Author : Charida Pukahuta

Summary : Lentinula edodes (berk.) Pegler or shiitake mushroom is a favorite and high-nutritional mushroom. Cultivation of this mushroom in Thailand was interfered by fungal contamination which caused a great damage. Among the fungal contaminant, Trichoderma was dominantly found. Therefore, the objectives of this work are to study the mechanism of invasion and growth inhibition of Trichoderma and L. edodes, resistant mechanism of L. edodes to Trichoderma and genetic improvement of L. edodes for Trichoderma resistance. From 38 samples of contaminated materials for shiitake cultivation, 43 fungal isolates were obtained and 22 of which were T. harzianum. Ten isolates of T. harzianum and eight strains of L. edodes cultivated in Thailand were randomly seleted to study the mechanisms of invasion and growth inhibition of T. harzianum on L. edodes. Hyphal interaction between these two fungi showed variation in microscopic structures and three types of hyphal interaction were observed. Volatile and non-volatile substances of some isolates of T. harzianum reduced growth of some strains of L. edodes. The degree of inhibition was different depending on isolates of T. harzianum and sensitivity of L. edodes strains. T. harzianum TB-3 and TL-6 showed strong inhibition on L. edodes growth through their non-volatile and volatile substances, respectively. Mycolytic enzymes of T. harzianum TB-3 and TL-6 released cell wall component from mycelia of L. edodes L-7. These evidences suggested that various mechanisms involving in invasion and growth inhibition of T. harzianum to L. edodes. The resistance of L. edodes to T. harzianum TB-3 and TL-6 in dual culture on synthetic medium was variable among strains of L-edodes and isolates of T. harzianum. L. edodes L-7 was selected to use for further study since it showed strong resistance to both isolates of T. harzianum in dual culture. Resistant mechanism of L. edodes L-7 to T. harzianum was expressed by formation of brownish pigment at the contact zone of two fungi. This pigment showed similar characteristics to melanin and had ability to decrease mycolytic activity of culture filtrate of T. harzianum TB-3 and TL-6 on L. edodes L-7 mycelia. There was antifungal substance produced produced by L. edodes L-7 that inhibited growth of both isolates of T. harzanum in the area of dark brownish color at the contact zone L. edodes and T. harzianum TB-3. To improve the T. harzianum resistance of L. edodes L-7, the protoplast of monokaryon no. 320 of L. edodes was mutated by UV-irridiation. Also the monokaryon no.1 of L. edodes CH-1 which has opposite mating type to monokaryon no.320 was mutated by the same method. The monokaryons from regenerated survived protoplasts were screened for the resistant mutant monokaryon by soaking in T. harzianum conidial suspension. Four resistant mutant monokaryons of L-7/320 obtained were crossed to a resistant mutant monokaryon of CH-1/no.1 and dikaryon Km-2, KM-3, KM-62, and KM-63 were attained. All of hybrid dikaryons fruited in supplemented sawdust medium in the laboratory. The basidiospores collected from each hybrid showed higher percentage of germination than the percent L-7. The number of monokaryon achieved from germinated basidiospores of each hybrid dikaryon which revealed the resistance to T. harzianum TB-3 and Tl-6 was also higher of the parent L-7. All hybrids and the parent L-7 could overgrow the colony of fungal contaminant in supplemented sawdust in induced contaminated condition. However only hybrids were able to form fruiting body while the parent L-7 was not Higher phenoloxidase activity was found in KM-63 than in the parent L-7.