Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2014, 4 (5):46-51
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ISSN : 2231 –3168

CODEN (USA) : JMBRB4

Biological control of disease complex involving Meloidogyne incognita and

Rhizoctonia solani on growth of okra through microbial inoculants
Safiuddin*, Sartaj A. Tiyagi, Rose Rizvi and Irshad Mahmood

Plant Pathology and Nematology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, India
_____________________________________________________________________________________________

ABSTRACT

A pot experiment was conducted to assess the potential role of biological control agents such as Trichoderma viride
and Azotobacter chroococcum individually and concomitantly against the disease complex involving Meloidogyne
incognita and Rhizoctonia solani in terms of growth and yield parameters as well as root-knot and root-rot
development on okra (Abelmoschus esculentus L.). Significant reduction was observed in root-knot and root-rot
development caused by M. incognita and R. solani due to the inoculation of T. viride and A. chroococcum
individually and concomitantly. The growth parameters of okra like plant length, fresh as well as dry weights,
chlorophyll content, ascorbic acid content, number of fruits per plant and fruit weight per plant improved
significantly due to these microbial inoculants but found more pronounced in A. chroococcum inoculated plants
than T. viride treated plants. Nematode population of M. incognita was also significantly reduced in most of the
treatments. This type of biological management of interacting pathogens in the rhizospheric soil proved to be a
important components of organic farming. Long term application of such organic components enhanced the crop
production as well as soil organic carbon for sustaining soil health.

Key words: Meloidogyne incognita, Rhizoctonia solani, Disease complex, Okra, Biological control agents,
Trichoderma viride, Azotobacter chroococcum.
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INTRODUCTION

Okra (Abelmoschus esculentus (L.) Moench) is grown as a summer crop in northern as well as southern states of
India. Seventy per cent of total world production of okra produced by India form 0.35 million ha dedicated land [1].
The production of this crop greatly affected due to some biological and agrochemical constraints in the recent years.
Improper and inadequate supply of nutrients and disease incidence are the major constraints in the way of
production of this crop. Among the biological constraints, root-knot nematodes (Meloidogyne spp.) are the most
important and serious pest [2, 3]. Estimated annual yield losses in okra due to root-knot nematode, Meloidogyne
incognita have been observed more than 14% [4] while Sasser [5] and Agrios [6] reported about 16.9% loss due to
this nematode. Root-knot nematodes cause reduction in crop growth and severe stunting besides the formation of
galls on roots of okra plant. Galls are tiny swelling lesions on the roots due to infestation by root-knot nematodes.
The other soil-borne pathogen like root-rot fungus, Rhizoctonia solani caused root-rot symptoms on okra. Crop
losses usually ranged from negligible to 50% depending on the extent of severity and different stages of crop [2, 7].
Their association further aggravates the grim situation for the growth of okra that severely affected the growth of
plants. To overcome this problem, the management of such important pathogens could be achieved with the use of
chemicals, fertilizers, broad spectrum pesticides, etc. Pesticides and chemical fertilizers are considered to be the
most effective control strategies to date. Their continuous use have resulted in the development of pesticide
resistance, caused direct toxicity to predators, fishes, man and cattle population and caused adverse effect on soil
health and environment [8]. More attention has been paid to safe and eco-friendly management of such soil-borne
pathogens in integrated manner. The excessive use of pesticides lead to emphasize on supplementation or
substitution of these hazardous chemicals fertilizers with low priced and easily available nutrient sources such as
organic and bio-organics components of environment. The organic matters like farm yard manure, composts and

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botanical resides are being used in various crops. These are store house of nutrients and found not only in enhancing
crop production [9], but also had the capability to increase soil fertility [10]. There is a need of integrated application
of organic and bio-control agents for sustaining the desired crop productivity. The use of biological control agents is
an obvious alternative and environmentally safe option for managing disease complex involving root-knot nematode
and root-rot fungus. Trichoderma spp. are among the most frequently isolated free living soil fungi and present in
plants root ecosystem [11]. Trichoderma viride is potential nematode biological agent [12] and also reduced
infection of R. solani [13]. The bio-fertilizer like Azotobacter harvest atmospheric N which is made available
directly to plants or released in soil.

Keeping in view of the importance of okra and associated pathogens, a preliminary soil-survey was conducted to
ascertain the presence of root-knot nematode (M. incognita) and root-rot fungus (R. solani) in okra infested field.
The aim of this study was to assess the effect of Trichoderma viride and Azotobacter chroococcum on disease
complex involving M. incognita and R. solani in okra in terms of growth parameters and disease intensity.

MATERIALS AND METHODS

A pot experiment has been carried out in greenhouse of the Botany Department, AMU, Aligarh to find out the
potential effect of individual and combined inoculations of M. incognita and R. solani on growth and yield
parameters of okra (Arka Anamika) in relation to their biological management by an antagonistic fungus,
Trichoderma viride and free-nitrogen fixing bacteria Azotobacter chroococcum.

Preparation of Nematode Inoculum

Root samples of eggplant infected with M. incognita were collected from single eggmass culture from mircroplots.
Distilled water was used to rinse the roots of infected plants, thereafter female and eggmasses from the galled tissue
were excised. The identification of the species was confirmed after the observation of perineal pattern of ten
females [14]. Freshly picked eggmasses from infected roots were taken and incubated on a coarse sieve lined with
cross layers of tissue paper placed in a Petridish filled with distilled water at 25±2°C for a week. The hatched second
stage infective juveniles (J2) were collected every 24hr and added more water. Second-stage juveniles of M.
incognita were counted in a counting disc and standardized to 2000J2/10Ml suspension. Two thousand J2 was
inoculated according to inoculation schedule.

Preparation of Trichoderma viride Inoculums

The bio-control fungus, Trichoderma viride was cultured on PDA medium in 9cm-diameter Petridishes at 26±2ºC.
Further it was raised on PDB (Potato Dextrose Broth) medium for inoculation purpose. The mycelial growth was
observed after two days and finally mat was formed after 15days on broth medium. Suspension was prepared by the
grinding the mixture of 90 ml distilled water and 10g mycelia mat (10:1). 2g mycelia mat of T. viride was used for
the each treatment.

Preparation of Rhizoctonia solani Inoculum

The root-rot fungus, Rhizoctonia solani exhibit the symptoms of root-rot on okra root, isolated and cultured on PDA
(Potato Dextrose agar Medium, 200g of peeled potato, 20g dextrose and 20g agar agar) and kept in incubator at
25ºC. After identification of the fungus it was raised and maintained on Richards liquid medium [15]. Mycelial mat
was collected from the Richard’s liquid medium and rinsed with sterile water, to remove the traces of medium
followed by remove the moisture by blotting paper. Suspension was prepared by adding the 90ml sterile water in
10g of mycelial mat (10:1) and grinded. 2g mycelial mat of R. solani was used for the each treatment.

Composition of Soil and Maintenance of test Plant

The fertile field soil was used, having pH 6.5, organic carbon 1.08% with available water holding capacity of
150mm in one meter soil depth. One kg steam sterilized soil was taken in 3:1 ratio filled in clay earthen pots of
15cm diameter. Four to five seeds of okra were surface sterilized in 2% mercuric chloride solution and sown directly
in each pot according to experimental design. The seeds of okra were soaked in Azotobacter chroococcum 25 g/kg
seed for 24h of that treatment in which Azotobacter was applied. Three weeks old seedlings of okra was treated with
Mi (Meloidogyne incognita), Rs (Rhizoctonia solani), Ac (Azotobacter chroococcum), Tv (Trichoderma viride),
Mi+Rs, Mi+Rs+Ac, Mi+Rs+Ac+Tv, Mi+Ac, Mi+Tv, Rs+ Ac, Rs+Tv, Ac+Tv, Ac+Tv+Mi, Ac+Tv+Rs and
Mi+Rs+Tv. Five replicates of each treatment were made and untreated plants served as control. Irrigation and
weeding were done whenever required and experiment was terminated after 90 days. Ten days after seed
germination thinning was done to retain one plant per pot. Another ten days later 2000 second stage juveniles of M.
incognita and 2g mycelial mat of R. solani inoculated as given in inoculation scheme. Pots were arranged in a
Completely Randomized Block Design (CRBD) kept in benches.

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Termination of Experiment
The experiment was terminated after 90 days of seed germination. Different growth parameters and yield attributes
such as plant height, fresh as well as dry weights, number of fruits per plant and green fruit weight per plant,
ascorbic acid content and chlorophyll content were determined from untreated as well as treated plants with bio-
control agents like Trichoderma viride and Azotobacter chroococcum. Numbers of eggmasses as well as root-galls
per plant were counted according to inoculation schedule.

Estimation of Chlorophyll Content and Ascorbic Acid Content

Chlorophyll content of leaf was estimated by the method of Hiscox and Israelstam [16]. One hundred milligrams of
leaf pieces was placed in a vial containing 7 ml of dimethyl sulfoxide (DMSO) and the chlorophyll was extracted
into the fluid by incubating for 60 min. The extracts was transferred to a graduated tube and made up to 10 ml with
DMSO and assayed immediately. A sample of 3 ml of chlorophyll extract was transferred to a cuvette and the
optical density (OD) values at 645 and 663 nm were read using a Spectrophotometer (Spectronic 1001) against a
DMSO blank. The ascorbic acid content of okra tissue was determined by the method based on the reduction of 2, 6-
dichlorophenol indophenols dye [17].

Number of root-galls, eggmasses and root-rot index

Root was gently rinsed under low stream of tap water and galls and eggmasses counted in each infected root of okra
plants. The root-rot index was also determined on the basis of 0-5 scale. Where 0= no root-rot, 1= very light root-rot,
2= light root-rot, 3= moderated root-rot, 4= heavy root-rot and 5= very heavy root-rot.

Determination of Nematode Population

Final soil population of Meloidogyne incognita was determined at the time of uprooting of roots from pots using
Cobb’s sieving and decanting method followed by modified Bearmann’s funnel technique [18]. The nematode
suspensions were examined in a counting dish with binocular microscope to quantify the number of J2 and the
population was determined per kg soil.

RESULTS

Effect on growth parameters and yield attributes

The results presented in Table 1 clearly revealed that the root-knot nematode, M. incognita and root-rot fungus, R.
solani caused significant reduction in growth and yield parameters such as plant length, fresh as well as dry
weights, ascorbic acid content, chlorophyll content, number of fruits/plant and fruit weight/plant. However, M.
incognita alone caused more reduction in growth and yield parameter than R. solani alone as compared to untreated
control. Combined inoculation of M. incognita and R. solani caused maximum reduction in plant length (29.78cm),
fresh weight (30.62g), dry weight (8.99g), chlorophyll content (1.027mg/g), ascorbic acid content (22.88 mg/100g),
number of fruits per plant (8.79) and fruit weight per plant (51.18g) as compared to uninoculated control where plant
length (75.50cm), fresh weight (87.62g), dry weight (28.52g), chlorophyll content (2.467mg/g), ascorbic acid
content (52.35mg/100g), number of fruits per plant (20.50) and fruit weight per plant (140.22g) were determined.
The individual inoculation of bio-control agents like T. viride and A. chroococcum caused significant improvement
in all the growth parameters as compared to untreated control. Azotobacter chroococcum alone improved the
different growth parameters such as (98.35cm), fresh weight (117.37g), dry weight (38.80g), number of fruit per
plant (27.16) and fruit weights per plant (184.61g) as compared to Trichoderma viride where plant length
(95.61cm), fresh weight (113.37g), dry weight (37.74g), chlorophyll content (3.220mg/g), ascorbic acid content
(67.34mg/100g), number of fruits per plant (26.51) and fruit weight (178.68g) recorded when inoculated alone
(Table. 1).

The combined inoculation of A. chroococcum and T. viride further improved the growth as well as yield parameters
of okra plants. The damaging potential of M. incognita and R. solani was found greatly reduced due to the individual
as well as concomitant inoculation of these two bio-control agents, however Azotobacter also acts as biological
nitrogen fixing organisms and add more N to the soil (Table 1). Root-rot fungus, R. solani was also greatly affected
due to these bio-control organisms as compared to M. incognita.

Effects on root-knot and root-rot development

The individual inoculation of T. viride and A. chroococcum significantly reduced the damaging potential of M.
incognita and R. solani in terms of disease incidence of root-knot and root-rot. The highest number of root-galls
(160.72) and eggmass per root system (229.40) were recorded in those plants where M. incognita inoculated alone.
The lowest number of root-galls (57.29) and eggmasses (93.26) were determined in a treatment where two bio-
control agents added, seems to be due to their synergistic effect. The effect of Azotobacter was found more
prominent as compared to Trichoderma individually as well as concomitantly in different combinations. Numbers of

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root-galls as well as eggmasses were found greatly reduced in all the treatments as compared to uninoculated
control. Some antagonistic effect between M. incognita and R. solani was also observed. Similarly, nematode
population was also greatly affected due to these biological control agents individually and concomitantly. The
highest number of nematode population was determined in individual inoculation of M. incognita, where the
population was 14516 while the least (2107) recorded in those treatments received inoculum of both the biological
control agents. Rhizoctonia solani also reduced the nematode population but bio-control agents T. viride and A.
chroococcum greatly affected its multiplication (Table 2).

Root-rot development caused by R. solani was found highest (5.00) in individually inoculated plants but greatly
reduced when both the bio-control agents inoculated where it was observed as 1.0. The root-rot index was
significantly reduced in other treatments as well where these biological agents inoculated individually as well as
concomitantly (Table 2).

Table.1. Effect of biological control agents on disease complex involving Meloidogyne incognita and Rhizoctonia solani on growth and
yield parameters of okra (Abelmoschus esculentus L.)*

Plant Plant Plant Chlorophyll Ascorbic acid Number of Fruit weight/

Treatments
length (cm) fresh weight (g) dry weight (g) content (mg/g) content (mg/100g) fruits/plant plant(g)
Control 75.50 g 87.62 e 28.52 ef 2.467 bcd 52.35 g 20.50 gh 140.22 gh
Mi 41.99 k 44.41 h 13.54 ij 1.214 f 27.54 i 10.62 j 74.04 j
Rs 46.12 j 49.73 h 15.46 i 1.374 ef 30.10 i 11.64 j 82.24 j
Ac 98.35 b 117.37b 38.80 ab 3.295 ab 68.98 b 27.16 b 184.61 b
Tv 95.61 bc 113.37bc 37.74 abc 3.220 ab 67.34 bc 26.51 bc 178.68 bc
Mi+Rs 29.78 l 30.62 i 8.99 j 1.027 f 22.88 ij 8.79 k 51.18 k
Mi+Rs+Ac 55.25 i 69.95 g 22.14 gh 1.960 de 42.75 h 16.56 i 114.69 i
Mi+Rs+Ac+Tv 88.58 de 69.30 g 34.89 bcd 2.969 abc 61.77 de 24.33 de 166.39 de
Mi+Ac 73.39 g 90.67 e 30.07 def 2.562 bcd 53.66 g 21.12 g 143.54 g
Mi+Tv 65.33 h 80.93 f 26.67 fg 2.356 cd 50.55 g 19.68 h 132.76 h
Rs+Ac 85.60 ef 102.13 d 33.75 bcd 2.850 abc 59.70 ef 23.52 ef 160.37 ef
Rs+Tv 81.43 f 97.71 d 32.78 cde 2.775 abc 57.79 f 22.85 f 154.24 f
Ac + Tv 106.25 a 126.79a 42.09 a 3.588 a 74.69 a 29.46 a 199.65 a
Ac + Tv + Mi 91.80 cd 109.44 c 36.11 bc 3.067 abc 64.16 cd 25.30 cd 171.49 cd
Ac + Tv + Rs 93.33 bcd 110.53 c 36.49 bc 3.131 abc 65.24 cd 25.63 cd 173.40 cd
Mi + Rs + Tv 53.77 i 68.09 g 21.59 gh 1.912 de 41.78 h 16.17 i 111.66 I
CD= (P= 0.05) 5.73 6.62 2.19 0.215 3.64 1.33 10.29
*Each value is an average of five replicates. Data labeled by the same letters did not differ significantly at P<0.05
Mi= Meloidogyne incognita, Rs= Rhizoctonia solani, Ac= Azotobacter chroococcum, Tv= Trichoderma viride.

Table.2 Effect of biological control agents on disease complex involving Meloidogyne incognita and Rhizoctonia solani on root-knot/root-
rot development affecting okra (Abelmoschus esculentus L.)*

Number of
Treatments No. of galls/root system Root-rot index Nematode population
eggmass/ root system
Control - - - -
Mi 160.72 a 229.40 a - 14516 a
Rs - - 5.0 a -
Ac - - - -
Tv - - - -
Mi+Rs 143.50 b 193.67 b 4.4 b 11762 b
Mi+Rs+Ac 122.48 d 174.33 cd 3.8 c 10326 d
Mi+Rs+Ac+Tv 075.40 g 111.57 f 1.4 f 4620 g
Mi+Ac 092.35 f 140.24 e - 6405 f
Mi+Tv 110.20 e 161.49 d - 8358 e
Rs+Ac - - 3.0 e -
Rs+Tv - - 3.5 d -
Ac + Tv - - - -
Ac + Tv + Mi 57.29 h 93.26 g - 2107 h
Ac + Tv + Rs - - 1.0 g -
Mi + Rs + Tv 130.64 c 179.12 c 4.0 c 10680 c
CD= (P= 0.05) 6.39 12.94 0.22 56.14
Each value is an average of five replicates. Data labeled by the same letters did not differ significantly at P<0.05
Mi= Meloidogyne incognita, Rs= Rhizoctonia solani, Ac= Azotobacter chroococcum, Tv= Trichoderma viride.

DISCUSSION

Root-knot nematode, M. incognita and root-rot fungus, R. solani are the most important pests of many vegetables
including okra caused reduction in growth and yield. Khan and Hussain [19] and Bhat [20] reported damage in okra
plant when these two pathogens introduced simultaneously. The effect was found synergistic in combined inoculated

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plants. Bhagwati et al. [21] reported synergistic interaction between M. incognita and R. solani on okra plant. The
synergistic nature of this association was most evident in combined inoculated plants. Plant growth was found
suppressed due to concomitant inoculation of M. incognita and R. solani as reported by Hussain et al. [22]. The
maximum reduction was recorded in growth as well as yield parameters of okra in combined inoculated plants. This
was probably due to predisposition of plants by nematode to fungal attack and thus increasing disease severity. Such
findings have also been observed earlier by Siddiqui et al. [23] and Prasad [24]. The nematode population was
greatly affected due to the effect of R. solani because of competition between space and food [2]. The population of
Meloidogyne incognita decreased due to effect of R. solani colonies of R. solani was found frequently observed in
the gall tissues of okra roots in the present investigation. Golden and Van Gundy [25] observed that R. solani was
found more attracted to the gall tissues as compared to non-galled tissues, because galled tissues released some
chemical substances on infection of M. incognita.

Trichoderma viride is the most frequently available soil fungi and present in plants root ecosystem [11]. These fungi
are opportunistic, avirulent plant symbionts and function as parasites and antagonists of many plant pathogens thus
protecting from disease causing organisms. Khan and Haque [26] reported that Trichoderma spp. reduced the effect
of Meloidogyne spp. in some other crops. Various isolates of Trichoderma has also showed potential effect against
root-knot nematodes and suppress their population [27]. Our results suggest that T. viride caused direct as well as
indirect effect on nematode population, egg and subsequently enhanced resistance in plants thus check the nematode
population, feeding and egg hatching. Trichoderma viride also reduced the damaging potential of R. solani. Our
results are in agreement with those of Khan and Sinha [13] and Vinale et al. [28]. In our study consistent root-knot
nematode suppression clearly demonstrated that potentiality of Trichoderma viride. Effects of biological control
organisms also emphasized in recent years by many research workers [29, 30]. The increase in growth attributed to
improved nutrient uptake and favorable environment prevalent in the rhizosphere to release of indole acetic acid,
gibberellins and cytokinins under bio-agent based treatments. In the present study, application of Azotobacter caused
higher growth. The pathogenic potential of pathogens has been reduced by the application of certain biological
control agents [31]. The effectiveness of bio agents like A. chroococcum and T. viride was found to be species
dependent. Use of Azotobacter and T. viride caused a greater reduction in root- galling, root-rot index and the
nematode population.

CONCLUSION

The inference drawn from the present investigation clearly revealed that the plants get ride from the harmful effect
of M. incognita and R. solani individually as well as concomitantly due to application of biological control agents .
The success of bio-control agents is dependent in recent years on the disease complex interactions that these
beneficial microbial populations established contact with pathogens and plants in soil ecosystem. The application of
A. choococcum and T. viride for the purpose of crop protection, such as the host defense mechanism and produces
antibiotics, may become a reality in near future when used as nutrient sources. They can be produced in large
quantities on an industrial scale and may be used for harmful effect caused by pathogenic fungi and plant-parasitic
nematodes including M. incognita. In continuation, further study should be conducted for better understand the
various mechanisms of action of Trichoderma and Azotobacter and their possible synergisms with other compounds
used in agriculture. Their population must be enhanced through the utilization of organic substrates. This will be
useful for sustainable production of many vegetable crops.

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