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荧光假单胞菌(Pseudomonas fluorescens)FD6分离自福建闽侯青口青菜根围土壤,采用凹玻片法和离体果实接种法测定菌株FD6对桃褐腐病菌(Monilinia fructicola)的抑制能力。PCR法克隆硝吡咯菌素合成基因簇结构基因(prn),利用同源重组构建prn A缺失突变体并分析其功能。结果表明,菌株FD6处理桃后可完全抑制桃褐腐病发生;细菌悬浮液对褐腐病菌分生孢子的萌发抑制率达93.18%,细菌培养滤液的抑制率为69.36%;细菌悬浮液对黄瓜根结线虫也具有较强的致死作用。序列分析表明荧光假单胞菌FD6硝吡咯菌素合成基因簇全长为5 868 bp,内含4个开放阅读框prn A、prn B、prnC和prn D,这4个基因组成1个共转录单元。系统发育进化树显示菌株FD6与P.protegens CHA0、Pf-5的prn基因相似性达94%。利用遗传学方法证实prn A基因是硝吡咯菌素合成的必需因子,此外该基因还影响2,4–二乙酰基间苯三酚和藤黄绿脓菌素的合成。
Pseudomonas fluorescens FD6 was isolated from the roots of cabbage in Minhoukou, Minhou, Fujian Province. The inhibitory effect of strain FD6 on Monilinia fructicola was determined by the method of concave slide and in vitro fruit inoculation. PCR was used to clone the pyrrolnitrin gene cluster gene (prn). The prn A deletion mutant was constructed by homologous recombination and its function was analyzed. The results showed that the treatment of peach FD6 could completely inhibit the occurrence of peach brown rot. The inhibition rate of bacterial suspension on the conidial germination of brown rot was 93.18% and the inhibition rate of bacterial culture filtrate was 69.36% Root-knot nematode also has a strong lethal effect. Sequence analysis showed that the fluorogenic Pseudomonas FD6 pyrrolnitrin synthetic gene cluster total length of 5 868 bp, contains four open reading frames prn A, prn B, prnC and prn D, the four genes form a co-transcription unit. Phylogenetic tree showed that the similarity of prn gene between strain FD6 and P. proteus CHA0 and Pf-5 was 94%. The use of genetic methods to confirm that the prn A gene is an essential factor for the synthesis of pyrrolnitrin, in addition to the gene also affects 2,4-diacetylphloroglucinol and pyocyanin pyocyanin synthesis.