Nematicidal Activity of Different Plants Extracts against Root Knot Nematodes

Javeria Afzal, Muhammad Abid, Faisal Hussain* and Alia Abbas

Dr. Abdul Ghaffar Laboratory of Aerobiology and Plant Pathology, Department of Botany, Federal Urdu University of Arts, Sciences and Technology, Gulshan-e-Iqbal Campus, Karachi, Pakistan.

Received | November 16, 2020; Accepted | January 07, 2021; Published | March 20, 2021

*Correspondence | Faisal Hussain, Dr. Abdul Ghaffar Laboratory of Aerobiology and Plant Pathology, Department of Botany, Federal Urdu University of Arts, Sciences and Technology, Gulshan-e-Iqbal Campus, Karachi, Pakistan; Email: [email protected]

Citation | Afzal, J., Abid, M., Hussain, F. and Abbas, A., 2021. Nematicidal activity of different plants extracts against root knot nematodes. Pakistan Journal of Nematology, 39(1): 1-7.

DOI | http://dx.doi.org/10.17582/journal.pjn/2021/39.1.1.7

Keywords | Nematicidal activity, Hatching, Mortality, Plant extracts, Root-knot nematodes, Karachi

Introduction

The root- knot (Meloidogyne spp.) plant parasitic nematodes have significant harmful properties against almost all field crops (Perry and Maurice, 2006; Adekunle and Akinlua, 2007). It causes approximately $100 billion every year worldwide including Pakistan (Khan et al., 2008). Root-knot nematodes damage vast range of plants and vegetables crops and plants predominantly in subtropical and tropical countries (Osman et al., 2012; Youssef et al., 2012). Damage and crop losses in tropics are more than the temperate region because of favourable environmental condition which support more pathogen diversity. Vegetable crops usually are among the most susceptible and worst affected by the nematodes (Sharma et al., 2006; Anwar and Mckenry, 2007; Singh and Khurma, 2007). Vegetable yield reductions have reached as high as 30% for susceptible genotypes in the presence of plant parasitic nematodes in some production areas (Anwar et al., 2009). The infected plants or crops appeared stunted or chlorotic patches were observed on foliar part of plants (Archana and Saxena, 2012).

Plant protection against nematodes is difficult because nematodes cannot be eradicated completely from the field (Budai et al., 2005). Nematode management strategies therefore need to involve manipulation of nematode densities to non-injurious and sub-economical threshold levels (Viaene et al., 2006). Pesticides are generally used for the control of pests and diseases but these chemical pesticides cause human health and environmental hazards. So, there is a need to find alternative methods which are safe and suitable for environment (Abid et al., 2005; Khalil and Darwesh, 2017). Plants having nematicidal properties have been used as bio-pesticides (Ibrahim et al., 2006; Khan et al., 2015; Farzana et al., 2016).

Plants are important sources of naturally occurring compounds having nematicidal properties, like alkaloids, diterpenes, fatty acids, glucosinolates, isothiocyanates, phenols, polyacetylenes, sesquiterpenes and thienyls (Chitwood, 2002). The ability of plant parts/ products to reduce crop damage caused by root knot nematodes, Meloidogyne spp., in amended soil is well documented (Begum et al., 2003; Jesse et al., 2006). Their ability, minimum toxicity to man and animal safety, safety to the environment and effectiveness in controlling the nematodes make plant parts indispensable to nematode control (Jesse and Jada, 2004).

Therefore, the aim of the present this study was to evaluate extracts of some plants commonly grown in Karachi for their nematicidal efficacy against root knot nematodes in vitro and check their antagonistic effects against root-knot nematode.

Materials and Methods

Site collection

Different sites including grass growing area, road side and garden area were studied and plant samples were collected from these sites in plastic bags.

Collection of plant material

Five plants viz., slender amaranth (Amaranthus viridis), bathwa (Chenopodium album), macko (Solanum nigrum), papaya (Carica papaya) and asthma-plant (Euphorbia hirta) were collected from different areas of Karachi.

Collection of plant samples

The collected samples were kept in polythene bags, tagged and brought to the Dr. A.G. Lab of Aerobiology and Plant Pathology, Department of Botany, FUUAST, Karachi. The collected plants were dried in screen house and ground in an electric mixture into powder separately, stored in polythene bags at room temperature until use.

Preparation of extraction

2, 6 and 10 grams of dried leaves, stem and fruits of A. viridis, C. album, S. nigrum, C. papaya and E. hirta were separately mixed with 100 ml distilled water and left for 24 hours at room temperature; then filtered from a Whatman No. 1 filter paper. The filtrate was considered as standard(S). Further dilutions were prepared from standard solution.

Culture of nematodes

The culture of root-knot nematode was maintained on host plants. The infected plants were uprooted; roots were thoroughly washed in running tap water then cut into 1 centimeter in length. Eggs masses were isolated using method described by (Hussey and Barker, 1973) in Petri dishes containing sterilized distilled water.

Egg hatching test

Effects of plant extracts on egg hatching were studied. One ml of root-knot egg suspension containing 50–56 eggs and 1 ml of each plant extract was transferred to each cavity block separately; treatments were replicated three times. All treatments were kept at 28±2°C and after 72 hours the numbers of hatched eggs were observed and counted.

Mortality test

The effect of plant extracts were checked for larval mortality. Eggs of root-knot were placed in distilled water and incubated at temperature 28 ± 2 °C for 72 hours. A suspension of freshly hatched juveniles in distilled water containing (10-50 juveniles/ml) was prepared. 1 ml of each plant extract was transferred to each cavity block separately; treatments were replicated three times Nematodes were considered dead if they did not move when probed with a fine needle was touched (Abbasi et al., 2008).

Analysis of data

The observed data were analyzed by using one and two way ANOVA according to the experimental design (Gomez and Gomez, 1984). The follow up of ANOVA were included with LSD and DMRT were also applied to compare treatment means.

Results and Discussion

Egg hatching test

The five plants were selected and tested @ 2, 6 and 10% concentrations for egg hatching of root-knot nematode (Table 1). The result indicated that egg hatching was decreased with the increase in concentration. Maximum reduction (24.33%) in egg hatching was observed in C. album stem extract at 2% concentration as compared to control. Statistically it was found highly significant (F=22.28, p<0.0003***) difference in mean. Highly significant result was observed (F=104, p<0.0000***) in leaf, stem (F=49.244, p<0.0000***) and fruit (F= 35.11, p<0.0001***). The stem extract of S. nigrum were observed highly significant (0.33±0.33) and showed antagonistic effect (F=32.73, p<0.0001***). In distilled water extract; result showed that extract of A. viridis was significant in the reducing of egg hatchability. Almost all plants extract inhibited the egg hatching.

The increasing of time period and addition in concentration also decrease in egg hatchability. The C. album stem extract has been observed to be less toxic effecting killing capacity. When compared to other extracts but in case of A. viridis has been observed highest killing capacity for egg hatchability as compared to other extracts and the egg hatchability has been found to be decreasing with increasing concentrations from 2% to 10% after 48 to 72 hours exposure time. Similarly, all the leaf extracts were found increasing of egg hatchability at low concentration and decreasing at high concentration. The egg hatchability was observed in the decreasing order is A. viridis> C. papaya > C.album. Higher concentrations were related with higher death rates. E. hirta has been observed killing capacity for egg hatchability. It was highly significant in fruit (F= 252.18, p<0.0000***), stem (F= 52.52, p<0.0000***) and leaf (F= 34.69, p<0.0001***). The statistical analysis, of C. papaya were non-significant (F= 3.79, p<0.0584 ns) in leaf extract and it showed highly significantly results in fruit (F=23.97, p<0.0003***) (Table 1).

The present results of Analysis of variance for antagonistic effect of various plants on root-knot nematode show the hatching (%) of leaf, stem and fruit extract of A. viridis and E. hirta showed highly significant differences. Similarly, S. nigrum and C. album showed significant differences. However, leaf extract of C. papaya showed non-significant results.

Larval mortality test

The freshly hatched root-knot larvae were exposed in water extract of different plants for 72 hours in laboratory at temperature (28 ± 2 ºC). The maximum larval morality (32% and 33%) was recorded in leaves and stem extract of C. album at highest concentration (10%) as compared to other plants (Table 2). The minimum larval mortality was observed in leaves and stems extract of E. hirta and S. nigrum, respectively. Statistically, S. nigrum was found highly significant (F= 56.05, p<0.0000***). Larval mortality was decreased with the increase of dilution of plants extracts. While, juvenile mortality was increased consequent to an increase of concentration. Other plants such as A. viridis caused 7% mortality in leaf extract, with non-significant (F=0.58, p<0.06436ns) results. All aqueous extracts showed potential nematicidal effects and caused mortality of juveniles (Table 2).

 

Table 1: Effect of different concentrations of water extracts of plants on egg hatching of root-knot nematode after 48 hours with mean and standard error.

Name of plants

Control*

Egg hatching % after 48 to 72 hours of exposure

Leaf

Stem

Fruit

2%

6%

10%

2%

6%

10%

2%

6%

10%

A. viridis

10 ±0.57

2.33 ±0.3

1 ±0

0.33 ±0.3

2.33 ±0.8

0.66 ±0.6

0.33 ±0.3

4 ±0.5

3.66 ±0.3

1.33 ±0.8

E. hirta

25 ±0.57

6 ±1.1

5 ±3.0

3.6 ±2.1

2 ±1.1

1 ±0.57

1 ±0

1.66 ±0.6

1 ±0.5

1 ±0

C. papaya

15 ±2.3

9.33 ±4.7

3.33 ±3.3

1.33 ±1.3

_

_

_

1.33 ±0.8

2.33 ±1.2

1 ±0.5

S. nigrum

12.6 ±1.2

2.66 ±0.8

1.66 ±1.2

0.66 ±0.3

2.33 ±0.8

0.33 ±0.3

0.33 ±0.3

_

_

_

C. album

28.3 ±1.4

7 ±4.16

5.33 ±2.3

2.66 ±0.3

24.3 ±10.7

6.66 ±2.3

1.33 ±0.8

_

_

_

 

*The control values are represented by mean number of eggs/juveniles.

 

Table 2: Effect of different concentrations of water extracts of plants on larval mortality of root-knot nematode after 72 hours with mean and standard error.

Name of lants	Control*		Larval mortality % after 72 hours of exposure
			Leaf			Stem			Fruit
			2%	6%	10%	2%	6%	10%	2%	6%	10%
A. viridis	10.6±1.4	3.33±2.3		7.33±4.3	6±3.05	3±1	2.33±1.2	2.66±1.3	1.33 ±0.3	2±1	3.66 ±0.3
E. hirta	12.3±1.4	1.66±0.3		1.33±0.3	2.33±1.4	0.66±0.6	0.66±0.6	1±1	0.66 ±0.3	1.33 ±0.3	1±0.5
C. papaya	11±0.5	2.33±0.8		2.33±0.8	4.66±2.0	_	_	_	1.33 ±0.8	1.66 ±0.8	1±1
S. nigrum	11±0.8	0.66±0.3		1±0.5	1.66±0.8	0.66±0.3	1±0.5	2±0.5	_	_	_
C. album	48±1.5	15±3		18.33±3.4	32±8.5	12.3±2.3	20±2.0	33±7.9	_	_	_

 

*The control values are represented by mean number of eggs/juveniles.

The exposure time period has significant role in mortality in the exposure of time period. The plant extract caused significant mortality as compared to control. The results of various parts of plant shows that treatment showed highly significant results in leaf, stem and fruit of E. hirta, C. papaya and S. nigrum against root-knot nematode. While in mortality percentage of A. viridis leaf extract shows non-significant results. However, C. album showed significant results, respectively.

Application of plant extracts for the control of nematode populations is cheap, easy to apply and has advantages over the standard nematicides regarding environmental safety (Zurren and Khan, 1984; Adegbite, 2003). Plant extracts contain some phenolic compounds, organic acids, terpenes and terpenoids, coumarin-like compounds and other secondary metabolites (Insunza et al., 2001; Shaukat et al., 2004).

Results indicate that plants extract of weed species including A. viridis, C. album, S. nigrum, C. papaya and E. hirta reduced egg hatching and induced deaths of juveniles. It has been observed that nematicidal activity could be related with the release of phenolic and other secondary metabolites which are present in plants. In previous research studies, phenolic compounds such as caffeic acid, benzoic acid and p-cumaric acid induced juvenile’s death in M. javanica (Shaukat and Siddiqui, 2001b). A. viridis was the significant in inhibiting the root-knot nematodes but had a negative effect on plant growth (Costa et al., 2003). In present study, leaf extracts of A. viridis were phytotoxic on root-knot nematode including all parts of plant. Plant powder extracts were toxic to larvae and cause of death in juveniles of M. javanica. Mortality was increased as the exposure of time increased. The plant extracts including Eucalyptus sp., Prosopis juliflora, Samanea saman, Azadirachta indica noted maximum juvenile’s mortality. Soil amendment with A. viridis inhibited the nematode population densities in soil (Shaukat and Siddiqui, 2001c). Different species of Eucalyptus possess few essential oils which have toxicity against nematodes, weeds, insects, fungi and bacteria (Batish et al., 2008).

Liu et al. (2014) reported that second stage of M. incognita juveniles (J2s) were inhibited by Triadica sebifera, Leptopus chinensis, Glochidion eriocarpum, Croton tiglium, Phyllanthus urinaria, Ricinus communis and Euphorbia spp., at the concentration of 1 mg/ml after exposure of 72 h. However, ethanol extracts with plants R. communis, L. chinensis,E. fischeriana and C. tiglium showed 100% mortality against M. incognita at the concentration of 1 mg/ml after 72 h exposure. In our studies, E. hirta aqueous extract of leaf, stem and fruit little bit toxic against juveniles. In our studies, while latex producing plant likes E. hirta suppressed egg hatching it was also phytotoxic against nematode. C.album and S.nigrum caused highest reduction in hatching as compared to the control and C.album caused highest mortality at highest concentration. Vpadhyay et al. (2003) reported that E. hirta, C. album and Ecliptaalba caused more than 80% reduction in hatching at 1:5 dilution. R. communis, A. sativum, S. nigrum, A. indica and A. cepa caused up to 90% reduction in hatching compared to the control.

In the case of screening of plants for nematicidal products against Meloidogyne incognita, the extract of seeds of C. papaya was found to be active. Methanol extracts of C. papaya seeds were higher nematicidal activity than petroleum ether extract but the activities of steam distilled oil from both fresh and dry seeds were by far the highest (Nagesh et al., 2002). In the present results indicate that the seeds of C. papaya possess excellent nematicidal activity towards both saprophytic and plant nematodes which are due to benzyl isothiocyanate. Isothiocyanates in plants are normally derived from the hydrolysis of glucosinolates (Daxenbichler et al., 1991). In our studies, C. papaya also showed nematicidal activity against juvenile and egg hatching. In different compounds of Azadirachta indicasuch as limonoids or triterpenes are recorded as vigorous factors for bioactivity of neem against pests and nematodes (Alam, 1993; Kraus, 1995). Guzman and Saxena (1997) reported that extracted oil of neem or neem cake have high nematicidal properties against the RKN (M. incognita) in green house as well as laboratory. Nimin, is considered a product of neem which can also significantly reduced the soil and RKN populations of M. inognita and promote the parameters of plant growth (Mojumder et al., 2004). The root exudates E. hirta exhibits nematicidal activity against juveniles of Meloidogyne incognita (Kumar et al., 2010).

Conclusions and Recommendations

It is concluded that all plant extracts indicates more than 30% nematicidal activity in treatment against RKN. However, extracts of Chenopodium album exhibited maximum nematicidal activity as compared to remaining plant species. Contemporary finding furthers more reported that nematicidal activity of all plants species dependent on kind of extract, time exposure and dose of extract. It is plainly proved in this study that activity increased in the increment of the extracts and in the concentration and time exposure. Our present study indicated that these plant species can be utilized for biocontrol of RKN and this method of management is cheap, environmentally friendly and free from any hazards.

Novelty Statement

This study revealed that nematicidal activity of all plants species dependent on kind and dose of extract. It is also indicated that these five selected plant species can be utilized for biocontrol of RKN and this method of management is cheap environment friendly and free from hazards.

Author’s Contribution

Javeria Afzal: Primary author who conducted the study.

Muhammad Abid: Supervised the research.

Faisal Hussain: Anlysed the data and wrote the manuscript.

Alia Abbas: Conducted the experiments and finalized the manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Abbasi, M.W., Ahmed, N., Zaki, M.J. and Shaukat, S.S., 2008. Effect of Barleria acanthoides Vahl. on root-knot nematode infection and growth of infected okra and brinjal plants. Pak. J. Bot., 40: 2193-2198.

Abid, M., Zaki, M.J., Khan, M.Q. and Sattar, A., 2005. Use of marine algae for the management of root-knot nematode (Meloidogyne javanica) in okra and tomato plants. Int. J. Phycol. Phycochem., 1: 187-192.

Adegbite, A.A., 2003. Comparative effects of carbofuran and water extract of Chromolaena odorata on growth, yield and food components of root-knot nematode-infested soybean Glycine max (L) Merrill. Ph. D. Dissertation, University of Ibadan, Ibadan, Nigeria.

Adekunle, O.K. and Akinlua, A., 2007. Nematicidal effects of Leucaena leucocephala and Gliricidia sepium extracts on Meloidogyne incognita infecting okra. J. Agric. Sci., 52: 53-63. https://doi.org/10.2298/JAS0701053A

Alam, M.M., 1993. Bioactivity against phytonematodes. In: Randhawa, N.S. and Parmar, B.S. (Eds.). Neem research and development. Soc. Pesticide Sci., New Delhi, India, pp. 123-141.

Anwar, S.A. and McKenry, M.V., 2007. Variability in reproduction of four populations of Meloidogyne incognita on six cultivars of cotton. J. Nematol., 39: 105-110.

Anwar, S.A., Mckenry, M.V. and Legari, A.U., 2009. Host suitability of sixteen vegetable crop genotypes for Meloidogyne incognita. J. Nemtol., 41: 64-65.

Archana, B. and Saxena, R., 2012. Nematicidal effect of root extract of certain medicinal plants in control of Meloidogyne incognita in vitro and in vivo conditions. Pak. J. Nematol., 30: 179-187.

Batish, D.R., Singh, H.P., Kohli, R.K. and Kaur, S., 2008. Eucalyptus essential oil as a natural pesticide. For. Ecol. Manage., 256: 2166-2174. https://doi.org/10.1016/j.foreco.2008.08.008

Begum, Z., Shaukat, S.S. and Siddiqui, I.A., 2003. Suppression of Meloidogyne javanica by Conyza canadensis, Blumea obliqua, Amaranthus viridis and Eclipta prostrata. Pak. J. Plant Pathol., 2: 174-180. https://doi.org/10.3923/ppj.2003.174.180

Budai, C., Szantone, V. and Nadasy, M., 2005. Harmful parasitic nematodes. Gyakorlati Agro Forum, 16: 34-46.

Chitwood, D.J., 2002. Phytochemical based strategies for nematode control. Annu. Rev. Phytopathol., 40: 221-249. https://doi.org/10.1146/annurev.phyto.40.032602.130045

Costa, S., DosS. Da R., Santos, M.S., Nde A. and Ryan, M.F., 2003. Effect of Artemisia vulgaris Rhizome extracts on hatching, mortality and plant infectivity of Meloidogyne megadora. J. Nematol., 35: 437-442.

Daxenbichler, M.E., Spencer, G.F., Carlson, D.G., Rose, G.B., Brinker, A.M. and Powell, R.G., 1991. Glucosinolate composition of seeds from 297 species of wild plants. Phytochemistry, 30: 2623-2638. https://doi.org/10.1016/0031-9422(91)85112-D

Farzana, Abid, M. and Hussain, F., 2016. Root-rot root-knot disease complex of vegetables in Karachi region and their non-chemical control. Int. J. Biol. Biotechnol., 13: 143-151.

Gomez, K.A. and Gomez, A.A., 1984. Statistical procedures for agriculture research. 2nd ed. Wiely, New York. pp. 680.

Guzman, R.S. and Saxena, R.C., 1997. Nematicidal activity of neem and its derivatives. J. Res. (Philippines), 4: 40-44.

Hussey, R.S. and Barker, K.R., 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Dis. Rep., 57: 1025-1028.

Ibrahim, S.K., Traboulsi, A.F. and El-Haj, S., 2006. Effect of essential oils and plant extracts on hatching, migration and mortality of Meloidogyne incognita. Phytopathol. Mediterr., 45: 238−246.

Insunza, V., Aballay, E. and Macaya, J., 2001. In vitro nematicidal activity of aqueous plant extracts on Chilean populations of Xiphinema americanum Sensu Lato. Nematropica, F1: 47-54.

Jesse, Y.A. and Jada, Y.M., 2004. Effect of water soluble extracts of shea butter (B. paraduxum) seeds on root-knot nematode (M. incognita). J. Sustain. Trop. Agric. Res., 9: 16-19.

Jesse, Y.A., Sule, H. and Philip, C.B., 2006. Soil amendment with Parkia biglobosa pods and Hyptis spicigera leaves for the control of Meloidogyne incognita on tomato in Nigeria. Int. J. Nematol., 16: 161-163.

Khalil, M.S. and Darwesh, D.M., 2017. Soil amendments the alternative approach in modern agriculture. Agric. Res. Technol. Open Access J., 4: 1-2. https://doi.org/10.19080/ARTOAJ.2017.04.555646

Khan, A., Sayed, M., Shaukat, S.S. and Handoo, Z.A., 2008. Efficacy of four plants extracts nematodes associated with papaya in Sindh, Pakistan. Nematol. Mediterr., 36: 93-98.

Khan, S.A., Abid, M. and Hussain, F., 2015. Nematicidal activity of seaweeds against Meloidogyne javanica. Pak. J. Nematol., 33: 195-203.

Kraus, W., 1995. Biologically active ingredients: azadirachtin and other triterpenoids. In: Schmutterer, H. and Weinheim, V.C.H. (Eds.) The neem tree, Azadirachta indica A. Juss., and other meliaceous plants: Sources of unique natural products for integrated pest management, medicine, industry and other purposes. Germany, pp. 35-74. https://doi.org/10.1002/3527603980.ch2a

Kumar, S., Malhotra, R. and Kumar, D., 2010. Euphorbia hirta: Its chemistry, traditional and medicinal uses, and pharmacological activities. Pharmacogn. Rev., 4: 58–61. https://doi.org/10.4103/0973-7847.65327

Liu, X., Chao, L., Zhou, and Liu, Z.L., 2014. Evaluation of nematicidal activity of ethanol extracts of Euphorbiaceae plants and constituents from Euphorbiafischeriana to Meloidogyne incognita (Kofoid and White) Chitwood. J. Entomol. Zool. Stud., 2: 311-317.

Mojumder, V., Pankaj, Chawla, G. and Singh, J., 2004. Effect of urea with neem formulations on root-knot and reniform nematodes in okra. Indian J. Nematol., 34: 60-63.

Nagesh, M., Chandravadana, M.V., Sreeja, V.G. and Babu, C.S.B., 2002. Benzyl isothiocyanate from Carica papaya seeds - a potential nematicide against Meloidogwe incognita. Nematol. Mediterr., 30: 155-157.

Osman, H.A., Youssef, M.M.A., El-Gindi, A.Y., Ameen, H.H., Abd-Elbary, N.A. and Lashein, A.M.S., 2012. Effect of salicylic acid and Pseudomonas fluorescens against Meloidogyne incognita in eggplant using split-root technique. Pak. J. Nematol., 30: 101-113.

Perry, R.N. and Maurice, M., 2006. Plant nematology. CABI Publishing, Wallingford, UK, pp. 109. https://doi.org/10.1079/9781845930561.0000

Sharma, A., Haseeb, A. and Abuzar, S., 2006. Screening of field pea (Pisum sativum) selections for their reactions to root-knot nematode (Meloidogyne incognita). J. Zhejiang Univ. Sci., 7: 209-214. https://doi.org/10.1631/jzus.2006.B0209

Shaukat, S.S. and Siddiqui, I.A., 2001b. Effect of some phenolic compounds on survival, infectivity and population density of Meloidogyne javanica in mungbean. Nematol. Mediterr., 29: 123-126.

Shaukat, S.S. and Siddiqui, I.A., 2001c. Effect of some weed extract on mortality and egg hatching of Meloidogyne javanica (Treub.). Pak. J. Biol. Sci., 4: 1251-1252. https://doi.org/10.3923/pjbs.2001.1251.1252

Shaukat, S.S., Siddiqui, I.A. and Zarina, B., 2004. Effect of some common weeds from Pakistan on plant -parasitic nematodes in vitro and population densities and survival of Meloidogyne incognita in okra and brinjal. Nematol. Mediterr., 32: 111-115.

Singh, S.K. and Khurma, R.K., 2007. Susceptibility of six tomato cultivars to the root-knot nematode Meloidogyne incognita. South Pac. J. Nat. Sci., 13: 73-77. https://doi.org/10.1071/SP07013

Viaene, N., Coyne, D.L. and Kerry, B.R., 2006. Biological and cultural management. In: Plant nematology (eds. Perry, R.N. and Maurice, M.). CAB Int., Wallingford. UK. pp. 346-369. https://doi.org/10.1079/9781845930561.0346

Vpadhyay, K.D., Dwivedi, K. and Vttam, S.K., 2003. Effect of some plant extracts on the mortality and hatching of Meloidogyne incognita and Heterodera calani infesting pigeon pea. Nematol. Mediterr., 31: 29-31.

Youssef, M.M.A., Mohammed, M.M.M. and Korayam, A.M., 2012. Effect of organic and inorganic fertilizers on Meloidogyne incognita infesting sugar beet. Pak. J. Nematol., 30: 143-149.

Zurren, S. and Khan, M.I., 1984. Nematicidal activity in some plant lattices. Pak. J. Nematol., 2: 69-77.