Evaluation of Biocontrol Agents for Augmentative Bio-Suppression of Pearl Millet Smut

Abstract

Plant diseases will remain a moving target as pathogens evolve new capabilities or move into niches released by the control of their bio-competitors. Developing and deploying control strategies had been an age old effort but very few efforts have been made in terms of biological control. The utilization of organic wastes against plant pathogens needs serious scientific attention to develop innovative and eco-friendly techniques ensuring better plant protection. In the present study investigations were made towards management of pearl millet smut caused by Tolyposporium penicillarie. Bio-control agents like raw cow milk, cow dung, neem extract, basil extract and cow urine were assessed to protect the crop from smut disease. Maximum protection over control was obtained by seed treatment with cow dung followed by cow urine. Basil extract and raw cow milk were almost comparable to the above treatments, however, basil extract provided minimum protection against the disease.

Introduction

Diseases in field crop are generally managed by the use of synthetic fungicides. Concern about possible risks associated with the use of fungicides have resulted in an intensive search for safer, effective and economic control options that pose minimum risk to the human health and the environment.

Seed borne diseases seriously affect the crop yield and quality. The most effective measures of their control are by the exclusion and reduction of inoculum during seed production. Though various chemical methods are being tried to reduce the disease intensity, yet it is important to establish certain organic or bio-control based approaches. Extensive studies are being carried out for devising safe and alternative resources, especially those based on organic matter as countless hazards are associated with the use of chemicals. Seed treatments are mostly favoured over sprays as it requires relatively low concentrations of active compounds. A large number of physical treatments such as hot water, electron beam and humid air treatments have been tried with a limited success as these were found detrimental to the seed viability (Forsberg et al, 2003).

Significant progress has been made in developing potential biological

alternatives to synthetic fungicides for the control of fungal diseases of plants

especially in legume crops (Arun Kumar, 2009). Many plant diseases have

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been successfully controlled using bio-control agents. Common bunt of wheat

caused by Tilletia caries has been controlled by mustard flour; milk powder

and acetic acid reducing the infection up to 96% (Borgen and Davanlou, 2000;

Borgen 2004). Extracts of many plants like garlic bulb, Strychnos nux-vomica,

ginger rhizome, basil (Ocimum sanctum) leaf and neem (Azadirachta indica)

kernel were used to control Alternaria padwickii in rice seed yield (Shetty,

1989). Organic wastes from animal origin were used for plant nutrient and

plant protection along with milk and ghee (butter oil) in the indigenous

knowledge system (Sadhale, 1999; Arun Kumar and Verma, 2005; Arun

Kumar, 2007). Raw milk of cow and goat have been used to control leaf curl

of chilli and downy mildew and smut diseases of pearl millet (Arun Kumar et

al, 2002; Arun Kumar et al., 2004; Dashora, 2005; Arun Kumar, 2006; Nene,

2007). The protection by raw cow milk in managing pearl millet downy

mildew is ascribed to induced systemic resistance due to increase in resistance

related enzymes (PPO, POX, PAL, CAT and β-1, 3-glucanase) and

metabolites such as phenols, ODP and amino acids such as L-phenylalanine,

L-isoleucine and Hydroxyproline(Arun Kumar et al., 2009).

Pearl millet is a staple cereal grain crop in all the arid and semi arid regions of

the world. It is grown as a rainfed crop with adequate fertilizer application and

despite of high potential, the crop yield is very low. In addition to harsh

climatic conditions, low soil fertility, ineffective nutrient management

practices, the attack of various pathogens still worsens the situation. In India it

is grown as a chief kharif crop in arid and semi arid zones. It is cultivated in

about an area of 50 million hectares with the production of approximately 20

million tones. It is susceptible to numerous diseases like green ear, dowry

mildew, ergot, smut etc, which cause considerable loss every year (Dashora,

2005).

Smut has been recognized as an important floral disease of pearl millet in

Africa and Asia since the early part of this century (Butler, 1918; Chevalier,

1931; Ajrekar and Likhite 1933; Ramakrishnan, 1971). The most obvious

symptom of this disease is the replacement of cereal grains by bright green,

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enlarged, oval to conical sori, projecting beyond the glumes. Initially the sori

are bright green and soft, which later turned brown to black and brittle in

texture.

Presently this disease holds the economic importance next only to downy

mildew in Indian arid zone and aggravates when the humid weather prevails

during the flowering period. The present study deals with the management of

pearl millet smut with the help of biological compounds of plant and cattle

origin.

Material and Method

Effect of seed treatment on pearl millet seed (cv. Nokha local) with different

natural compounds against T. penicilliarie was assessed. The set of 100 seeds

per treatment were taken and soaked in 7.0 ml of each treatment. The

uniformity was ensured by keeping the beaker on an electrical shaker for

overnight at room temperature. After the treatment the seed were transferred to

the sieve and washed thoroughly with running tap water for removing excess

of treating agents. The treated seeds were then placed in Petri dish to dry

overnight at 30˚C temperature.

A field experiment was conducted during the rainy season with Nokha local, a

smut susceptible pearl millet cultivar was used. The experiment was

conducted with seven treatments in a randomized block design (RBD) with

three replications:

1 Seed treatment with neem extract

2 Seed treatment with raw cow milk (50% dilution with water for 18h)

3 Seed treatment with cow urine

4 Seed treatment with cow dung

5 Seed treatment with basil extract

6 Control (No seed treatment)

The plant extracts were prepared by the method suggested by Ezhilan et al.

(1994). Fresh plant parts were collected washed in tap water followed by

double distilled sterile water. The leaves were ground thoroughly and

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extracted with 80% (v/v) ethanol by adding 10 times the volume of leaf

material. The mixture was vigorously shaken and centrifuged for 5 min at

5000 rpm. The supernatant was concentrated for 1.5 h in a laboratory

evaporator. This concentrate was diluted with 20% (v/v) ethanol in water to

the starting volume and again centrifuged for 5 min. at 10,000 rpm to remove

the debris. The efficacy of cow dung, cow urine, raw goat milk, neem and

basil extracts were studied against the pathogen. A set with no treatment was

treated as control.

Each plot measured 3m X 2m, with 4 rows and each row had 20 plants. The

crop was fertilized with diammonium phosphate (40 kg ha-1) as basal dose. No

insecticides or herbicides were applied. Smut incidence was recorded twice,

30 days after sowing (DAS) and at soft dough stage (60 DAS). Inhibition of

fungal growth was calculated by following formula:

I = C - T x 100

C

Where I = Inhibition per cent, C = Growth rate of the control, T=Growth rate of fungus in treatment.

Inoculations for smut incidence were made following standard inoculation

technique at the boot stage by injecting 5.0 ml of spore suspension obtained by

soaking sporeballs overnight in water. The suspension was filtered through a

double-layered muslin cloth (Dashora, 2005).The inoculated ear heads were

covered with polythene bags for ensuring high humidity. During the period

between inoculation and expression of symptoms the plants were daily

irrigated for 3 times. The polythene bags were opened after 7days of

inoculation and the formation of bright green, enlarged, oval sori were

observed.

Result

Smut management requires reduction of primary inoculum from seed and soil

and secondary infection in plants later during crop growth. Data in table

showed that all the treatments significantly reduced the fungal disease

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incidence. This study clearly indicates that all the biological components were

effective against T. penicillariae. Application of cow dung for 24 h appeared

most effective with 24% disease incidence followed by cow dung treatment

for 16 h with 28% disease incidence and cow dung treatment for 8 h giving

28% disease incidence. The next better alternate was of cow urine for 24 h

giving 36% disease incidence as compared to 69 % in control. Cow urine for

16 h and raw cow milk for 24 h were at par giving 44% disease incidence.

Neem extract for 16 h and 24 h gave 46% disease incidence followed by neem

extract for 8 h giving 48% disease incidence. This makes the neem extract

superior option as compared to raw cow milk where 16 h and 8 h gave 54%

disease incidence which is at par with basil extract treatment for 16 h and 24 h.

Cow urine for 8 h can also be considered as a good treatment which gave 51%

disease incidence. Least effective treatment was of basil extract for 8 h which

gave 56% disease incidence as compared to 58% in control. The best result

was seen in treatment of seeds with cow dung which is a very ecologically and

economically friendly option with very easy availability.

Discussion

In general, we abide by the hypothesis that only chemical fungicides can

provide substantial protection against fungal pathogens. However, it is clear

from the present experiment that alternate mode of plant safety can be tried

with moderate to high level of success. The motivation of our study was not to

identify a hazardous or non-feasible fungicidal agent, but rather to establish a

strategic mode of crop protection, which is safe to environment and at the

same time feasible to farmer in terms of economy and availability. The result

of our study revealed that treating seeds of pear millet with cow dung might

have some post infection efficacy in the field in the similar environmental

conditions. Recently, one of the studies related with downy mildew (DM)

(Sclerospora graminicola) management by treating pearl millet seed with

amino acids showed that amino acids like Serine, Tryptophan , Leucine and

Isoleucine reduced by DM by 57 % as a result of amino acid – mediated

induced systemic resistance (ISR) in pearl millet (Shetty et al , 2005). Here it

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is important to note that cow and goat milks contain all the amino acids with

potassium phosphate help boosting plant’s immune system through systemic

acquired resistance (SAR) (Kumar et al, 2002; Bettiol, 1999). Thus, seed

application of the biocontrol agents can be taken as beneficial components of

integrated disease management in pearl millet under arid farming system.

The composition of cow dung is of crude fibre, crude proteins and materials

like cellulose, hemi-cellulose, micronutrients, metabolic nitrogen, epithelial

cells from animal, bile salts, potash, sulphur, large number of bacteria

fermenting cellulose, hemi-cellulose and pectin. Cow dung has emulsifying

properties. Presence of bacteria may antagonize potential pathogens ready to

attack seeds. These bacteria quickly colonize the area around sown seeds and

thus compete with potentially pathogenic fungi and bacteria and prevent them

from attacking seeds. According to van Loom et al (1998) the cow dung

possess siderophores that are low molecular weight high affinity iron (III)

chelators, therefore possess the ability to induce systemic disease resistance.

Its availability will also be a matter of convenience for farmers.

Cow urine consists of nitrogen, potash and sulphur along with traces of

phosphorus. The nitrogen compound present in cow’s urine improves the

efficacy of the compound against the pathogen attack. Mixing medicines in

cow’s urine is a very common practice in Ayurveda for increasing the

resistance in plants.

Milk and Milk products: Milk has been traditionally used for crop protection.

Recently it has been validated by the contemporary workers (Arun Kumar,

2005; Dashora, 2008; Shetty, 2005). About forty percent of total amino acid in

milk are glutamine, leucine and proline. These amino acids are mainly

responsible to promote resistance against pathogens. Niranjan raj and Shetty

(2002) have found amino acid proline to systemically induce resistance in

plants and stimulate production of antimicrobial phenolics.

Neem and basil extracts find their age old and time tested significance for

antimicrobial properties. The role of these organic extracts as anti fungal

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agents need to be explored scientifically and develop novel and eco-friendly

methods of manuring and plant protection through research.

Increased activities of enzymes (PAL, POX and β-1, 3-glucanase) in

both RCM and amino acids treated plants suggest their active role in inducing

defense responses in host suppressing downy mildew disease in pearl millet.

Raw cow milk has emerged as non-phytotoxic natural resource, which activate

the host defense responses during pathogenesis. In this case activation of

induced resistance can be correlated with amino acid-mediated

phenylpropanoid pathway (Arun-Kumar and Mali, 2007; Arun Kumar et al.,

2009).

Reference:

Arun Kumar (2007). Traditional plant protection management practices of Rajasthan. LEISA India, 9: 29-30.

Arun Kumar and Verma, S. K. (2005). Milk in the management of plant diseases. In: Proc. National Conference on “Bridging gap between ancient and modern technologies to increase agricultural productivity, (S L Chaudhary, R C Saxena and Y L Nene Eds.), December 16-18, CAZRI, Jodhpur, India, pp. 67-73.

Arun Kumar, Sudisha, J. and Shetty, H. S. (2009). Elicitation of resistance and defense related enzymes by raw cow’s milk and amino acids in pearl millet against downy mildew disease caused by Sclerospora graminicola. Proc. International Conference on Nurturing Arid Zones for People and the Environment: Issues and Agenda for the 21st Century, held at CAZRI, Jodhpur, India, Nov. 24-28, 2009, pp…..

Ezhilan, G. J., Chandrashekhar, V. and Kurucheve, V (1994): Effect of six selected plant products and oil cakes on the sclerotial production and germination of Rhizoctonia solani. Indian phytopathology 47: 183-185

Borgen, A. and Davanlou, M. 2000. Biological control of common bunt

(Tilletia tritici). Journal of Crop Production, 3: 157-171.

Borgen, A. (2004): Strategies for regulation of seed borne diseases in organic farming. Seed testing international, 127, 19-21

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Forsberg,G.; Kristensen,L.; Eibel,P.;Titone,P. and Hartl,W. (2003) : Sensitivity of cereal seeds to short duration with hot humid air. Journal of plant diseases and protection (110), Pp1-16

Shetty, S.A.; Prakash, H.S. and Shetty, H.S.;(1989) : Efficacy of certain plant extracts against seed borne infection of Trichoneilla padwickii in paddy (Oryza sativa). Canadian journal of Botany, No. 57, Pp 1956-1958

Dashora,Kavya (2005) : Biology, epidemiology and control of smut (Tolyposporium penicillariae) on pearl millet in Rajasthan. PhD thesis, Jai Narain Vyas University, Jodhpur, Rajasthan, India.

Nene, Y.L. (2007): Scope for organic wastes as ecofriendly materials in crop protection. Indian journal of plant protection. Vol 35 (2) Pp 161-167

Sadhale,N (1999): Krishi parashara (Agriculture by Parashara). Agri-History Bulletin No.2, Asian Agri history foundation, Secunderabad 500009, India, Pp 94.

Van Loon, L.C., Bakker, P.A.H.M and Pieterse, C.M.J. (1998) Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology 36: 453-483

NiranjanRaj,S. and Shetty,H.S. (2002): Proline-A novel inducer of resistance in pearl millet against downy mildew disease caused by Sclerospora graminicola.Abstract; Asian congress of Mycology. University of Mysore, and Indian Society of Mycology and Plant pathology, Udaipur, India,Abstract 142 Pp.

Arun Kumar,R.Raja Bhansali and Mali P.C.. (2002): Response of biocontrl agents in relation to acquired resistance against leaf curl in chilli .Abstract; Asian congress of Mycology. University of Mysore, and Indian Society of Mycology and Plant pathology, Udaipur, India,Abstract 266 Pp

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