In Vitro Antagonism of Native Isolates of Tricoderma spp Against Sclerotium rolfsii

Vol. 4 (3) Jul– Sep 2013 www.ijrpbsonline.com 886

International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

_________________________________________Research Article

In Vitro Antagonism of Native Isolates of Tricoderma spp Against

Sclerotium rolfsii

M. Padmaja, K. Narendra, J. Swathi, KM. Sowjanya, P. Jawahar Babu and

A. Krishna Satya*

Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur-

522510, India. ABSTRACT The antagonistic potential of native Tricoderma isolates was investigated in vitro against S. rolfsii the causal agent of stem rot disease in groundnut. Ten native Tricoderma isolates and a commercial formulation of Tricoderma were screened. Eight isolates inhibited the pathogen from 60 to 80%. Two isolates showed maximum growth inhibition (80, 78%). Volatile & non volatile compounds produced from these isolates were evaluated along with the commercial formulation through poisoned food technique and culture filtrate studies. Both the isolates proved to produce volatile and non-volatile compounds that inhibited the pathogen effectively. Even at 20% concentration of culture filtrate, these native isolates inhibited the test fungi up to 60% and at 50% concentration recorded 100 % inhibition of the test fungi. Both the native isolates proved effective in controlling the pathogen in-vitro than the commercial agent, indicating their superiority in the bio control of phyto pathogens especially the one under the test. Keywords: Sclerotium rolfsii, Tricoderma, volatile and non-volatile compounds. INTRODUCTION Soil born, plant pathogenic fungi such as Sclerotium, Rizoctonia, Pithium, Fusarium cause diseases in most of the economically important crops. Sclerotium rolfsii Sacc., is a serious soil borne plant pathogen of worldwide importance with a host range of over 500 species belonging to one hundred plant families. The disease causes severe damage to the crop, at any stage of its growth and yield losses of over 25 percent have been reported (Mayee and Datur, 1988)1. Control of these plant pathogens is very difficult because of their survival capacity. It is also not practicable owing to high cost of chemicals besides problems of environmental pollution and development of resistance in target fungus as opined by Uma Maheswari et al., 20022 and Rudresh et al., 20053. Biological control offers a novel approach, as an alternative solution for long term sustainability and is a promising disease management technology against soil borne plant pathogens, when applied either alone or in combination with other management practices. Tricoderma spp., which is a common saprophytic filamentous fungus, in almost all soil and rhizosphere microflora, it is recognized as a bio-control agent against various plant pathogenic fungi. They are commercially applied as biological control agents based on different mechanisms, such as the production of antifungal metabolites,

competitor for space and nutrients and through mycoparasitism.( Pandey et al., 2005)4. Different native antagonists from rhizosphere of groundnut were tested invitro against S.rolfsii, it reported 60 -70% inhibition in mycelial growth by Sai et al 2010(5) and Faheem Amin 20106. Several reports indicated variation among the isolates of Tricoderma in their antagonistic potential, as variation exists between strains/ species within a genus, isolated from different climatic conditions(Tripathi and Khare (2005)7. Biological control agent is necessary in terms of possible number of antagonistic traits such as overgrowth, production of volatiles, non-volatiles and lytic enzymes, to exploit the full potential of the agent. R.K.Srivastava, et.al, 20128 tested different Tricoderma isolates and clearly indicated that the efficacy of different isolates of same species also showed variation when tested against the same pathogen. Therefore, before to field application, the potentiality of every strain should be tested against target pathogen. In the present investigation, in vitro efficacy and mode of antagonism of native Trichoderma spp. against Sclerotium rolfsii Sacc. was elucidated. MATERIALS AND METHODS Isolation of pathogen from groundnut plants The fungal pathogen (Sclerotium rolfsii Sacc.) was isolated from affected plants of groundnut



ISSN: 2229-3701

Rangaswami and Mahadevan (1999)9 and identified based on the morphological characters. Isolation of bio-control microorganisms Pure culture of the antagonist (Trichoderma sp.) isolated from the rhizosphere soil using Serial dilution technique (Johnson and Curl, 1972)10 and maintained in agar slants. Screening of antagonists by dual plating Ten isolates of Trichoderma were tested by dual culture technique of Mortan and Straube, (1955) 11. A 2 mm mycelial disc of Sclerotium rolfsii and Trichoderma were placed opposite to each other near the periphery of the Petri plate and incubated at room temperature (28±2°C). S. rolfsii alone inoculated plate served as control. Mycelial growth of the pathogen was measured in dual culture plates as well as in control plates. Observations were recorded of inhibition zone. Detection of volatile and non-volatile metabolites Efficacy of volatile metabolites of selected Trichoderma isolates was tested by using paired plate technique. (Dennis and Webster, 1971b)12. Sterile PDA plates were inoculated with 2mm culture disc of either S. rolfsii or individual Trichoderma spp. at the centre. These two bottom plates of Petri dish were paired together, sealed and incubated. Only test pathogen was maintained in the Control Plate and Observations were recorded on the radial growth of S. rolfsii. Culture filtrate studies were carried out to study the effect of diffusible nonvolatile metabolites of isolates against S. rolfsii. Initially seven day old culture broth of Trichoderma isolates were filtered and prepared 20, 30, 40, 50 percent concentrations by diluting with PDA which was poured into Petri plates and allowed to solidify and inoculated with 2mm discs of S. rolfsii culture and incubated. Plates inoculated with S. rolfsii on PDA alone (without culture filtrate) served as control. Observations were recorded on the radial growth of S. rolfsii. Percent inhibition of the pathogen over control was calculated by the formula adopted by Nene and Thapliyal (1982)13.

I (%) = (C-T)/ C x 100 I = Percent growth inhibition C= Growth in control T= Growth in treatment. RESULTS In vitro antagonism of Trichoderma against S. rolfsii The antagonistic potential of the ten native Tricoderma isolates and the commercial Tricoderma isolate, tested through dual culture studies presented in Table – 1

Observations on qualitative parameters studied in dual culture are recorded in Table - 2 The interaction of Test Isolates on the pathogen is present in Fig.1 and Fig. 2. The effect of volatiles on the growth of Sclerotium rolfsii is presented in Table-3 The inhibition of growth in Sclerotium rolfsii with varying concentrations of Tricoderma culture filtrates are presented in Table-4 Effect of non-volatiles on S. rolfsii Fig. 3. DISCUSSION Varying degrees of antagonism were exhibited by fungal antagonists against S. rolfsii in dual culture plates. For the first two days Tricoderma presence did not show much effect on S.rolfsii radial growth and prolonged incubation beyond 3 days resulted in significant differences in the growth of the pathogen. Radial growth of S.rolfsii in check plate (4.0) significantly differed with that of interaction plates 4 days after inoculation. Observations at the end of six days after inoculation indicated maximum reduction in the radial growth of S.rolfsi in T1 isolate with 80% inhibition followed by T5 (78%). Further, isolates T2, T8 and T9 were on par with T1 in terms of percent inhibition in growth. T6 and T10 also inhibited up to 67%. These 7 antagonists completely over grew the pathogen. Least reduction in the radial growth of S. rolfsii (3.7cm) was observed in T3 with 53 per cent growth inhibition. T4, T7, T11 were on par with T3 and are not considered effective in controlling the pathogen. Interactions between S. rolfsii and test Trichoderma isolate revealed formation of zone of inhibition, lysis of either of the isolates and/or overgrowth. Incubation beyond 3 days resulted in joining of both the test fungi in dual cultured plates. This was considered as zone of inhibition. Formation of inhibition zone at the contact between Tricoderma and S. rolfsii could be explained on the basis of production of volatile and non-volatile metabolites as well as the production of extra-cellular hydrolytic enzymes by Tricoderma. Similar opinion was expressed by EI-Katatny(2001)14. Zone of inhibition clearly show on day 4 in 6 plates. Observations after 7 days of interactions revealed that Trichoderma isolates T1, T2, T5 and T8 caused lysis and over growth on S. rolfsii, and isolates T9 and T10 shown only over growth. T6 (Commercial) inhibited the pathogen initially but later shown static growth. Negative radial growth of S. rolfsii with T1, T2, T5 and T8, five days after inoculation, is an indication of its lysis at interaction zone. Lysis of pathogenic hyphae by Trichoderma is evident due to antibiosis, mediated through cellulolytic enzymes and toxins as reported by Jacobs, (2007)15. Observations indicated that the Trichoderma isolates T1, T2, T5, T8, T9 and T10 overgrew S. rolfsii and Trichoderma



ISSN: 2229-3701

isolates T3, T4, T7 and T11 were overgrown by S. rolfsii. Based on the qualitative and quantitative parameters, two Trichoderma isolates viz., T1, T5 with fast radial growth, simultaneous lysis and over growth on S. rolfsii were chosen for testing volatiles and non-volatile products in comparision with Commercial isolate (T6). Volatiles It was found that all the three isolates T1, T5 and commercial inhibit S. rolfsii through their volatile compounds. Maximum inhibition in the radial growth of S.rolfsii could be seen in T1. By 4th day, the entire plate of 9cm was covered by the pathogen and 5.8 cm in T5 and 4.5 cm consisting commercial Tricoderma isolate. It was the least in T1 with only 2.7 cm radial growth. Thus, T1 was more effective with 70% inhibition in the radial growth of S.rolfsii compared to 50% inhibition with commercial isolate. This is due to the involvement of volatiles in Tricoderma antibiosis as reported by Uma Maheshwari et al., (2002)2. Trichoderma spp. produces over 40 different secondary metabolites that may contribute to their mycoparasitic and antibiotic action. Large number of volatile secondary metabolites produced by Tricoderma such as Ethylene, Hydrogen cyanide, Aldehydes and Ketones play an important role in controlling plant pathogens and metabolites such as harzianic acid, alamethicins and tricholin that hinders the growth of other microorganisms vey et.al (2001)16. Isolate variation among Trichoderma spp. in inhibiting pathogenic fungi is due to production of volatiles Srinivasulu et al. (2005)17. Non volatiles. From the data presented, the culture filtrates from T1 to T5 could inhibit radial growth of S.rolfsii through their non-volatile compounds. Complete inhibition of 100% occurred at 40% and 50% concentrations of both the isolates. It is interesting to note that even at lower concentrations of 20% both the native isolates recorded significant inhibition compared to check whereas commercial isolate has no effect at all. This indicates that non volatiles produced by both these isolates are very effective. Simultaneously lysis and over growth of these two isolates could be through production of strong non volatiles. Reports on inhibitory effect of Trichoderma culture filtrate and non-volatiles on the growth of S. rolfsii were reported earlier by Rudresh et. al., (2005)3. Antibiotics secreted by antagonistic agents may be volatile or non-volatile and Trico produce both the types. They exert strong inhibitory action with

Isopropyl alcohol, caryophyllene, pentadecane, demadin tricodedrmin, viridepyronone Chakraborty et.al (2004)18. The results conclude the strong correlation between bio efficacy and production of metabolites by Mishra et al (2011)19. In this study the non-volatile secondary metabolites from Trichoderma species were found more effective in suppressing the mycelial growth of S.rolfsii, compared to volatile compounds. There is need for further studies in-vivo as complete inhibition could be noticed in-vitro even at low concentrations. Faheem Amin 201020 reported 67% growth inhibition of S.rolfsii by the Trichoderma isolates from dual culture studies and rated it as the best isolate. Anand 200921 identified two best performers from 42 Trichoderma strains, which inhibited the growth of S.rolfsii up to 42 and 40%. Rahel Ratnakumari et.al 201122 tested the volatile and non-volatile metabolites of Trichoderma against S.rolfsii and shown 42-63% inhibition in dual culture and 33-46% with culture filtrate studies. E.I. Eziashi 200623 concluded from his studies that the inhibition varies depending on the Trichoderma species producing the metabolites from 2.0% to 64% in volatile, 0.0% to 74% in non-volatile form. B.K. Mishra 201119 tested several isolates and found his best isolate showing 68% growth inhibition and 100% in culture filtrate study and suggested it for commercial use. Asma Murtaza etal 2012(24) tested the effect of tricoderma culture filtrate with different concentrations grown in different media and reported 60 to 80% inhibition at 100% concentration and very less effect at lower concentrations. In the present investigation, the two native isolates T1 & T5 have shown 80% & 78% growth inhibition in dual culture studies and 100% inhibition even with 50% culture filtrate. The volatiles are very effective. Hence these two native isolates are far more superior in their antagonistic effect against S.rolfsii, compared to the results obtained by earlier researchers. CONCLUSION Tricoderma isolates are more effective and show excellent control of S. rolfsii, responsible for groundnut rot. These isolates could be exploited on a large scale for field application in local conditions. The two isolates will be promising bio control agents against rots caused by S. rolfsii in field crops with best outcome in future plant biotech applications.



ISSN: 2229-3701

Table 1: Radial growth of S. rolfsii dual cultured with

different isolates of Trichoderma spp S .No Isolate Radial growth of S. rolfsii (cm) Percent

inhibition 2 DAI 3 DAI 4 DAI 5 DAI 6 DAI 1 T1

0.8 1.2 1.5 1.9 1.6 80 2 T2 0.9 1.8 2.1 2.0 1.9 76 3 T3 0.9 2.3 2.5 3.0 3.7 53 4 T4 1.2 1.9 2.9 3.5 3.5 56 5 T5 0.9 1.4 1.4 1.8 1.7 78 6 T6 (commercial) 1.0 1.8 1.9 2.1 2.6 67 7 T7 1.0 1.9 2.6 3.3 3.6 55 8 T8 0.8 1.0 1.4 2.4 2.0 75 9 T9 0.9 1.1 1.3 1.9 1.9 76 10 T10 0.9 1.9 2.0 2.4 2.6 67 11 T11 1.1 2.5 3.0 3.2 3.3 59 12 Check 1.1 2.9 4.0 5.6 8.0 __

SEm+ 0.03 0.05 0.07 0.06 0.06 CD (p=0.01) 0.13 0.16 0.2 0.2 0.16

Table 2: Qualitative parameters observed in dual culture of S. rolfsii with different isolates of Trichoderma spp

Treatments Over growth Lysis Zone of inhibition T Sr T Sr

Sr+T1 + _ _ Y P Sr+T2 + _ _ Y P Sr+T3 _ + Y _ A Sr+T4 _ + Y _ A Sr+T5 + _ _ Y P Sr+T6 _ _ _ _ P Sr+T7 _ + Y _ A Sr+T8 + _ _ Y P Sr+T9 + _ _ _ P Sr+T10 + _ _ _ A Sr+T11 _ + _ _ A

+: indicates over growth Y: indicates Lysis P: Presence of zone of inhibition between the isolates

Fig. 1: Interaction of S. rolfsii and isolates

of Tricoderma in dual culture



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Interaction of Tricoderma with S.rolfscii

0123456789

DAI2 DAI3 DAI4 DAI5 DAI6

Days of Inoculation

Rad

ial g

row

thT1T2T3T4T5T6T7T8T9T10T11CHECK

Fig. 2: Interaction of Tricoderma with S.rolfsii

Table 3: Effect of volatiles on the growth of Sclerotium rolfsii Treatments Radial growth of mycelium

(cm) Percent growth inhibition

S.r + T1 2.7 70 S.r + T5 5.8 36 S.r +T6 4.5 50 Control 9.0 _

Table 4: Effect of culture filtrate of tricoderma on Sclerotium rolfsii growth (inhibition %)

Conc Per cent growth inhibition

Day1 Day2 Day3 Day4 T1 T5 T6 T1 T5 T6 T1 T5 T6 T1 T5 T6

20 48 57 17 40 45 7 46 49 8 50 54 11 30 61 70 57 55 69 31 69 75 20 82 84 28 40 78 87 52 79 88 24 85 92 25 97 96 35 50 87 91 44 93 93 33 97 97 44 100 100 50

effect of non volatiles

0

20

40

60

80

100

120

20 30 40 50

concentration of culture filtrate

grow

th in

hibi

tion

T1T5com

Fig. 3: Effect of non-volatiles on S.rolfsii



ISSN: 2229-3701

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In Vitro Antagonism of Native Isolates of Tricoderma spp Against Sclerotium rolfsii

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