3.5.5
MOLECULAR MARKERS FOR MONITORING BIOCONTROL STRAINS OF TRICHODERMA HARZIANUM IN NATURAL ENVIRONMENTS


Department of Microbiology and Genetics, CSIC/University of Salamanca, Spain

Background and objectives
Trichoderma harzianum Rifai is a species aggregate which includes a plethora of strains that can be used as biological control agents of plant pathogenic fungi and viral vectors. The taxonomic status of this species is imprecise and the criteria used to classify and identify strains so far do not provide sufficient discrimination, specially with those isolates of interest in biocontrol programmes. Given the variability within T. harzianum, this name involves a species concept that is too wide for practical situations, and a particular strain may be a good or bad biological control agent depending upon the intended target and the functions required. It will therefore be necessary to select the most efficient strain for each individual pathosystem.
We have recently supported the existence of cryptic species with similar morphologies, and DNA-based species, and proposed attributes which may be used for the selective choice, as biological control agents, of new strains of T. harzianum between four cluster groups described [1].
The sequencing of rDNA 5.8 S gen gave us the possibility of separating strains within every cluster group. However, identical ITS 1-ITS 2 sequences of isolates enabled them to be separated with this molecular approach and further molecular markers were selected for those strains using the SCAR (sequence characterized amplified region) technologie from RAPD-PCR products.

Materials and methods
We have cloned and sequenced two RAPD fragments obtained from T. harzianum 11 using OPF-10 (10 nucleotides) . Two specific oligonucleotides were designed containing the RAPD primer at the 5' termini followed by the 16 adjacent nucleotides, in each fragment: A1-A1c and A2-A2c. These primers were used in a PCR procedure which was performed as described by Williams [2] except that the annealing temperature was 68°C for primers A1-A1c and 65°C for A2-A2c.

Results and conclusions
Two pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. The SCAR marker (950 bp approximately) obtained with primers A1-A1c was present in only one strain (11) from 17 strains tested. The SCAR marker (350 pb approximately) obtained with primers A2-A2c was present in only one strain (11) but another single band (400 pb approximately) was amplified in 13 from 17 strains tested. The SCAR marker amplified with A1-A1c is a useful tool to differentiate the isolate T. harzianum 11 from other closely related strains. This will enable monitoring of the specific isolates in field trials as a biological control agent.

References
1. Grondona I, Hermosa R, Tejada M, Gomis Md, Mateos PF, Bridge PD, Monte E, Garcia-Acha I, 1997. Applied and Environmental Microbiology 63, 3189-3198.
2. Williams J F, 1989. Bio Techniques 7, 762-768.