International Journal of Chemical Studies 2019; 7(1): 2498-2501 P-ISSN: 2349–8528 E-ISSN: 2321–4902 IJCS 2019; 7(1): 2498-2501 © 2019 IJCS Received: 06-11-2018 Accepted: 10-12-2018 Vignesh P Department of Plant Pathology, Central Agricultural University, Imphal, Manipur, India LNK Singh Department of Plant Pathology, Central Agricultural University, Imphal, Manipur, India Bireswar Sinha Department of Plant Pathology, Central Agricultural University, Imphal, Manipur, India Ph Sobita Devi Department of Plant Pathology, Central Agricultural University, Imphal, Manipur, India K Sarda Devi Department of Plant Pathology, Central Agricultural University, Imphal, Manipur, India V Bhuvaneswari Department of Plant Pathology, Central Agricultural University, Imphal, Manipur, India Correspondence Vignesh P Department of Plant Pathology, Central Agricultural University, Imphal, Manipur, India In-vitro evaluation of Trichoderma spp. for the management of Rhizoctonia oryzae-sativae causing aggregate sheath spot of rice in Manipur Vignesh P, LNK Singh, Bireswar Sinha, Ph Sobita Devi, K Sarda Devi and V Bhuvaneswari Abstract Present research was carried out to understand antagonistic potentiality of six native Trichoderma spp. against Rhizoctonia oryzae-sativae causing Aggregate sheath spot of rice. Dual culture technique revealed the inhibition percentages of R. oryzae-sativae by T. asperellum (KU933475), T. koningiopsis (KU904460), Hypocrea lixii (KX0113223), T. harzianum (KU904458), T. ovalisporum (KU904456) and T. atroviridae (KU933472) of 58.43%, 68.23%, 60.0%, 72.94%, 64.31%, and 70.19% respectively. Bell’s scale study showed class III category by Trichoderma spp and class II showed by T. harzianum (KU904458) against Rhizoctonia oryzae-sativae. Among the different biological agents Trichoderma harzianum is the most effective one for reducing rapid growth of the pathogen. Further, all Trichoderma isolates significantly inhibited the mycelial growth of the pathogen. Keywords: rhizoctonia oryzae-sativae, dual culture, trichoderma spp Introduction Rice (Oryza sativa L.) is the most prominent crop of India as it is the staple food for most of the people of the country. It is one of the major food crops of India. More than 90% of the world’s rice is grown and consumed in Asia where 60% of the earth’s people live (Mahajan et al., 2017) [13]. Rice is the principal food crop in Manipur. It is widely cultivated in both hill and valley areas of Manipur occupying nearly 1.80 lakh ha of the total cropped area in the state (Goud et al., 2018) [8]. It suffers from many fungal and bacterial diseases resulting in heavy losses of grain yield. Rhizoctonia oryzae-sativae (Sawada) Mordue causes aggregate sheath spot in rice. R.oryzae-sativae was first described in Taiwan as Sclerotium oryzae-sativae (Sawada, K., 1922) [19]. Since then, the disease has been reported in Iran (Rahimian, 1989) [16] Chile (Ricardo-Madariaga et al. 1999) [17] and recently in Australia (Lanoiselet et al. 2001) [12]. In the USA, the disease was reported in California (Gunnell and Webster, 1984) [7]. The teleomorph of R. oryzae-sativae was described as Ceratobasidium oryzae-sativae (Gunnell and Webster, 1987) [6]. Aggregate sheath spot lesions first appear on the lower leaf sheaths at the water line during the tillering stage. Lesions are circular to elliptical with grey-green to strawcoloured centers surrounded by distinct brown margins. Frequently, additional margins from around the initial lesion, producing a series of concentric bands. A strip of light-coloured necrotic cells runs down the lesion centre. Later in the season, secondary infections frequently occur well above the water line. Trichoderma is a fungal genus that is found in the soil. It is a secondary fast growing opportunistic invasive, which produces large numbers of spores, enzymes able to degrade the fungal cell wall (chitinases, glucanases, and proteases) and compounds with antimicrobial activity. Many Trichoderma species are also well known as biocontrol agents (BCA) of important phytopathogenic fungi. The primary mechanisms of biocontrol used by Trichoderma in direct confrontation with pathogenic fungi are the mycoparasitism (Papavizas, 1985) [15] antibiosis, and competition for nutrients with the pathogen (Harman and Kubicek, 1998) [9]. The present research work carried out to understand the effect of Trichoderma spp. On the growth of Rhizoctonia oryzae-sativae in in-vitro. ~ 2498 ~ International Journal of Chemical Studies Materials and Methodologies Isolation of fungus The infected rice plant showing typical symptoms of aggregate sheath spot were collected and were examined under microscope in Department of Plant Pathology, College of Agriculture, Central Agricultural University, Imphal laboratory. Later the collected samples were lacerated to small pieces (<1.0 cm) and were washed under tap water twice to remove soil particles and other debris. Surface sterilization was done by dipping the cut pieces in 1%Sodium hypo chloride (NaOCl) solution and through a series of sterile distilled water at 3 times for one minute intervals respectively. The treated sample pieces were blot dried and then transferred to petri plates containing sterilized potato dextrose agar medium with four pieces per plate using sterile forceps. The isolated fungus was identified as Rhizoctonia oryzae-sativae (MH255604 & MH255605). All plates were kept at 25 ± 2°C for 3-4 days and from these plates pure cultures of R. oryzaesativae isolates were maintained. The fungus was then sub cultured whenever needed during the present study. In-vitro Evaluation of bio control agents against growth of Rhizoctonia oryzae-sativae Dual culture method: In vitro antagonistic activity of Trichoderma spp. against Rhizoctonia oryzae-sativae was studied in dual culture technique by following the method by Kucuk and Kivanc (2003) [11]. Petridishes containing sterile PDA were inoculated with a 5mm diameter plug of 4- dayold pure culture of antagonistic fungi and pathogens. One mycelial disc of each fungus was placed on opposite poles of PDA plates using sterile cork borer and sterile needle and incubated at 25°C in incubator and radial growth of pathogen was measured at 24hr intervals. A petridish without antagonist served as control. Each treatment was replicated thrice. The bio control agents used in this study was collected from department of Plant Pathology, College of Agriculture, Central Agricultural University, Imphal. The per cent inhibition of mycelial growth of test fungus over control was calculated by using the formula suggested by Dennis and Webster (1971) [2]. Where, I = C = T = I = C−T x 100 C Per cent inhibition, linear growth of the fungus in control, linear growth of the fungus in treatment. Bell’s scale with slight modification Class I: The antagonist completely overgrew the test pathogen (100 % overgrowth). Class II: The antagonist overgrew at least 2/3rd of the test pathogen surface (75% over growth). Class III: The antagonist colonized on half of the growth of the test pathogen surface (50% over growth). Class IV: The test pathogen and the antagonist locked at the point of contact. Class V: The test pathogen overgrew the antagonist. Class VI: The test pathogen and antagonist form inhibition zone. The list of Bio-control agents used is listed with Isolate code and Accession number 1. CAUNCIPM-7 (T. asperellum) - KU933475 2. 3. 4. 5. 6. CAUNCIPM-18 (T. koningiopsis) - KU904460 CAUNCIPM-48 (Hypocrea lixii) - KX0113223 CAUNCIPM-78 (T. harzianum - KU904458 CAUNCIPM-96 (T. ovalisporum) - KU904456 CAUNCIPM-118 (T. atroviridae) - KU933472 Results and Discussions Differential biocontrol ability among the antagonists was noticed against Rhizoctonia oryzae-sativae (Fig.1) and percent inhibition also calculated (Fig.2). Results showed that among the six potential antagonists, T. atroviride and T. harzianum proved to be the most potent bioagents against R. oryzae-sativae. 6 days of incubation showed different degrees of mycelial growth inhibition of R. oryzae-sativae (Fig.3). T. harzianum was most potent in reducing growth of the pathogen than T. atroviride (Saikia et al., 1995) [18]. However all the species showed a considerable mycelial growth inhibition i.e., T. harzianum by (75.68%), T. atroviradae by (70.19%), T. koningiopsis by (68.23%), T. ovalisporum by (64.31%), Hypocrea lixii by (60.0%), T. asperellum by (58.43%) and respectively. The highest percent of inhibition 75.68% was shown by T. harzianum and the least percent inhibition of 58.43% was shown by T. asperellum. According to Papavizas and Lumsden (1982) [14]; (Devaki et al. 1992) [4], the mechanisms involved in the control of pathogens by Trichoderma spp. are probably due to antibiosis, lysis, competition and mycoparasitism. The mode of mycoparasitism was observed to be entirely different between T. viride and R. solani (Pandey et al., 2005) [10]. T. harzianum hyphae coiled around the pathogen. Later on pegging started and knob like haustoria formed inside the hyphae of R. solani. The cytoplasmic contents may be taken through the haustoria and only cell wall was clearly visible without cytoplasm. Baker and Cook (1979) [1] have reported that the enzymes may be produced that digest the mycelial walls and septal walls or antibiotics may be formed that inhibit growth or cause endolysis. Dennis and Webster (1971) [3] have reported that Trichoderma spp. are known to produce a number of antibiotics such as Trichodermin, Trichodermol, Harzianum A and Harzianolide as well as some cell wall degrading enzymes such as Chitinases, glucanases that break down polysaccharide, chitins and β-glucans, thereby destroying cell wall integrity (Elad, 2000; Devaki et al., 1992) [4, 5]. These may also play a major role in mycoparasitism because of changes in cell wall integrity. The Bell’s scale classified the antagonism nature of T. harzianum to class II where the antagonist over grew at least two thirds of the pathogen surface. And the rest other antagonists T. atroviridae, T. koningiopsis, T. ovalisporum, Hypocrea lixii and T. asperellum are classified into Class III because the antagonist colonized on half of the growth of the pathogen. Conclusion The present investigation showed that tested Trichoderma isolates reduced the growth of R. oryzae-sativae pathogen significantly. However, this study was conducted in laboratory conditions and it will be effective for future plant disease management to control aggregate sheath spot of rice with proper field studies. The degree of antagonism varied between and within species of Trichoderma against the pathogen. Biocontrol agents can be incorporated for integrated disease management of many soil borne plant pathogens for sustainable crop production. ~ 2499 ~ International Journal of Chemical Studies Table 1: Evaluation of Trichoderma isolates against R. oryzaesativae using dual culture Sl. No. 1. 2. 3. 4. 5. 6. Bio control agent T. harzianum (KU904458) T. atroviridae (KU933472) T. koningiopsis (KU904460) T. ovalisporum (KU904456) Hypocrea lixii (KX0113223) T. asperellum (KU933475) SE (d) CD(P=0.05) *Mean of three replications Bell’s scale Inhibition (%)* Class II 75.68 Class III 70.19 Class III 68.23 Class III 64.31 Class III 60.0 Class III 58.43 1.32 2.97 Fig 2: Percent inhibition of mycelial growth in culture plate Fig 1: Dual culture technique of bio control agents against the growth of R. oryzae-sativae Fig 3: Growth pattern of R. oryzae-sativae and the biological control agents in terms of radius of the colony ~ 2500 ~ International Journal of Chemical Studies References 1. Baker FK, Cook RJ. Biological control of plant pathogens. S. Chand and Company Limited, New Delhi, India, 1979, pp433. 2. Bell DK, Wells HD, Markham CR. In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology. 1982; 72(4):379-382. 3. Dennis C, Webster J. Antagonistic properties of speciesgroups of Trichoderma. I. Production of non-volatile antibiotics. Trans Brit Mycol Soc. 1971; 57:25-39. 4. Devaki NS, Bhat SS, Bhat SG, Manjunath KR. Antagonistic activities of Trichoderma harzianum against Pythium aphanidermatum and Pythium myriotylum on tobacco. Journal of Phytopathology. 1992; 136:82-87. 5. Elad Y. Biological control of foliar pathogens by means of Trichoderma harzianum and potential modes of action. Crop Protection. 2000; 19:709-714. 6. Gunnell PS, Webster RK. Ceratobasidium oryzae-sativae sp. nov., the Teleomorph of Rhizoctonia oryzae-sativae and Ceratobasidium setariae comb. nov., the Probable Teleomorph of Rhizoctonia fumigata comb. nov. Mycologia. 1987; 79(5):731-736. 7. Gunnell PS, Webster RK. Aggregate sheath spot of rice in California [Rhizoctonia oryzae-sativae]. Plant Diseases, 1984. 8. Goud ER, Ram D. Comparative profile of Communication Behaviour among the Rice growers in Imphal West District of Manipur, India. Int. J. Curr. Microbiol. App. Sci. 2018; 7(5):2273-2279. 9. Harman GE, Kubicek CP. Trichoderma and Gliocladium: Enzymes, Biological Control and Commercial Applications, 1998, 2. 10. Pandey KK, Pandey PK, Upadhya JP. Mycoparasitism of Trichoderma spp. on Fusarium and Rhizoctonia. Journal of Mycology Pathology. 2005; 35:174-176. 11. Kucuk C, Kivanc M. Isolation of Trichoderma spp. and determination of their antifungal, biochemical and physiological features. Turkish Journal of Biology. 2003; 27:247-253. 12. Lanoiselet VM, Ash GJ, Cother EJ, Priest MJ, Watson A. First report of Waitea circinata causing sheath spot and Rhizoctonia oryzae-sativae causing aggregate sheath spot on rice in south-eastern Australia. Australasian Plant Pathology. 2001; 30(4):369-370. 13. Mahajan G, Kumar V, Chauhan BS. Rice production in India. In Rice Production Worldwide, 2017, 53-91. 14. Papavizas GC, Lumsden RD. Improved medium for isolation of Trichoderma spp. from natural soil. Plant Disease. 1982; 66:1019-1020. 15. Papavizas G. Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol. 1985; 23:23-54. 10.1146/annurev.py.23.090185.000323. 16. Rahimian H. Occurrence of aggregate sheath spot of rice in Iran. Journal of Phytopathology. 1989; 125(1):41-46. 17. Ricardo Madariaga B, Ximena Morales C, Roberto Alvarado A. Rhizoctonia oryzae-sativae (Sawada) Mordue, causal agent of aggregate sheath spot of rice (Oryza sativa L.) in Chile. Agricultura Tecnica. 1999; 59(2):77-84. 18. Saikia MK, SK Gandhi. Comparative Activities of Three Antagonistic Fungi against Cauliflower Stem Rot Pathogen Rhizoctonia solani. Journal of Mycology Pathology. 1995; 33:138-140. 19. Sawada K. Descriptive catalogue of Formosan fungi II. Dept Agri Gov Res Inst Formosa Japan Rept. 1922; 2:27-31. ~ 2501 ~