USE OF Trichoderma harianum KRL- AG 2 AGAINST PHYTOPATHOGENIC FUNGI, LIBYA

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1 USE OF Trichoderma harianum KRL- AG 2 AGAINST PHYTOPATHOGENIC FUNGI, LIBYA Zahra Ibrahim El-Gali Department of Plant Protection, Faculty of Agriculture, Omer Al-Mukhtar University, El-Beida, Libya: Article Information Abstract Article history: Received: 30 April Received in revised form: 22 May Accepted: 25 May Available online: June Keywords: Trichoderma harianum soil-borne fung Phytopathogenic fungi inhibitory effect Corresponding Author: Zahra Ibrahim El-Gali Zelgali@yahoo.com 2015 Danish Journals All rights reserved In this study, isolate of T. harzianum was tested for antagonistic action against four soil borne phytopathogenic fungi using dual culture assay. T. harzianum grow faster than the isolated fungi considerable antagonistic effect on mycelium growth of the pathogens in dual culture compared to the control. Maximum inhibition occurred in Botrytis cinerea interactions (67.5%) followed by Macrophomina phaseolina (66.1%), Fusarium solani (51.1%) and Sclerotinia sclertiorum (44.4%) with zone inhibition was observed in S. sclerotiorum only. To Cite This Article: Zahra Ibrahim El-Gali,Department of Plant Protection, Faculty of Agriculture, Omer Al-Mukhtar University, El-Beida, Libya Danish journal of psychology,6-10, 2015 Introduction Soil borne pathogens cause important plant diseases, which play a direct role in the destruction of natural resources in agriculture. The distribution of several phytopathogenic fungi, such as Botrytis cinerea, Fusarium solani f. sp. phaseoli, Macrophomina phaseolina and Sclerotinia sclerotiorum, have spread during the last few years due to changes introduced in farming, with detrimental effects on crops of economic importance (Baker and Paulitz, 1996; El-Gali, 2003; El-Gali, 2008). Fungal antagonist that is, Trichoderma was evaluated as potential bio-control agent against number of fungal phytopathogens. Species of the genus Trichoderma are well documented fungal biocontrol agents (Elad and Kapat, 1999; Howell, 2002). The antagonistic action of Trichoderma species against phytopathogenic fungi might be due to either by the secretion of extracellular hydrolytic enzymes (Di Pietro, et al., 1993; Schirmbock et al., 1994), by the production of antibiotics (Howell, 1998), by competition (Harman, 2004; Howell, 2003) or by predator (El-Gali, 2015). The objective of the present investigation was isolation and screening of effective Trichoderma harianum KRL-AG 2 against some of plant pathogenic fungi. MATERIAL AND METHODS Isolation of phytopathogenic fungi. Diseased plant tissues were obtained from vegetables local field, in El-Beida city. Sclerotinia sclerotiorum was isolated from Cucumber (Cucmis sativus), Botrytis cinerea from Onion (Allium cepa), Macrophomina phaseoli and F. solani f. sp phaseoli from Beans (Phaseolus vulgaris). Diseased plant tissues were washed under running tap water to remove surface soil, dust and other contaminants. Tissue pieces were cut out from the leading edge of lesion, and placed in one percent sodium hypochlorite for two minutes, then washed in sterile distilled water and dried on sterile filter paper. The dried pieces were cut into approximately one centimeter pieces, plated onto PDA and incubated at 25 o C. Isolation and identification of Trichoderma harzianum Trichoderma was isolated from Apple fruits (Golden delicious) are selling in local market and identification in plant pathology lab., College of Agriculture, University of Omer Al-Mukhtar, Libya. Evaluation of Dual culture on agar plates Antifungal activity of of T. harzianum against B. cinerea, F. solani f. sp. phaseoli, M. phaseolina and S. sclerotiorum. was tested on dual culturing method (Rao, 2003). All the isolates were grown on sterilized standard PDA (Potato Dextrose Agar) at 25ºC in an incubator for 5 days in order to obtain juvenile colonies for the studies of antagonism. After the incubation period of 5 days, five millimeter diameter mycelial plugs of each isolated fungi were placed at the periphery of culture plates and on the same day. Antagonist T. harzianum was placed on the opposite side of the same previous petri- dishes, incubated at 25 ± 2 o C. The antagonism was evaluated after 5 days, and one isolate was done with three repetitions, Observation of antagonism was recorded 6

2 by antagonism index and also by recorded per cent growth inhibition according to formula as proposed by Datta et al (2004): %I = [(C-T)/C] x100). Where: I= percentage inhibition of pathogen by antagonists. C= radial growth in control. T= radial growth in the treatment. The degree of antagonistic activity was estimated as follows (Sookchaoy, et al., 2009): 4 very high antagonistic activity (R > 75), 3 high antagonistic activity (R = 61-75), 2 moderate antagonistic activity (R = 51-60), 1 low antagonistic activity (R < 51). And the width of zone of inhibition (ZI) measured as the smallest distance between the colonies in the dual culture plate (Reddy and Hynes, 1993). Statistical analysis Data were converted by using the formula Arc sin x (YazdiI Samadi, 1997). Complete randomized design was used and the treatments were replicated three times. The data on effect of the treatments on the growth of pathogens was analyzed by analysis of variance (ANOVA), and treatment means were compared by using Duncan s multiple range test (DMRT) and least significant difference test (LSD) at P = RESULTS AND DISCUSSION Experiment was conducted in order to study the behavior of T. harianum against the tested fungi. M. phaseolina showed initial faster growth and covered more than half of the plate within 2 days Later, Trichoderma overgrew on Macrophomina (at red arrow) and destroyed it completely after 5 of incubation (Fig.1- a). In this study I observed no inhibition halo between M. phaseolina, B. cinerea, F. solani and T. harzianum colonies, which suggests that the antagonistic effect of T. harzianum isolates is based on the competition for niche and nutrients and not on a chemical aggressiveness or classic antibiosis. This result is harmony with (Fiume and Fiume, 2006) reported no inhibition halo between B. cinerea and T. harzianum colonies, Fig. 1. Plate assay for antagonistic activity of T. harzianum on fungal pathogens; (a) : Trichodermal growth and inhibition of M. phaseolina, (b): F. solani, (c): B. cinerea and (d): S. sclerotiorum. (white line marks show Trichodermal growth), (red arrow marks show: Trichodermal overgrowth), (yellow arrow marks show inhibition zone between them), initial sporulation (at green arrow) by Trichoderma is seen on and in vicinity of Sclerotinia colony. 7

3 From data presented in (Table 1and Fig. 2) it was evident that T. harzianum significantly decreased the mycelial radial growth of isolated pathogenic fungi. T. harzianum retarded colony growth of the pathogen and inhibited its growth. 67.5%, 66.1%, 51.1% and 44.4% in case of B. cinerea, M. phaseolina, F. solani and S. sclerotiorum respectively. The antagonistic T. harzianum which caused reduction by growing over the pathogen did so by fast growing and better saprophytic activity than the pathogen which could not complete. However, according to the scale used by Sookchaoy et al. [20], moderate antagonistic activity (3 points on a 1-4 scale) was shown by T. harzianum + M. phaseolina and T. harzianum + B. cinerea (Table 3, Fig. 2). Table 1. Inhibition of the growth of B. cinerea, M. phaseolina,, F. solani and S. sclerotiorum by T. harzianum isolate and its antagonistic activity against these pathogens in dual culture test. antagonistic zone Mean Linear Average degree of Treatments activity (on 1- Inhibition Trichoderma colonization growth (cm) growth inhibition (%) 4 scale)* mm (ZI) Complete colonization over the M. phaseolina + T. harzianum 3.5 c 66.1 (54.39) b 3 - M. phaseolina control 9.0 a e - - F. solani + T. harzianum 4.4 c 51.1 (45.63) c 2-2/3 colonization over the F. solani control 9.0 a e - B. cinerea + T. harzianum 3.0 c 67.5 (55.24) a 3-2/3 colonization over the B. cinerea control 9.0 a e - S. sclerotiorum + T. harzianum 5.0 b 44.4 (41.78) d 1 5 1/2 colonization over the S. sclerotiorum control 9.0 a e LSD at Each value is a mean of 5 replicates. *1 = low antagonistic activity (R < 51), 2 = moderate antagonistic activity (R = 51-60), 3 = high antagonistic activity (R = 61-75), 4 = very high antagonistic activity (R > 75) Values between brackets are arcsine square root of transformation percentage of inhibition. Values within column followed by the same letter(s), are not significantly different at (p= 0.05). T. harzianum inhibited the growth of the target organisms through its ability to grow much faster than the pathogenic fungi thus competing efficiently for space and nutrients. Starvation was the most common cause of death for microorganisms, so that competition for limiting nutrients resulted in biological control of fungal phytopathogens. Fig. 2. Effect of T. harzianum on growth of Macrophomina, Fusarium, Botrytis and Sclerotinia. Antibiotic production, mycoparasitism, the production of cell wall-degrading enzymes and competition for nutrients or space are considered as the actions involved in biocontrol of pathogen (Vinale, et al., 2008; Zeilinger, and Omann, 2007). During mycoparasitic interactions between Trichoderma and fungal pathogen, diffusible factor released from the host before physical contact was responsible for induction of hydrolytic enzymes (Cortes, et al., 1998; Zeilinger, and Omann, 2007; Zeilinger, et al., 1999).. During direct contact, lectins in the host s cell wall can induce coiling of the Trichoderma around the host hyphae andmycoparasite 8

4 can produce appressorium-like structures to destroy the pathogen (El-Gali, 2015; Zeilinger, and Omann, 2007). According to Zeilinger and Omann (2007), both enzyme production and infection structure formation are induced responses activated by the diffusible factor. Benhamou and Chet (1993) illustrated many interactions of Trichoderma with pathogens (Rhizoctonia and Pythium), such as Trichoderma had grown parallel to pathogen, grown along the pathogen and grown around the pathogen. Normal degradation of pathogen mycelia would take place after penetrating with appressorium-like structures and they had observed the growth of Trichodermal hyphae within the pathogen Benhamou and Chet, 1993). Claydon et al. (Claydon, et al., 1987) reported inhibition due to antibiotics trichodermin, harzianum A and harzianolide. These initial results indicated that present strain of Trichoderma can be used as a biocontrol agent against the tested fungal pathogens. REFERENCES 1- Baker R and Paulitz T.C Theoretical basis for microbial interactions leading to biological control of soilborne plant pathogens In: Principles and Practice of Managing Soilborne Plant Pathogens. Hall, R. (ed.). The American Phytopathol. Soc. St. Paul, MN., pp Benhamou, N., Chet, I., Hyphal interaction between Trichoderma harzianum and Rhizoctonia solani: ultrastructure and gold chemistry of the mycoparasitic process. Phytopathology 83: Claydon, N., Allan, M., Hanso, J. R. and Avent, A. G Antifungal alkyl pyrones of Trichoderma harzianum. Transactions British Mycological Society, 88: Cortes, C., Gutierrez, A., Olmedo, V., Inbar, J., Chet, I., Herrera-Estrella, A., The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor. Mol. Genet. Genomics 260: Datta, B.S., A.K. Das and S.N. Ghosh, Fungal antagonists of some plant pathogens. J. Mycol. Plant Pathol., 42: Di Pietro A, Lorito M, Hayes C, Broadway K, Harman GE (1993). Endochitinase from Gliocladium virens. Isolation, characterization, synergistic antifungal activity in combination with gliotoxin. Phytopathology 83: Elad Y, Kapat A (1999). The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. Eur. J. Plant Pathol. 105: El-Gali, Z.I Histopathological and biochemical studies on bean seeds infected by some seed-borne fungi. PhD. Thesis. Department of Agricultural Botany. Alexandria University. 9- El-Gali, Z.I Evaluation susceptibility of some chick-pea cultivars to root- rot and damping-off disease caused by Macrophomina phaseolina Arab J. Plant Prot., 26: (In Arabic). 10- El-Gali, Z.I Antagonism capability in vitro of Trichoderma harzianum against Alternaria alternata on Ceratonia siliqua. EJPMR, 2(2): Fiume F. and Fiume G., Biological control of Botrytis gray mould on tomato cultivated in greenhouse. Commun Agric. Appl. Biol. Sci. 71(3 Pt B): ] Harman, G. E. (2004). Trichoderma species opportunistic, avirulent plant symbionts. Nature Reviews Microbiology 2: Howell CR (1998). The role of antibiosis in biocontrol. In: Harman GE, Kubicek CP(eds) Trichoderma and Gliocladium, vol. 2.Taylor & Francis, Pads tow, pp Howell CR Cotton seedling pre-emergence damping-off incited by Rhizopus oryzae and Pythium spp. and its biological control with Trichoderma spp. Phytopathology 92: Howell, R.C Mechanisms employed by Trichoderma species in the biological control of plant diseases, the history and evolution of current concepts. Plant Disease 87: Rao, N.S.S., Methods used in soil Microbiological studies. Soil Microbiology. 4 th Edition. pp: (Oxford and IBH Publishing Co. Pvt. Ltd. New Delhi. 17- Reddy, M.C., and Hynes, R.K Relationship between in vitro growth inhibition of pathogens and suppression of percentage damping off and post emergence root rot of white bean seedlings in the green house by bacteria. Can. J. Microbiol. 40: Schirmbock M, Lorito M, Wang YL, Hayes CK, Arisan-Atac I, Scala F, Harman GE, Kubicek CP Parallel formation and synergism of hydrolytic enzymes and peptaibolantibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl. Environ. Microbiol. 60:

5 19- Sookchaoy K., Panthhachode S., Thipchu J., Screening of Trichoderma spp. for Phytophthora root and foot rot on Citrus sinensis biocontrol. Intl Conf. on the Role of Universities in Hands-On Education, August, Thailand: Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Marra, R., Barbetti, M.J., Li, H., Woo, S.L., Lorito, M., A novel role for Trichoderma secondary metabolites in the interactions with plants. Physiol. Mol. Plant Pathol. 72: YazdiI Samadi B, Rezaei A, Valyzadeh M Statistical Design in Agricultural Research. Teh. Uni. Pub. 234 pp. 22- Zeilinger, S., Omann, M., Trichoderma biocontrol: signal transduction pathways involved in host sensing and mycoparasitism. Gene Regul. Syst. Biol. 1: Zeilinger, S., Galhaup, C., Payer, K., Woo, S.L., Mach, R.L., Fekete, C., Lorito, M., Kubicek, C.P., Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host. Fungal Genet. Biol. 26: