Metabolites released from Trichoderma viride, T. polysporum, T. hamatum and T. aureoviride were tested in culture medium against Ceratocystis paradoxa, which causes black seed rot in oil palm sprouted seeds. The Trichoderma metabolites had similar fungistatic effects on the growth of C.paradoxa except those from T. aureoviride. The inhibition varied depending on the Trichoderma species producing the metabolites; from 2.0% to 64% in volatile, 0.0% to 74% in non-volatile and 0.0% to 81% from direct-diffusible metabolites. C. paradoxa growth was significantly reduced in the presence ofmetabolites produced by T. viride and T. polysporum than the other species. T. aureoviride had the least growth inhibition, and medium containing direct-diffusible metabolite supported highest inhibition of C. paradox

Adekunle, AA

Airede, CE

Eziashi, EI

Uma, NU

English

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African Journal of Biotechnology Vol. 5 (9), pp. 703-706, 2 May 2006 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2006 Academic Journals       Full Length Research Paper  Effect of metabolites produced by Trichoderma species against Ceratocystis paradoxa in culture medium  E.I. Eziashi1*, N.U. Uma2, A.A. Adekunle2, C.E. Airede1  1Nigerian Institute for Oil Palm Research (NIFOR), Plant Pathology Division, P.M.B. 1030 Benin City, Edo State. Nigeria. 2Department of Botany and Microbiology, University of Lagos, Akoka, Yaba, Lagos State. Nigeria.  Accepted 4 April, 2006  Metabolites released from Trichoderma viride, T. polysporum, T. hamatum and T. aureoviride were tested in culture medium against Ceratocystis paradoxa, which causes black seed rot in oil palm sprouted seeds. The Trichoderma metabolites had similar fungistatic effects on the growth of C. paradoxa except those from T. aureoviride. The inhibition varied depending on the Trichoderma species producing the metabolites; from 2.0% to 64% in volatile, 0.0% to 74% in non-volatile and 0.0% to 81% from direct-diffusible metabolites. C. paradoxa growth was significantly reduced in the presence of metabolites produced by T. viride and T. polysporum than the other species. T. aureoviride had the least growth inhibition, and medium containing direct-diffusible metabolite supported highest inhibition of C. paradoxa.  Key words: Metabolite, Trichoderma, Ceratocystis paradoxa, Fungistatic.   INTRODUCTION  The oil palm (Elaeis guineensis Jacq.) is said to be indigenous to West Africa (Zeven, 1965; Corner, 1966) but also can be found throughout a belt of land extending approximately between latitude 16oN and 10oS where annual rainfall ranges from 1524-3048 mm and temperatures average 30oC (Billows and Beekwith, 1913). The oil palm industry provides direct employment to about four million Nigerian people in about twenty oil palm growing states in Nigeria, and indirectly to other numerous people involved in processing and marketing (Ahmed, 2001). Despite the enormous potential of the oil palm, there is problem with soil borne fungus Ceratocystis paradoxa causing black seed rot disease in oil palm sprouted seeds. The cause of the dry basal rot in adult palm, conducted by Robertson (1962), was also shown to be due to the Ascomycete, C. paradoxa of which the imperfect stage is known as Thielaviopsis paradoxa. It  is       *Corresponding authors E-mail: eziashius@yahoo.com. a soil inhabitant, widely distributed throughout the tropics of Africa and  Asia  and  causes disease of  several  other crops (Rajagopalan, 1965). Biological control of plant pathogens by micro-organisms has been considered a more natural and environmentally acceptable alternative to the existing chemical treatment methods (Baker and Paulitz, 1996). Trichoderma species produce both volatile and non-volatile metabolites that adversely affect growth of different fungi (Bruce et al., 1984; Corley et al., 1994; Horvath et al., 1995; Moses et al., 1975). Dennis and Webster (1971b) found that some Trichoderma isolates produced volatile components, which were inhibitory to the growth of other fungi. Acetaldehyde was identified tentatively as one of the metabolites of Trichoderma viride inhibitory to other fungi. Dennis and Webster (1971a) also found that many isolates of Trichoderma species produced non-volatile antibiotics, which were active against a range of fungi. The relative abilities of the three biotypes of T. harzianum to colonize compost in competition with Agaricus bisporus and their influence on A. bisporus growth may be associated with secondary metabolite production (Seaby, 1987). Harman et al. (1980)  and  Nelson et al.  (1988) r eported  the   use   of 704       Afr. J. Biotechnol.    Table 1. Growth of C. paradoxa in the presence of volatiles released by Trichoderma species                    Mean values followed by * are significant (p<0.05) according to oneway analysis of variance as compared to the inoculated control. Data were expressed as % of control colonies without antagonist and values are average of 3 replicates.   Table 2. Growth of C. paradoxa in the presence of non-volatiles released by Trichoderma species.          Mean values followed by * are significant (p<0.05) according to oneway analysis of variance as compared to the inoculated control. Data were expressed as % of control colonies without antagonist and values are average of 3 replicates.    Trichoderma hamatum for the control of Pythium seed rot and Rhizoctonia root rot in pea.  Although, benomyl is recommended for the control of C. paradoxa (Omamor, 1985), information on the use of bioagent for control is lacking. This paper present different effect of metabolites produced by T. viride, T. polysporum, T. hamatum, and T. aureoviride against C. paradoxa, which causes black seed rot in oil palm sprouted seeds.   MATERIALS AND METHODS  The effect of volatile metabolites from Trichoderma viride, T. polysporum, T. hamatum, and T. auroeviride against C. paradoxa were tested in the assemblage described by Dennis and Webster (1971b). Two bottons of Petri dishes containing PDA were individually inoculated with a disc of pathogen and antagonist, and bottons were adjusted and attached by tape. Trichoderma species were inoculated 48 h earlier, since growth of C. paradoxa was more rapid. The control sets did not contain the antagonist. The effect of non-volatile metabolites from Trichoderma species against C. paradoxa was by the method described by Lundberg and Unestan (1980) and Dennis and Webster (1971a). Initially, mycelial agar plugs (4 mm diameter) removed (with a no 2 cork borer) from the edge of a young culture Trichoderma species were transferred to the center of Petri dishes (90 mm diameter) containing PDA and a sterilized cellophane disc (Courtauld Films: 50mm thick) adjusted on the medium surface, where the antagonist was grown for 3 days. Then the cellophane containing the Trichoderma growth was removed and on the same medium a disc of the pathogen was placed. The control treatments had C. paradoxa growing similarly on PDA medium where previously there was a cellophane disc without antagonist. The effect of direct-diffusible metabolites from T. viride, T. polysporum, T. hamatum, and T. aureoviride against C. paradoxa were determined by transferring seven days old mycelial plug (4 mm diameter) removed from growing edge each of Trichoderma species to the center of Petri dishes containing PDA, previously left overnight to allow excess water to evaporate. The uninoculated Petri dishes were placed over the inoculated Petri dishes of Trichoderma species and attached firmly by tape. The Petri dishes were untapped after 3 days. Seven old mycelial disc (4 mm diameter) of C. paradoxa was inoculated on previously uninoculated Petri dishes. The control sets did not contain the antagonist, only had the uninoculated Petri dishes placed over the same uninoculated Petri dishes and attached also firmly by tape.  The studies of volatile, non-volatile and direct-diffusible metabolites were conducted in three replicates repeated twice. The cultures were incubated at 27-28oC. Growth rates were recorded daily by measuring colony diameter according to Lilly and Barnett (1951). The inhibition percentage was obtained using the formula 1% = [(C2-C1)/ C2)] x 100 (Edington et al., 1971).    RESULTS AND DISCUSSION  Volatile, non-volatile and direct-diffusible metabolite tests showed that all the Trichoderma species except T. aureoviride inhibited the growth of C. paradoxa. The inhibition varied from 2.0% to  64%  in  volatile  (Table 1), Radial growth (cm per day) of C. paradoxa Days Inhibition rate of mycelial growth  Treatment 1 2 3 (%) T. viride 2.8 0.2 0.2 64* T. polysporum 3.0 0.1 0.1 63* T. hamatum 3.1 1.7 2.8 16* T. aureoviride 3.9 1.9 2.9 2 Control 4.1 2.2 2.7 0.0 Radial growth (cm per day) of C. paradoxa Days Inhibition rate of mycelial growth  Treatment 1 2 3 (%) T. viride 1.3 0.4 0.6 74* T. polysporum 1.3 0.7 0.6 71* T. hamatum 1.6 0.7 0.9 64* T. aureoviride 4.1 1.7 3.2 0.0 Control 4.2 2.4 2.4 0.0 Eziashi  et  al.        705                          Table 3. Growth of C. paradoxa on medium containing direct-diffusible metabolite produced by Trichoderma species.          Mean values followed by * are significant (p<0.05) according to oneway analysis of variance as compared to the inoculated control. Data were expressed as % of control colonies without antagonist and values are average of 3 replicates.   0.0% to 74% in non-volatile (Table 2) and 0.0% to 81% in direct-diffusible metabolite (Table 3). The results of volatile and non-volatile indicated that mycelial growth of C. paradoxa was significantly (p<0.05) inhibited by T. viride than the other species tested. The results of direct-diffusible metabolites showed that mycelial growth of C. paradoxa was significantly (p< 0.05) inhibited by T. polysporum up to 81% (table 3). T. aureoviride recorded the least inhibition in all the tests from 0.0% to 2.0%. In all the tests conducted, T. polysporum, T. viride, and T. hamatum significantly (p<0.05) reduced mycelial growth of C. paradoxa in medium containing direct-diffusible metabolite when compared with media containing volatile and non volatile metabolites, and the control.  Biological activity of antagonist fungi and bacteria may partially be associated with production of antibiotic (Etebarian et al., 2000; Faull et al., 1994; Pusey and Wilson, 1984). The production of antibiotics; Trichodermin (Godtfredsen and Vangedal, 1964), ergokonin (Kumeda et al., 1994), viridin (Grove et al., 1996; Grove et al., 1965) and viridin fungin A, B and C (Harris et al., 1993) by Trichoderma viride have been involved in biological control. However, it has not been possible to extract these substances from Trichoderma species tested in this study. The metabolites produced by T. viride in these experiments are likely similar to metabolites produced by T. viride by the other investigators mentioned above. This investigation suggests that metabolites released by these Trichoderma species are toxic and fungistatic to C. paradoxa. Medium containing direct-diffusible metabolites should be re-employed for further studies since it gave the best results compared to volatile and non-volatile tests. Trichoderma species involved in this study should be investigated for their ability to control C. paradoxa in commercial situation.   ACKNOWLEDGEMENTS  E.I. Eziashi wish to thank the Director, Board and Management members of Nigerian Institute for Oil Palm Research for the award of scholarship and provision of oil palm sprouted seeds.   REFERENCES  Ahmed SA (2001).  Agriculture and Food Security in Nigeria. Paper prepared for a forum with Mr. President on Agriculture and Food Security. Council chambers, Presidential Villa, Abuja. January 2001. Baker R, Paulitz TC (1996).  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. 50-79. 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Effect of metabolites produced by Trichoderma species against Ceratocystis paradoxa in culture medium

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Effect of metabolites produced by Trichoderma species against Ceratocystis paradoxa in culture medium

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