论文部分内容阅读
由尖镰孢古巴专化型1号生理小种Fusarium oxysporumf.sp.cubenserace1(Focr1)引起的香蕉枯萎病是粉蕉类香蕉品种(AAB)不能规模化种植的最主要因素,其致病机理至今尚不十分清楚。本实验室前期通过T-DNA插入获得Focr1致病性丧失突变体Focr1-328,从Focr1全基因组序列中定位了因T-DNA插入失活的基因,并从野生型菌株Focr1-N2中敲除了该致病相关基因,获得了敲除突变体△Focr1-328。为了明确该致病相关基因的生物学功能,通过活体叶片、活体根部、孢子悬浮液等接种方法对敲除突变体△Focr1-328的致病性进行了测定,研究了该突变体与野生型菌株Focr1-N2在PDA培养基上菌落、菌丝和分生孢子形态上的差异;在不同碳、氮源培养基上的生长速率及形态差异;在培养不同时间后菌体生物量、pH值、OD值的变化差异及玻璃纸穿透能力的差异等。致病性测定结果显示4种接种方法都未见△Focr1-328发病症状,表明其丧失了致病力;表型测定结果显示:在PDA培养基上,△Focr1-328的生长速率、分生孢子产量、分生孢子萌发率、培养不同时间的菌丝干重均明显低于野生型菌株;在不同培养时间的培养液中,△Focr1-328的pH值、OD值均极显著低于野生型菌株Focr1-N2;敲除突变体和野生型菌株在不同碳、氮源上生长差异显著,最适碳源分别为山梨醇和麦芽糖,最适氮源均为硝酸钠;突变体菌株不能穿透玻璃纸生长,而野生型可以。上述实验结果表明:T-DNA插入失活的致病相关基因与菌株的碳源利用、产酸调控及菌丝穿透能力有关。
The banana wilt disease caused by the Fusarium oxysporumf.sp.cubenserace1 (Focr1), a Fusarium oxysporum f.sp.cubenserace1 strain of Fusarium oxysporum f.sp., is the most important factor for the banana banana variety (AAB) Not yet very clear. In our laboratory, Focr1-328, a Focr1 pathogenicity-loss mutant, was obtained by T-DNA insertion in the past, and the gene inactivated by T-DNA insertion was located in the whole Focr1 genome sequence and knocked out from the Focr1-N2 wild-type strain The pathogenicity-related gene, knockout mutant △ Focr1-328 was obtained. In order to clarify the biological function of the pathogenicity-related genes, the pathogenicity of △ Focr1-328 knockout mutant was determined by inoculation methods of live leaves, living roots and spore suspension, The morphological differences of colony, mycelium and conidia of Focr1-N2 on PDA medium were observed. The growth rate and morphological difference of Focr1-N2 on different carbon and nitrogen sources were observed. The biomass, pH value , The difference of OD value and the difference of cellophane penetration ability. The results of pathogenicity test showed that none of the four inoculation methods showed the symptom of △ Focr1-328, indicating that it lost its virulence. The phenotypic results showed that on the PDA medium, the growth rate of △ Focr1-328, The spore yield, germination rate of conidia and the dry weight of mycelium at different time were significantly lower than those of the wild-type strain. The pH value and OD value of △ Focr1-328 in the culture medium with different culture time were significantly lower than those in the wild Focr1-N2; Mutant and wild-type strains showed significant differences in growth on different carbon and nitrogen sources. The optimal carbon sources were sorbitol and maltose, respectively, and the most suitable nitrogen source was sodium nitrate. Mutant strains could not penetrate Cellophane grows while wild type can. The above experimental results show that: T-DNA insertion-inactivation of the disease-related genes and strains of carbon utilization, acid production and mycelial penetration ability.