Abstract: Investigation was undertaken to screen the potential native isolate of Trichoderma viride for bio suppression of corm rot pathogen complex, as Trichoderma viride are the most successful and widely used biocontrol agents. Taking the advantage and constraints of Trichoderma viride into consideration, efforts were made to encourage the native isolate against corm rot pathogens. Nine isolates of Trichoderma viride namely TK1, TK3, TK4, TK6, TK8, TK9, TK10, TK11 and TK15 were isolated from soils of different orchard plantations of Kashmir valley on modified Trichoderma Specific Medium (TSM). The isolates were studied for their cultural, morphometric characters and antagonistic potential against six newly recorded major fungal pathogens of saffron viz. sterile Basidiomycetes fungus, Rhizoctonia solani, Phytophthora sp., Fusarium oxysporum f. sp. gladioli, F. oxysporum and F. solani individually on Potato Dextrose Agar, the culture morphology of all the isolates was found to be similar. The isolate TK1, TK3, TK4, TK8, TK9, TK11 and TK15, were found fully overgrown on all corm rot Pathogens of saffron, where as the isolates TK13 failed to inhibit the Phytophthora sp. Efforts are onto evaluate the performance of promising isolate in field by soil and seed application methods.
INTRODUCTION
During last fifteen years, saffron crop has been affected by severe rotting caused by sterile Basidiomycetes fungus, Rhizoctonia solani, Fusarium f. sp. gladioli, Fusarium oxysporum and Fusarium solani (Mir and Devi, 2004) and reduction in yield has been reported. In 1980 the yield per hectare was 5.66 kg ha-1 (Mir, 1992) and now its present productivity is 1.53 kg ha-1 (Anonymous, 2009) which is the lowest in the world.
In recent years, attempts were also made to use a consortium of biocontrol agents to get persistent control of plant pathogens (Chaube and Sharma, 2002). Biological control, therefore, holds promise as a strategy for disease management and it is environment friendly too. Antagonistic fungi - 38 - especially Trichoderma spp. has been widely used against a number of phytopathogens (Rini and Sulochana, 2006) and parasitized hyphae of other fungi in vitro and brought about several morphological changes during destruction (Anitha and Murugesan, 2001). Screening of potential Trichoderma strains was done by Bandopadhyay et al. (2003) against major root pathogens and it was found that more or less all the strains checked the growth of the pathogen and stimulate plant defensive mechanisms (Hanson and Howell, 2004; Harman et al., 2004; Yadav et al., 2011).
Trichoderma harzianum is one efficient biocontrol that is commercially produced to prevent development of several soil pathogenic fungi (Jegathambigai et al., 2009). Biocontrol is an important approach for plant disease management under changing food habits and commercialization of agriculture (Manczinger et al., 2002).
Therefore, keeping in view medicinal importance and to remove the pesticidal residue of such valuable medicinal crop, the present study was undertaken for screening of several local antagonistic isolates of T. viride, obtained from different orchards of Kashmir valley, under in vitro conditions against few pathogens sterile Basidiomycetes fungus, Rhizoctonia solani, Fusarium oxysporum f. sp., gladioli, Fusarium gladioli, Fusarium solani, Phytophthora sp. causing corm rot syndrome of saffron.
MATERIALS AND METHODS
Collection of pathogen: Four pathogenic isolates namely sterile Basidiomycetes fungus, Rhizoctonia solani, Fusarium oxysporum f. sp. gladioli, Fusarium gladioli, Fusarium solani and Phytophthora sp. were isolated, from infected corms from Kashmir valley from saffron growing area. The pathogens were maintained on PDA medium at 4°C.
Isolation of Trichoderma spp. (TK1, TK3, TK4, TK6, TK8, TK9, TK10, TK11 and TK15) was done from randomly collected soils from different vegetable fields and orchards of Kashmir valley by dilution plate technique using Trichoderma specific medium TSM (Elad et al., 1981) modified by Saha and Pan (1997).
Antagonistic potential of Trichoderma viride isolates on saffron pathogens: The antagonistic properties of fifteen isolates of Trichoderma viride were tested on PDA by dual culture plate technique. Paired cultures were observed for a total of 12 days before being discarded. All the ratings were done after contact between pathogen and the antagonist using a Bells scale (Bell et al., 1982) which is slightly modified (Class 1-7) as follows.
| • | S1=The pathogen and the antagonism locked at the point of contact |
| • | S2=The antagonism starts overgrowth on pathogen. |
| • | S3=The pathogens starts overgrowth on mycoparasite |
| • | S4=The antagonist overgrew at least 15% of pathogen |
| • | S5=The antagonist overgrew of least 30% of pathogen |
| • | S6=The antagonist overgrew at least 60% of pathogen |
| • | S7=The antagonist completely overgrew the pathogen (100% overgrowth) |
RESULTS AND DISCUSSION
Identity of isolates of Trichoderma spp.: In general, colony morphology of all the isolates was more or less similar showing sparse to thin colony mycelial mass with whitish border in some cases. Sporulation started after 48h of incubation at 28±1°C for all the isolates (Table 2).
Micrometric measurements of Trichoderma viride (Table 1) showed that phialospore length ranged between 2.98-5.52 μm and, breadth ranged from 2.71-4.6 μm and phialides length 9.22-12.56 and breadth 1.3-2.5. These characteristics, particularly the trifid phialophore with short phialides clearly resembled the identical characters of Trichoderma viride (Rifai, 1969).
Antagonistic potential of Trichoderma viride isolates against corm rot pathogens of saffron.
Phytophthora sp.: The results showed that isolate TK1, TK8, TK4 and TK11 were antagonistic to Phytopthora by totally overgrowing the pathogen within seven, nine and eleven day respectively. Isolate TK10, TK15 and TK3, TK6 and TK9 were antagonistic to Phytopthora overgrowing 75, 45 and 60%, respectively.
F. oxysporum f. sp. gladioli: The results showed that isolate TK1, TK4, TK6, TK8, TK9 and TK11 were antagonistic to F. oxysporum f. sp., gladioli by totally overgrowing the pathogen within 8 to 12 days. Isolates TK3 and TK10, were overgrowing the pathogen 90 and 45%, respectively.
Sterile Basidiomycetes fungus: The results against Basidiomycetes fungus showed that five isolates TK1, TK8, TK15 and TK9 and TK6, totally overgrowing within nine, eight and 12 day, respectively. The remaining isolate TK11, TK4, TK3 and TK10, overgrew 90, 75, 45 and 15%, respectively.
| Table 1: | Micrometric measurement of phialospores, phialides and chlamydospores of isolate |
| Table 2: | Colony characters of Trichoderma viride isolates |
| Table 3: | Hyperparasitic potential of T. viride wild isolates on fungal pathogens of saffron |
| D: Days before contact, R: Rating, **: An average of five individual observation. *: The numerical value represents the days required for attaining S1 to S7 stage of modified Bell’s scale | |
Rhizoctonia solani: The results showed that all isolates were antagonistic to Rhizoctonia solani by totally overgrowing the pathogen with six to nine day except isolate TK15 it overgrew only 75% even after day.
F. oxysporum: The results showed that isolate TK1, TK3, TK4, TK8 and TK15 were antagonistic to F. oxysporum by totally overgrowing the pathogen within 6 to 9 days. Isolates TK9, TK10 and TK11 did not progress beyond 30% ever after day. The remaining isolate TK6 totally fails to overgrow the host pathogen even upto 12 days of inoculation inspite of attaining the point of contact of the third day.
F. solani: The result shows that five isolates TK1, TK3, TK4, TK8 and TK15 were highly antagonistic to Fusarium solani, totally overgrowing the pathogen within 6 to 11 days. Isolates TK9 and TK11 were overgrew the pathogen 30% whereas TK10 15% and TK6 failed to overgrew the host pathogen even after 12 days of inoculation, in spite of attaining the point of contact on the 4th day of inoculation.
The overview of the results (Table 3) showed that the isolates TK1, TK3, TK4, TK8, TK9, TK11 and TK15, were found fully overgrown on all corm rot Pathogens of saffron, where as the isolates TK13 failed to inhibit the Phytophthora sp. To identify then, isolates of Trichoderma spp. have been listed in the tables that reached class-I (S7) stage within 6-11 days of inoculation. However, based on this information the antagonistic Trichoderma viride did not allow an early selection of isolates, as the variability in the antagonistic characteristic within the isolate and isolate-pathogen interaction was very high.
The above observations established the fact that Trichoderma isolates existing in their natural conditions in natural ecosystem do differ with respect to their growth and antagonistic potential. Similarly Li et al. (2001) studied eighteen isolates of Trichoderma spp. of these isolates, TR13 showed greatest antagonists effects against Rhizoctonia solani. Several research papers that have appeared in the literature do reveal the fact that various species and isolates of fungal antagonist Trichoderma suppress mycelial growth, reduce root rots, increase plant growth and induce resistance in various crops with which Sclerotium rolfsii (Tian et al., 2001; Das and Dutta, 2002; Palomar et al., 2002), Rhizoctonia solani (Li et al., 2001; Burgess and Hepworth, 1996; Zapata et al., 2001; Ziedan and Mahmoud, 2002; Gaikwad and Nimbalkar, 2003; Yossen et al., 2003; Fravel and Lewis, 2004; Hajlaoui et al., 2001; Singh et al., 2003; Huang and Erickson, 2004; Salehpour et al., 2005) are associated. It is clear that the success of bioagents introduces in soil does not guarantee the control the target pathogen(s) because plants, physicochemical and biological factors of soil affect establishment, proliferation and antagonistic activities of the introduced bioagents. It is necessary that, identified antagonist efficiency against foot, root rot and damping off should be investigatited and examined in vivo conditions also, the results of such survey would be reported by the authers in near future (Shaigan et al., 2008).
It is in this context that to ensure success of introduced bioagents, they should be isolated for the local areas where they exit. Since, they have already faced various processes of evaluation, their application would be feasible and result oriented. We reviewed the literature to find out that have others worked on these aspects. Literature analysis revealed that comparative studies have been done with various species (Kucuk and Kivanen, 2003; Chang et al., 2006) studied Trichoderma isolates from different soil sampled and grouped them according to their antagonistic potential and chitin utilization.
CONCLUSION
The overgrowth by the antagonist under in vitro conditions may be good criteria of selecting an isolate shows good performance under in vitro conditions. The trend of the results also indicated that there was not only variability amongst the isolates of Trichoderma viride with differential degree of a ntagonism towards a single pathogen but also towards different pathogens.
The results of the study are the pointer to the fact that the antagonists should be isolated from different systems and locations to create a huge genetic pool and tested for their antagonistic potential against variety of the targeted plant pathogens and recommended specifically for different locations and systems. The present study clearly indicates the high potential of biocontrol agent, Trichoderma viride isolates for different plant pathogens. Efforts are onto evaluate the performance of promising isolate in field by soil and seed application methods.
ACKNOWLEDGMENTS
I am greatly thankful to Department of Science and Technology, New Delhi for providing fully financial assistance under Young Scientist Scheme to carry out the work. I am grateful to DADA for his technical help during the research equally I wish to thank my advisor Prof. L.S. Devi and my dear teachers Prof. P. Gupta, Prof. P. Williams, Prof. Shafat Ahmad, Dr. V. Manoj Kumar, Prof. G. Abraham and Prof. Ram Lal and also appreciate Mithai Lal jee, Maqsood sahib, Amrit Lal jee, Subash jee, Gyan Chand jee, Rajkaran jee and Kanaya lal jee, AAI-DU, Allahabad, U.P. India, for their support through out the research period.