessary to avoid the infection. With the deviation of above mentioned ranges of the air

temperature numbers of the fungicidal sprays can be adjusted accordingly. Rainfall (at

least 1.10 mm) during the early booting stages provides additional critical circumstances

for the multiplication and dispersal of sporadia and hence in the progress of the disease.

Protocol

pre- combi 33.39 ab 1.53 30.10 abc 3.15

Thiomil 32.43 ab 4.36 28.87 bc 7.08

Control 33.91 a 0.00 0.00 abc 0.00

Chpapter 5

DISCUSSION

1- Survey of Karnal bunt disease During the comprehensive survey it was assessed that Karnal bunt disease was

found in all the wheat growing areas of the Punjab. However its’ incidence varied from

district to district. Distinction in the incidences of the disease among the districts may be

attributed to the differences in the climatic factors, as the specific weather conditions

affect on the life cycle of the fungus (Diekmann, 1998) and soil type play an important

role in the survival and germination of teliospores (Babadoost, et al., 2004). According to

Sansford et al., (2006) effect of T. indica on yeild and quality varied from year to year

and location to location. Because the wheat kernels as well as the grains present in them

are partially infected therefore, the incidence and severity of the disease differ from

location to location; even within a field it may be patchy. Management/ production

practices, adoptation of different technologies by farmers, sowing of newly evolved

commercial varieties, diversity in local climate especially from flag leaf stage to maturity

of the crop and degree of susceptibility of cultivars are the factors which may provide

justification to variation in disease incidences in various localities of a district.

In the past, Karnal bunt was known to occur in Sialkot district of the Punjab in

1950 in Northern areas of the country (Saleem and Akhtar, 1988) and was considered to

be a disease of minor importance as it occurred in traces in foothill districts of Punjab

(Munkdur, 1943). According to (Warham 1986, 1992), Karnal bunt affects wheat crop in

the northern regions of Pakistan and India. Increasing incidence, severity and distribution

of the disease toward the Southern Punjab have provided a great threat and alarming

situation for the Plant Pathologists and Plant Breeders as these areas were considered free

of this disease due to different climatology.

During survey it came to know that seed corporations and seed companies

were the main source of spreading the disease, because most of these companies get the

infected grains from affected areas at low rates, mix it with normal healthy seeds and sell

it at high price during growing season. The survey results were close to that of Mirza,

(2005) who pointed out that in Punjab MH 97, Inqilab-91 and Wattan varieties were

mostly affected with Karnal bunt disease. Average infection in grain markets and storage

godowns of main districts of the Punjab coincided with survey conducted by Mirza,

(2005) on farm disease assessment in the Punjab conducted during growing season 2003-

04 by Mirza, (2005). However, the survey results vary year to year as the climatic

conditions seldom remain same in every year during the growing season of wheat.

Normally the nature of the Karnal bunt disease is sporadic but in epidemic

years it causes substantial losses to crop in the Punjab, Sindh and North Western Frontier

Province (Ilyas et al., 1989). The disease remained endemic to Sialkot for a considerable

time (Hassan, 1971) but with the passage of time it had spread to new sites extending

from Sialkot to Mardan in the north and towards Jhang, Khanewal and Muzaffargarh in

the south (Bhatti and Ilyas, 1986). During a Karnal bunt survey of the wheat crop carried

earlier (Ilyas et al., 1989) the disease was reported to be absent in wheat samples

collected from Multan, D.G. Khan and other districts of the southern Punjab but now it is

prevalent in these districts. This spread of Karnal bunt in all 17 surveyed districts of the

Punjab is of great quarantine concern. This aggravating condition and high wheat grain

infection by Karnal bunt disease at farmer’s fields, research stations/institutes, seed

corporations and Model Farms may serve as a source of inoculum to other unaffected

areas of the Punjab for next year’s crops and may further increase the incidence of Karnal

bunt disease in the Punjab, thus aggravating more and more quarantine status of the

wheat produce of the Punjab.

The biodiversity of the pathogen, its successful establishment and gradual

distribution in various agro-ecological zones may further be attributed to physiological

races. Aujla et al., (1987) reported the existence of four pathotypes, K1, K2, K3 and K4

among 21 N. indica isolates or field collection from different geographic locations of

Punjab and Himachal Pradesh on a set of 17 differentials. As the N. indica is a

heterothallic fungus and it survives through sexual reproduction, there is no well-

developed race concept based on virulence patterns of different N. indica isolates on

different host genotypes and genetics of pathogen (Datta et al., 1999). Singh et al., (1995)

and Boned et al., (1996) considered it appropriate to classify the variation in the isolates

as aggressiveness.

Biological analysis of the N. indica isolates was done by field inoculations to

check the level of virulence of the isolates (Datta et al., 1997). A substantial differential

reaction was noted between wheat host plants and fungal isolates representing genetic

distinction among N. indica isolates. The order of the virulence pattern of N. indica

isolates obtained on the basis of susceptible reactions on the host plants for each isolate

was as follows – Ni4>Ni5>Ni1>Ni7>Ni8>Ni6>Ni2. An individual isolate showed a

differential reaction against two host cultivars and similarly, an individual host cultivar

gave different reactions against two N. indica isolates. Thus, Datta et al., (1999) reported

that different Karnal bunt resistance genes perhaps existed in these cultivars and that

there was genetic variability for pathogenicity in the isolates. However, they also

suggested that though the virulence typing explains the pathogenic behaviour of fungal

isolates, it is a rough estimate. Further, it was influenced by environmental conditions

which made the reproducibility of the results difficult in successive seasons even under

controlled conditions.

Screening of advanced lines/ cultivars of wheat against the disease Breeding for resistance has been a successful research activity and certainly is

the best control method in the long term. However, variation in the pathogen may

sometimes pose a threat to wheat production. Therefore, it needs a continuous breeding

programme for the source of resistance against the diseases.

The susceptibility of bread wheat to Karnal bunt disease of wheat has been well

documented (Fuentes-Davila et al., 1992, 1993) reaching infection levels greater than

50% under artificial condition; therefore it is important to continue evaluating new

advanced lines and commercial cultivars against the disease in various agro-ecological

regions. The identification and tagging of K-B resistant genes is important for further

breeding and developing resistant wheat cultivars because of quarantine restrictions on

the pathogen and the large effect of the environment on the disease development. The

most reliable and sufficient levels of genetic resistance to KB, have been observed among

Chinese, Indian and Brazilian wheat cultivars. (Gill et al., 1993; Fuentes-Davila et al.,

1995 Singh et al., 1995b). Wheat line HD29 is potentially an important source of genetic

resistance to Karnal bunt providing resistance to diverse isolates of T. indica (Gill et al.,

1993). A true breeding mapping population (RILs) was developed from the cross of

WL711x HD29, because Karnal bunt screening over a number of replications and years

to obtain reliable phenotypic data was required to establish reliable markers/traits

associations. In experiments during 1998-99, little disease occurred because of warm

temperature during flowering, illustrated well the large influence of environment on KB

disease even in inoculated nurseries. The distribution of KB disease severity on the RILs

was skewed toward that of resistant parent in three experiments, suggesting the

segregation of multiple genes with dominant complementary gene action in wheat line

HD29. This was consisted with the results of previous studies (Morgunov et al., 1994).

Karnal Bunt of wheat is such a disease which is very effective in low quantity. In views

of its importance Fuentes-Davila 1996, reported that 1-4 % of kernels are enough to

render the wheat grains unacceptable for human consumption. According to Sekhon et

al., (1992) the flour from grains with over 3 % bunted kernels imparts an off colour and

unpleasant odour. Therefore according to modern requirement a modified rating scale of

(Aujla et al., 1989) was used and lines or varieties having 2.1-5 % grain infection were

considered as moderately susceptible and lines/ cultivars with more than 10 % infected

grains were declared as highly susceptible.

The highly resistant advanced lines of wheat identified in the present

screening can further be exploited, as resistant sources against Karnal bunt disease, in

breeding programmes for the development of disease resistant commercial cultivars after

determining their genetics or these advanced lines can be released directly as commercial

cultivars if these were found to possess other desirable agronomic characters.

The sources of resistance in the wheat germplasm against Karnal bunt disease

are not uncommon. Aujla et al., (1980) reported ten wheat lines with only 1-5 percent

grain infection among 286 lines screened by them. Earlier Gautam et al., (1977a)

screened 96 wheat lines and reported lines with less than 1 percent infection. In a field

screening trial of superior genetic stocks consisting of 350 lines, Gautam et al., (1977b)

found 160 lines with no infection. Aujla et al., (1985) screened germplasm under

artificial epiphytotic conditions against Karnal bunt and reported 26 lines which remained

disease free and 58 lines having 0-5 percent infection. The screening of 38 wheat lines,

included in NUWYT of 2004-05 at NARC, Islamabad revealed that all the lines were

highly susceptible except 5 lines that remained free from grain bunt infection

(Anonymous, 2005).

As regards the commercial cultivars, there is scarcity of resistance in them

except a few moderately resistant cultivars. The scarcity of resistance in the commercial

cultivars is a serious threat as regards the quarantine status of wheat of Pakistan. This

scarcity of resistance in commercial cultivars against Karnal bunt disease across the

border (Krishna and Singh, 1983) and in the country has already been reported (Iftikhar

et al., 1988; Anonymous, 2005).

Weather conditions and disease predictive models A part from the importance of weather conditions or climatic factors, in

creating epidemics and distribution of Karnal bunt disease these are interlinked/

interrelated with each other to certain extant. Three environmental factors i. e. maximum

air temperature, minimum air temperature and rain fall were tabulated to get a better

scenario in Karnal bunt over all correlations. Maximum air temperature and minimum air

temperature play a key role with disease development and influenced significantly almost

all the lines/ cultivars.

Various models to estimate risk of establishment of Karnal bunt in

different countries have been developed (Kehlenbeck et al., 1997; Diekmann 1998;

Murray and Brennan 1998; Sansford 1998; Baker et al., 2000). This study was taken

into consideration and concentrated only on phenological timing. Although this

timing is acknowledged to fluctuate, we used the weather parameters within a sowing

region in order to forecast the timing of the reproductive period; wich is the only

wheat stage vulnerable to infection by Karnal bunt. As the disease is believed to be a

dynamic process and temperature is a key factor in disease tetrahedron in the

presence of host followed by a certain period of time.

Development of Karnal bunt depends on favourable weather conditions

for infection and disease development from heading to flowering (anthesis) of the

wheat crop. Moderate temperature, high relative humidity or free moisture,

cloudiness and rainfall during anthesis favour disease development (Fuentes-Davila,

1996), which is also reported by our results to some extent as max. and min. air

temperature had influenced directly on 62 and 58 lines and cultivars respectively in

the month of February and March, the flowering and heading stages of wheat.

Rainfall had influenced 21 lines/ varieties. Results of Workneh, et al., (2008),

revealed that temperature and rainfall were the main factors affecting the Karnal bunt

occurrence in Texas. A weather based model was developed for the prediction of

disease to evaluate bunt-negative and bunt-positive fields. The model accurately

forecasted the occurrence of disease except for the year 2004, when the climatic

conditions were predicted favourable for the development of the disease. However,

according to their arguments wet and cool weather during vulnerable period of crop

received rainfall seven times during eighteen day period in April 2004. Thus greater

frequency with twice amount of rainfall as compared to last years was too wet for the

development of disease. The view is supported by remarks of Sharma and Nanda

(2003), reported that prolonged wet conditions are unfavourable for Karnal bunt

development in India. Jhorar et al., (1992), also made the observation that conditions

for the establishment of Karnal bunt disease are unfavourable when HTI values are

too high, and the large HTI values are the result from high relative humidity and cool

temperature. The excessive rainfall wash off the sporadia from the plants

consequently, fungus infection is reduced. One possible explanation as pointed out by

Indian situation is that excessive rainfall may have produced conducive weather

circumstances for the germination of spores before the onset of vulnerable stage of

the host, consequently exhausting the inoculm potential.

There is conflicting information available on how abiotic conditions

during the rest of year affect the survival of pathogen and development of Karnal

bunt, summarized by Warham (1986). For Karnal bunt of wheat to develop,

conditions must be suitable for teliospore of T. indica to germinate to produce

sporidia between the flag leaf emergence and anthesis stages of the growing wheat

plant and for these spores to infect the developing ears (Nagarajan et al., 1997).

Relationship exists between condition at anthesis and development of Karnal bunt for

sites in India.

Mavi et al., (1992) studied the relationship of weather conditions during

the reproductive stage of wheat crop to disease intensity over a 19 year period in

Ludhiana, in India and developed a model based on the average maximum

temperature during mid to late anthesis (-ve correlation), the “evening relative

humidity” (presumably 2:30, +ve correlation) and sunshine duration (-ve) during

early to late anthesis, and number of rainy days in early anthesis (+ve). As the

climatic conditions and the wheat growing season in Indo-Pak. have no big

difference, therefore vulnerable stages in main wheat growing regions of both

countries are the same (exist mostly in Feb. and March). Therefore, environmental

conditions including maximum air temperature, minimum air temperature, rainfall

relative humidity and wind speed were employed for the assessment of Karnal bunt

disease occurrence. Using the climatological data and stepwise discriminant analyses,

Diekmann, (1993) conducted a study to determine areas of the world that were at low

risk for Karnal bunt. Although recognizing limited Karnal bunt distribution that could

be due to the difficulty of recognizing the disease in the field, Diekmann believed the

limited distribution was largely due to the very specific environmental requirements

for disease development.

During our observations when the data were split by varieties to develop

model for each variety it could not be done, because parameter estimates were biased

and these data did not fit well to the model. More number of observations were

needed to get goodness of fit. These environmental parameters had high degree of

multi-colinearity and are sensitive to their order or sequence of entry into the model.

However, it is evident that all the five environmental parameters had significant

influence on disease development. Air temperature (max. / min.) and wind speed

should be studied critically with great detail and care in future to reach towards a

sound biological conclusion for Karnal bunt disease prediction. Present studies

provide some base line information in this regard.

Disease-risk locations were characterized by a high difference between daily

maximum and minimum temperatures in the planting month, a relatively low daily

maximum temperature in the month of flowering, and mild winter. Jhorar et al.,

(1992) found a high correlation between a humid thermal index (HTI), defined as the

high RH in mid-afternoon divided by maximum daily temperature, calculated at

wheat ear emergence, and the disease incidence over a19-year period in the central

Punjab in India.

Evaluation of fungicides against the Karnal bunt disease of wheat Protection of the wheat from Karnal bunt infection by applying a systemic

fungicide to the seed has no value due to two reasons: i) systemic fungicides did not

persist long enough to inhibit floret infection and (ii) the inoculum for Karnal bunt is

primarily soil-borne and not seed-borne like that of dwarf bunt (Hoffmann, 1982;

Brooks and Buckley, 1977). Therefore, an unfortunate consequence of applying

systemic fungicides to wheat at late growth stages give rise to the apprehension of

high level of residue in the grain at harvest. Contact fungicides, because they do not

redistribute systemically within the spike, would probably have insignificant residues,

as the residual chemical would be removed when the grain is threshed. In this

experiment discovery of Shelter and Dolomite in which mancozeb is main portion of

active ingredient, is a ray of hope for controlling the Karnal bunt disease as the

mancozeb belong to Ethylenebisdithiocarbamates (EBDCs) group of non-systemic

(surface acting) fungicides and could be used on wheat crop without acute toxicity.

Karnal bunt disease of wheat has assumed an alarming situation in the

Punjab during the previous two to three decades (Anon, 2005) and has been

reported to cause, depending upon the cultivar affected, upto 30 percent grain losses

(Anon, 1986; Ilyas et al., 1989). This calls for control of disease either by the use of

host resistance or through the use of chemotherapy. Since resistance to karnal bunt

disease in available commercial wheat cultivars is absent (Anon., 2005), the

chemotherapeutic control of the disease can be achieved by foliar application of

fungicides at proper plant stage growth (Singh and Prasad, 1980; Singh et al., 1985;

Ilays et al., 1989a). Foliar spray of fungicides may protect the plants from infections

or eradicate the established infection (Vyas, 1984).

There are few contradictory reports on the chemotherapeutic control of

Karnal bunt disease of wheat. According to Bedi, (1980) fungicides are not

effective against Karnal bunt disease. Although reduction in the disease was

achieved on two occasions by the application of carbendazim (Krishna and Singh

1982, Singh and Prasad, 1980). Seed treatment was not preferred on the basis of

some sound reasons. Fungicides applied as seed treatment had controlled neither

artificial nor natural infection by Karnal bunt pathogen, they reduced the tillering

capacity of plants, also delayed heading by about one week. (Smilannick et al.,

(1987). Secondly the infection in the spike of wheat is initiated by airborne sporadia

which may come from other fields of remote areas and the effect of seed dressing

fungicide persist no longer upto the heading or booting stage of wheat crop.

Fungicides effective in inhibiting the in vitro colony growth of T. indica

were also equally effective for in vivo control of the karnal bunt infection of wheat

grains. However, the protective applications of these fungicides were comparatively

more effective in controlling wheat grain infection than their eradicative

applications. Regarding the time of application (Protective or curative spray) our

results are very similar to that of Ammerman et al., 1992., Baldwin et al., 1996.,

Gold et al., 1996 and Mitsuhiro et al., 1999. Application of Mancozeb and Metalaxyl

provide the best control against the Downey mildews when applied before inoculation

rather than post infection (curative application). They also reported that these

fungicides possess a broad spectrum activity against fungal pathogen from all the

three major divisions of fungi: Oomycota, Ascomycota and basidiomycota. Dolomite

and Shelter which are basically a combination of Mancozeb and Metalaxyl provided

the better control when used as a protective spray in the field against the Karnal bunt

disease of wheat.

Under the present situation of scarcity of resistance to Karnal bunt

disease the control of the disease, through chemotherapy is not uncommon (Singh

and Prasad, 1980; Singh et al., 1985; Ilyas et al., 1989a; Krishna and Singh, 1982).

Krishna and Singh, (1982) reported that Bavistine (Derosal-60) and Bayleton, when

sprayed at boot leaf growth stage prior to inoculation (protective spray) controlled

the karnal bunt disease. Singh and prasad (1980) found that a single spray of

Benomyl or Bevistine or Dithane M-45 at boot leaf growth stage was effective

against Karnal bunt disease. Singh et al., (1985) also reported that spray of either

Mancozeb or Carbendazim can prevent karnal bunt disease of the wheat plants.

Ilays et al., (1989a) reported that Spotless, Tilt or Baycor were the most effective

fungicides that significantly reduced the grain infection, the least effective spray

were that of Bayleton, Topas C-50 and Dithane M-45 while that of Brassicol

(PCNB), Bayfidan and Bavistine were intermediate in their effectiveness for

reducing karnal bunt disease over the unsprayed control.

Chapter 6

SUMMARY Karnal bunt (partial bunt) disease caused by Tilletia indica being a floret-

infecting is sporadic in nature. For the successful infection, coincidence between

conducive weather conditions and susceptible stages of wheat crop is utmost necessary.

Vulnerable period for the invasion of fungus remains for a short time about 3-4 weeks.

Emergence and existence of vulnerable stages (starting early booting to milky) vary in

each germlasm line and cultivar. It is also dependent on date of sowing, production

technology and other agronomic practices to some extent. During the survey it was

assessed that the disease is extending its broad-spectrum activity from north of the Punjab

(Sialkot) toward the south of Punjab (Multan, Bahawalpur and D. G. Khan divisions).

This indicates that the hot and dry climatic conditions of south Punjab exert no adverse

effect on hardy nature teliospores of the pathogen. These conditions provide security to

teliospores from suicidal germination. Consequently during the susceptible period of next

growing season teliospores release sporadia to infect the host plant.

During the epidemiological studies it was concluded that Karnal bunt disease

severity was higher in 2007 as compared to 2006 in about all the entries except with a

deviation of few entries. After temperature the other environmental factor that exerted a

significant influence is rainfall. More rainfall with comparative low air temperature

(min./ max.) during month of Feb. and March 2007 (1.99 & 1.33mm) rendered more

disease incidences as compared to (0.52 & 1.19mm) year 2006 respectively. There was a

significant correlation of maximum air temperature with 62 entries, minimum air

temperature and rain fall exerted a significant influence on 58 and 21 entries respectively.

In screening trials a large number of germplsm lines were categorized between

moderately resistant to highly susceptible. Only 16 lines were immune or highly resistant

and 9 were resistant. None of the cultivars which are being practically sown in the Punjab

was found immune or resistant. Only seven varieties (BWP-97, SA-42, SH-2002, Blue

silver, Farid 2006, Shafaq-06 and Manthar) were found moderately resistant to Karnal

bunt disease of wheat. The protective spray of Dolomite and Shelter were most effective

and reduced the disease upto 62.45 and 63.90 % respectively, while the eradicative spray

of these fungicides caused 38.84 % and 40.48 % reduction in the disease.

Chapter 7

CONCLUSIONS 1. Karnal bunt disease is rapidly spreading from East and North Punjab to

southern

areas of the Punjab and Sindh.

2. Seed corporations including private seed companies are the main source of

spreading the disease, as these companies mix the diseased grains with

healthy seeds and sell it during next growing season.

3. At present, a few varieties i.e Blue silver, Menthar, Farid-06 and BWP-97

which

were evolved from Regional Research Institute Bahawalpur, and Shafaq-06,

SA-

42 and SH-2002 evolved from Wheat Research Institute Faisalabad are

avaible.

4. Out of 200 germplasm lines obtained from Wheat Research Institute

Faisalabad only 16 lines were highly resistant and 9 were resistant all the

other lines fall in the rank of moderately resistant, moderately susceptible,

susceptible, and highly susceptible.

5. During the correlation analysis of environmental factors, max. air

temperature

and min. air temperature had a significant influence on all the line/ varieties.

6. Based upon the environmental conditions of two years data combined

stepwise regression indicated that a model combining of air

temperature (max. /min.) relative humidity and rainfall expained 84% 0f the

variability in disease development.

7. When the data were split by year a model consisting of min./max. air

temperature, relative humidity, rainfall and wind speed explained 81 percent

of the variablity in disease development during wheat growing season 2005-

06.

8. In the second year (2006-07) a model consisting of min. air temperature wind

speed max. air temperature explained 90 percent of the variability in Karnal

bunt disease development.

9. Dplomite and Shelter were the best fungicides to control the disease and were

staticially equally effective against the disease.

10. Protective spray at early booting stage was more helpful than curative spray.

Chapter 8

RECOMMENDATIONS 1. Farid-06, Menthar, Bwp-97, Shafaq-06 and blue silver must be grown in

the affected areas.

2. Seed corporations and private seed companies should not be allowed to

sell the seeds until they got the certificate of disease free seed from plant

pathologist.

3. Plant protection department must be provided with necessary facilities and

options to check the storage godowns freely to get rid of the problems of

adulterations.

4. Local quarantine measures should be strictly adopted so that the pathogen

could not enter in the disease free areas.

5. Protective spray of Shelter and Dolomite at the recomended doses during

the

early booting stage of wheat crop provide the best control against the

disease.

6. Research workers, extension workers and Agricultural university staff

should

work in collaboration with each other. At least fortnightly meeting must be

held between these personells to exchange views for the solution of

farmers,

problems.

.

Chapter 9

FUTURE STUDIES 1- Assessment of precise number of protrctive and curative foliar sprays

to attain 100 % control of Karnal bunt of wheat disease with Dolomite

and Shelter fungicides.

2- Survival of teliospores in different soils containing various

proportions of sand, silt and clay with various combinations of macro

and micronutrient.

3- Germination of teliospores to release the primary and secondary

sporadia at various levels of soil moisture and temperature.

4- Survival and longivity of secondary sporadia under dry and wet

conditions and its virulence/ aggressiveness with passage of time.

5- As the heterothallism has been reported in the pathogen, there is dire

need to study the infection process in detail and histology of infection

of wheat by Tilletia indica.

6- Biological control of the Karnal bunt disease of wheat.

Chapter 10 REFERENCES

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treated with fungicides. FAO Plant Protection Bulletin 40, 148–153. Agarwal, V.K., D. V. Singh and S.B, Mathur. 1993. Seed-borne diseases and seed health

testing of wheat,. S.B.Mathur and B. M. Cungfer, eds., Seed borne diseases and seed health testing of wheat. Institute of Seed Pathology for developing countries. Danish Government. ABC Grafik, Fredeirksberg, Denmark. 3 31-43

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