Specific PCR Analysis And Antifungal Effect Of Endophytic Fungi On Brachiaria

Brachiaria is predominately an African genus of grasses with about 100 species. Some species of them are economically important forages in tropical America. The fungus Acremonium implication can develop an endophytic association that is mutually beneficial with Brachiaria species. Diversity exists among 11 A. implication isolates showed in cultural characters, RAPD and AFLP analysis. On PDA medium, some isolates are protruding while others are effuse, some isolates are pink while some isolates are white or yellowish milky, and textures of most of the isolates are loose while some are hard and close. In RAPD analysis with 11 arbitrary 10-bp primers, the 11 isolates were clustered into 7 groups based on their similarities. The similarity coefficients are 0.23-0.93 among groups. Results of AFLP analysis, with 4 selective primer pairs, are consistent with that of RAPD. Based on genetic diversity, natural strains of A. implicatum, that contained plant fitness-enhanced characters but toxin-free to livestock, would be able to selected for Brachiaria breeding.Based on RAPD analysis, a common product about 500-bp to all isolates of A. implicatum that amplified with primer OPAK10 was cloned and named as p Ai AK10-500. The product was sequenced. Based on the sequence data, primers were synthesized and used to amplify genomic DNA from isolates of A. implicatum, pathogenic (Drechslera sp. et. al) and non-pathogenic fungi (Colletotrichum gloeosporioides et. al), endophytes on tall fescue and ryegrass, and endophyte-infected or -free Brachiaria plants. Among of the primers, PI (STICGAATGATAAGGCAGATC3) and P4 (5'ACGCATXCCACTGTATGCTAC3') was highly specific to A. implicatum with an about 450bp amplified product. A PCR method was developed for specific detection for the A. implicatum. Compositions of the specific PCR (20 mL) were 1xPCR buffer, 3mM MgCl2, 0.25mM dNTPs, 0.5 uM primer PI and P4, 1U TaqDNA polymerase, and 30ng template DNA. Cycling parameters were Step 1, 94 C 3min; step 2, 94 C 30 sec; step 3, 65 C 1 min; step 4, 72 C 1 min; step 5, go to step 2 for 45 cycles; then 72 C 10 min. A. implicatum could distribute in leaf sheaths, leaf blades, stems, roots and seeds, and could be transmitted by plant seeds, proved by the PCR detection method. And with the PCR method, the effect ofelimination of A. implicatum from species of Brachiaria using fungicides was evaluated. Endophyte-infected Brachiaria young tillers were treated using fungicide Benomyl, Propiconazole, or Folicur. Each of the treatments could kill A. implicatum in some plants, but not completely eliminate in some plants. It was better to eliminate A. implicatum from young tillers that cultured in sand inundated in low concentration of fungicide (10-25 ug/L) for a long time (35 days) than that soaked in higher concentration(75-150 mg/L)of fungicide water solution for a short time (5 hours).Most of the endophyte strains showed inhibition to Drechslera sp. and Rhizoctonia solani on PDA medium. It was different in anti-fungal effects among endophyte strains against the same pathogen. And it was also different in the inhibitory effects of the same endophyte strain to different pathogens. EB 6780.501 and EH 32a showed strong inhibition to both Dre. sp. and Rhi. solani. Plant protection in greenhouse from foliar blight by the endophyte was clearly in the early stage after inoculation with Rhi. solani CIAT 6780. But the resistance became weak as time went on. There was correlation between in vitro and in vivo tests, in that the isolates with strong in vivo inhibitory effects showed strong disease resistance.