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Effect of Steinernema glaseri and Heterorhabditis indica on the Plant Vigour and Root Knot Nematodes in Tomato Roots at Different Densities and Time of Applications.

Byline: Nazir Javed, Sajid Aleem Khan, Imran-ul-Haq, M. Atiq and M. Kamran

Abstract

Steinernema glaseri and Heterorhabditis indica applied at different inoculum levels before and simultaneously with root knot nematodes were investigated for invasion of Meloidogyne spp. in tomato. Suppression of M. incognita varied with application rate of S. glaseri, and H. indica The high application rates of, S. glaseri and H. indica applied both same and before reduced invasion of M. incognita. The low rate of S. glaseri and H. indica were not as effective as high rates. Both entomopathogenic nematodes when applied at 1250 and 2500/pot 24 h before or at the same time reduced the invasion of root knot nematodes in tomato root. Whereas S. glaseri applied at 500 /pot at the same time with the root-knot nematodes reduced the invasion. Both the entomopathogenic nematodes (EPN) applied 24 h before or at the same time with root knot nematodes at 2500/pot reduced the invasion as compared with 500/pot.

Only H. indica significantly reduced invasion when it was applied at 1250/pot 24 h before the root knot nematodes. Whereas S. glaseri applied at the same time and H. indica 24 h before at 2500/pot significantly reduced the invasion as compared with 1250/pot. S. glaseri was more effective in reducing the invasion as compared to H. indica. Recovery of both the entomopathogenic nematodes when they were applied alone was comparatively more than when applied with the root knot nematodes. But in case of H. indica its recovery was more when it was applied at the same time with the M. javanica.

Key words. Steinernema glaseri, Heterorhabditis indica,root knot nematodes, entomopathogenic nematodes.

INTRODUCTION

Root-knot nematodes (RKN) are notoriously difficult to manage because of their high reproductive potential. Economic damage on tomato can occur with root-nematode densities of 0.1-1.0 nematodes per cm3 soil at planting (Sikora and Fernandez, 2005). Whitehead (1998) suggested that 99.9% control is required in order to prevent the subsequent build-up of damaging population because of the reproductive potential of Meloidogyne spp. On tomato these nematodes can cause 24-38% loss, where sequential cropping of one susceptible crop after another is practiced with up to four per year. In the absence of effective control it would lead to total crop failure. Plant parasitic nematodes cause global losses to crop plants with an estimated loss of $ 125 billion per year in the tropics (Chitwood, 2003). The nematode infected plants show poor growth and become less productive because the damaged root systems are less efficient in absorbing water and nutrients.

Current dissatisfaction with chemicals nematicides, due to safety issues, environmental concern and limited use of many products (e.g., methyle bromide) has stimulated interest in control strategies that are ecologically compatible with current production system. In fact developing alternative to hazardous chemical nematicides is one of the top priorities for the future of nematology. Several control strategies, such as host plant resistance, rotation with non- hosts, sanitation and avoidance, destruction of residual crop roots, and judicious use of nematicides have been reported to effectively control RKNs (Whitehead, 1998).

Entomopathogenic nematodes (EPN) of Steinernematidae and Heterorhabditidae families have been used as biological control agents against different insect pests. These nematodes are soil- dwelling organisms, and are obligate parasites of insects. The only stage which lives freely on the soil is the infective juvenile or J3 and to complete their life cycles, those J3s must find a suitable host. When they locate the host, penetrate into them through the body natural openings (Poinar, 1979; Burnell and Stock, 2000) or also through the cuticle in Heterorhabditidae (Bedding and Molyneux, 1982), reaching the hemocoel and releasing a symbiotic bacterium (Xenorhabdus in Steinernema and Photorhabdus in Heterorhabditis) which starts reproducing and finally kills the insect by septicaemia between 24 to 72 h.

Some species of EPN such Steinernema glaseri, S. carpocapsae and Heterorhabditis megidis have been reported following root plants (Bird and Bird, 1986; Kanagy and Kaya, 1996) or their exudates when insect consume them (Rasmann et al., 2005), probably as a result of a defensive strategy used by plants to protect themselves from insect attacks.

Due to environmental concerns and increased regulations on use of chemical fumigants, more management strategies for management of RKNs (Meloidogyne spp.) are currently being investigated (Nico et al., 2004). Biological control using EPN is one potential alternative to chemical nematicides. Recently it has been demonstrated that EPNs can affect the number of RNK infecting plants, or the number of eggs produced when they are applied near the root system (Fallon et al., 2002; Perez and Lewis, 2002; 2004). The explanation of this effect has been theorized as a result of an allelopathic response produced by the symbiotic bacteria of EPNs which is repellent to RKNs (Grewal et al., 1999), however the application of the bacterium has not shown a consistent suppression of RKN in some studies (Fallon et al., 2004).

The objective of the recent study is to determine the effect of different inoculums levels and time of application for 2 species of EPNs (Sg and Hi) on the invasion of RKN to tomato roots and their effect on plant vigour improvement.

MATERIALS AND METHODS

Culture of EPNs

The greater wax moth Gallaria mellonella (L.) were obtained from bee hives infected with G. mellonella. Last instars larvae of G. mellonela were separated for nematode culture, leaving small sized larvae for moth emergence and egg laying. Fresh laid eggs were transferred to modified artificial diet prepared by mixing oat, wheat, rice and maize porridge (20 g), yeast granules (50 g) in solution of 80 ml warm honey and (100 g) glycerol (Alrubei and Al-Izzim, 1986). Diet with Gallaria was then kept at 27oC in an incubator. After reaching last instars, they were taken out from the diet and used for storage and nematode isolation/multiplication. Steinernema glaseri and Heterorhabditis indica were multiplied and harvested from greater wax moth larvae (Lepidoptera: Pyralidae) (Woodring and Kaya, 1988; White, 1927) and later stored at 10oC. Infective juveniles of these nematodes were harvested two weeks after incubation using white traps and washed in three changes of distilled water (Dutky et al., 1964).

These juveniles were stored at 10oC and before use they were left over night at 20+-3oC.

Culture of RKN M. javanica

The RKN M. javanica was maintained on tomato (Lycopersicon esculentum) plants cv., Tiny Tim in a glass house at 28+-4+-C. Eggs of M. javanica were collected using a modification of the technique described by McClure (1977). Galled roots with egg masses were washed free of soil, cut into 2-cm long pieces and after placing in 0.26% sodium hypochlorite (commercial bleach) were triturated at 30 s intervals at maximum speed in a two-speed blender. To separate the organic debris from eggs this suspension was poured through a series of sieves. The eggs were collected on 38mm- pore sieve and washed carefully with tap water. The egg suspension was poured on to cotton-wool filter paper (modified Baermann) and incubated at 28degC. The hatched second stage juveniles (J2) were collected daily. Only freshly hatched J2 collected within 48 h were used for experiments.

Effect of S. glaseri and H. indica on the plant vigour and development of M. javanica in tomato roots

One month old tomato plants c.v, Tiny Tim was maintained in 80 ml modules. S. glaseri and H. indica were applied separately at 500, 1250, 2500 individuals per pot (80 ml) at various time intervals (24 h before, at the same time with or without root- knot). Plants receiving only RKNs were kept as control. Each time and application dose of entomopathogenic nematodes had its respective control. These plants were completely randomised in a glass house in controlled conditions where temperature was 22-38oC and each treatment was replicated five times. Plants were kept in green house for one week after exposure of plants to root- knot nematodes. Plants were not watered one day before harvesting. Then they were removed from pots and the root balls were shaken until most of the soil had been dislodged from the root. From this soil the EPNs were recovered by plating the soil for 48 h using Baermans funnel method.

After washing and taking root and shoot weight these roots were stained in acid fuchsin (Byrd et al., 1983) and macerated. Then total number of nematodes was counted.

In one set of treatment where S. glaseri and H. indica were applied at the same time with root knot nematodes, the experiment run for 31 days. The number of egg masses and females were counted on the whole root system (Holbrook et al., 1983). Experiment was repeated twice to confirm the data.

Data was analyzed using ANOVA by using SAS statistical software (SAS Institute, Cary, NC, USA). The significance of differences within treatments was separated by using Least Significant Difference test at 5%.

RESULTS

There was no significant effect of any treatment on the root and shoot weight of tomatoes. The root weigh where S. glaseri applied at 2500 with root knot nematodes at the same times was low and had a significant effect, but this effect could not be repeated again as the experiment was repeated twice (Table I). Similarly shoot weight where S. glaseri were applied at 1250 with root knot 24 h before was comparatively lower than other treatments (Table I).

Recovery of S. glaseri from soil after one week of its application was significantly different (P less than 0.05) from H. indica. S. glaseri and H. indica recovered from soil were significantly different from each other at different densities and time of application (24 h before or after application). Similarly it was insignificant (P = 0.54) at its method of application (with or without RKN). Recovery of S. glaseri was more when it was applied without root-knot nematodes in contrast to H. indica. Recovery of both EPNs when they were applied alone were comparatively more than when they applied with the RKNs. In case of H. Indica, the recovery was more when it was applied at the same time with RKN (Table II).

Both the EPNs when applied at 1250 and 2500/pot 24 h before or at the same time significantly (P less than 0.05) reduced the invasion of RKNs in tomato root. Whereas only S. glaseri applied at 500 /pot at the same time with the RKNs significantly reduced the invasion (Table III). Both the EPN applied 24 h before or at the same time with RKNs at 2500/pot significantly (P less than 0.05) reduced the invasion as compared with 500/pot. Only H. indica significantly reduced invasion when it was applied at 1250/pot 24 h before the RKNs as compared with 500/pot.Whereas S. glaseri applied at the same time and H. indica 24 h before at 2500/pot significantly reduced the invasion as compared with 1250/pot (Table IV). S. glaseri was more effective in reducing the invasion as compared to H. indica.

Table IV indicating that both the EPN differed significantly (P less than 0.05) with control in reducing the number of egg masses at all levels of their application (500, 1250 and 2500).There was no significant difference between the EPN at various doses expect S. glaseri at 500/pot which differed significantly with H. indica at same and 2500/pot level of application. Both the EPN at 2500/pot differed significantly (P less than 0.05) with other densities in reducing the number of egg masses. They also significantly (P less than 0.05) reduced the number of females as compared with control.

DISCUSSION

The effect of S. glaseri and H. indica was investigated on M. javanica in tomato roots

Different factors are responsible for the suppressive effects of EPN on plant-parasitic nematodes as competition between the nematode groups for space in rhizosphere (Bird and Bird, 1986; Tsai and Yeh, 1995), attraction towards the CO2 and other root exudates (Robinson, 1995), increased density of predators resulting from the application of nematode biomass to the soil (Ishibashi and Kondo, 1986), behavioral response

Table I.- Effect of EPN on root weights and shot of tomato plant.

Treatments###Time of EPN###Densities of EPNs (IJ/pot)

###500###1250###2500

###application###Alone###With RKN###Alone###With RKN###Alone###With RKN

Root weight

S. glaseri###24 h before###1.57+-1.26###1.90+-0.21###1.72+-0.06###1.64+-0.13###1.49+-0.15###1.51+-0.07

###Simultaneous###1.56+-0.075###1.47+-0.19###1.97+-0.15###1.93+-0.16###1.78+-0.14###1.43+-0.15

H. indica###24 h before###1.55+-1.16###1.76+-0.17###1.54+-0.10###1.73+-0.11###1.48+-0.13###1.77+-0.07

###Simultaneous###1.76+-0.05###1.59+-0.14###1.82+-0.11###1.71+-0.22###1.53+-0.09###1.71+-0.22

Control (Root-knot)###1.782+-0.089

Healthy plants###1.786+-0.188

Shoot weight

S. glaseri###24 h before###4.39+-0.06###4.62+-0.35###4.49+-0.17###3.98+-0.26###4.43+-0.05###4.28+-0.21

###Simultaneous###4.93+-0.12###4.11+-0.20###4.97+-0.20###4.55+-0.17###4.83+-0.31###4.65+-0.19

H. indica###24 h before###4.83+-0.23###4.53+-0.29###4.64+-0.35###4.99+-0.42###4.52+-0.16###4.92+-0.56

###Simultaneous###4.75+-0.21###4.60+-0.27###5.37+-0.23###4.86+-0.21###4.98+-0.27###5.04+-0.34

Control (Root-knot)###4.21+-0.34

Healthy plants###4.87+-0.39

Table II.- EPN (actual values and log values) recovered from soil at the time of harvesting (one week after application)

Treatments###Time of EPN###Densities of EPNs (IJ/pot)

###500###1250###2500

EPN (actual values)###application###Alone###Pathogen###Alone###Pathogen###Alone###Pathogen

S. glaseri###24 h before###152.40+-24.80###172.2+-46.2###325.6+-102.1###298.8+-90.5###715.2+-73.5###557.0+-131.4

###Simultaneous###148.60+-28.58###116.2+-34.75###268.6+-58.0###215.6+-38.8###684.2+-101.4###600.8+-128.2

H. indica###24 h before###49.0+-14.44###55.8+-23.99###137.2+-29.66###142.0+-20.73###236.4+-58.1###243.0+-45.3

###Simultaneous###44.6+-9.45###71.8+-27.89###118.4+-35.15###198.8+-58.1###335.0+-69.4###362.8+-63.9

EPN (log values)

S. glaseri###24 h before###2.15+-0.07###2.17+-0.12###2.42+-0.13###2.40+-0.12###2.84+-0.04###2.69+-0.10

###Simultaneous###2.13+-0.09###2.0+-0.10###2.38+-0.09###2.30+-0.08###2.81+-0.07###2.73+-0.10

H. indica###24 h before###1.59+-0.15###1.62+-0.15###2.10+-0.08###2.13+-0.06###2.30+-0.12###2.35+-0.08

###Simultaneous###1.60+-0.10###1.72+-0.17###2.01+-0.11###2.22+-0.12###2.47+-0.11###2.52+-0.08

Densities of EPN; P less than 0.05; SED; 0.05 Treatments; P less than 0.05; SED; 0.045; Time; P =0.08; SED; 0.045; Method (alone or with pathogen); P = 0.54; SED; 0.045; Treatments x Densities x Time x Method); P = 1.0; SED; 0.15

Table III.- Effect of H. indica and S. glaseri applied 24 h before or at the same time with root-knot nematodes on the invasion of root-knot nematodes within tomato root.

###Densities of EPNs ( IJ/pot)

Treatments###500###1250###2500

###24 h before###Simultaneous###24 h before###Simultaneous###24 h before###Simultaneous

S. glaseri###110.0+-17.40###93.0+-8.58###84.4+-9.26###86.8+-5.70###67.8+-11.21###54.2+-9.59

H. indica###129.0+-5.73###110.4+-13.12###90.0+-9.34###81.0+-16.95###62.4+-10.78###61.6+-13.03

Control (RKN)###131.20+-15.05

Treatments; P less than 0.05; SED; 12.73; Densities of EPN; P less than 0.05; SED; 13.17; Time: P=0.35; SED; 12.73; Treatments x Densities x Time; P = 0.76; SED; 16.66

Table IV.- Effect of EPN on number of females and egg masses.

Treatment###Densities of Entomopathogenic nematodes ( IJ/pot)

###500###1250###2500

###Egg masses###Females###Egg masses###Females###Egg masses###Females

S. glaseri###43###94###72###97###15###80

H. indica###78###147###73###108###9###33

Control (root-knot nematodes###127.0###225.7###127.0###225.7###127.0###225.7

Egg masses:###Females;

Treatments; P less than 0.05 SED: 16.41###P less than 0.05; SED; 16.81

Densities; P less than 0.05 LSD; 16.41###P less than 0.05; SED; 16.81

Treatments x Densities P= 0.36; SED; 28.42###P=0.09; SED; 29.12

and increased natural enemies (Grewal et al., 1999) and production of allelochemicals by the EPNs symbiotic bacteria complex (Grewal et al., 1999; Hu et al., 1999; Samaliev et al., 2000; Lewis et al., 2001). Nematicidal properties of metabolites of symbiotic bacteria Xenorhabdus spp. associated with Steinernema spp. (Grewal et al., 1999; Hu et al., 1999; Samaliev et al., 2000) and P. temperate and P. luminescens with H. megidis and H. bacteriophora (Boemare, 2002) might be responsible for the suppressive effect of EPNs on root knot nematodes. The difference in the suppressive effect might be due to the difference of the associated bacteria and its toxic metabolites. Cell-free extracts of Xenorhabdus spp. were found to be toxic and repellent to M. incognita juveniles and inhibited its egg hatching (Grewal et al., 1999).

EPNs belonging to Steinernematids were found in tomato roots. Steinernema spp. has ability to enter in roots by following infecting root-knot nematodes (Fallon et al., 2002). M. incognita suppression using Heterorhabditids was less consistent than steinernematids. Our results are in confirmitty with the work done by different workers (Grewal et al., 1999; Fallon et al., 2002, 2006; Jagdale and Grewal, 2008). It can be concluded that the Steinernema spp. were more efficient in suppressing M. incognita due to their ability to enter the roots and release associated bacteria inside the roots. The bacteria inside the root tissue release allelochemicals those are toxic and repellent to RKNs (Grewal et al., 1999; Fallon et al., 2002). EPNs can be successfully applied as the environment safe management approach for RKNs.

REFERENCES

ALRUBEI, H.F. AND AL-IZZIM, M.A.J., 1986. Laboratory rearing of Gallaria mellenella on artificial diet. J. biol. Sci. Res., 17: 57-64.

BEDDING, R.A. AND MOLYNEUX, A.S., 1982. Penetration of insect cuticle by infective juveniles of Heterorhabditis spp. Nematologica, 28:354-359.

BIRD, A.F. AND BIRD, J., 1986. Observations on the use of entomopathogenic nematodes as a means of biological control of root-knot nematodes. Int. J. Parasitol., 16:511-516.

BOEMARE, N., 2002. Biology, taxonomy and systematics of Photorhabus and Xenorhabdus. In: Entomopathogenic nematology (ed. R. Gaugler). CABI Publishing, Wallingford, UK, pp. 35-56.

BURNELL, A.M. AND STOCK, S.P., 2000. Heterorhabditis, Steinernema and their bacterial simbiont-lethal pathogen of insects. Nematology, 2:31-42.

BYRD, D.W.JR., KIRKPATRICK, T.J.R. AND BARKER, K.R., 1983. An improved technique for clearing and staining plant tissues for detecting of nematodes. J. Nematol., 15:142-143.

CHITWOOD, D.J., 2003. Research on plant parasitic nematode biology conduct by the United State Department of Agriculture- Agriculture Research Service. Pest Manag. Sci., 59: 748-753.

DUTKY, S.R., THOMPSON, J.V. AND CANTWELL, G.E., 1964. A technique for the mass propagation of the DD-136 namatode. J. Insect Pathol., 6: 417-422.

FALLON, D.J., KAYA, H.K. AND SIPES, B.S., 2006. Enhancing Steinernema spp. suppression of Meloidogyne javanica. J. Nematol., 38: 270-271.

FALLON, D.J., KAYA, H.K., GAUGLER, R.G. AND SIPES, B.S., 2004. Effect of Steinernema feltiae-Xenorhabdus bovienii insect pathogen complex on Meloidogyne javanica. Nematology, 6: 671-680.

FALLON, D.J., KAYA, H.K., GAUGLER, R.G. AND SIPES, B.S., 2002. Effect of entomopathogenic nematodes on

Meloidogyne javanica on tomato and soyabeans. J. Nematol., 34: 239-245.

GREWAL, P.S., LEWIS, E.E. AND VENKATACHARI, S., 1999. Allelopathy: A possible mechanism of suppression of plant-parasitic nematodes by entomopathogenic nematodes. Nematology, 1:735-743.

HOLBROOK, C.C., KNAUFT, D.A. AND DICKSON, D.W. 1983. A technique for screening peanut for resistance to Meloidogyne arenaria. Pl. Dis., 67: 957-958.

HU, K., LI, J. AND WEBSTER, J.M., 1999. Nematicidal metabolites produced by Photorhabdus luminescens (Enterobacteriaceae), bacterial symbiont of entomopathogenic nematodes. Nematology, 1: 457-469.

ISHIBASHI, N. AND KONDO, E., 1986. Steinernema feltiae (DD-136) and S. glaseri persistence in soil and bark compost and their influence on native nematodes. J. Nematol., 18: 310-316.

JAGDALE, G.B. AND GREWAL, P.S., 2008. Influence of the entomopathogenic nematode Steinernema carpocapsae in host cadavers or extracts from cadavers on the foliar nematode Aphelenchoides fragariae on Hosta. Biol. Contr., 44: 13-23.

KANAGY, J.M.N. AND KAYA, H.K., 1996. The possible role of marigold roots and aterthienyl in mediating host- finding by steinernematid nematodes. Nematologica, 42: 220-231.

LEWIS, E.E., GREWAL, P.S. AND SARDANELLI, S., 2001. Interaction between the Steinernema feltiae- Xenorhabdus bovienii insect pathogen complex and root-knot nematode Meloidogyne incognita. Biol. Contr., 21:55-62.

McCLURE, M.A., 1977. Meloidogyne incognita: A metabolic sink. J. Nematol., 9: 88-90.

NICO, A.I., RAFAEL, R.M., JIMeNEZ-DAZA, M. AND CASTILLO, P., 2004. Control of root-knot nematodes by composted agro-industrial wastes in potting mixtures. Crop Prot., 23: 581-587.

PEREZ, E.E. AND LEWIS, E.E., 2002. Use of entomopathogenic nematodes to suppress Meloidogyne incognita on greenhouse tomatoes. Biol. Contr., 30:336-341.

PEREZ, E.E. AND LEWIS, E.E., 2004. Suppression of Meloidogyne incognita and Meloidogyne hapla with entomopathogenic nematodes on greenhouse peanuts and tomatoes. Biol. Contr., 30:336-341.

POINAR, JR. G.O., 1979. Nematodes for biological control of insects. CRC Press, Inc. Boca Raton, Florida. pp. 277.

RASMANN, S., KOLLNER, T.G., DEGENHARDT, J., HILTPOLD, I., TOEPFER, S., KUHLMANN, U., GERSHENZON, J. AND TURLINGS, T.C.J., 2005. Recruitment of entomopathogenic nematodes by insect- damaged maize roots. Nature, 434: 732-737.

ROBINSON, A.F., 1995. Optimal release for attracting M. incognita, Rotylenchus reniformis and other nematodes to carbon dioxide in sand. J. Nematol., 27: 42-50.

SAMALIEV, H.Y., ANDREOGLOU, F.I., ELAWAD, S.A., HAGUE, N.G.M. AND GOWEN, S.R., 2000. The nematicidal effects of the Pseudomonas oryzihabitans and Xenorhabdus nematophilus on the root-knot nematode Meloidogyne javanica. Nematology, 2: 507-514.

SIKORA, R.A. AND FERNANDEZ, E., 2005. Nematode parasites of vegetables. In: Plant-parasitic nematodes in subtropical and tropical agriculture (eds. M. Luc, R.A. Sikora and J. Bridge.), second ed. CABI Publishing, Wallingford, UK, pp. 319-392.

TSAI, B.Y. AND YEH, H.L., 1995. Effect of Steinernema carpocapsae Weiser on the infectivity of Pratylenchus coffeae (Zimmermann) FilipJev & Schourmans Stekhoven and Meloidogyne javanica (Treub) Chitwood. Pl. Prot. Bull., 4: 106.

WHITE, G.F., 1927. A method for obtaining infective nematode larvae from culture. Science, 66: 302-303.

WHITEHEAD, A.G. 1998. Plant nematode control. Cab International, Wallingford, UK, pp. 384

WOODRING, J.L. AND KAYA, H.K. 1988. Steinernematid and Heterorhabditid nematodes. In: A handbook of techniques. Southern Cooperative Series Bulletin 331, Arkansas Agricultural Experiment Station.

Department of Plant Pathology, University of Agriculture, Faisalabad, Corresponding author: Nazirpp2003@yahoo.com
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Author:Javed, Nazir; Khan, Sajid Aleem; Imran-ul-Haq; Atiq, M.; Kamran, M.
Publication:Pakistan Journal of Zoology
Article Type:Report
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Date:Aug 31, 2012
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