Factors Favoring Mycoparasitism of Sclerotinia Sclerotiorum by Trichoderma spp.

Transcription

1 Indian Res. J. Genet. & Biotech. 9(2) May: (2017) Factors Favoring Mycoparasitism of Sclerotinia Sclerotiorum by Trichoderma spp. Gopi Kishan *, Rahul Tiwari and Pratibha Sharma Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi ABSTRACT Trichoderma is a biocontrol agent which controls the pathogens by various mechanisms viz. hyper-parasitism, antibiosis, competition, systemic induced resistance etc. of which hyperparasitism is the most important mechanism employed by Trichoderma against several plant pathogens. An investigation on factors favoring mycoparasitism of S. sclerotiorum by Trichoderma spp. shows that both temperature and ph have great effect on mycoparasitism. At 15 C temperature T. viride and at 25 C T. harzianum (Th3) were most effective by inhibiting up to per cent and per cent mycelial growth respectively. At 35 C pathogen growth was not observed in most of the dual culture plates as well as in control. At different ph Trichoderma behaves differently as it was less effective at ph 9 as compared to ph 5 and 7. At ph 5 Th3 inhibited up to 92 per cent growth of some the isolates of S. sclerotiorum while at ph 7 it inhibited up to 79 per cent of growth of pathogen. At combination of different temperature and ph, Th3 inhibited up to 81 per cent 73 per cent and 60 per cent growth at ph 5, 7 and 9 at 15 C. At 25 C Th3 inhibited 86 per cent, 82 per cent and 55 per cent growth of pathogen at ph 5, 7 and 9 respectively. Th3 had a very effective biocontrol potential at temperature 15 C, 25 C and ph 5 and 7. At ph 9 it was able to inhibit the mycelial growth of pathogen up to 55 per cent. Key words: Biocontrol agents, confrontation assay, Mycoparasitism, Sclerotinia sclerotiorum, Trichoderma spp. Introduction White rot of mustard caused by Sclerotinia sclerotiorum, has become important in India and elsewhere with high disease incidence and causes up to 40 per cent yield losses leading to discouragement of growers of the crop (Chattopadhyay et al., 2003). Trichoderma species (Teleomorph: Hypocrea) is a filamentous ascomycetous fungus widely used as biocontrol agent (BCA) against many plant pathogens. It included different economically important species viz.,t. harzianum, T. asperellum, T. viride, T. atroviride, and T. virens and T. reesei (Sharma et al., 2014). Trichoderma antagonize the many phytopathogenic fungi, bacteria and invertebrates through mechanism of hyperparasitism, antibiosis, competition and induced systemic resistence. Mycoparasitism is the most considered and direct form of antagonism. It involves tropic growth of biocontrol agent towards the target organism (Tewari, 1996). Mycoparasitism by Trichoderma is affected by many soil and environmental parameters, among them temperature and ph are the two most important. The optimum temperature and ph for growth differs among the Trichoderma species. Most Trichoderma strains are mesophilic and grow well at neutral to slightly acidic ph (Huang and Erickson 2008). MATERIALS AND METHODS Pathogen cultures Survey of major mustard growing areas in different regions of Delhi and adjoining areas was conducted at post-flowering to pod formation stage of the crop, to record stem rot Published by Indian Society of Genetics, Biotechnology Research and Development, 5, E Biotech Bhawan, Nikhil Estate, Mugalia Road, Shastripuram, Sikandra, Agra Online management by

2 May 2017 Factors Favoring Mycoparasitism of Sclerotinia 11 incidence and collection of the diseased plant material. Samples were collected during the Rabi crop season ( ) at Mustard field, ICAR-Indian Agricultural Research Institute, New Delhi, Rajasthan, Himachal Pradesh and Pantnagar. The fungal pathogens were isolated, identified and purified on PDA plates and were further grouped into four different isolates viz. SS26, SS41, SS43, SSAco on the basis of morphology, cultural characteristics and sclerotial bodies. The fungal pathogens was isolated, identified and purified on PDA plates and named as isolates SS26. Cultures were routinely grown in potato dextrose agar (PDA) (HIMEDIA Laboratories Pvt. Ltd., India) plates and incubated at 28±2 C for 5-7 days. Trichoderma cultures All the Trichoderma spp. were procured from ITCC and Biological Control Laboratory, Division of Plant Pathology, IARI, New Delhi mentioned as follows: Trichoderma harzianum (Th3) ITCC-5593; T. viride ITCC-8315; Trichoderma harzianum (Th5) collected from IARI field; T. konongi - Biocontrol lab; T. atroviride ITCC- 7445; T. asperellum ITCC-8940; T. virens Biocontrol lab and T. longibrachiatum ITCC Among these, three spp. viz. Trichoderma harzianum (Th3), T. viride and Trichoderma harzianum (Th5) were selected for this particular study because these three were found more effective then others in previous study. Cultures were routinely grown in potato dextrose agar (PDA) (HIMEDIA Laboratories Pvt. Ltd., India) plates and incubated at 28±2 C for 5-7 days. Confrontation assay Antagonistic potential of Trichoderma spp. was studied by dual culture assay against Sclerotinia sclerotiorum by keeping the pathogen inoculated PDA plates as control (Dennis and Webster, 1971). Three replications of the each culture plates were incubated at different temperature level of 15 C, 25 C and 35 C for measuring colony growth both in control and dual culture plates. The plates were evaluated based on the grading method (Bell et al., 1982). The radial growth of the pathogen was measured after full growth and the percent inhibition was calculated as follows: PI = (C - T) 100 / C, where PI is the percent inhibition of mycelia growth; C is the radial growth of pathogen in control plates (cm) and T is the radial growth of pathogen in dual culture (cm). To study the effect of ph on mycoparasitism, PDA medium was prepared and autoclaved. ph of media was adjusted to 5, 7 and 9. On the media the pathogen and selected species of Trichoderma were dual cultured and kept at 25 C. Three replications of each Sclerotinia sclerotiorum isolates with each Trichoderma spp. were kept. These dual cultured plates were kept for 7 days and observed for growth of pathogen. The radial growth of pathogen in treatment and control were measured and percentage mycelium growth inhibition was calculated by using previously mentioned formula. To study the effect of combination of temperature and ph, media of different ph (5, 7 and 9) were prepared and the pathogen and selected species of Trichoderma were dual cultured and kept at different temperature level of 15 C, 25 C and 35 C. The radial growth of pathogen in treatment and control were measured and percentage mycelium growth inhibition was calculated by using previously mentioned formula. Statistical analysis All experiments were repeated atleast once. Data were analyzed by one-way analysis of variance (ANOVA) using OPSTAT software ( Differences between treatment mean values were determined following least significant difference (LSD) test at P <0.05. RESULTS Effect of different temperature levels on mycoparasitism Temperature has a direct effect on growth of Trichoderma, Sclerotinia sclerotiorum and mycoparasitism activity of Trichoderma. The results show that at different temperature level Trichoderma mycoparasitize Sclerotinia sclerotiorum in different ways. At 15 C temperature all the three species of Trichoderma inhibited all the isolates of Sclerotinia sclerotiorum. Trichodermaviride, Th3 and Th5 inhibited per cent, per cent and per cent growth of SS26 respectively.

3 % inhibition 12 Gopi Kishan et al. [Vol.9 No.2] Dual culture at 15 C shows that T. viridewas better than other two Trichoderma harzianum isolates. At 25 C temperature both the pathogen and Trichoderma grow very well and rapidly. At this temperature T. viride, Th3 and Th5 inhibited per cent, per cent and per cent growth of Sclerotinia sclerotiorum isolate SS26 respectively. At 35 C growth of all Trichoderma spp. was good but most of isolates of Sclerotinia sclerotiorum were unable to grow. At this temperature per cent inhibition of Sclerotinia sclerotiorum was observed to be from per cent to 90 per cent (Figure 2). The very poor growth of Sclerotinia sclerotiorum at 35 C was due to effect of high temperature. So inhibition by Trichoderma is not responsible for low growth. The results on effect of different temperature levels on mycoparasitism of Sclerotinia sclerotiorum by Trichoderma spp. shows that at temperature 15 C T. viride was significantly different than other two. But at 25 C Th3 was significantly higher than T. viride and Th5 (Figure 1) C 25 C 35 C Temperature T.viride T.harzianum(Th3) T. harzianum(th5) Fig. 1- Percent inhibition activity of Trichoderma spp. at different temperature against SS26 isolate of Sclerotinia sclerotiorum Fig 2 : Confrontation test of Trichodermaspp. against Sclerotinia sclerotiorum isolates at 15 0 C temperature Effect of different ph levels on mycoparasitism Trichoderma grow very well at ph 5 to 8 and control the pathogens effectively. Results on

4 % inhibition May 2017 Factors Favoring Mycoparasitism of Sclerotinia 13 effect of different ph levels on mycoparasitism of Sclerotinia sclerotiorum by Trichoderma shows that at ph 5 all the Trichodermaspp. inhibit all the isolates of Sclerotinia sclerotiorum ranging from per cent to per cent. At this ph all the Trichoderma spp. grow very well but isolates of Sclerotinia sclerotiorum were unable to grow effectively. Trichoderma viride, Th3 and Th5 inhibited per cent, per cent and per cent growth of Sclerotinia sclerotiorum isolate SS26 respectively. At ph 7 both Trichoderma and Sclerotinia sclerotiorum grow very well and growth inhibition of Sclerotinia sclerotiorum isolates by Trichoderma spp. recorded between range of per cent to per cent. Trichoderma viride, Th3 and Th5 inhibited per cent, per cent and per cent growth of sclerotinia sclerotiorum isolate SS26 respectively. At ph 9 isolates of Sclerotinia sclerotiorum grow very well but Trichoderma spp. were recorded to grow at somewhat slowly and growth inhibition of Sclerotinia sclerotiorum isolates by Trichoderma spp. recorded between range of per cent to per cent (Figure 3 and 4). At ph 9 Trichoderma viride, Th3 and Th5 inhibited per cent, per cent and per cent growth of Sclerotinia sclerotiorum isolate SS26 respectively ph T. viride T. harzianum(th5) T. harzianum(th3) Fig.3- Percent inhibition activity of Trichoderma spp. at different ph against SS26 isolates of Sclerotinia sclerotiorum Fig 4: Confrontation test of Trichodermaspp. against Sclerotinia sclerotiorum isolates at different ph level.

5

6 Indian Res. J. Genet. & Biotech. 9(2) May: (2017) Effect of combination of temperature and ph on mycoparasitism Results of this experiment shows that at different combination of temperature and ph Trichoderma inhibited growth of Sclerotinia sclerotiorum isolates differently. At 15 C temperature Trichoderma spp. react differently at different ph against all the isolates of Sclerotinia sclerotiorum. At ph 5 Trichoderma harzianum(th3) inhibited all the isolates of Sclerotinia sclerotiorum effectively. Trichoderma harzianum(th3) inhibited per cent growth of the isolates of Sclerotinia sclerotiorum. At ph 7 Trichoderma harzianum(th3) inhibited per cent growth of the isolates of Sclerotinia sclerotiorum and at ph 9 Th3 inhibited per cent growth (Table 1). So at 15 C temperature Th3 were efficient in controlling all the isolates of Sclerotinia sclerotiorum at different ph 9. At 25 C Th3 were good in controlling Sclerotinia sclerotiorum at different ph. At ph 5 Th3 inhibited per cent growth of Sclerotinia sclerotiorum. At ph 7 Th3 inhibited per cent growth of the isolates of Sclerotinia sclerotiorum. At ph 9 Th3 inhibited per cent growth of Sclerotinia sclerotiorum. At 35 C the growth of Sclerotinia sclerotiorum was not so effective but results of growth inhibition vary at different ph. At ph 5 Th3 inhibited per cent growth of Sclerotinia sclerotiorum. At ph 7 it s inhibited per cent growth of Sclerotinia sclerotiorum. At ph 9 Th3 inhibited per cent growth (Figure 5). Fig 5 : Confrontation test of Trichoderma spp. against Sclerotinia sclerotiorum isolates at combination of different temperature and ph level. Table 1: Combined effect of temperature and ph on mycoparasitism activity of T. harzianum (Th3) against SS26 isolate of Sclerotinia sclerotiorum Inhibition of mycelialgrowth (%) ph/ Temperature C 81.30(64.37) 73.24(58.83) 59.54(50.48) Published by Indian Society of Genetics, Biotechnology Research and Development, 5, E Biotech Bhawan, Nikhil Estate, Mugalia Road, Shastripuram, Sikandra, Agra Online management by

7 May 2017 Factors Favoring Mycoparasitism of Sclerotinia C 86.34(68.32) 81.56(64.58) 54.96(47.83) 35 C 90.04(71.66) 83.97(66.39) 65.02(53.72) * Value in parenthesis representing arc sin transformed value Factors C.D. SE(d) Temperature(A) ph(b) Factor(A X B) DISCUSSION White rot caused by Sclerotinia sclerotiorum is an important disease of mustard crop. As chemical methods of disease management are not ecologically and economically safe and sound, so we have to explore our biocontrol agents to control disease like this. Investigations were made to study the factors favoring the mechanism of mycoparasitism by Trichoderma spp. against white rot pathogen. Studies are available on the effects of temperature on the spore germination, germ-tube growth, mycelial growth, competitive saprophytic abilities, volatile and non-volatile metabolite production of Trichoderma strains. The optimum temperature for growth differs among the Trichoderma species. Most Trichodermastrains are mesophilic. T. viridewas the most abundant species in early spring and autumn in a spruce forest soil. The lower proportion of cold-tolerant strains in T. harzianumspecies aggregate (6 of 142) than in T. viridespecies aggregate (7 of 78) appears to be consistent with the higher optimum growth temperature of the T. harzianum (Widden and Abitbol,1980). Abiotic and biotic environmental parameters may have negative influence on the biocontrol efficacy of Trichoderma strains (Kredicset al., 2003). Trichdermaasperellum, TaDOR673 was highly tolerant to heat shock of 52 C with a mean spore count (log c.f.u/ml) of 4.33 (Pusapati et al., 2014). T. harzianum isolates grew well at temperature 20 C, ph 5 and molasses agar medium (Meenaet al., 2014). As compare to these, results of the present study also shows that T. harzianum (Th3) grow very well at 15 C and 25 C and inhibit the Sclerotinia sclerotiorum very effectively which is also a low temperature loving pathogen. T.viride also shows very good results at 15 C and 25 C. At 35 C pathogen growth was not observed in dual culture plates as well as in control. So we can say that the absence of growth at 35 C is mainly due to the effect of temperature and it is not due to mycoparasitic activity of Trichoderma. Both Trichoderma spp. and Sclerotinia sclerotiorum are influenced by different levels of ph.mycoparasiticactivity of Trichodermachanges at different level of ph.trichodermastrains were able to grow at a wide range of ph from with an optimum at 4.0. However, the mycelial growth of some of the examined plant pathogenic fungi had ph-optima at alkali values ( Kredics., 2004). Jackson et al., (1991) have found that optimum biomass production of three Trichodermaisolates occurred at ph ranges between 4.6 and 6.8. Similarly, this study showed that at all the three ph (5, 7 and 9) T. harzianum (Th3) grow very well and inhibit the Sclerotinia sclerotiorum. At ph 5 and 7, T.viridealso showed good results by inhibiting pathogen up to per cent and per cent respectively. At ph 9 both Th3 andt.viride showed pathogen inhibition upto per cent and per cent which is not considered to be effective as biocontrol agents. Results of effect of combination of temperature and ph show that at 15 C, Th3 was most effective at all the three ph. It parasitizes the

8 16 Gopi Kishan et al. [Vol.9 No.2] pathogen very effectively at ph 5 and 7 but at ph 9 it inhibits the pathogen up to per cent. The temperature 25 C favours the growth of both pathogen and Th3 at ph 5 and 7 but at ph 9 the growth is suppressed. At 35 C growth of Sclerotinia isolates was suppressed at ph 5 and 7 but at ph 9 pathogen grows very well in control plates but in dual culture plates it shows REFERENCES 1. Arunachalam, M.K. and Sharma, P. (2012).Confrontation assay for Trichodermaas a potential biocontrol agent against Pythium aphanidermatum and Sclerotinia sclerotiorum. Pest Management in Horticultural EcosystemsVol. 18, No. 1 pp Chakravarthy, S., Nagamani, K., Ratnakumari, A.R. and Bramarambha, Y.S. (2011).Antagonistic ability against Rhizoctonia solani and pesticide tolerance of Trichodermastrains.Advances in Env. Biol. 5(9): Chattopadhyay, C., Meena, P.D. and Kumar, S. (2002). Management of Sclerotinia rot of Indian mustard using ecofriendly strategies. J. Mycol. Pl. Pathol. 32: Das, M.G., Das, B.C. and Sarmah, D.K. (2002).In-vitro studies of some antagonists against Sclerotinia sclerotiorum (Lib) de Bary. Journal of the Agricultural Science, Society ofnorth-east India15 (1): Dennis, C. and Webster,J. (1971). Antagonistic properties of species-groups of Trichoderma: I. Production of non-volatile antibiotics. Trans. Br. Mycol. Soc. 57: Dos Santos, A.F. and O.D. Dhingra, O.D. (1982). Pathogenicity of Trichoderma spp. on the sclerotia of Sclerotinia sclerotiorum.canadian journal of botany. 60(4): Harman,G.E. (2000). Myths and dogmas of biocontrol: changes in perceptions derived from research on Trichoderma harzianum T- 22. Plant Disease 84: Hung-Chang, Huang. and Scott Erickson, R.( 2008). Factors Affecting Biological that pathogenic growth inhibited up to 65% by Th3. ACKNOWLEDGMENTS The authors are grateful to Head, Division of Plant Pathology, ICAR-IARI, New Delhi for providing facilities to carry out this work. First author is thankful to ICAR-IARI, New Delhi, India for funding fellowship for the academic study. Control of Sclerotinia sclerotiorum by Fungal Antagonists. Phytopathology vol 156: Inbar, J., Menendez, A. and Chet, I.(1996). Hyphal interaction between Trichoderma harzianum and Sclerotinia sclerotiorum and its role in biological control. Soil Biol. Biochem. 28: Knudsen, G.R., Eschen, D.J., Dandurand, L.M. and Bin, L.(1991). Potential for biocontrol of Sclerotinia sclerotiorum through colonization of sclerotia by Trichoderma harzianum. Plant Dis. 75: Meena, P. D., Chattopadhyay, C., Meena, P. S., Goyal, P. and Kumar, V. R.(2014). Shelf life and efficacy of talc-based bioformulations of Trichoderma harzianum isolates in management of Sclerotinia rot of Indian mustard (Brassica juncea). Annals of Plant Protection Sciences 22(1): Mukherjee, P.K. (2015). Trichoderma Genetics, Genomics and Beyond. Indian Phytopath. 68: Sharma, P., Sharma, M., Raja, M. and Shanmugam, V. (2014). Status of Trichoderma research in India: A review. Indian Phytopath. 67(1): 1-19.