Soil Biology & Biochemistry 40 (2008) 20162020

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Soil Biology & Biochemistry

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Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum

N. Sahebani*, N. Hadavi
Department of Plant Pathology, University College of Abureihan, University of Tehran, Enghelab, Tehran 324354, Iran

a r t i c l e i n f o
Article history: Received 12 August 2007 Received in revised form 14 November 2007 Accepted 21 March 2008 Available online 12 May 2008 Keywords: Biological control Meloidogyne javanica Trichoderma harzianum Chitinase Peroxidase Polyphenol oxidase Phenylalanine ammonia lyase

a b s t r a c t
The lamentous fungi Trichoderma spp. is currently developed as biocontrol agents against many plant pathogens. Recent studies have shown that these fungi are able to infect nematode eggs and juveniles. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Trichoderma harzianum BI was investigated in greenhouse and laboratory experiments. Results showed that different concentrations (102108 spores/ml) of T. harzianum BI decreased nematode infection and other parameters signicantly, compared to control. T. harzianum BI was able to penetrate nematode egg mass matrix and signicantly decreased nematode egg hatching level. Specic activities of resistance-related enzymes, namely peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased signicantly in T. harzianum BI inoculated plants. Maximum activities of POX, PPO and PAL were observed at the 5, 5 and 6 days after inoculation, respectively. Chitinase activity was also increased in culture ltrates of T. harzianum BI grown on wheat bran moistened with salt solution supplemented with colloidal chitin or nematode eggs. Maximum activity of chitinase was recorded at the 4 days after inoculation, in media supplemented with colloidal chitin (1.15 U/min per ml) and nematode eggs (0.85 U/ min per ml). Results suggested that direct parasitism of eggs through the increase in extracellular chitinase activity, which would be indicator of eggs infection capability, and inducing plant defense mechanisms leading to systemic resistance are two main suppression mechanisms used by T. harzianum BI against nematode. 2008 Elsevier Ltd. All rights reserved.

1. Introduction The root knot nematodes (Meloidogyne spp.) are sedentary endoparasites and are among the most damaging agricultural pests attacking a wide range of crops (Mai and Abawi, 1987). Due to the problems caused by chemical control, mainly their deleterious effects on human health and environment, development of alternative control methods is of great importance. Trichoderma spp. has been widely studied as a biological control agent against microbial diseases of crops. (Cherif and Benhamou, 1990; Chet, 1987; Chet et al., 1981; Elad et al., 1980, 1983). Several attempts have been made to use Trichoderma spp. to control plant parasitic nematodes. T. harzianum Rifai has been reported to be an effective bioagent for the management of the citrus nematode (Parvatha Reddy et al., 1996; Rao et al., 1998; Seifullah and Thomas, 1996; Sharon et al., 2001). Windham et al. (1986) reported reduced egg production in the root-knot nematode Meloidogyne arenaria following soil treatments with T. harzianum and T. koningii preparation. Rao et al.

* Corresponding author. Tel.: 98 91 2292 0211; fax: 98 29 2302 5292. E-mail address: sahebani@ut.ac.ir (N. Sahebani). 0038-0717/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.03.011

(1998) evaluated aqueous extracts of neem (Azadirachta indica), caster (Ricinus communis) and pingamia (Pingamia harzianum) as substrates for the mass production of T. harzianum which was used in the management of Meloidogyne incognita in eggplant under eld conditions. They reported that caster cake extracts showed the best biocontrol activity. Seifullah and Thomas (1996) studied the parasitism of Globodera rostochiensis by T. harzianum using low temperature scanning electron microscopy. Rao et al. (1998) and Sharon et al. (2001), showed that T. harzianum isolates can reduce the M. javanica infection. There is little information on the mechanisms of Trichoderma species against nematodes. Two mechanisms of action are thought to be responsible for the reduction in nematode infection following root treatment with Trichoderma spp.: (1) direct parasitism of eggs and larva through the increase in chitinase and protease activities, which would be indicators of eggs infection capability (Sharon et al., 2001; Suarez et al., 2004); and (2) inducing plant defense mechanisms leading to systemic resistance. Extracellular enzymes such as chitinase and protease which display antifungal activities appear to participate in the Meloidogyne javanica Trichoderma spp. interaction (Sharon et al., 2001). Chitin is a homopolymer, b-1,4-linked of N-acetylglucosamine, the second most abundant

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polymer in nature and also a common structural component (40% w/w) of a nematode eggshell (Wharton, 1980). Nematophagous egg-parasitic fungi, such as Pochonia chlamydosporia, must penetrate the nematode eggshell during infection (Lopez-Llorca and Duncan, 1988; Lysek and Krajci, 1987). This structure is formed by several layers (Wharton, 1980), including the chitinous layer, composed of a protein matrix (5060% of the composition) embedding chitin microbrils (Kim et al., 1992; Meyer and Wergin, 1998). This layer is probably the major barrier against invasion by soil microorganisms. Furthermore, the collagen-containing cuticle of plant parasitic nematodes also acts as an effective structural barrier to infection (Tikhonov et al., 2002). Parasitic microorganisms must be able to penetrate these barriers for their successful establishment in the host. Suarez et al. (2004) characterized a trypsin-like protease, PRA1, from the culture ltrates of T. harzianum CECT2413 grown in liquid medium supplemented with fungal cell wall or chitin. They showed that the number of hatched eggs of root knot nematode Meloidogyne incognita was reduced after incubation with pure PRA1 preparation. Khan et al. (2003) showed that the lamentous fungus Paecilomyces lilacinus as a biocontrol agent against plant parasitic nematodes penetrates nematode eggs and cuticles through the production of lytic enzymes. A serine protease and chitinase were puried from a culture medium and were further conrmed by in vitro tests. In the present study, we used greenhouse experiments to show the ability of Trichoderma harzianum BI to: (1) suppress the root knot nematode, Meloidogyne javanica infection and nematode egg hatching on tomato plant; and (2) laboratory experiments to monitor the induction of some resistance-related enzymes in plants and production of extracellular chitinase using colloidal chitin as the substrate. 2. Materials and methods 2.1. Nematode inoculum preparation Infected sample was collected from a tomato eld in Shiraz (Fars province, Iran) and single egg mass was used to establish a population on Rutgers tomato variety for the experiment. Eggs were extracted from infected tomato roots using 1% NaOCL. Extracted eggs were gently washed with tap water to remove NaOCL (Hussey and Barker, 1973). The species of nematode was identied as M. javanica according to morphological and morphometrical characters (Eisenback, 1985). 2.2. Fungal inoculum preparation Trichoderma harzianum BI was obtained from plant pathology department, agriculture faculty of Tehran University and was cultured on potato dextrose agar (PDA, Oxoid). Six days after incubation (27 C), the puried fungi were used to produce spore suspension for inoculation. 2.3. Plant material Experiments were carried out with tomato (Lycopersicon esculentum var. Roma VF, susceptible to M. javanica) grown in a controlled-environment cabinet. The air temperature was maintained at 27 C. 2.4. Effect of various concentrations of T. harzianum BI on M. javanica infection Seeds of tomato were surface sterilized with 1% sodium hypochlorite (5 min) and sown in pots (1 kg) containing sterile soil [Mixture of eld soil (silty clay; 78% organic matter) and sand

(1:1 v/v)]. Tomato seedlings at four-leaf stage were inoculated with various concentrations of Trichoderma spores (at 102108 spores/ ml) by root dipping (5 min) respectively. After 2 days, seedlings in each treatment were inoculated with 2000 M. javanica Juveniles per individual seedling. Thirty days after seedling inoculation with nematode, number of galls and egg masses per seedling, and number of eggs per individual egg mass were evaluated. Tomato seedlings inoculated with nematode without fungal inoculation [inoculated with sterile distilled water (SD/water)] were used as control. The experiment was performed with 10 replications. 2.5. Effect of T. harzianum BI on nematode egg hatching Tomato seedlings at four-leaf stage were inoculated with 2000 nematode J2 per individual seedling. After 2 days (for penetration of nematode J2 into roots), plant roots were washed with top water and were inoculated with 106 spores/ml of fungus (SD/water as control) by root dipping (5 min) and transplanted in sterile soil. After 20 days, nematode eggs were extracted and were hatched according to Hussey and Barker (1973). Nematode eggs hatching percentage were evaluated for 9 days, and 1 day intervals in treatments and their controls. 2.6. Resistance-related enzymes assays Seeds of tomato were surface sterilized as mentioned above, and sown in pots containing sterile soil. The seedlings were inoculated with 106 spores/ml of fungus at four-leaf stage by root dipping (5 min) and transplanted to sterile soil, and were irrigated with SD/ water. Two days after inoculation with fungus, plant roots were inoculated with 2000 nematode J2/plant. Root sampling were done 7 days with 1 day intervals. Fresh tomato roots were washed and dried with lter paper after sampling and homogenized with liquid nitrogen in an ice cold mortar and pestle. The homogenized tissue was rinsed with the same volume of 10 mM sodium phosphate buffer (PH 6.0) at 4 oC, and was ltered through a 0.2 mm nylon lter into a centrifuge tube. The tissue extracts were centrifuged at 12,000 rpm for 20 min at 4 oC. The supernatant was used for the enzymatic activity assay. The experiment was performed with ve replications. 2.7. Phenylalanine ammonia lyase function assay Phenylalanine ammonia lyase (PAL) function was assayed according to Chen et al., (2000). The standard Bradford assay was employed to test the protein concentration of root extracts in each sample. 2.8. Peroxidase and poly phenol oxidase function assay Peroxidase (POX) and poly phenol oxidase (PPO) function were assayed according to Liu (2002). 2.8.1. Evaluation of T. harzianum BI for chitinase production Seven grams of dry wheat bran in a 250 ml Erlenmeyer ask and was supplemented with 3 ml salt solution containing 0.5% NH4NO3, 0.2% KH2PO4, 0.1% NaCl, 0.1% MgSO4 7H2O. The initial moisture level in the substrate was adjusted by adding adequate amount of SD/water. The substrate was mixed thoroughly and autoclaved for 20 min at 121  C (1.5 Atm.) and cooled to room temperature before inoculation. The sterilized solid substrate medium was inoculated with 1ml fungal spore inoculum (106 spores/ml) under aseptic conditions. The contents were mixed thoroughly and the asks were placed in an incubator at 30  C for 10 days and 1 day intervals. Flasks were removed every 24 h and the enzyme extraction and Chitinase assay was done as described below. An adequate amount

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of distilled water containing 0.1% Tween 80 was added to the substrate to get a total extraction volume of 100 ml. The contents were mixed thoroughly by keeping the asks on a rotary shaker at 150 rotations per minute for 30 min. The mixture was centrifuged at 10,000 rpm for 10 min at 4  C. The supernatant was collected and used for Chitinase assay. 2.8.2. Chitinase assay Chitinase activity was assayed by measuring the release of reducing saccharides from colloidal chitin as follows (Miller, 1959): a reaction mixture containing 1 ml of culture supernatant, 0.3 ml of 1 M sodium acetate buffer, pH 4.7, and 0.2 ml of colloidal chitin was incubated at 50  C for 1 h and then centrifuged at 10,000 rpm for 5 min at 4  C. After centrifugation, an aliquot of 0.75 ml of the supernatant, 0.25 ml of 1% solution of dinitrosalycilic acid in 0.7 M NaOH, and 0.1 ml of 10 M NaOH were mixed in 1.5 ml Eppendorf tubes and heated at 100  C for 5 min. Absorbance of the reaction mixture at 530 nm was measured after cooling to room temperature using UV spectrophotometer (Shimadzu, Japan). A calibration curve with N-acetyl-D-glucosamine (NAGA) as a standard was used to determine reducing saccharide concentration. Under the assay conditions described, a linear correlation between A530 and NAGA concentration was found in the interval of 40800 mg/ml NAGA. One unit (U) of the chitinase activity is dened as the amount of enzyme that is required to release 1 mmol of NAGA per minute from 0.5% of dry colloidal chitin solution under assay conditions. 2.9. Nematode eggs as a chitinase inducer Erlenmeyer asks (1 L) containing 150 ml growth medium (0.03% NaCl, 0.03% MgSO4 7H2O, 0.03% K2HPO4, 0.02% yeast extract, and 1% (w/v) glass wool) were sterilized by autoclaving at 120  C for 30 min. Sterile nematode egg (150 eggs/ml) as the main source of N and C and seven agar plugs (5 mm diameter) taken from the edges of 34 days-old colonies grown on PDA (seven plugs of PDA medium without fungus as control) and were used to inoculate the asks and these were incubated at 30  C in dark for 9 days and 1 day intervals. Culture medium was ltered through a Whattman Paper No. 3 lter followed by ltration through 0.2 mm Millipore membranes. The ltrate obtained was analyzed for chitinase activity as described above. All experiments were conducted in greenhouse and Laboratory Conditions, in department of plant pathology, University college of Abureihan, University of Tehran, in the years of 2006 and 2007. 2.10. Statistics Growing, inoculation and sampling of plants were done in two independent experiments. Results were tested with SPSS version 11 for windows (SPSS Inc., Chicago, IL) by one-way analysis of variance (ANOVA). Signicantly differences between treatment means were separated by using T test at the P < 0.05 level. 3. Results 3.1. Effect of various concentrations of T. harzianum BI on nematode infection Tomato plants from nematode J2-infested soils treated with fungus exhibited a signicant reduction in disease level (number of galls per plant), number of egg masses per plant and number of eggs per individual egg mass (Fig. 1ac), (P 005). There was signicant reduction in number of eggs per egg mass in comparison to their controls in all concentrations. Concentrations more than 107 spores/ml exhibited no signicant difference in all parameters in comparison to 106 spores/ml.

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Fig. 1. a: Effect of various concentrations of T. harzianum BI spore on M. javanica infection (No. of gall/plant) in tomato (var. Roma VF) (Dark columns), sterile distilled water (Light columns) as control. Each value represents the mean of ten replicates from two paralleled experiments. The bars correspond to the standard error. b: Effect of various concentrations of T. harzianum BI spore on M. javanica infection (No. of egg mass/plant) in tomato (var. Roma VF), (Dark columns), sterile distilled water (Light columns) as control. Each value represents the mean of ten replicates from two paralleled experiments. The bars correspond to the standard error. c: Effect of various concentrations of T. harzianum BI spore on M. javanica infection (No. of egg/individual egg mass) in tomato (var. Roma VF) (Dark columns), sterile distilled water (Light columns) as control. Each value represents the mean of ten replicates from two paralleled experiments. The bars correspond to the standard error.

3.2. Effect of T. harzianum BI on nematode egg hatching Under in vitro condition, T. harzianum BI showed signicantly effect on nematode egg hatching. Whereas maximum of egg hatching percentage in control was at 3rd day, in treatments was at 6th day and was more or less 20% lower than their controls. So, T. harzianum BI not only reduced nematode egg production (as inoculum for second generation) but also caused egg weakness and mortality (Fig. 2).

3.3. PAL, POX and PPO enzymes activities in roots inoculated with T. harzianum BI and Meloidogyne javanica Inoculation of tomato roots with T. harzianum BI signicantly increased PAL, POX and PPO enzyme activities compared to control (inoculated with nematode). Maximum (peak) level of enzyme

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Egg hatching (%)

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time (day)
inoculated with T. harzianum + M. javanica control (inoculated with M. javanica)
Fig. 4. Peroxidase specic activity in tomato roots (var. Roma VF) inoculated with T. harzianum BI and Meloidogyne javanica. Each value represents the mean of ten replicates from two paralleled experiments. The bars correspond to the standard error.

Time (day)
Inoculated with T. harzianum Control (inoculated with dd/water)
Fig. 2. Effect of T. harzianum BI (106 spores/ml) on nematode eggs hatching(percentage). Each value represents the mean of ten replicates from two paralleled experiments. The bars correspond to the standard error.

activities were at 6, 5 and 5 days after inoculation, respectively, and then slowly decreased (Figs. 35). 3.4. Chitinase production and activity Wheat bran, colloidal chitin and nematode eggs induce the growth and chitinase production. Results showed that in liquid minimal medium supplemented with colloidal chitin, the maximum chitinase (1.15 U/min per ml) production per day was observed at 4th day, but enzyme production at the same level was continued until 5 days after incubation and then started to decrease. Activities between 0.1 and 1.2 U/min ml were found among these experiments. In liquid minimal medium supplemented with nematode eggs, the maximum chitinase (0.85 U/min per ml) production per day was observed at 4th day, but enzyme production at the same level was continued until 7 days after incubation and then started to decrease (Fig. 6). 4. Discussion Our results indicate that T. harzianum BI have signicant potential as biocontrol agent against the root-knot nematode M. javanica in greenhouse experiment. Inoculation of tomatoes with T. harzianum can signicantly reduce population of this nematode and disease severity. Suitable rate of T. harzianum BI for suppressing nematode activities such as nematode infection, egg mass

Microgr. trans-cinnamic acid

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production and number of eggs per egg mass was observed in 106 spores/ml concentration. Results also showed that T. harzianum BI signicantly increased PAL, POX and PPO activities compared to control and this suggest that T. harzianum BI can induce such defense enzymes and probably other defense compounds leading to systemic resistance in plants. Several studies have shown that root colonization by T. harzianum strains increased level of defenserelated plant enzymes including various peroxidase, chitinase, b-1,3-glucanases, lipoxigenase and phenylalanine ammonia lyase (Howell et al., 2000; Yedidia et al., 1999; Evans et al., 2003). Direct parasitism of eggs and larva through the increase in chitinase and protease activities, which would be indicators of eggs infection capability, is another mechanism of T. harzianum against this nematode. Sharon et al. (2001) showed direct parasitism of T. harzianum (T-203) on M. javanica under in vitro condition. They showed extracellular protease enzyme secreted by the fungus, but the percentage of direct parasitism was very low. Because of T. harzianum takes place in the soil, out of plant roots or in the cortex tissue (Larito et al., 1993; Cherif and Benhamou, 1990) and has no direct relationship with nematode in the host tissue, so an inducedresistance cascade can probably be excluded or fungal metabolites with anti-nematode activity. Our results suggest that T. harzianum BI caused direct and indirect effect on nematode eggs, juveniles and inducing resistance in plant, and it can reduce nematode penetration, nematode feeding and egg hatching. Chitinases, which have been assumed to be required for hyphal growth (Takaya and Yamazaki, 1998), are a kind of inducible enzyme catalyzing chitin that is one of the important components of fungal cell wall, nematode and insect eggshell and insect cuticle. Role of chitinase in infecting nematode eggs were reported in Paecilomyces lilacinus and Pochonia spp. isolated from infected

changes in A(515nm)/ min/mg protein

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T. harzianum + M. javanica inoculated Control (M. javanica inoculated)
Fig. 3. Phenylalanine ammonia lyase specic activity in tomato roots (var. Roma VF) as release of trans-cinnamic acid from phenylalanine, inoculated with T. harzianum BI and Meloidogyne javanica. Each value represents the mean of ten replicates from two paralleled experiments. The bars correspond to the standard error.

0 1 2 3 4 5 6 7

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inoculated with T. harzianum + M. javanica control (inoculated with M. javanica)
Fig. 5. Polyphenol oxidase specic activity in tomato roots (var. Roma VF) inoculated with T. harzianum BI and Meloidogyne javanica. Each value represents the mean of ten replicates from two paralleled experiments. The bars correspond to the standard error.

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Chitinase activity (U/min ml)

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Liquid medium+colloidal chitin Liquid medium+nematode egg

Fig. 6. Chitinase activity in culture ltrates of T. harzianum BI growing in liquid medium induced by colloidal chitin (Dark columns), Nematode eggs (Light columns). Each value represents the mean of ve replicates from two paralleled experiments. The bars correspond to the standard error.

nematode eggs (Khan et al., 2003; Tikhanov et al., 2002). We showed that the proportion of infected nematode eggs increased concurrently with enhancement of chitinase activity. Because of the proteinaceous and chitinous nature of nematode eggshell, T. harzianum like other nematophagous fungi must be able to produce extracellular chitinase and protease enzymes. It is also possible that other lytic enzymes be involved in egg penetration. The ratio of the chitinase and total protein content of T. harzianum medium extract, indicate that other extracellular protein nature have been induced by colloidal chitin which may be involved in nematode egg penetration. 4.1. Conclusion The results of our study suggest that T. harzianum BI can be used as a bioagent for the sustainable management of root knot nematodes on selected crops and in particular circumstances. Future studies on nematode biocontrol will focus on the effect of biocontrol agent on other plant defense mechanisms and extracellular enzymes. References
Chen, C., Belanger, R., Benhamou, N., Paulitz, T.C., 2000. Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Phytium aphanidermatum. Physiological and Molecular Plant Pathology 56, 1323. Cherif, M., Benhamou, N., 1990. Cytochemical aspect of chitin breakdown during the parasitic action of a Trichoderma sp. on Fusarium oxysporum f.sp. lycopersici. Phytopathology 80, 14061414. Chet, I., 1987. Trichoderma-application, mode of action and potential as biocontrol agent of soilborn plant pathogenic fungi. In: Chet, I. (Ed.), Innovative Approaches to Plant Disease Control. John Wiley & Sons, New York, pp. 137160. Chet, I., Harman, G.E., Barker, R., 1981. Trichoderma hamatum its hyphal interaction with Rhizoctonia solani and Pythium spp. Microbiology and Ecology 7, 2938. Eisenback, J.D., 1985. Detailed morphology and anatomy of second-stage juveniles, males, and females of the genus Meloidogyne (root-knot nematode). In: Sasser, J.N., Carter, C.C. (Eds.), An Advanced Treatise on Meloidogyne, Vol. 1. Biology and Control, North Carolina state Univ., Graphics, Raleigh. Elad, I., Chet, P., Katan, J., 1980. Trichoderma harzianum: a biological agent effective against Sclerotinia rolfsii and Rhizoctonia solani. Phytopathology 70, 119121. Elad, I., Chet, P., Henis, Y., 1983. Parasitism of Trichoderma spp. on Rhizoctonia solani and Sclerotinia rolfsii scanning electron microscopy and uorescence microscopy. Phytopathology 73, 8588. Evans, H.C., Holmes, K.A., Thomas, K.A., 2003. Mycobiota of an indigenous Theobroma species (Sterculiaceae) in Ecuador: assessing its potential for biological control of cocoa diseases. Mycology Progress 2, 149160. Howell, C.R., Hanson, L.E., Stipanovic, R.D., Puckhaber, L.S., 2000. Induction of terpenoid synthesis in cotton roots and control of Rhizoctonia solani by seed treatment with Trichoderma virens. Phytopathology 90, 248252.

Hussey, R.S., Barker, K.R., 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Reporter 75, 10251028. Khan, A., Williams, K., Molloy, M.P., Nevalainenc, H., 2003. Purication and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels. Protein Expression and Purication 32, 210220. Kim, D.G., Riggs, R.D., Kim, K.S., 1992. Ultrastructure of Heterodera glycines parasitised by Arkansas Fungus 18. Phytopathology 82, 429433. Larito, M., Harman, G.E., Hayes, C.K., Broadway, R.M., Di Pietro, A., 1993. Chitinolytic enzymes produced by Trichoderma harzianum: antifungal activity of puried endochitinase and chitobiosidase. Phytopathology 83, 302307. Liu, T.G., 2002. Induced resistance of tobacco to TMV with non-fungicidal chemical compounds. Journal of Northeast Agricultural University 33, 129133. Lopez-Llorca, L.V., Duncan, G.H., 1988. A study of fungal endoparasitism of the cereal cyst nematode Heterodera avenae by scanning electron microscopy. Canadian Journal of Microbiology 34, 613619. Lysek, H., Krajci, D., 1987. Penetration of ovicidal fungus Verticillium chlamydosporium through the Ascaris lumbricoides egg-shells. Parasitology 34, 5760. Mai, W.F., Abawi, G.S., 1987. Interactions among root-knot nematodes and fusarium wilt fungi on host plants. Annual Review. Phytopathology 25, 317338. Meyer, S.L.F., Wergin, W.P., 1998. Colonisation of soybean cyst nematode females, cysts and gelatinous matrices by the fungus Verticillium lecanii. Journal of Nematology 30, 436450. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31, 426429. Parvatha Reddy, P., Rao, M.S., Nagesh, M., 1996. Management of citrus nematode, Tylenchulus semipenetrans, by integration of Trichoderma harzianum with oil cakes. Nematologia Mediterrannea 24, 265267. Rao, M.S., Reddy, P.P., Nagesh, M., 1998. Evaluation of plant based formulations of Trichoderma harzianum for management of Meloidogyne incognita on egg plant. Nematologia Mediterrannea 26, 5962. Seifullah, P., Thomas, B.J., 1996. Studies on the parasitism of Globodera rostochiensis by Trichoderma horzianum using low temperature scanning electron microscopy. Afro-Asian Journal of Nematology 6, 117122. Sharon, E., Bar-Eyal, M., Chet, I., Herrera-Estrella, A., Kleifeld, O., Spiegel, Y., 2001. Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum. Phytopathology 91, 687693. Suarez, B., Rey, M., Castillo, P., 2004. Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Applied Microbiology and Biotechnology 65, 4655. Takaya, N., Yamazaki, D., 1998. Intracellular chitinase gene from Rhizopus oligosporus: molecular cloning and characterization. Microbiology 144, 26472654. Tikhonov, V.E., Lopez-Llorca, L.V., Salinas, J., Jansson, H.B., 2002. Purication and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium. Fungal Genetic and Biology 35, 6778. Wharton, D., 1980. Nematode eggshells. Parasitology 81, 447463. Windham, G.L., Windham, M.T., Williams, W.P., 1986. Effect of Trichoderma spp. On maize growth and Meloidogyne arenaria reproduction. Plant Disease Reporter 73, 493494. Yedidia, I., Benhamou, N., Chet, I., 1999. Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Applied Environmental Microbiology 65, 10611070.