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AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA
ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2014.5.1...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
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2ºC for 5-7 days. The colonies which appeared were
transplanted to PDA medium...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
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maintained for each treatment. The percent growth
inhibition was calculated b...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
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Brunner et al., 2003). The diffusion of these enzymes
dissolves cell fragment...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
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Effect of filterate concentrations of T. harzianum that
collected at differen...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
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Table 4. Effect of different filtrate concentrations of T. harzianum grown on...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
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Table 6. Effect of different filtrate concentrations of T. harzianum grown on...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
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produce various defense enzymes and secondary
metabolite containing antibioti...
Agric. Biol. J. N. Am., 2014, 5(1): 15-23
23
Monte E (2001). Understanding Trichoderma: between
biotechnology and microbia...
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Study of antagonistic capability of trichoderma harzianum isolates against some pathogenic soil borne fungi

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Study of antagonistic capability of trichoderma harzianum isolates against some pathogenic soil borne fungi

  1. 1. AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2014.5.1.15.23 © 2014, ScienceHuβ, http://www.scihub.org/ABJNA Study of antagonistic capability of Trichoderma harzianum isolates against some pathogenic soil borne fungi Sajid Salahuddin AL-Saeedi Bihar Moqdad AL-Ani Department of Biology, College of Science, Anbar University, Ramadi, Iraq. ABSTRACT In this study in vitro potential of two isolates of T.harzianum (T1, T2) were evaluated against seven soil borne pathogenic fungi isolates ( Acremonium sp. , Alternaria sp. , Aspergillus sp., Penecillium sp. , Pythium sp. , Rhizoctonia sp. and Verticillium sp.) in dual culture teqniques and through production of volatile inhibitors at two incubation periods (5 and 7 days)and non-volatile inhibitors collected at four incubation periods (3,6,9 and 12days) and different concentrations (25,50,75 and 100%) were investigated to evaluate their effects on pathogens mycelia. T2 isolate showed highest inhibitory effect of mycelial growth of pathogens (45.99%). Maximum effect noticed in Alternaria-T2 interaction (51.18%). Effect of volatile metabolites at 7day seems to be more effective on mycelial growth of all pathogens than the 5 day periods. Maximum growth inhibition noticed against Rhizoctonia sp. by T2 isolates (53.66%). A variability among the collection periods of T2 cultural filtrates and the fungal pathogens was evident. Generally, the had concentration (100%) of T2 isolate had highest growth reduction in all pathogens. Keywords: bio control, Trichoderma harzianum , soil borne fungi . INTRODUCTION Soil borne plant pathogens such as bacteria, fungi and nematodes annually create a major economically losses in many important crops. Toxicological environmental and sociological concerns have led to drastic reduction in the availability of efficient commercial compounds, and also the use of fungicides may lead to the appearance of new resistant strains of pathogens. The biological control is the best alternative especially against soil borne pathogens. Biological control of pathogens, i.e., the total or partial destruction of pathogen populations by other organisms, occurs routinely in nature (Hajiehgrari et al, 2008). Among the various antagonists used for the management of plant diseases. Trichoderma spp. have been widely studied as potential biocontrol agents for controlling many plant pathogens (Agrios, 2005; Lynch,1990).Most of the studies on Trichoderma species have been conducted with respect to their activity as biological control agents against fungal pathogens (Papavizas,1992, Sahebani and Hadavi , 2008, Hanada,2009). The antagonistic activity of Trichoderma depends on multiple synergistic mechanisms (Nallathambi et al, 2009; Howell, 2003). The various mechanisms include antibiosis, parasitism, inducing host-plant resistance, competition, secretion of chitinolytic enzymes, mycoparasitism and production of inhibitory compounds (Harman et al., 2004). Trichoderma produces a number of secondary metabolites with biological activity(Harman, 2006; Ghisalberti and Sivasithamparam, 1991). Many strains are strong opportunistic invaders, fast growing, prolific producers of spores and powerful antibiotic. Consequently, these fungi are ecologically very successful since strains have been found in all climatic zones (Monte, 2001). The aim of this study was to evaluate the biological potential of Trichoderma harzianum gainst some soil borne plant pathogens. MATERIALS AND METHODS: Isolation and identification of Trichoderma species : Two isolates of Trichoderma harzianum were used in this study. The first one (T1) was isolated from polluted cultures of the fungus Pleurotus sp. and the other isolate (T2) was isolated from palm debris, on Potato Dextrose Agar (PDA) and incubated at 26±
  2. 2. Agric. Biol. J. N. Am., 2014, 5(1): 15-23 16 2ºC for 5-7 days. The colonies which appeared were transplanted to PDA medium for purification using the method of Raper and Fennell (1965). Isolation and identification of phytopathogenic fungi : cremonium sp. , Alternaria sp., Aspergillus sp., Penicillium sp. were isolated using dilution plate technique (Johnson et al., 1959) on PDA with Rose Bengal 0.035 g/L and Streptomycin 1/1000M (3ml/L) and incubated at 26±2ºC for 10 days .The colonies were counted and identified. Pythium sp. and Verticillium sp. were isolated from Cress roots while Rhizoctonia sp. was isolated from Okra roots. Diseased root 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 five minutes; they washed in sterile distilled water and dried, sterile filter paper. Then pieces 1cm long plated onto PDA and incubated at 26± 2ºC for 5-7 days. The colonies which appeared were transplanted to PDA medium for purification. Stock cultures of fungi were maintained on PDA slants and stored at 4°C in refrigerator. Fungi were identified based on the microscopic observations and colony characteristics following the taxonomic key of Watanabe, (2002). Microscopic examination was carried out by mounting the culture in lacto phenol cotton blue. Evaluation of Dual culture on agar plates: To evaluate the activity of Trichoderma isolates(T1,T2) against pathogenic fungi, two discs (5mm) obtained from one week old culture on PDA, the first one carrying the stock of the antagonistic agents (T1 and T2) and the other of the pathogenic agents were placed at the periphery of Petri plate (9cm in diameter) at the distance . One disc of each pathogenic agent was maintained as control (alone culture). Each replicates have three plates. Both the dual and alone cultures were incubated at 26±2°c for one week and measurement of radial mycelial growth of the fungus was taken every 24 hours. The percentage inhibition of growth (I) was calculated using the formula given below: [I (%) = (1 – T /C) x 100]…...1 Where: I= Inhibition Percentage of pathogen growth by antagonists. C=Radial growth in control. T=Radial growth in the treatment (Mokhtar and Aid ,2013). Evaluation of dual culture using slide methods: For each pathogen-T.harzianum interaction, a clean slide was placed in 9 cm diameter plates and sterilized. Then a small amount of autoclaved melted potato dextrose agar was spread over the slide to make a thin PDA film on the slide. The 5 mm discs of one week old of each pathogen and T. harzianum isolates were placed on the opposite sides of the slide 3 cm apart on the PDA surface. Then 5ml of distilled water was added to the plate to prevent drying and then incubated at 26 ± 2ºC for a week. At the end of incubation period, region of contact between T. harzianum–Pathogen hyphae was stained with lactophenol and cotton blue and examined under a light microscope (Hajieghrari et al., 2008). Effect of volatile metabolites: The effect of volatile metabolites produced by the antagonistic microorganisms on pathogens, mycelial growth was determined by the method described by Dennis and Webster (1971) and Goyal et al. (1994). The antagonistic fungi were centrally inoculated by placing 5 mm diameter mycelia disc taken from one week old culture on the PDA plate and incubated at 26±2°C for 2 days. The top of each Petri dish was replaced with bottom of the PDA plate inoculated centrally with the pathogen. Two plates were sealed together with paraffin tape and further incubated at 25ºC. In control treatment, T.harzianum isolates replaced with inoculum of 5 mm of sterile PDA medium only . Colonies diameter of the pathogen was measured at 5 and 7 days after incubation and the inhibition of mycelial growth was calculated. The percent growth inhibition was calculated by using equation 1(Dolatabadi et al., 2011). Effect of non-volatile inhibitors : To determine the effects of nonvolatile metabolites of T.harzianum isolate on mycelia growth of the pathogens, three discs of mycelial agar plugs (5 mm diameter) were removed with a cork borer from the edge of the young culture. T.harzianum isolates were inoculated in 100 ml sterilized potato dextrose broth (PDB) in 250 ml flasks and incubated at 26 ± 2ºC on a rotary shaker set at 150 rpm for 3,6,9 and 12 days. The culture was filtered through Millipore filter to removing mycelial mats and then sterilized through biological membrane filter (0.2 μm). Certain numbers of wells were prepared on PDA cultured with the pathogenic fungi (after 2 days for Alternaria sp., Pythium sp. , Verticillium sp. and 4days for Aspergillus sp. and 5days for Rhizoctonia sp.) with the help of sterile cork borer (9 mm in diameter), 0.2 ml of the Trichoderma culture filterate concentrations (25,50, 75,100%) were introduced into the wells . Controls were made by using the distilled water. Plates were then incubated at 26±2°C for 7days. Three replications were
  3. 3. Agric. Biol. J. N. Am., 2014, 5(1): 15-23 17 maintained for each treatment. The percent growth inhibition was calculated by using equation (1) (Dolatabadi et al., 2011). Statistical analysis: Complete Randomized Design (C.R.D.) was used as an experimental design. Data were analyzed by using statistical analysis system- IBM SPSS statistics 21. Least significant difference (LSD) was used to compare the significant difference between means at P≤ o.o5. RESULTS AND DISCUSSION: Both of Trichoderma isolates were identified as T. harzianum according to the taxonomic key of Watanabe (2002). The results of dual culture indicated that both isolates of T.harzianum inhibited the growth of pathogenic fungi at varying degrees (Table1). It was found that T.harzianum2 inhibited the growth of pathogens (45.99%) more than T.harzianum1 isolate (41.08%). Hence this isolate was selected for subsequent investigations. 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 ( Siameto et al.,2011). The variation in fungicidal activity among the two T.harzianum isolates could be attributed to the presence of different types of chemical constituents in different isolates (Wang et al., 2003; Zhou et al., 2008; Eneyskaya et al., 2009; Yang et al., 2009).On the other hand the pathogenic fungi affected by the antagonistic fungi at different rates, maximum growth inhibition was recorded by Aletrnaria sp. (51.15%) followed by Pythium sp. (46.77%) and Verticillium sp. (46.23%) and Rhizoctonia sp. (45.11%) respectively. Whereas Acremonium sp. was less affected fungi (25%) (table1). The differences in average responses to antagonism by T.harzianum isolates between the different genera of pathogens indicated that several gens of both the antagonist and pathogen must be involved in regulating the different levels of antagonism observed in the study (Bell et al., 1980). Table 1: Inhibition percentage of the mycelia growth of isolated fungi by Trichoderma harzianum, in the dual cultures, on PDA medium. Mean ebarnrireb nIitIbihnI Pathogenic fungi T.harzianum 2T.harzianum 1 25.00 C 28.9521.05* Acremonium sp. 51.18 A 52.6549.71 Alternaria sp. 45.11 AB 46.3243.89 Aspergillus sp. 43.74 B 50.3237.15 Penicillium sp. 46.77 AB 47.0646.47 Pythium sp. 46.23 AB 47.2545.20 Rhizoctonia sp. 46.77 AB 49.4144.12 Verticillium sp. 45.99 A 41.08 B Mean * values are average of 3 replicates. Means with the same letter in each column are not significantly different in L.S.D. at (P ≤.05). The direct confrontation of T. harzianum against the different fungal isolates in vitro on PDA medium showed that when the mycelium of both cultures came in contact with each other the hyphal growth of the pathogenic fungus were found to be inhibited by the hyphae of T. harzianum. A clear zone of interaction was formed in all Trichoderma-pathogen combination except in the interacting between T.harzianum and Aspergillus sp., an inhibition zone without physical contact between the colonies around the pathogen colony was observed. The presence of an inhibition zone in dual culture without the hyphae contact suggests the secretion of diffusible non-volatile inhibitory substance by the T.harzianum isolate. Previous studies have demonstrated that before mycelia of fungi interact, Trichoderma sp. produces low quantities of extracellular exochitinases (Kullnig et al., 2000;
  4. 4. Agric. Biol. J. N. Am., 2014, 5(1): 15-23 18 Brunner et al., 2003). The diffusion of these enzymes dissolves cell fragments of host cells. These cell fragments in turn induce the production of further enzymes and trigger a cascade of physiological changes, stimulating rapid and directed growth of Trichoderma sp. (Zeininger et al., 1999). Microscopically observation of hyphal interaction indicated that antagonistic hyphae coiled around the hyphae of pathogen, mycelia denatured and inhibited them. T.harzianum either formed hook or bunch like structure around the hyphae of pathogens before penetration or entered directly. No hyphae coiling was observed in Aspergillus-Trichoderma interaction. T.harzianum prevented the spore's formation of the Alternararia sp. and Aspergillus sp. and seclerotia in Rhizoctonia sp. in compare with controls (fig.1). Fig 1:Microscopic observations showed the effect of T. harzianum on mycelia of Pthogenic fungi by:Coiling the hyphae of T. harzianum (black arrows) around the hyphae of:A,B= Alternaria sp., ,C=Penicillium sp., D,E= Pythium sp.,F=Verticillium sp. and on spore formation (white arrows) of: G=Alternaria sp. Compared with control (H), I=Aspergillus sp. Compared with control (J), K=the effect of T. harzianum on cytoplasm (voculation) in Rhizoctonia sp. (red arrow) as compared with control (L). Microscopic observation of the interaction region between T.harzianum-pathogens revealed that T.harzianum has a high inhibitory effect against the different isolated fungi, with a several biological modes like mycoparasitism and antibiosis. This results has been reported and confirmed by Siameto et al. (2011); Mokhtar and Aid (2013). Effect of volatile metabolites on the growth of pathogens presented in table 2 showed that the volatile metabolic substances of antagonistic fungus (T2) inhibited the mycelia growth of the different pathogenic fungi, with different rates over the five days of treatment, the percentage inhibition of mycelia growth reached 43.75% for Rhizoctonia sp. and decreased to 35.86%, 29.62% and 26.8% for Aspergillus sp., Alternaria sp. and Pythium sp. respectively, the minimum ratio was recorded in Verticillium sp.(23.7%). After seven days of incubation, more effects of mycelial growth inhibition were observed, T. harzianum (T2) caused maximum growth inhibition against Rhizoctonia sp. (53.66%) followed by Aspergillus sp. and Alternaria sp. (39.3% and 36.28% respectively), but in the Pythium sp. and Verticillium sp. have recorded the lowest inhibition percentages (19.61% and 18.43% respectively) (table 2). These observations are similar to those of Ayoubi et al. (2012) findings. The earlier studies also revealed that antimicrobial metabolites such as aromatic pyrone antibiotics (Keszler et al., 2000). A E I B C D F G H J K L
  5. 5. Agric. Biol. J. N. Am., 2014, 5(1): 15-23 19 Effect of filterate concentrations of T. harzianum that collected at different times on mycelial growth of test pathogenic fungi was revealed that the aqueous extracts of T. harzianum reduced the mycelial growth of the target fungal pathogens. However, variability among the collection times of cultural filtrates and the fungal pathogens was evident. Table 2: Inhibition percentage of the mycelia growth of isolated fungi by the volatile substances of T. harzianum, in the dual cultures, on PDA medium. Inhibition percentage of mycelia growth after: Pathogenic fungi 7days5days 36.28 B 29.62* AB Alternaria sp. 39.30 B 35.86 AB Aspergillus sp. 19.61 C 26.80 AB Pythium sp. 53.66 A 43.75 A Rhizoctonia sp. 18.43 C 23.70 B Verticillium sp. 7.19610.391LSD * values are average of 3 replicates. Means with the same letter in each column are not significantly different in L.S.D. at (P ≤.05). Maximum growth reductions in all studied pathogenic fungi were observed with increasing concentrate- ions (Tables 3,4,5,6,7). A similar effects were observed by Anita et al. (2012) when studying the effect of secondary metabolites secreted by T. atroviride at different concentrations and found rapid decrease observed in the linear growth of the pathogen with increased concentration of the metabolites. In Alternaria sp. there was remarkable increase in growth inhibition at the treatment with T.harzianum filtrates that collected after 6 and 9 days of incubation which were 58.75% and 56.43% respectively. Whereas, less inhibition effect was recorded at the treatment with filtrates that collected after 12 days of incubation (48.57%) (Table 3). In Aspergillus sp. highest growth inhibition was noticed at the treatment with T.harzianum filtrates that collected after 3and 9 days of incubation (46.91% and 48.85% respectively). Less inhibition effect was recorded at the treatment with filtrates that collected after 6 and 12 days of incubation (42.09% and 45.47%)(Table 4) .The maximum inhibition percentage was recorded in Pythium sp. by treated with collected T.harzianum filtrates after 6 days (71.39%) and minimum inhibition percentages were observed by treated the same fungus with collected T.harzianum filtrates after 9 and 12 days (60.31% )(table 5), while in Rhizoctonia sp. no significant differences noticed in response to tested T.harzianum filtrate(table 6). The maximum inhibition percentage was observed by treated Verticillium sp. with filtrate collected at 6 and 3 days (63.53% and 60.94%) and the activity was decreased at 9 and 12 days which were 58.13% and 57.81% respectively(table 7). Table 3. Effect of different filtrate concentrations of T. harzianum grown on P.D medium at different incubation times on the growth of Alternaria sp. Mean Concentration / inhibition Percentage Incubation time 100% 75%50%52% 53.44 B 61.2552.55050* 3days 58.75 A 62.5063.7556.2552.5 6 days 56.43 AB 61.4357.1454.2952.86 9 days 48.57 C 48.5748.5748.5748.57 12 days 58.44 A 55.49 A 52.28 B 50.98 B Mean * values are average of 3 replicates. Means with the same letter in each column are not significantly different in L.S.D. at (P ≤.05).
  6. 6. Agric. Biol. J. N. Am., 2014, 5(1): 15-23 20 Table 4. Effect of different filtrate concentrations of T. harzianum grown on P.D medium at different incubation times on the growth of Aspergillus sp. Mean Concentration / Inhibition Percentage Incubation time 100%75%50%52% 46.91 A 53.67 a 45.95 b 45.95 b 42.08 b 3days 42.09 B 51.74 ab 45.95 b 40.15 b 30.50 c 6 days 48.85 A 49.81 ab 49.81 ab 49.81 ab 45.95 b 9 days 45.47 B 49.81 b 44.02 b 44.02 b 44.02 b 12 days 51.26 A 46.43 AB 44.98 B 43.58 C Mean * values are average of 3 replicates. Means with the same letter in each column are not significantly different in L.S.D. at (P ≤.05). Table 5. Effect of different filtrate concentrations of T.harzianum grown on P.D medium at different incubation times on the growth of Pythium sp. Mean Concentration / Inhibition Percentage Incubation time 100%75%50%52% 66.18 B 68.2468.2464.7163.53* 3days 71.39 A 76.6772.2268.8967.78 6 days 60.31 AB 61.2561.2560.0058.75 9 days 60.31 C 63.7560.0044.2057.50 12 days 67.48 A 65.43 B 63.40 BC 61.89 C Mean * values are average of 3 replicates. Means with the same letter in each column are not significantly different in L.S.D. at (P ≤.05).
  7. 7. Agric. Biol. J. N. Am., 2014, 5(1): 15-23 21 Table 6. Effect of different filtrate concentrations of T. harzianum grown on P.D medium at different incubation times on the growth of Rhizoctonia sp. Mean Concentration / Inhibition Percentage Incubation time 100%75%50%52% 48.0350.9849.0149.0143.13 3days 51.2252.9450.9851.9649.01 6 days 43.1447.0645.1045.0935.29 9 days 47.4049.0247.0646.4447.06 12 days 50.0048.0448.1343.62 Mean Table 7. Effect of different filtrate concentrations of T. harzianum grown on P.D medium at different incubation times on the growth of Verticillium sp. Mean Concentration / Inhibition Percentage Incubation time 100%75%50%52% 60.94 A 63.7562.5060.0057.50 3days 63.53 A 64.7164.7162.3562.36 6 days 58.13 B 60.0057.5057.5057.50 9 days 57.81 B 61.2560.0055.0055.00 12 days 62.43 A 61.18 A 58.71 B 58.09 B Mean * values are average of 3 replicates. Means with the same letter in each column are not significantly different in L.S.D. at (P ≤.05). The results of non-volatile substance against the pathogens that were more effective support this hypothesis. The results were in agreement with Hajieghrari et al. (2008) who evaluated six isolated of Trichoderma sp. against five isolates of soil borne phytopathogenic fungi in dual culture techniques and through production of volatile and non-volatile inhibitors. The different effects of T.harzianum culture filtrates collected at different incubation times between tested pathogenic fungi due to T.harzianum ability to
  8. 8. Agric. Biol. J. N. Am., 2014, 5(1): 15-23 22 produce various defense enzymes and secondary metabolite containing antibiotics with varied nature, quantity and quality at different incubation times (Anita et al,2012; Perveen and Bokhari, 2012). Selection of biocontrol agents as well as understanding the mechanisms involved in the antagonistic effect of Trichoderma spp. on plant pathogens are important in designing effective and safe biocontrol strategies. Different isolates of Trichoderma have different combative ability for pathogen; their indirect effects may also vary. Therefore, one of the most interesting aspects of biology is the study of the mechanisms employed by biocontrol agents to affect disease control. Possible mechanisms of antagonism employed by Trichoderma spp. includes nutrient and niche competitions, antibiosis by producing volatile components and non-volatile antibiotics that are inhibitory against a range of soil borne fungi, as well as parasite- ism. ACKNOWLEDGEMENT We would like to thank Mrs. Mustafa (Department of Biology, College of Science, Anbar University, Ramad,Iraq) for contributing to this work. REFERENCES: Agrios,George N (2005). Plant Pathology. 5 th .ed. Academic Press,INC.Pp903. Anita S., Ponmurugan P. and Ganesh Babu R (2012). Significance of secondary metabolites and enzymes secreted by Trichoderma atroviride isolates for the biological control of phomopsis canker disease. 11(45): 10350-10357. Ayoubi N., Doustmorad Z. and Mansoureh M (2012). Combination of Trichoderma species and Bradyrhizobium japonicum in control of Phytophthora sojae and soybean growth. J. Crop Prot. 1(1): 67- 79. Bell D.K., Wells H.D. and Markham C.R (1980). In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology .72:379-382. Brunner K., Peterbauer CK., Mach RL., Lorito M., Zeilinger S. and Kubicek RL. (2003). The Nacetylgucosaminidase of Trichoderma atroviride is essential for chitinase induction by citin of and major relevance to bio-control. Curr. Gen. 43:289-295 Dennis C. and Webster J (1971). Antagonistic properties of species group of richoderma III. Hyphal interaction. Trnas. Briticsh Mycological Soceity. 57:363-369. Dolatabadi K.H., Goltapeh E.M., Varma A. and Rohani N (2011). In vitro evaluation of arbuscular mycorrhizal- like fungi and Trichoderma species against soil borne parhogens. J. Agric. Technol. 7(1): 73-84. Eneyskaya EV., Sundqvist G., Golubev AM., Ibatullin FM., Ivanen DR., Shablin KA., Brumer H., Kulminsskaya AA (2009). Transglycosylating and hydrolytic activics of the ß-mannosidase from Trichoderma reesei. Biochimie. 91:632-638. Ghisalbert EL and Sivasihmparm K (1991). Antigungal Trichoderma spp. Soil antibiotics produced by Biol. Biochem. 23:1011-1020 Goyal S.P., Jandiak C.L. and Sharma V.P (1994). Effect of weed fungi metabolites on the mycelial growth of A. bisporus (Lang.). Imbach. Mushroom Research. 3: 69: 69-74. Hajiehgrari Behzad, Mousa Torabi-Giglou, Mohammed Mahdi Davari Reza Mohammadi and (2008). Biological potential of some Iranian Trichoderma isolates in the control of soil borne plant pathogenic fungi. African Journal Bioteol. Control. 50: 143-149 Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004). Trichoderma species-opportunistic, a virulent plant symbionts. Nat. Rev. Microbiol. 2:43-56. Harman GE (2006). Overview of mechanisms and uses of Trichoderma spp. J. Phytopathol. 96: 190-194 Howell CR (2003). Mechanisms employed by Trichoderma species in the biological control of plant disease: the history and evolution of current concepts. Plant Dis. 87:4-10. Johnson LE., Curl J., Bond, Fribourg, H (1959). Methods for studying soil microflora-plant disease relationships. Minneapolis: Burgess Publishing Company.. Keszler A., Forgacs E., Kotai L., Vizcaino JA., Monte E. and Garcia-Acha I (2000). Separation and identification of volatile components in the fermentation broth of Trichoderma atroviride by solid-phase extraction and gas chromatography-mass spectrometry. J. Chromate Science. 38:421-424.. Kullnig C., Mach RL., Lorito M. and Kubicek CP (2000). Enzyme diffusion from Trichoderma atroviride to Rhizoctonia solani is a perequisite for triggering of Trichoderma ech 42 gene expression before mycoparasitic contact. Appl. Environ. Microbiol. 66:2232-2234. Lynch, J. M. Fungi antagonists. In: R. R. Baker, P. E. DUNN(eds.) (1990). New directions in biological control: Alternatives for suppressing agricultural pests and disease, 243-253. Alan R. Liss, New york. Mokhtar H. and Aid D (2013). Contribution in isolation and identification of some pathogenic fungi from wheat seeds, and evaluation of antagonistic capability of Trichderma harzianum against those isolated fungi in vitro. Agric. Boil. J. N. Am. 4(2): 145-154.
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