African of chemicals in the control of rubber
African Journal of Biotechnology Vol.
7 (15), pp. 2569-2572, 4 August, 2008 Available online at http://www. academicjournals. org/AJB ISSN 1684–5315 © 2008 Academic Journals Full Length Research Paper Use of phylloplane fungi as biocontrol agent against Colletotrichum leaf disease of rubber (Hevea brasiliensis Muell. Arg. ) Evueh, G.
A. and Ogbebor, N. O. * Plant Protection Division, Rubber Research Institute of Nigeria, PMB 1049, Benin City.
Accepted 4 March, 2008 Phylloplane fungi were used as biocontrol agent against Colletotrichum leaf disease of rubber (Hevea brasiliensis Muell. Arg. ). Aspergillus sp. ysed the cytoplasm of Colletotrichum gloeosporioides on Potato Dextrose Agar. Trichophyton sp. and Gliocladium sp.
antagonised C. gloeosporioides by overgrowing on it. Other phylloplanes used in this study such as Botrytis sp. , Pleurothecium sp. and Staphylotrichum sp. exhibited weak antagonism on the pathogen while Gonatorrhodiella sp. and Syncephalastrum sp.
showed different levels of zones of inhibition with the pathogen. Metabolites produced by Gonatorrhodiella sp. and Syncephalastrum sp. affected the pathogen by antibiosis.
This finding showed that Trichocladium sp. and Trichophyton sp. exhibited the highest antagonistic effects on C.
loeosporioides. Key words: Para rubber, phylloplane fungi, biological control, disease management. INTRODUCTION Hevea brasiliensis (Muell. Arg. ) commonly called para rubber, is an economic crop whose healthy existence is significant to its productivity (Rao, 1965). Rubber diseases are mainly caused by fungal pathogens (Igeleke, 1988; Begho, 1990). Colletotrichum gloeosporioides (Penz.
) Sacc. is the causal agent of Colletotrichum leaf fall of rubber tree. It is one of the serious leaf diseases which affects the new flushes produced following the ‘wintering effect’, when the rubber tree loses its leaves during the dry season.The disease also affects young rubber plants under nursery conditions and when severe can lead to shoot die back (Rao, 1965; Webster and Baulkwill, 1989).
The successful use of chemicals in the control of rubber diseases has been extensively reported by various scientists. However, the high cost of chemical fungicides limits its availability and use by small – scale farmers. Biological control for plant diseases is now receiving increasing attention, although the potential of biological control through the effect of phyllosphere antagonists has been realized for sometime.Several workers have investigated the use of biological control of plant diseases (Osando and Waudo, 1994; Tewari, 1995; Ogbebor and Adekunle, 2005). Osando and Waudo (1994) used various isolates of Trichoderma to control Armillaria root rot fungus of tea.
They found that different isolates of Trichoderma sp. exhibited different level of antagonism against Armillaria root rot fungus. This study seeks to find possible phylloplane fungi as biocontrol agents against Colletotrichum leaf disease of rubber.
MATERIALS AND METHODS Isolation of leaf pathogen Leaves of H. brasiliensis infected with C. loeosporioides were collected from Rubber seedlings in Rubber Research Institute of Nigeria.
Bits of 1 x 1 cm cut across lesions were surfaced sterilized by submerging in 0. 1% of mercuric chloride for 1 min, after which it was rinsed in five changes of sterile distilled water. Then, they were placed on PDA. Isolation of phylloplane fungi *Corresponding author. E-mail: [email protected] com. Phylloplane fungi were isolated from healthy leaves of rubber plant 2570 Afr.
J. Biotechnol. in RRIN nursery through leaf washing technique (Blakeman, 1981). Dilution method of Pelczar and Chan (1972) was employed and dilution factor of 10-4 was used.
Pure isolate were established on antibiotic-amended PDA. Dual inoculation of leaf pathogen and potential antagonist on PDA Dual inoculation of the pathogen and an antagonist was set up. A 10 mm disc of the pathogen with similar size of each potential antagonist was taken from the edge of a 5-day-old pure culture using a cork borer and plated 20 mm apart respectively on PDA medium. Potential antagonists tried are Trichoderma sp. , Aspergillus sp. , Gliocladium sp. , Pleurothecium sp.
, Botrytis sp. , Staphylotrichum sp. , Trichocladium sp. , Gonatorrhodiella sp. , Trichophyton sp. and Syncephalastrum sp.
The control plates were plated with the pathogen and antagonists separately. Four replications per treatment were set up for each pathogen and antagonist combinations. Petri-plates were incubated at room temperature. Daily growth measurement towards and away from the opposing fungal colony was taken for 10 days. The percentage inhibition of radial growth of the pathogen was calculated using a formula by Vincent (1927) and Jacob et al. (2006).
The mode of interaction was rated using the method adapted by Fokkema (1973): 0: No visible sign of inhibition of pathogenic fungi, the mycelium of which overgrew the test organism. : Mutual inhibitions. Both organisms stopped growing on contact at the center or close to the center of the Petri-plate. 2: Inhibition of pathogen with inhibition zone > 1cm in width. 3: Inhibition of pathogen with inhibition zone < 1cm in width. 4: Inhibition of pathogen by overgrowth or displacement of pathogen. Test isolate with ratings of 2 to 4 were further observed microscopically to determine their effects of antagonism on the hyphal growth of the pathogenic fungi.
plate when they came in contact with C. gloeosporioides forming a straight line at their meeting point. Their mode of interaction were rated 5.
Trichoderma sp. and Aspergillus sp. both rated as 3 inhibited the growth of C. gloeosporioides by 90% and did not overgrow it till the end of the experiment.
Microscopic observation of mycelial interactions Microscopic observation was carried out on the interaction of C. gloeosporioides with Trichoderma sp. , Trichocladium sp. , Aspergillus sp. , Gonatorrhodiella sp. , Syncephalastrum sp.
and Trichophyton sp. When Aspergillus species came in contact with hypha of Colletotrichum sp. , lysis of the hypha occurred and the pathogen could not be re-isolated from the point of contact. In the case of Trichocladium sp. when it came in contact with that of the pathogen, lysis occurred at the point of septa formation.
Trichoderma sp. and Trichophyton sp. did not show any clear pattern of hyphal interaction. In the interaction between C.
gloeosporioides with Syncephalastrum sp. and Gonatorrhodiella sp. , there were mutual inhibition of both the pathogen and antagonists. DISCUSSION Antagonistic effects of different phylloplane fungi indicated the importance of many such fungi as a possible biocontrol agent.
Trichocladium sp. completely overgrew C. gloeosporioides in culture and eventually displaced it.This could possibly be as a result of competition between saprophytic and pathogenic microorganisms for nutrients. Interaction between the fungal colonies of C.
gloeosporioides with Gonatorrhodiella sp. and Syncephalastrum sp. respectively showed that they did not meet in culture. C.
gloeosporioides was inhibited and this is as a result of the production of metabolites, possibly antibiotics, by Gonatorrhodiella sp. and Syncephalastrum sp. in the medium.
Antibiotics have been shown to be involved in disease suppression (Anjaiah et al. , 1995; Handelman and Stabb, 1996). Anjaiah et al. 1995) demonstrated that phenazine-1-carboxamide and anthranilate are antibiotics involved in disease suppression by Psuedomonads. Trichophyton sp.
and Gliocladium sp. antagonized C. gloeosporioides by overgrowth mechanism.
Only Trichophyton sp. showed appreciable reduction in the growth of C. gloeosporioides. Botrytis sp. , Pleurothecium sp. and Staphylotrichum sp.
did not show appreciable antagonistic effect. From the observation of ‘direct’ post contact inhibition, Aspergillus sp. caused damage to C. gloeosporioides by coagulating its cytoplasm and also caused lysis and tip burst of the pathogen.
This could be as a result of antagonism due to parasitism or and antibiosis as lytic activity has been demonstrated to be involved. Jayasu- RESULTS Antagonism between colonies of Colletotrichum gloeosporioides and phylloplane fungi The mycelial growth measurement of C. gloeosporioides and the ten antagonists towards each other on Potato Dextrose Agar on the tenth day after inoculation and percent inhibition of C. gloeosporioides are summarized in Table 1. A significant interaction was exhibited by Trichocladium sp. , in which the growth of C.
gloeosporioides was much affected by hyphal interference.The antagonistic fungus grew over the colony of C. gloeosporioides and completely inhibited its growth. The interaction was rated 5. Syncephalastrum sp. and Gonatorrhodiella sp.
inhibited the growth of C. gloeosporioides by 84. 1 and 80. 9% respectively. An inhibition zone of 0. 16 cm with Gonatorrhodiella sp.
and 0. 43 cm with Syncephalastrum sp. were observed. Trichophyton sp.
and Gliocladium sp. overgrew C. gloeosporioides and their mode of interaction was intermingling and was rated 5 and 2 respectively. Botrytis sp.
, Pleurothecium sp. and Staphylotrichum sp. Stopped growth at the center of the Evueh and Ogbebor 571 gloeosporioides. Table 1. The mycelial growth measurement of Colletotrichum gloeosporioides and ten antagonists towards each other on Potato dextrose Agar on the tenth day after inoculation and percent inhibition of C.
S/N 1 2 3 4 5 6 7 8 9 10 • Pathogen/Antagonist C. gloeosporioides Botrytis sp. * C. gloeosporioides Trichoderma sp C. gloeosporioides Gliocladium sp. * C. gloeosporioides Staphylotrichum sp.
* C. gloeosporioides Trichocladium sp. * C. gloeosporioides Gonatorrhodiella sp. * C.
gloeosporioides Aspergillus sp. * C. gloeosporioides Pleurothecium sp. * C. gloeosporioides Trichophyton sp. * C.
loeosporioides Syncephalastrum sp. * Mycelial growth ±SE (Treatment) cm 1. 13 ± 0. 04 0. 88 ± 0.
04 0. 55 ± 0. 05 1. 45 ± 0. 05 1. 40 ± 0. 00 2.
00 ± 0. 07 0. 93 ± 0. 04 1. 73 ± 0. 04 0. 00 ± 0.
00 3. 50 ± 0. 07 1.
05 ± 0. 05 0. 78 ± 0.
04 0. 55 ± 0. 05 1. 48 ± 0.
08 0. 83 ± 0. 04 1. 18 ± 0.
04 0. 93 ± 0. 04 5. 50 ± 0. 08 0. 88 ± 0.
04 0. 70 ±0. 00 Mycelial growth (Control) cm 5.
50 5. 50 5. 50 5. 50 5. 50 1.
70 5. 50 5. 50 5. 50 5. 50 5. 50 1. 30 5.
50 5. 50 5. 50 5. 50 5.
50 5. 50 5. 50 1. 50 Percent inhibition (%) 69. 20 90.
90 74. 50 83. 18 100. 00 80.
90 99. 09 85. 00 83.
18 84. 09 = Antagonist. riya and Deacon (1995) demonstrated B. inerea to cause explosive hyphal lysis or coagulation of cytoplasm, often penetrating the hyphae of Rigidoporus lignosus 4 7 min after contact. Jacob et al. (2006) suggested that lysis of cell wall of C.
gloeosporioides is involved in the biological control of mango anthracnose disease by Pseudomonas fluorescence. This study demonstrates Trichocladium sp. and Trichophyton sp. to possess higher antagonistic effect on the pathogen. Integrated approach of disease management using the interaction of chemical means of control with a biological component is likely to be more attractive to the grower than a biological approach alone.Munnecke et al. (1981) used this approach to control Armillaria mellea in soil by weakening the pathogen with methyl bromide so that natural occurring antagonist could easily invade the weakened pathogen inoculum’s.
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