哈茨木霉LTR-2双价基因重组菌株的构建及其防治效果

Transformants of Trichoderma harzianum LTR-2 with the Bivalent Genes and Their Disease Control Efficiency

本文利用限制性内切酶介导法转化哈茨木霉LTR-2,获得具有双价基因(glu14、chi42)的重组生防菌株。试验得到的木霉转化子具有遗传稳定性,于无药PDA平板上连续转接6次后,在Hyg 200μg/m L的PDA平板上仍可继续生长。PCR扩增及Southern杂交证实目的基因已随机插入木霉基因组DNA上,本试验转化子均为单拷贝插入。转化子的β-1,4-葡聚糖酶和几丁质酶水解活性较出发菌株LTR-2显著提高(P<0.01),其中转化子L-15的两种水解酶活性均最高。转化子在温室条件下对番茄晚疫病、茄子猝倒病和黄瓜灰霉病均有较好的防治作用,但不同处理间存在显著性差异(P<0.01),其中转化子L-15最高,对3种病害的防治效果分别达到了91.5%、94.9%和83.8%,较出发菌株LTR-2处理分别提高了53.5%、55.9%和33.5%。结果表明,利用REMI技术,将β-1,4-葡聚糖酶基因glu14和几丁质酶基因chi42重组到木霉染色体DNA上,是获得高效重组菌株的有效手段。

Trichoderma harzianum LTR-2 is a kind of plant disease suppressive fungus. We constructed a expression vector named p C1300-2-G14-C42 of β-1,4-glucanase gene glu14 which was isolated from Bacillus megaterium strain Ap25 and the chitinase gene chi42 was from T. harzianum LTR-2, and used the vector to transform the strain LTR-2 by restriction enzyme mediated integration(REMI). Transformants stability were determined by six successive transfers of conidia on potato dextrose agar(PDA), followed by plating of each isolate onto PDA plates containing 200 μg/m L of hygromycin B. Compared with original strain LTR-2, most transformants changed in growth rate, colonial colour, subiculum density and the conidium quantity. Chromosomal integration was confirmed by PCR amplification and Southern blotting. All transformants not only expressed higher β-1,4-glucanase and chitinase hydrolytic activity, but also high disease suppressive efficacy against tomato late blight(Phytophthora infestans), eggplant damping-off(Pythium ultimum) and cucumber gray mold(Botrytis cinerea) in greenhouse(P<0.01). Transformant L-15 showed the highest biocontrol efficacy with 91.5%, 94.9% and 83.8%, respectively, against the diseases. Disease suppression rates were increased by 53.5%, 55.9% and 33.5%, respectively in comparison with that of original strain LTR-2. The results indicated that Trichoderma transformants with enhanced biocontrol efficiency could be effectively constructed by inserting β-1,4-glucanase gene glu14 and chitinase gene chi42 into chromosome DNA with REMI method.