基质中添加西兰花残体对大丽轮枝菌GFP标记菌株在棉花体内扩展的影响

 由大丽轮枝菌引起的棉花黄萎病是一种难以防治的土传病害,西兰花残体还对其有一定的防治作用,防效为62.82%。为解析西兰花残体对棉花黄萎病的防治机制,本研究通过土壤杆菌转化的方法构建了大丽轮枝菌的绿色荧光标记菌株,采用共聚焦显微镜观察标记菌株在添加西兰花残体营养基质中和空白对照营养基质种植的棉花体内的侵染和扩展情况。结果表明构建的标记菌株gfp-wx-1与野生菌株wx-1的生物学特性无显著性差异。同时发现大丽轮枝菌在添加西兰花残体营养基质中棉花体内扩展较慢,具体表现为:在空白营养基质种植的棉花体内,大丽轮枝菌在接种第2 d时就可侵染根尖表层及根内部,第3 d到达茎部维管束,第5 d叶片可观察到少量病原菌,第7 d第一片子叶呈现大量病原菌,后期迅速扩展,叶片出现黄萎病斑;而在营养基质中添加西兰花残体种植的棉花体内,大丽轮枝菌在接种第2 d时就可侵染根尖表层,第3 d侵染根尖内部,第5 d侵染茎部维管束,直到第7 d第一片子叶才出现少量病原菌,后期扩展慢,叶片病斑较少。本研究通过荧光定量PCR方法检测了大丽轮枝菌在两个处理棉花根部定殖情况,结果表明西兰花残体能够显著降低大丽轮枝菌在棉花根部定殖的量。该研究初步明确了西兰花残体对棉花黄萎病的防治机制,为棉花黄萎病的绿色防治提供了一条新的途径。

Effect of growth substrate amended with broccoli residues on the spread of GFP-labeled Verticillium dahliae strain in cotton plant

Cotton verticillium wilt caused by Verticillium dahliae Kleb. is a soil-borne fungal disease, which is very difficult to control. We found that broccoli residues retention showed promising control effect on the disease, and the control effect reached to 62.82%. To understand the control mechanism of broccoli residues against cotton verticillium wilt, we generated a green fluorescent protein (GFP)-labeled strain, gfp-wx-1, of V. dahliae by the method of Agrobacterium transformation. The localizations of the GFP-labeled strain in cotton plant grown in growth substrate amended or un-amended with broccoli residues were observed under a confocal microscope. Results showed that there was no significant differences on biological characteristics between the strain gfp-wx-1 and a wild-type strain wx-1. But we found that the broccoli residues significantly inhibited the spread ofV. dahlia. In the cotton plant grown in growth substrate un-amended with broccoli residues, V. dahliae infected the surface and interior of the root tip on the second day after inoculation and penetrated into the stem vascular bundles on the third day. A limited amount of the pathogen was found in the first cotyledon on the 5th day, and a large number of the pathogen was found in the first cotyledon on the 7th day. It showed a rapid spread of V. dahliae in cotton plant in the late stage. However, in the cotton plant grown in growth substrate amended with broccoli residues, V. dahliae only infected the root tip surface layer on the 2nd day. The pathogen started to infect the interior of root tip on the 3th day, and it infected the stem vascular bundle on the 5th day. A limited amount of pathogen could be found in the first cotyledon on the 7th day. The pathogen expanded very slowly in the cotton plants at the late stage, and the sizes of the lesions on cotton leaves were smaller. Colonization of V. dahliae in cotton grown in growth substrate amended or un-amended with broccoli residues was detected by fluorescence quantitative PCR, and the results showed that the broccoli residues significantly reduced the colonization of V. dahliae in cotton roots. This study suggests that inhibition of the colonization of V. dahiliae in cotton plant is probably the mechanism responsible for suppression of cotton verticillium wilt by broccoli residues.