3.5.19

PCR-BASED IDENTIFICATION AND CHARACTERIZATION OF TRICHODERMA SPP. FOR RICE SHEATH BLIGHT DISEASE MANAGEMENT IN THE PHILIPPINES


CJR CUMAGUNl , J HOCKENHULL2 and M LUBECK2

lDepartment of Plant Pathology, University of the Philippines, Los Banos, College, Laguna 4031, Philippines; 2Plant Pathology Section, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaidsensvej 40, DK-1871 Frederiksberg C., Copenhagen, Denmark

Background and objectives
There is a great difficulty in identifying Trichoderma spp. by morphological observations because its morphological characters are unreliable and there is inconsistent phenotypic concept of the fungus. Correct identification of Trichoderma is important due to the increasing interest in the organism as a biological control agent of plant diseases. For example, pellet formulation of T. harzianum has been proven to be effective in controlling sheath blight of rice caused by Rhizoctonia solani in non-sterile soil. To aid in further identification of strains that are morphologically similar, molecular approach such as ITS1 ribotyping and Universally Primed PCR (UP-PCR) were developed. ITS1 (internal transcribed sequence 1) ribotyping is the analysis of the ribosomal DNA region by PCR. The RDNA region are attractive as targets for PCR because they are present in up to 220 copies in the fungal genome and they possess abilities to differentiate between genera, species, and subspecies. Universally Primed PCR (UP-PCR) is a PCR fingerprinting method that amplifies DNA from any organism without previous knowledge of DNA sequences and to generate multi-banding profiles (fingerprints) following gel electrophoresis [2]. The objectives of this study are to identify, characterize isolates of Trichoderma with the UP-PCR method and ribotyping, compare results to morphological data and develop specific molecular markers for promising fungal isolates against R. solani.

Results and conclusions
Forty-two isolates of Trichoderma coming from four provinces of the Philippines were analyzed using ribotyping and Universally Primed PCR (UP-PCR). Identical size and restriction pattern in their internal transcribed spacers (ITS) region of the ribosomal DNA and UP-PCR banding profiles using a UP primer (L45) occurred in all isolates except isolates 94-054 and 94-055. Four isolates (94-012, 94-015, 94-016 and 94-022) known for their high cellulolysis adequacy indices (CAI) and competitive saprophytic abilities (CSA) were previously identified as T. harzianum Rifai by morphological observations. Based on visual inspection of the aligned banding profiles, the majority of the isolates were also presumed to belong to the same species. The Danish isolate U2 was the basis for classifying them into T. harzianum group. Isolates 94-054 and 94-055 were classified by morphological observations as T. viride in accordance with the ITS1 ribotyping and their distinct UP-PCR banding profiles. The four isolates of T. harzianum mentioned previously were further analyzed for identification of UP primers in order to distinguish them from the 38 isolates. A diagnostic marker was obtained from each of these isolates. Isolate 94-016 was of particular interest because it possessed the highest CAI and CSA among the four and was found to be a potential biological control agent against R. solani causing sheath blight of rice [1]. However, isolate 94-012 seemed to be clone of isolate 94-016 because no difference could be detected between them using all primers and primer combinations. A clear distinct band was obtained from these two isolates using AA2M2 + AS15 primer combination. Among the 40 isolates, only one isolate- 94-017 showed the same band but it was differentiated using either of the primer combinations AS15 + ASinv, AS15inv + Fok 1 or L21 + Fokl. Using these primer combinations to recognize isolate 94-016 could be a valuable tool in monitoring this specific isolate upon release into the field to study its population dynamics and biological control abilities.

References
1. Cumagun CJR, Liag LL, Manalo JO, 1996. Proceedings Asian International Mycological Congress, 1996. p. 111.
2. Lubeck M, Lubeck PS, 1996. Developments in Plant Pathology 8,113-122.