Abstract: The efficacy of Trichoderma viride, T. harzianum, T. hamatum, T. koningii and T. pseudokoningii was tested for the control of Meloidogyne javanica, root knot nematode in okra and mungbean. Culture filtrates of Trichoderma spp., significantly reduced egg hatching and showed nematicidal activity by killing second stage juveniles of M. javanica. Soil application with conidial suspension of T. harzianum significantly reduced nematode population densities and root knot development in okra and mungbean. Apart from suppressing root knot nematode, T. harzianum also elevated plant height and fresh shoot weight of both okra and munhbean.
Introduction
Fungi and plant parasitic nematodes being common inhabitants of crop rhizosphere show synergetic relation (Inagaki and Powell, 1969; Jorgenson, 1970). Since they occur together in the rhizpsphere, the toxic metabolites naturally produced by microorganisms may be responsible for keeping low level of nematode populations. Filtrates of nematophagous or entomophagous fungi have been found to be active against free living nematodes (Alam et al., 1973; Cayrol et al., 1989; Ali, 1990).
Among the fungal antagonists, various species of Trichoderma have shown promising results in the control of soilborne plant pathogens (Elad et al., 1983). Use of Trichoderma species in the biological control of soilborne root-infecting fungi like Sclerotium rolfsii Sacc., Macrophomina phaseolina (Tassi) Goid., Rhizoctonia solani Kühn and Fusarium spp., has also been reported (Chet et al., 1981; Elad et al., 1971; Bell et al., 1982; Wells et al., 1972; Ghaffar, 1992). Beside control of root-infecting fungi, T. harzianum has also been found to antagonize plant-parasitic nematodes (Saifullah, 1996a; Dos Santos et al., 1992). It shows good potential for the control of Globodera rostochiensis, G. pallida and M. javanica (Saifullah, 1996b; Saifullah and Thomas, 1996). Experiments were therefore carried out to examine the ability of Trichoderma spp., to control M. javanica, root-knot nematode in okra and mungbean.
Materials and Methods
Cultures of Trichoderma viridi, (Karachi University Culture Collection (KUCC-656), T. harzianum (KUCC-195), T. koningii (KUCC-427), T. pseudokoningii (KUCC-93) and T. hamatum (KUCC-29) were multiplied on potato dextrose agar (PDA) medium and were grown in Erlenmyer flasks containing Czapek’s Dox liquid medium. The pH of the medium was adjusted to 6.7 before autoclaving. A5-mm diam., disc from a 5-day old culture of the fungus was inoculated in each flask. The flasks were kept at 28±2 °C in an incubator and 2 weeks later the liquid was passed through Whatman No.1 filter paper several times to remove any spores. The solution obtained was stored in refrigerator before use. One ml of the spore free culture filtrate of Tricoderma spp., was transferred to watch glasses separately in which two hand picked medium sized egg masses were placed. Egg masses kept in 2 ml Czapek’s Dox liquid medium without the fungus served as control. After 48 h, the number of hatched juveniles were counted under a stereo microscope. The egg masses from culture filtrate were then transferred to sterile distilled water and their hatching in water was recorded to ascertain whether the egg masses kept in the filtrate had been permanently or temporarily inactivated. The emergence of juveniles was again recorded after 48 h.
To study the effects of Trichoderma spp., on mortality of M. javanica larvae, one ml of the culture was transferred into watch glasses. One ml juvenile suspension was added to each watch glass (at 30-45 juveniles/watch glass). Juveniles kept in 2 ml Czapek’s Dox liquid medium without the fungus served as control. Number of dead juveniles were counted after 24 and 48 h intervals. The nematodes were considered dead if they did not move when probed with a fine needle (Cayrol et al., 1989).
Sandy loam soil (pH 8.1) was filled in 8-cm diam., plastic pots (350 g/pot). The soil was excavated to a depth of 3 cm and a conidial suspension of Trichoderma spp., viz., T. viride (cfu 3.6 x 107 ml-1); T. harzianum (cfu 2.8x 07 ml-1); T. koningii (cfu 1.8x 07 ml-1) and T. pseudokoningii (cfu 3.6x107 ml-1) were drenched separately in each pot @ 25ml/pot. After treatment, 8 seeds of okra and mungbean were sown in each pot. After germination only four seedlings were maintained in each pot. There were three replicates of each treatment and pots were randomized on the green house bench of Soil-borne Diseases Research Laboratory, Department of Botany, University of Karachi. The soil in each pot was kept at 50% W.H.C. After one week of the seedling emergence, roots in each pot were inoculated with 2000 freshly hatched, second stage juveniles of M. javanica. Plants were uprooted 45 days after the addition of nematode and plant height, root length and fresh weight of shoot and root were recorded. Number of galls induced by M. javanica and nematode populations in soil and roots were recorded. The data were subjected to analysis of variance (ANOVA). Treatments means were compared using Duncan’s multiple range test (Sokal and Rolf, 1995).
Results and Discussion
Trichoderma species showed significant (p<0.05) inhibition in egg hatching and caused larval death of M. javanica. Maximum inhibition in egg hatching (44%) was recorded after treatment with culture filtrate of T. viride followed by T. harzianum which resulted in 40% inhibition in egg hatching as compared to untreated controls Table 1. Likewise culture filtrates of T. harzianum at 48 h caused 41% mortality of M. javanica compared to T. viride which produced 31% juveniles death (Table 2).
Soil drenches with conidial suspension of Trichoderma spp., significantly reduced root knot formation due to M. javanica (p<0.01) and root knot nematode population in soil (p<0.001) and root (p<0.05).
| Table 1: | Effects of culture filtrates of Trichoderma spp., on egg hatching of Meloidogyne javanica. |
| * After a 48 hour exposure period in culture filtrates, egg masses were transferred to distilled water. Data represent mean of three replicates; Means followed by the same letters in each column are not significantly different at p<0.05 according to Duncan’s Multiple Range Test. | |
| Table 2: | Effects of culture filtrates of Trichoderma spp., on mortality of Meloidogyne javanica. |
| Data represent mean of three replicates; Means followed by the same letters in each column are not significantly different at p<0.05 according to Duncan’s Multiple Range Test. | |
Soil application with T. harzianum resulted in the greatest reduction in nematode populations in soil (27% and 37%) and root (36% and 42%) and subsequent root knot disease severity (46% and 38%) in okra and mungbean respectively, as compared to controls (Table 3). Similarly T. harzianum significantly (p<0.05) increased plant height (51%) and >47%) and fresh weight of shoots (71% and 50%) of okra and mungbean respectively, in comparison with controls. The longer roots compared with untreated controls in both okra and mungbean were produced in treatments where T. hamatum was used whereas fresh weights of root did not differ significantly from those of controls in any of the treatments (Table 4).
Trichoderma spp., significantly reduced egg hatching, caused mortality of M. javanica juveniles and when incorporated in soils as drench significantly suppressed nematode populations in soil and root and subsequently reduced root knot development in both okra and mungbean. The production f antibiotics (Denis and Webster, 1971) and extra cellular lytic enzymes (Elad et al., 1982) by Trichoderma spp., are known to be involved in the antagonism. There are reports where use of T. harzianum significantly suppressed root knot disease in maize (Windhum et al., 1989). T. harzianum has also been found as an egg parasite of M. incognita race-3 killing 53% of eggs in vitro (Dos Santos et al., 1992). Khan et al. (1977) reported a significant control of M. incognita and Fusarium solani disease complex in papaya with T. harzianum.
| Table 3: | Effects of Trichoderma spp., on the development of root knot infection and nematode population in soil and root of okra and mungbean. |
| Table 4: | Effects of Trichoderma spp., on growth of okra and mungbean plants. |
Similarly Trichoderma spp. used individually or in combination with Pseudomonas aeruginosa, a pant growth promoting rhizobacterium, significantly controlled root rot-root knot disease complex in chili (Siddiqui et al., 1999). Further investigation is needed for the isolation and characterization of nematicidal compound (s) produced by Trichoderma spp.
Acknowledgments
This work was carried out under the grants of the University Grants Commission, which is sincerely acknowledged.