Journal of Applied Environmental and Biological Sciences ... Vol. 9, No.3, March, 2019.pdf · Associate Professor, Molecular Biology, Plant Molecular Pathology & Arid Lands Institute,

Volume 9, Issue 3, March 2019

Journal of Applied Environmental

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J. Appl. Environ. Biol. Sci., Vol. 9 No. 3: pp. 1-19, Year 2019

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Table of Contents, March 2019 Humaira Gul, Mamoona Arif, Husna, Yaseen Khan and Aqib Sayyed

Effect of Boron, Manganese and Iron on Growth, Biochemical Constituents and Ionic Composition of

Cowpea Grown under Salinity

J. Appl. Environ. Biol. Sci. 2019 9(3): 1-12. [Abstract] [Full-Text PDF] [Full-Text XML]

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Musarrat Ramzan, Tabassum Ara Khanum and Umber Batool

Incidence and New Record of Aphelenchoides perietinus (Bastian, 1865) Steiner, 1932 in Rice Fields

of Punjab, Pakistan

J. Appl. Environ. Biol. Sci. 2019 9(3): 13-19. [Abstract] [Full-Text PDF] [Full-Text XML]

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J. Appl. Environ. Biol. Sci., 9(3)1-12, 2019

© 2019, TextRoad Publication

ISSN: 2090-4274


and Biological Sciences www.textroad.com

*Corresponding Author: Humaira Gul, Department of Botany, Abdul Wali Khan University, Mardan, Pakistan

Effect of Boron, Manganese and Iron on Growth, Biochemical Constituents

and Ionic Composition of Cowpea Grown under Salinity

Humaira Gul*, Mamoona Arif, Husna, Yaseen Khan and Aqib Sayyed

Department of Botany, Abdul Wali Khan University, Mardan, Pakistan

Received: November 29, 2018

Accepted: February 27, 2019

ABSTRACT

Boron, Manganese and iron effect on cowpea (Vigna unguiculata L.) growth, ionic composition and

biochemical aspects were examined under sea salt. Micronutrients (Boron, Manganese and iron) were applied in

a pot experiment via foliar-spray at 0 and 5ppm concentration with or without (EC 2.5 dS.m-1 & EC 5 dS.m-1) sea salt concentration. The effects of these treatments on chlorophyll a, b, total chlorophyll, RWC, carbohydrate,

protein, shoot and root length, fresh and dry biomass, and sodium and potassium ions were studied. Sodium ion

concentration and total carbohydrates in different plant parts were directly proportional to sea salt concentration

in irrigation water. The inhibitory effects of salt on growth were observed in all the measured growth

parameters. Micronutrient exogenous application stimulates all growth parameters so these micronutrients are

effective to enhance cowpea growth under salt stress conditions.

KEYWORDS: Micronutrients, chlorophyll, Boron, Iron, Salinity, carbohydrates, sodium.

INTRODUCTION

In the Indo-Gangetic, arid and semi-arid tropics irrigated areas are in salinity stress due to over accumulation of

NaCl. Salinity is considered as a severe problem and major control in the production of crops where 50 mM of

NaCl can cause crop losses ≥70% [1, 2]. Every year 1-3% of the agronomic land is ceaselessly changing into

saline, either because of regular saltiness or because of human impedance which accounts about 20% of the flooded farming area. Due to bad agricultural practices and climate changes by the middle of the twenty-first

century salinity is expected to have devastating global effects on cultivated land [3, 4, 5].

Plant yield and quality can be disrupting by micronutrient deficiency. Boron is a fundamental complement to the

typical development of all plants, and its availability in water and soil is a key factor of agriculture. Inadequacy

of B causes diverse impacts on pollen tube development sugar movement, nucleic acid synthesis, starch

digestion, indoleacetic acid oxidase action and root elongation. Deficiency of Boron affect plasmalemma-bound

enzymes and plasma membrane potential [6, 7, 8, 9, 10]. Boron plays important role in vital process of

cytoskeletal proteins, phenolics accumulations, nitrogen and polyamines metabolism [11, 12].

Manganese is an important micronutrient and affects the chloroplast structure and photosynthesis. At plant

cellular level Mn is involved in various functions such as binding lamellae with the thylakoid membranes outer

surface, effecting photosynthesis and chloroplast structure [13]. All plants require Mn for Hill reaction, where the water decomposition and oxygen generation systems are involved in photosynthesis. Manganoprotein in

Photosystem II have manganese which catalyzes the initial stages of O2 evolution. Increasing the amount of

manganese produced by leaf under salt stress increased the photosynthetic activity and growth rate of barley

cells [14]. Iron is a vital nutrient for plants. Iron is involved in the manufacturing process of chlorophyll and

requires for some enzyme to function. Photosynthetic cells contain approximately 80% of the iron which plays

an essential role in the synthesis of iron containing hemeproteins (cytochrome) and other haem bonded to a

protein, including construction of chlorophyll, ETC (electron transport systems) and Iron–sulfur clusters during

oxidation-reduction reactions of mitochondrial electron transport [15]. Plant grown in saline environment

showed reduction in growth and many strategies have been adopted to grown them with higher yield in the same

environment. Many techniques are designed these days to apply foliar minerals in the form of fertilizers, to

encounter plants specific requirements for micro or macro nutrients and trace minerals. These techniques benefit

in growing healthier plants, speed up growth of crops, helps in strengthen damaged & weak crops, helps in micronutrient deficiencies and helps plants to grow better under stress condition. Keeping this view point in

mind, current research was designed to evaluate that whether foliar application of micronutrients was

compelling in prompting salinity in Vigna unguiculata L.

1

Citation: Humaira Gul, Mamoona Arif, Husna, Yaseen Khan and Aqib Sayyed; 2019, Effect of Boron, Manganese and Iron on Growth,

Biochemical Constituents and Ionic Composition of Cowpea Grown Under Salinity; Journal of Applied Environmental and

Biological Sciences, 9(3)1-12, 2019.

MATERIAL AND METHODS

Seeds of cowpea (Vigna unguiculata L.) were obtained from ICBA (International Center for Biosaline

Agriculture, UAE). Total of 48 pots divided into further 4 sets were used for this experiment. Details of Four

sets were as follows:

Set I: Non-Saline control with two saline treatment of (EC 2.5 dS.m-1 & EC 5 dS.m-1) sea salt irrigation and

without micronutrients. Set II: Non-saline control, two saline treatments with following sea salt irrigation (EC 2.5 dS.m-1 & EC 5 dS.m-

1) and B source copper sulfate.

Set III: Non-saline control, two saline treatments with following sea salt irrigation sea salt irrigation (EC 2.5

dS.m-1 & EC 5 dS.m-1) and Fe source molybdenum oxide.

Set IV: Non-saline control, two saline treatments with following sea salt irrigation sea salt irrigation (EC 2.5

dS.m-1 & EC 5 dS.m-1) and Mn source zinc sulfate.

For drainage, the pots had basal outlet. Out of forty-eight (48) pots 12 pots present in each set and for each

treatment three replicates were maintained:

i) Control

ii) Sea salt concentrations with EC 2.5 dS.m-1

iii) Sea salt concentrations with EC 5 dS.m-1

iv) Micronutrients at 5ppm. Each pot is filled with thoroughly washed sandy loam soil. Hoagland’s solutions are used for saturation of soil

in each pot. Uniform size seeds were surface sterilized for 1 minute with 0.1% HgCl2 and then washed with

distilled water. In each pot five seeds were sown; pots were then irrigated with equal amount of tap water daily.

At three leaves stage, Sea salt treatment was started and growing of sea salt were slowly increase in irrigation

water until the salinity required for each treatment is reached. Completely randomized design (CRD) was

assigned for all 48 pots in the Botanical Garden of Department of Botany, University of Karachi. All pots were

irrigated with sea salt solution and tap water twice a week. Different concentration of micronutrients was

applied foliarly and salinity was maintained in various sets. At the end of the research leaves, shoots, roots, fresh

biomass and dry biomass were noted in developed plants, and the number of pods per plant. For biochemical

analysis and RWC (relative water content) leaf samples were collected.

Relative water content: (RWC)

[16] method was used for determining Relative water content. Leaves

The fresh weight (FW) was measured and rehydrated in distilled water for 2 hours and their turgor weight (TW).

To obtain dry weight the samples were placed in a preheating oven for 48 hours at 80°C. Use the following

formula to calculate the relative water content (RWC).

RWC (%) = (FW-DW)/ (TW-DW) *100

Biochemical Analysis

Chlorophyll content:

Protocol of [17] was followed to determine the Chlorophyll concentration (Chl) in fresh leaves sample.

Total Carbohydrate Content: Total carbohydrates content in plants was determined by using Anthron Reagent from [18] method.

Analysis of total Protein: Bradford method [19] was used for extraction and analysis of total protein content.

Vegetative Parts Mineral Estimation:

Samples were used for mineralization estimation during growth and analysis of different cations (Na+ and K+).

For ash, the sample was dried and each dry sample of 0.5 gm was used for analysis. For mineral analysis, the

ash solution was then prepared in 50 ml of deionized water and then diluted in deionized water. PFP1 flame

photometer was used for measuring the concentration of cations in the sample.

Statistical analysis and Experimental design:

The experimental design was Completely Randomized Design (CRD) bearing three salt levels as well three

replicates. Results were analyzed by one-way ANOVA using SPSS 21.0 statistical software and Duncan’s

Multiple Range Test (P < 0.05) was used for determining differences between the means of parameters.

2


RESULT AND DISCUSSION

The positive impacts of Boron, Iron and Manganese in enhancing plant efficiency could be ascribed to their

improvement consequences for expanding plant metabolic action. Micronutrients fill in as chemicals factors.

Foliar application of fertilizer had dominant impact on growth of crops [20]. Salinity showed significant reduction in root length, fresh and dry biomass and plant height (Figures 1-4). Results obtained were like the

results of [21, 22]. Salt effect the enzymatic process of cell and interacts with the organic substances in cell of

crops, effecting the growth of cops [23]. Moreover, during saline conditions the osmotic potential of the roots

increases which decreases the crops productivity and brings physiological changes in plant which ceases its

growth crop [24]. Plants showed significant improvement in Height, weight and other growth parameters when

micronutrients were applied foliarly in salt stress. Several researchers reported the positive effect of

micronutrients on stress plants. [25] reported that exogenous application of chelated Zinc, Manganese and Iron

increase the root & shoot dry weight of sugar beet. The addition of boron increases the growth of rice in the

nutrient solution [26]. Tobacco plants shoot and root mass was decreased in Boron low concentration compared

to plants provided with enough Boron [27].

Salinity affects the stomatal opening, leaf growth, photosynthetic process and many other physiological and morphological process [28]. Research clearly showed reduction in Relative water content of plants during salt

stress. Relative water content was significantly reducing in plants during saline condition (Figure 5). According

to [29] the reduction in RWC specifies a loss of turgor, which would have resulted in inadequate water

availability for the cell extension process.

Observations of some of the results of early studies were observed to increase the leaf K + or maintain leaf

expansion under leaf stress conditions and RWC [30-, 31]. Exogenous application of micronutrients

significantly (P<0.001) improves this parameter. The data shown in Figure 6-9 show that chlorophyll a, b total

chlorophyll and ab ratios were significantly higher (P <0.001) than salt-treated pants compared to control non-

salt plants. These results confirmed by [32, 33]. This reduction can be ascribed to the inhibition of chlorophyll

activity involved in synthesis and destruction of chlorophyll in plant tissues. Exogenous application of trace elements such as boron, iron and manganese showed a significant (P<0.001) increase in the synthesis

chlorophyll a and Chlorophyll b, total chlorophyll content was increased compared with those under saline and

control conditions.

Iron plays a positive effect in synthesis of chlorophyll. Iron is a vital nutrient for plant development and growth,

involving biosynthesis of chlorophyll, thylakoid and development of chloroplast. Therefore, plants with iron

deficiency have chlorosis in newly formed leaves and less developed chloroplast [34, 15]. Manganese is

involved in the process of photosynthesis and act as activator in different enzyme reactions [35]. They further

added that the lack of Manganese induced chlorosis, necrosis, growth inhibition, and structure of chloroplast

was significantly impaired. Our results are consistent with the results found by [36, 35].

Different concentrations of sea salt solution significantly (P<0.0001) increased the amount of carbohydrate in the leaves (Figure10). Under sea salt irrigation, the increase in carbohydrate in plants is highest due to plants

insufficiency of using the stored carbohydrate, also supply of assimilates to growing areas were reduced due to

slow photosynthetic activity [37, 38]. Boron, iron and manganese were applied to the foliage of the salt-treated

plants, and the total carbohydrate was significantly (P<0.0001) improved under saline and non-saline conditions.

Exogenous application of potassium, Iron and boron collective treatment reduced the accumulation of

carbohydrate. In this treatment consumption of sugar is high in vegetative growth as yield production was

maximum [39]. According to [40] iron, zinc and manganese when applied in combine treatment it has a key

effect on carbohydrate production and photosynthesis process.

Data presented in Figure 11 exhibited that total protein of the plant treated with salinity was significantly

reduced compared to control. The reduction in total protein may be due to the proteolytic process under high saline condition. Many salt tolerant plants have stated a reduction in leaf protein content under saline condition,

due to breakdown of protein [41, 42, 43]. The current study also showed that iron, boron and manganese foliar

application exhibited significantly (P<0. 0001) enhancement in total protein content in control and saline

conditions. Foliar application micronutrients increased total protein content of canola plant [44]. [45] revealed

that protein content in Vigna radiata was significantly increased by spraying micronutrients.

Plants irrigated with different concentration of sea salt exhibited significant (P <0.0001) increase in NA, while

significant decreases in potassium were observed in different parts (Figure 12-14). [46] reported that nutrient

uptake by plants were decreased by increasing soil salinity. This showed increased amount of sodium and

3




chloride in soil have harmful effect on movement of essential nutrients like Phosphorus, Nitrogen and Potassium

[47]. Exogenous application of micronutrients improved Potassium in plant parts and decreased the amount Na

concentration in cowpea. The decrease in Na concentration in plants is important for reducing the harmful

effects of Sodium, and reports are available, Boron plays a vital role during stress condition by decreasing the

effect of sodium in plant [48, 49]. This help micronutrients to have control mechanisms or regulatory functions

for Na uptake and translocation rates [47]. Leaf feed with micronutrients can partially offset the negative effects

of NaCl on nutrient uptake by improving root growth and preventing plants nutritional uptake order, leading to an increase in nutrient movement and uptake by roots [50].

Figure 1. Effect of foliarly applied micronutrients (Boron, Manganese and Iron)

on plant height (cms) of Vignaunguiculata subjected to seasalt stress.


on root length (cms) of Vignaunguiculata subjected to seasalt stress.

0

5

10

15

20

25

30

35

Control 5PPM Boron 5 PPM Iron 5 PPM Manganese

Pla

nt

Heig

ht

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.001

Control 2.5 dS/m 5 dS/m

0

2

4

6

8

10

12


Root

Len

gth

Treatments

Salinity= P<0.001

Boron= Non-Significant

Iron= Non-Significant

Manganese= Non-Significant


4



on fresh biomass (gms) of Vignaunguiculata subjected to seasalt stress.


on dry biomass (gms) of Vignaunguiculata subjected to seasalt stress.

0

1

2

3

4

5

6

7

8

9


Fresh

Bio

mass

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.001


0

0.5

1

1.5

2

2.5

3

3.5


Dry B

iom

ass

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.001


5





on relative water content of Vignaunguiculata subjected to seasalt stress.


on chlorophyll a (mg/gmfr.wt) of Vignaunguiculata subjected to seasalt stress.

0

10

20

30

40

50

60

70

80


Rela

tive W

ate

r C

on

ten

t

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.001


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Ch

lorop

hyll

a

Treatments

Salinity= P<0.001





6



on chlorophyll b (mg/gmfr.wt) of Vignaunguiculata subjected to seasalt stress.


on total chlorophyll of Vignaunguiculata subjected to seasalt stress.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Ch

lorop

hyll

b

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.01


0

0.2

0.4

0.6

0.8

1

1.2

1.4


Tota

l C

hlo

rop

hyll

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.01


7





on chlorophyll a b ratio (a/b) of Vignaunguiculata subjected to seasalt stress.


on total carbohydrates (mg/gmfr.wt) of Vignaunguiculata subjected

to sea salt stress.

0

0.2

0.4

0.6

0.8

1

1.2

1.4


a/b

Treatments

Salinity= P<0.05





0

2

4

6

8

10


Tota

l C

arb

oh

yd

rate

s

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.001


8



on total proteins (mg/gmfr.wt) of Vignaunguiculata subjected to seasalt stress.


on sodium ion concentration of different plant parts (stem, root and leaves)

ofVignaunguiculata subjected to seasalt stress.

0

5

10

15

20


Tota

l P

rote

ins

Treatments

Salinity= P<0.001

Boron= P<0.001

Iron= P<0.001

Manganese= P<0.001


0

2

4

6

8

10

12

Co

ntr

ol

5P

PM

Bo

ron

5 P

PM

Iro

n

5 P

PM

Mang

anese

Co

ntr

ol

5P

PM

Bo

ron

5 P

PM

Iro

n

5 P

PM

Mang

anese

Co

ntr

ol

5P

PM

Bo

ron

5 P

PM

Iro

n

5 P

PM

Mang

anese

Stem Root Leaves

So

diu

m

Salinity= P<0.001

Boron= P<0.05

Iron= P<0.05

Manganese= P<0.05


9





on potassium ion concentration of different plant parts(stem, root and leaves)

of Vignaunguiculata subjected to seasalt stress.


on potassium sodium ratio (K+/Na+) of different plant parts (stem, root and leaves)

of Vignaunguiculata subjected to seasalt stress.

CONCLUSION

Vigna unguiculata showed reduced growth with respect of all studied parameters when grown in saline

environment. Foliar application of micronutrients (Boron, Iron and Manganese) improved growth of this plant

under both normal and saline irrigated condition.

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© 2019, TextRoad Publication

ISSN: 2090-4274


and Biological Sciences www.textroad.com

*Corresponding Author: Musarrat Ramzan, Department of Botany, The Islamia University of Bahawalpur, Pakistan. Email: [email protected]

Incidence and New Record of Aphelenchoides perietinus (Bastian, 1865)

Steiner, 1932 in Rice Fields of Punjab, Pakistan

Musarrat Ramzan1*, Tabassum Ara Khanum2 and Umber Batool3

1Assistant Professor, Department of Botany, the Islamia University of Bahawalpur

2Assistant Professor, National Nematological Research Centre University of Karachi 3Researcher, Department of Botany, the Islamia University of Bahawalpur

Received: November 2, 2018

Accepted: February 7, 2019

ABSTRACT

Surveys were conducted to document infestation of rice by plant parasitic nematodes especially emphasized on Aphelenchoides nematodes during 2016-2018 at harvest stage. Results indicate that incidence of Aphelenchoides perietinus in rice fields of Punjab was high in both years. Among the district surveyed, Lahore, Sheikhupura and Narowal were severely infested. The samples from these three districts showed 88.8%, 75% and 53.5% respectively. Moreover, resistance also evaluated by the symptoms of cultivated rice varieties. Among the tested cultivars on the bases of symptoms and incidence of A.perietinus, Lateefy, Dilrosh-92 and DR-8 showed high resistance. Moderate resistance was identified with Sugdasi Bengalo, IR-6 and Kanwal-95. While Shua -92, Sharshar, Sada hayat, Kangni-27, and Jajai-27 were susceptible to A.perietinus nematodes. Susceptible plant expresses varies symptoms such as shortening of plant, reduction of Tiller and grains per plant and low yield (100 seed weight). In few infested fields symptomless but infested plants were also found. In the present survey it is noted that A.perietinus was first time recorded from rice fields and first time evaluate resistance source in rice varieties against this nematodes. This survey also confirmed that this nematode is one of the most important and damaging pest which occurred in those fields where fungi infestation was measureable. Further the area infested with this nematode species are potential source for estimation of interaction between fungi and A.perietinus nematodes. KEY WORDS: Survey, resistance, Aphelenchoides spp, rice varieties and infestation.

INTRODUCTION

Cultivated rice, Oryza sativa L., represents the world’s most important staple food crop, feeding more than half of the human population (Lundo et al., 2006). Rice is an important food and cash crop, is the third largest crop of Pakistan after wheat and cotton. Pakistan is famous for growing and exporting long grain aromatic Basmati rice. The country ranked 14th in terms of rice production and 6th in rice export in the world.

Rice growing areas have different environments due to this in different countries and regions incidence of nematode disease was different because rice crops attacked by different nematode species. Generally, in irrigated rice, Hirschmanniella and Aphelenchoides species are frequently found. Nematodes not also causing direct crop loss, nematodes also cause indirect economic losses resulting from export/trade restrictions imposed due to the presence of quarantine nematode pests. Agreement of World Trade Organization revised their regulations and included several pests in the regulatory lists. Varaprasad et al., 2006 reported that amongst main nematode diseases of rice, Ufra and white-tip causing nematodes find a place in regulatory pest lists of numerous countries. Fungal feeding nematodes definitely affect the balance of endophytic fungi in host plant. Thus, in case of nematode disease incidence cannot depend on plant genotypes but also depend on as much or more on composition of endophyte species which production is enhance by Aphelenchoides nematodes (Raghavendra and Newcombe, 2013). Throughout the world, Aphelenchoides spp is widely distributed and now occurs in mainly of the rice growing areas (Ou, 1985). However, nematode found in maximum density even a weekly parasitic species like Aphelenchoides

parietinus might kill epidermal cells to cause a young luscious root a yellowish to brownish colour. It is external discoloration of the tissues.

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Citation: Musarrat Ramzan, Tabassum Ara Khanum and Umber Batool; 2019, Incidence and New Record of Aphelenchoides perietinus (Bastian, 1865) Steiner, 1932 in Rice Fields of Punjab, Pakistan; Journal of Applied Environmental and Biological Sciences, 9(3)13-19, 2019.

In Pakistan, very rare work have done on nematode disease of rice. Anwar and Khan (1973) first time reported A.

bessyi nematodes from rice fields of Punjab, Pakistan. Khan et al., 1990 studied on seed born fungi, bacteria and nematodes of rice in the Punjab and found 13 seeds out of 170 were infected with white tip disease. Musarrat et al., 2015 conducted an extensive survey of rice growing areas of Pakistan except Balochistan and reported different plant parasitic nematodes from rice fields including Aphelenchoides peritineous and A. bessyi. The main intentions of this study were construct data on population dynamics and assessing the prevalence and distribution of A. peritinus in rice growing localities of Punjab province. Different plant parasitic taxa were occurred but A.paritinus found in high density. In many fields pure population of this nematode was found. The species, Aphelenchoides paritinus was reported in Pakistan for the first time. While it was identified for the first time in association with rice throughout the world. Earlier, A.paritinus had been reported from different regions of world from rhizosphere of different host, the incidence and distribution of it in all rice cultivated areas is unknown. Therefore a survey of Aphelenchoides paritinus in Pakistan was undertaken and estimated its symptoms under field conditions, the extent of its distribution in some selected rice areas, the levels of infestation and its effects on yield components were investigated. The main aim of this research was to evaluate resistant rice cultivars and measure infestation of this nematode first time. The data of resistance variety can be useful to find source of resistance.

MATERIAL AND METHODS

Survey: Extensive surveys were taken throughout the consecutively two growing seasons of rice at harvest level. These rice growing areas of Punjab province of Pakistan can be divided in different zones such as Barani area, Mixed zone and Rice -Wheat zone. The main purpose of the sampling was to observe the incidence and prevalence of A.parietinus plant root feeder parasitic nematodes. Survey area 25 x 25 m was marked off by plastic ropes and collected soil and root samples. For this purpose plant was uprooted and plant shoot was cut off. Roots with rhizosphere soil were placed in plastic bags and used for the extraction of root feeder nematodes. Estimated 390 randomly selected fields from 10 locations were surveyed and soil samples were taken from each field of rice. Screening of soil and root samples for Aphelenchoides spp

The root samples were washed in soil suspension having bucket and thoroughly mixed. Extraction of plant parasitic nematodes from soil and rhizosphere of rice was made by Cobb’s sieving and Baermann funnel techniques (Baermann, 1971 and Cobb, 1918, respectively). The nematodes which had moved through the sieve into the water were collected after 24 hrs, and pour off excess amount of water finally 50 mL suspension was obtained, after that the suspensions were observed under a stereoscopic microscope ,juvenile and adult Aphelenchoides parietinus were counted. To estimate the susceptibility and resistance of the varieties, the prominence value (PV = nematode population density x √ (frequency of occurrence/10) of A. parietinus was measured (De Waele et al., 1998). Quantitative and Qualitative analysis

Nematodes were counted in an open counting chamber by the pouring of 5 ml extracted nematode suspension and repeat three times. For qualitative analysis, concentrated water transferred into glass cavity block and nematodes were killed by pouring hot water. For qualitative analysis heat killed nematode suspension was placed by a dropper on a glass slides and cover with cover slip and sealed with transparent nail polish. Nematode populations were counted on this temporary slide under the compound microscope and identify the juvenile and adult nematodes of Aphelenchoides genus.

Identification of Aphelenchoides spp

Suspension of killed nematodes immediately fixed and preserved in a TAF solution for 24 hrs. After 24 hrs they were washed with distilled water 3 times. Excess amount of supernatant water were poured off and further processing into glycerine by a slow dehydration method for which specimens were place in a cavity block containing 2 ml of 1.25% glycerine solution and kept in an incubator at 40-55 C for 5-6 days. Processed nematodes were picked with hair needle and placed in a small drop of glycerine on the slide. Paraffin wax was placed as three small lumps around the drop then placed cover slip on the wax lumps. Slide was gently heated on hot plate to melt the wax and filled the space between the slide and cover slip.

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Measurement and line drawing of nematodes:

Measurement and line drawing of the nematodes were done by the method given by de Man (1880) ad Hooper (1986) respectively.

Statistical analysis: Community analysis of phytoparasitic nematodes in rice fields of Pakistan was done by the using of Norton techniques (1978).

RESULTS AND DISCUSSION

Incidence of Aphelenchoides parietinus

The total incidence percentage of A.parietinus was determined from all soil samples of rice from major rice growing province (Punjab) of Pakistan. Present analysis showed that rice nematode Aphelenchoides parietinus was more prevalent (85.61%) in soil (Table.1).

Table 1. Incidence of Aphelenchoides parietinus nematodes from rice localities. Localities No of fields samples

(A)

Extraction of

A.perietinus by roots

(B)

Total incidence

After bioassay (C)

D/F in incidence

(C-B)

% incidence (C/A)

x 100

Sialkot 25 04 26 22 16.0 Lahore 36 32 34 2 88.8 Gujranwala 20 08 21 13 40.0 Sheikhupura 32 24 29 05 75.0 Okara 48 15 28 13 31.2 Jhang 39 13 19 06 33.3 Narowal 18 10 09 01 53.5 Kasur 20 06 14 08 30.0 Sahiwal 105 35 73 38 33.3 Faisalabad 47 15 23 08 31.91 390 123 (31.53%) 252 (64.61 %) 33.58%

The fields with high incidence of Aphelenchoides parietinus were easily detected through direct examination of root symptoms. In the present study different root symptoms were detected such as surface necrosis of roots as minor symptoms. While in major symptoms yellowish to brownish color of young succulent root. It might be due to the high density of A.parietinus. The present investigations indicated that two localities such as Lahore and Sheikhupura were highly infested out of ten surveyed localities of Punjab. The plants on fields with moderate level of infestation did not always show clear above ground symptoms but soil had few numbers of studied nematodes. Localities i.e Okara, Gujranwala, Jhang, Sahiwal and Faisalabad out of total localities were moderately infested with A. parietinus. Whereas in Sialkot fields with low level of infestation i.e 16% often had plant which did not showed any above and underground symptoms and roots are healthy not had Aphelenchoides nematodes after direct examination under binocular microscope, but nematodes also found in rhizosphere.

Table 2. Community analysis, Population and nematodes density/250 cm3 soil after Robbins et al (1989). S.NO Localities Nematode

population

Absolute

frequency

Relative

frequency

Mean

density

Relative

density

Prominence value

1 Sialkot 160 4.7 3.62 0.83 18.4 39.9 2 Lahore 1600 65.1 15.18 6.15 50.5 407.5 2 Gujranwala 396 54.6 12.7 1.61 30.5 219.5 3 Sheikhupura 600 72.5 16.91 2.30 39.8 350.5 4 Okara 120 11.3 2.63 1.46 21.4 71.9 5 Jhang 80 14.6 3.40 0.30 14.6 55.8 6 Narowal 620 74.5 57.4 3.22 40.8 352.2 7 Kasur 500 50.5 30.9 2.60 21.3 150.6 8 Sahiwal 421 54.6 12.70 1.61 30.5 225.4 9 Faisalabad 340 50.9 11.8 1.30 20.2 144.1

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Based on the prominence value, frequency of occurrence and abundance, Lahore, Sheikhupura and Narowal was the most infested region. In these regions Rice- Wheat is most common cropping sequence and farmer use the same rice varieties. These both factors might have contributed to the high prominence value of A. parietinus. During the survey it was found that most common growing varieties of rice are same in surveyed localities of Punjab. While other varieties grown locally different from region to region based on local market demand and preference.

According to community analysis Population density could be categorized as high in the localities of Lahore, Sheikhupura and Narowal (Table.3).In case of frequency among the all rice fields, the most infested samples encountered from Narowal followed by Sheikhupura and Lahore. Locality Lahore had the highest absolute density of A.peritinus, while the least density was observed in Jhang .The prominence value (PV) of Aphelenchoides genus varied among all infested zones (Table.2). Lahore (PV= 407.5) was the most infected locality followed by Narowal (352.2) and Sheikhupura (350.5). Whereas the least PV was recorded from Faisalabad i.e 39.9 as compared to other localities of Punjab. It should be noticed that A.parietinus was reported for the first time from rice fields in Pakistan.

Table 3. Rating of resistance and quantitative analysis of Aphelenchoides parietinus occur in soil

around the Oryza Sativa L. plant in fields of Punjab. Cultivars Reaction Damage

Index

Nematodes/250 g soil Symptoms in Fields

Mean localities

Jajai-77 S 5.0 1433 2,4,7 Stunted growth, low yields, hollow tillers

Khushboo-95 MS 4.5 1089 3,5,6,9,10 Not accountable symptoms

Lateefy H.R 0.8 22 1,8,9,10 Healthy Sonahri-Sugdasi R 1.5 83 3,5,9 Healthy Sugdasi bengalo MR 2.6 255 3,5,6,9,10 Healthy Sugdasi sadagulab R 1.8 87 1,8 Healthy Super basmati R 2.0 99 1 Healthy Dilrosh 97 MS 4.0 600 9,10 Not accountable

symptoms Dilrosh-92 H.R 1.0 20 1,8,10 Healthy DR-8 R 1.1 63 1,8,10 Healthy DR-83 R 1.7 88 1,8,10 Healthy IR-6 MR 3.0 499 3,5,6,9 Healthy Kangni-27 S 5.0 1600 2,4,7 Stunted growth, low

yields, hollow tillers Kanwal-95 MR 2.8 368 3,5,6 Healthy Sada hayat S 3.6 544 2,4,7 Not accountable

symptoms Sarshar S 4.0 600 2,4,7 Stunted growth, low

yields, hollow tillers Sugdasi ratria R 2.0 99 1,10 Healthy Shua 92 H.S 5.0 1488 2,4,7 Reduced plant growth

without matured tillers and grains

Shadab R 1.4 74 1,8,9,10 Healthy Localities: 1. Sialkot, 2. Lahore, 3. Gujranwala, 4. Sheikhupura, 5. Okara, 6. Jhang, 7. Narowal, 8. Kasur, 9. Sahiwal, 10. Faisalabad

Ranking of Resistance: In the present study the most common cultivasr Jajai- 77, Kangni-27, Shua-92 and Shadab were frequently grown in the highly infested localities. While moderate susceptible varieties were Khushboo, Dilrosh-97. Other remaining varieties were ranked as moderately resistant and resistant. The level of resistance varies from region to region. According to damage high damage index was recorded in Jajai-77, Kangni-27 and Shua -92 varieties (Table-3). While low damage index was recorded in Lateefy which ranked as highly resistant variety. With respect to nematode population, maximum population was found from Lahore, Sheikhupura, and Narowal localities where commonly grown varieties were Shua -92, Kangni-27 and Jajai-77. Whereas lowest and ignorable population of A.parietinus was recorded from Sialkot, Kasur, Sahiwal and Faisalabad localities. Plant growth and yield assessment: Data has been analyzed by the factorial test (F-test) at p< 0.05 level and presented in table.4. According to plant length and tillers per plant non significant results were observed in all resistant varieties. In case of susceptible genotypes, significant results were obtained in terms of plant length and tillers/plant, the variation in number of nematodes was significant. Jajai-77, Kangni-27 and Shua-92 exhibited more

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numbers of nematodes in soil as compared to other susceptible rice genotypes (Table.4). In case of yield assessment significant results were obtained in the weight of 100 seed in all susceptible varieties as compared to control. While maximum 100 seed weight were recorded in resistant varieties viz., Lateefy and Dilrosh-92. Significant results were also observed in moderate resistant varieties. Significantly maximum yield was recorded in all highly resistant varieties followed by resistance varieties. While minimum yield was recovered in five susceptible varieties followed by Shua-92 > Jajai-77 > Sadahayat> Sarshar >Kangani-27.

Table 4. Calculated F values and mean comparison of plant growth and yield parameters of rice

by applying ANOVA for single factor. Cultivars Plant length Tillers/plant Yield assessment

100 Seed wt Total yield

Jajai-77 7.3* b 7.9*b 8.7*a 12.4*a Khushboo-95 9.8* ab 9.8*b 6.6*b 8.7*b Lateefy 3.2cd 3.3d 0.2g 2.1d Sonahri-Sugdasi 3.3cd 1.5e 3.1cd 1.9de Sugdasi bengalo 4.1c 2.1e 1.5ef 2.1d Sugdasi sadagulab 3.8c 2.9d 3.0cd 2.5d Super basmati 4.2c 3.9c 2.6e 3.0c Dilrosh 97 8.7*ab 9.8*b 4.8c 2.0de Dilrosh-92 2.6d 3.4c 4.0c 3.3c DR-8 3.5cd 2.5d 1.0ef 1.3e DR-83 4.1c 3.6c 1.0ef 2.3d IR-6 3.8c 4.1c 1.0ef 3.2c Kangni-27 14.3*a 20.6*a 6.8*b 10.9*a Kanwal-95 4.0c 2.8d 1.6ef 2.3d Sada hayat 8.8*ab 6.5*b 10.9*a 14.3*a Sarshar 8.6*ab 12.3*a 9.4*a 13.8*a Sugdasi ratria 3.2cd 4.1c 1.0ef 2.2d Shua 92 20.3*a 6.8*b 2.2e 4.4*c Shadab 3.3cd 2.1e 1.0ef 3.4c

Morphological identification: Aphelenchoididae family is identified by its large metacorpus and pharyngeal glands not usually enclosed in a bulb (overlapping) just like other phytoparasitic nematodes. The main morphological characteristic shared by the Aphelenchoididae family members is short stylet while other plant parasitic nematodes having large stylet with well developed knobes. Description: Female body slightly ventrally accurate when relaxed. Cuticle finely annulated. Head slightly rounded. Lateral field marked by four incisures. Stylet 8-8.8 µm long, with small basal swellings, conus a little shorter than shaft. Oesophagous 176-205 µm long. Metacarpus 7.2-11.2 µm wide and 9.6-13.6 µm long. Excretory pore 50-52 µm from anterior end. Nerve ring immediately behind metacarpus. Hemizonid not seen. Ovary single, outstretched, oocytes in a single row except at the tip, vulva transverse. Spermatheca elongate oval filled with rounded sperms, vagina directed anterial. Occupying about 1/3 of vulva body width. Intestine straight, rectum 14.4-22.4 µm long. Post vulval sac elongate, 32-44 µm long, usually containing sperms, tail conoid, ventrally curves, 22.4-26.4 um long with terminal mucro (Table.5 ).

Table.5. Measurements (µm) of Aphelenchoides parietinus (Bastian, 1865) Steiner, 1932. Morphological characters Range (Mean ± Sd)

Female N= 20 Male= 20

L 410.4-506.4 (457.0±58.9) 376.8-464 (418.3±49.5) A 24.4-32.8 (27.7±2.4) 21.5-34 (25.6±1.4) B 6.0-8.8 (7.4±0.86) 6.6-8.2 (6.9±0.50) C 13-20 (17.3±1.4) 14.7-20.6 (15.8±1.44) c’ 2.1-3.3 (2.7±0.34) 2.1-3 (2.4±0.25) V 55.9-71.4 (67.5±5.6) - Stylet 5.6-8.8 (7.5±0.98) 8.0 Spicules - 11.2-16 (14.3±1.36) Tail 22-28.8 (26.0±2.5) 21.6-28.8 (26.1±2.44) Rectum 13-22.4 (19.3±2.7) - Post vulval sac 32-45 (40.5±10.3) -

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Male body curved ventrally more so in the posterior region. Cuticle finely striated. Head offset from the body. Stylet with prominent basal thickening. Nerve ring and excretory pore as in female. Testis single, outstretched reaching interiorly, upto oesophageal gland. Spicules are typically of Aphelenchoides, gubernaculums absent. Tail 24.4-28.8 µm, shape of tail similar to female tail. Three pairs of caudal papillae are present, the first pair near cloacal opening, the second one mid way along the tail and the third at the base of mucro. Remarks: Specimens were collected from rhizosphere of rice (Oriza sativa L.) from Punjab, Pakistan. The population of A.parietinus resembles to general morphological and morphometrical characters corresponding to those of the type population (Bastian, 1865) Steiner, 1932. The present population have slight differentiation in some measurements i.e., the length of tail smaller than tail of A. parietinus (22.4-28.8 µm vs 32.9 µm), c ratio is slightly greater than A. parietinus (15-17 vs 13-19). Tail with mucro and ventrally curves as compare to A.parietinus that slightly terminally curved (Fig.1). Description and illustrations were given for the first time from Pakistan.

DISCUSSION

Aphelenchoides are large nematodes genus and economically important pests of several crops especially in rice crop. In previous studies mostly researchers reported White tip disease of rice which causing agent is Aphelenchoides besseyi and next spp is Hirschmanniella graminis that frequently prevalent in rice fields. While other Aphelenchoides spp are not frequently found in rice fields but when present in large numbers even a weekly parasitic species like Aphelenchoides parietinus may kill enough epidermal cells to give a young succulent root a yellowish to brownish color. It is superficial discoloration of the tissues. In the present study high incidence and abundance of Aphelenchoides perietinus was observed in Punjab province of Pakistan. This study exhibited that prevalence of this weak nematode was due to same cropping system and grown same varieties in these localities due to the local market demand. In surveyed areas many researchers focused on reproduction of crop which determining resistance against phytoparasitic nematodes but nematode pathogenicity is not the major criteria. In this case symptom development should be evaluated. Plant length, tillers per plant and yield of 19 rice cultivars indicated that specific cultivars grown in upper Punjab of Pakistan are susceptible to Aphelenchoides perietinus. The results of present survey indicate and emphasize the need for breeding local cultivars. In many European countries A.besseyi has been controlled principally through the use of resistant cultivars (2, 13). The present surveys were carried out at harvesting of rice and, therefore, the early symptoms were not encountered. Khan (2010) reported that development of white tip disease and deformation of rice grain is dependent on population density of nematodes in rice plant and other many environmental factors. In the present investigation, population densities were varied among the rice sampling sites. The A.perietinus nematode in rice is widely distributed might be due to infested seedlings and soil through water between locations and regions within the same province. Jamali et al (2006) conducted survey from rice fields of Iran and he observed that incidence and severity of white tip disease varies from year to year and from variety to variety he concluded that these variations were found due to environmental factor, cultural practices and local rice cultivars. The present study further confirmed incidence and widespread of A.perietinus among all surveyed rice fields under Punjab conditions. Conclusion: The results of the present survey investigations indicate that weak nematodes such as A.perietinus present in excess then cause a disease. During the survey highly infested localities were point out to farmers and also confirm the resistant genotypes of rice that grower should be cultivated in infested fields. This nematode first time reported from rice fields of Punjab and first time gives detailed of infestation, symptoms and infested districts.

Acknowledgement: This research was not funded by any funding agency. Compliance with ethical standards Disclosure of potential conflicts of interest. We have no potential conflicts of interest. Research involving human participants and / or animals: No human participants or animals were involved in this study. Informed Consent: Not applicable.

18


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3. Bridge, J., R. A. Plowright, and D. Peng, 2005. Nematode parasites of rice. pp. 87-129. In “Plant parasitic nematodes in subtropical and tropical agriculture” (M. Luc, R.A. Sikora, J. Bridge, eds.). 2nd Edition. Wallingford, UK, CAB International Publishing.

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7. Hooper, D.J., 1986. Handling, fixing, staining and mounting nematodes. 59-80pp. In: Laboratory methods for work with plant and soil nematode. Reference book402. (Ed.) J.F. Southey. Her Majesty’s stationary Office, London, UK.

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10. Khan M.R., White tip nematode infestation in rice, pp.140-170, In: Khan, M.R. and Jairajpuri, M.S. (Eds.).Nematode Infestation, Part-I: Food Crops, The National Academy of Sciences, India. 2010, 325.

11. Musarrat, A.R., F. Shahina, A.A. Shah, R. Saba, and K. Feroza, 2016. Community analysis of plant parasiticand free living nematodes associated with rice and soybean plantation from Pakistan. Applied Ecology and

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12. Ou, S.H., 1985. Rice Diseases, 2nd edn. Commonwealth Mycological Institute, Kew, UK. 13. Popova, M.B., G.L. Zelenskii, and S.A. Subbotin 1994. An assessment of resistance in cultivars of Oryza

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