Abstract: The effect of Trichoderma species in control of root- rot fungus, Fusarium solani, root-knot nematode, Meloidogyne javanica or reniform nematode, Rotylenchulus reniformis disease complex and on growth of sunflower plant was studied under greenhouse conditions. Treating two weeks old sunflower seedlings cv. Giza 1 with Trichoderma harzianum, T. viride, T. koningii, T. reesei or T. hamatum gave highly significant effect in control of fusarium-rot disease incidence and nematodes infection on sunflower roots. Infection of F. solani was highly increased in M. javanica infested soil than R. reniformis. Treatments of Trichoderma species led to decrease Fusarium cfu counts in soil infested with either M. javanica or R. reniformis and also significantly improved the plant growth parameters. T. hamatum, T. harzianum and T. koningii gave the greatest reduction in disease incidence caused by Fusarium, M. javanica or R. reniformis infestation. Generally, there was highly significant reduction (P< 0.01) in the number of fusarium-wilt disease and nematode population and increases in plant growth parameter of sunflower when treated with Trichoderma species.
Introduction
Sunflower plants, Halianthus annus L. attack by a number of infections microorganisms mostly fungi, bacteria and nematodes, which significantly reduce the yield quantity and quality (Amin and Youssef, 1997).
Root-rot diseases are still the most important diseases-affecting sunflowers. Fusarium solani is the main pathogen of these diseases (Bhutta et al., 1997). Fusarium-nematode interaction are known to decrease the quantity and quality of major world crops included tomato (Stephan et al., 1996), cotton (Colyer et al., 1997), vegetables (Ghaffar, 1995) and soybean (Mousa, 1994). Because of hazards involved in the use of pesticides, biological control of plant disease has received increasing attention as a promising supplement and now capturing the imagination of plant parasitic nematodes and plant pathogen (Amin, 1999). Of the various bio-agents, fungi of Trichoderma species have been known to suppress many soils borne fungi and nematode diseases under greenhouse and field conditions. T. harzianum, T. hamatum has been found to antagonize fungal plant pathogen and parasitic nematodes (Siddiqui et al., 1999).
The present study aimed to study the effect of Trichoderma species in control of F. solani-M. javanica or R. reniformis disease complex and on plant growth of sunflower seedlings.
Materials and Methods
The fungus, Fusarium solani (Mart.) was isolated from diseased sunflower plants and cultured on Potato Dextrose Agar (PDA). Spore suspension of the fungi was harvested and adjusted to 3×103 spores /mi. Seeds of sunflower (Hellanthus annus L.) cv. Giza 1 were sown in 15 cm diam., pots filled with one kg sandy loam soil (1:1 v/v) free of plant pathogen and parasitic nematodes. After germination (about two weeks), the plants in each pot were thinned to one plant/pot. The pots were divided to two groups, one for root-knot nematode, Meloidogyne javanica (Treub) Chitwood, 1949 and the other group for reniform nematode, Rotylenchulus reniformis. Two days before nematodes inoculation pots were infested with 3×103 colony forming units (cfu) g–1 soil of mixed population of F. solani , as assessed soil dilution technique. Ten ml of spores suspension was pipetted around seedling roots in pots. Each pot was inoculated with 2000 infective stages of either M. javanica (eggs and juveniles) or R. reniformis (juveniles and unswollen females). Lin-inoculated - untreated four pots were served as check plants and other four pots inoculated -untreated served as check nematode. Two days after nematode inoculation seedlings were treated with conidia suspension of Trichoderma harzianum (Raifi), T. viride (Pers.), T. koningii (qudem), T. reesei (Simmons) and T. hamatum (Bon.) at a rate of 10 ml (5×105 cfu/g soil /seedling). There were four replicates of each treatment. The pots were arranged in a completely randomized design in a greenhouse at 30 ± 5°C. The pots were watered daily. After nine weeks of nematodes inoculation, sunflower plants were carefully uprooted and nematodes in roots were counted. Disease incidence caused by F. solar), during growth period was recorded. The incidence of Fusarium pathogen and Trichoderma species in soil rhizosphere was recorded as cfu/g soil and population counts respectively. The number of galls, females and egg-masses as compare to untreated pots was calculated for root-knot nematode and the number of females and egg-masses for R. reniformis. Length and weight of shoots and roots and flowering disc weight were recorded. Data was statistically analyzed using New Least Significant Difference (New LSD).
Results
The effect of Trichoderma species in control of fusariumrot in infested soil with nematodes on sunflower data in (Table 1) indicated that, percentages of root-rot disease caused by F. solani alone were 48%, while it was 88 and 68.6% in case of presence of M. javanica or R. reniformis respectively after 9 weeks of inoculation.
| Table 1: | Effect of Trichoderma species in control of fusarium root-rot in infested soil with Meloidogyne javanica and Rotylenchulus reniformis on sunflower |
| Table 2: | Effect of Trichoderma species in control of Meloidogyne javanica and Rotylenchulus reniformis in infested soil with Fusarium solani on sunflower |
| Table 3: | Effect of Trichoderma species in the growth of Fusarium solani in soil rhizophere of sunflower grown in infested soil with Meloidogyne javanica or Royenchulus reniformis |
Data also showed that, application of antagonistic microorganisms Trichoderma species resulted in decrease of Fusarium infection. A complete control of fusarium root-rot infection was observed in case of T. hamatum treatment. T. harzianum was highly effective in controlling fusarium root-rot , while T. viride and T. reesei were less effective. Data also showed that, the effect of Trichoderma species on fungi-nematodes interaction.
| Table 4: | Populatin Count of Trichoderma species in soil rhizosphere of sunflower grown in infested soil with Fusarium sawn', Meloidogyne javanica or Rotylenchulus reniformis |
| Table 5: | Effect of Trichoderma species in the growth of sunflower in infested soil with Fusarium solani and/or Rotylenchulus reniformis |
It means that the percentage of root-rot was great decreased in soil infested with either M. javanica or R. reniformis. T. hamatum and T. harzianum showed more effective in reducing disease incidence in the soil infested with M. javanica or R. reniformis. While, T. reesei showed less effective in reducing the pathogen incidence.
Use of Trichoderma species alone or in combination with F. solani gave a highly significant (p<0.01) reduction of female and egg-masses of root-knot and reniform nematode (Table 2). Great reduction in root-knot nematode females (more than 50%) was recorded where T. hamatum, T. harzianum and T. koningii were used alone followed by T. viride and T. koningii when treated in combination with F. solani. The same trend was noticed in the number of galls and immature stages in roots.
Highly significant reductions were observed on female reduction of R. reniformis (Table 2) where T. harzianum and T. koningii were used followed by T. viride and T. hamatum when used alone. Whereas, Trichoderma species treated with F. solani, a highly significant reduction in reniform nematode, was observed in case of T. hamatum and T. harzianum. It also noticed that, use of F. solani with either M. javanica or R. reniformis gave 55.1 and 43.5% female reduction respectively.
| Table 6: | Effect of Trichoderma species in the growth of sunflower in infested soil with Fusarium solani and / or Meloidogyne javanica |
Due to the effect of Trichoderma species on Fusarium population treated in M. javanica or R. reniformis infested soil. Population density of F. solani was increased in soil rhizospere until 9 weeks of plant growth by 9.4×104 cfu/g soil (Table 3). Also, data showed that population density of F. solani was greatly increased in soil infested with either M. javanica or R. reniformis. Inoculation soil with M. javanica increased F. solani population counts in soil than did reniform nematode, R. reniformis. Treatment of Trichoderma species recorded minimum population density of F. solani. T. hamatum and T. harzianum gave highly reduction in F. solani by 0.6×104 and 1.3×104 cfu/g soil. Moreover, these antagonisms gave greatly effect in reducing Fusarium count in infested soil with either M. javanica or R. reniformis.
Concerning Trichoderma population in fungal-nematodes infested soil, data indicated that population density of Trichoderma species was increased in soil rhizospere until 9 weeks of plant growth (Table 4). T. hamatum and T. harzianum counts were higher in soil rhizosphere during 9 weeks compare with other Trichoderma species. Moreover, T. koningii colonization was multiplied three times from one to 9 weeks in soil rhizosphere. The infested soil with both Fusarium and nematodes, the result, indicated that increased in population of Trichoderma in soil rhizosphere. The highest population counts was obtained in infested soil with M. javanica alone or with F. solani. Also, the addition of T. harzianum or T. hamatum in infested soil with F. solani and M. javanica or R. reniformis yielded the highest population in soil rhizosphere.
Due to the effectiveness of Trichoderma species on plant growth in infested soil with fungal-nematodes disease complex, results present in (Table 5, 6) revealed that, soil infested with F. solani significantly (p<0.05) decrease plant height and weight. Also, soil infested with F. solani
when mixed with M. javanica led to lowest value of plant length, weight and flower disc weight. But the treatment of Trichoderma species alone or in combination with root-rot fungus or nematodes was significantly increased plant growth parameters. T. hamatum, T. harzianum and T. koningii gave the best result in plant growth parameter. While, a slight increase was observed in T. reesei.
Discussion
In the present study, data showed that F. solani treated with root-knot or reniform nematodes increased root-rot disease incidence to a great extend than plant infested with the pathogen alone (Table 1). Also, plant infection was greatly increased in soil infested with M. javanica than R. reniformis. This result can be explained that nematodes predispose the plant pathogen invasion, as has been reported (Siddiqui et al., 1999). The interaction between the pathogen fungi and the endo-parasitic nematode, M. javanica gave a great damage to plant growth than the semi-endo parasite nematode, R. reniformis (Mousa, 1994). The addition of Trichoderma species in controlling disease complex caused by root-rot and root-knot or reniform nematodes, data indicated that, significant reduction in the pathogen and nematodes population and increased plant growth parameter. Several species of Trichoderma have been reported to suppress soil borne disease fungi included Fusarium and parasitic nematodes (Siddiqui et al., 1999). Trichoderma species are known to produce other secondary metabolites such as enzymes (Di Pietro, 1995). These enzymes in biocontrol can often assigned in both mechanism parasitism and antibiosis. In particular cell wall degrading enzymes such as chitinase, B-1,3-glucanases and cellulase, but only important features of micoparasites for colonization of their pathogens, but exhibit considerable antifungal activity on their one. Chitinase enzymes have been consider important in the biocontrol pathogenic fungi at low concentration because of their ability to degrade fungal cell walls of which a major component is chitin (Lorito et al., 1993). Also, most plant parasitic nematode eggs have a second layer is a thick chitins layer of eggshell, which is secreted by the egg (Lee, 1965). Recently, has been achieved for various chitinases, B-1, Bglucanase and cellulase from biocontrol fungi, T. harzianum, T. hamatum and T. koningii (Cotes et al., 1996). Whereas cellulase , glucanase and glucosidase are main enzymes produce by T. viride and T. reesei (Kanotra and Mathur, 1995). Lorito et al. (1993) found that two purified chitinase from T. harzianum inhibited F. solani growth at 30 mg/ml concentration. Besides parasitism of root-knot nematode it also hypothesized that the production of nematicidal compounds by Trichoderma species directly affected the nematode or made rootless attractive which might have resulted the reduction in the nematode population. These, results confirm the report of Siddiqui et al. (1999). Moreover, many authors recorded that, Trichoderma species as nematophagous fungi on eggs, larvae and females of cyst nematodes (Meyer et al., 1990).
In this study, treatment of Trichoderma species successfully reduced fungal-nematodes disease complex and increased plant growth parameter. In conclusion, the present study demonstrated the important of using biocontrol agents in reducing the damages of fungi-nematodes disease complex in order to manage the disease on economic crops.