甜椒根际合成抗生素假单胞菌遗传多样性分析及GP72菌株生防机制研究
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摘要
本实验室的已有研究表明植物根际合成吩嗪类以及聚酮类抗生素的假单胞菌对植物病原菌起着天然的生防作用。本课题通过在华东地区六个不同的地点采集甜椒根际土壤样品,以假单胞菌株M18中吩嗪-1-羧酸(Phenazine-1-carboxylic acid, PCA)和藤黄绿菌素(Pyoluteorin, Plt)合成基因簇中保守基因为分子探针,运用原位杂交及检测发酵产物的方法,从土壤样品中筛选出48株能够合成PCA与Plt的假单胞菌群,大致占了通过King’s B (KMB)培养基筛选菌株总数的0.4%。其中合成PCA的菌群(45个)占到了分离菌株总数的94%,分布的地点也相当广泛,而同时合成PCA及Plt的菌群(3个)只占总数的4%,分布范围也相对比较狭窄,在上述采样点中没有发现单独合成Plt的菌株存在。利用限制性片段长度多态性的分析(Restriction Fragment Length Polymorphism, PCR-RFLP)方法,根据该菌群核糖体16S rDNA以及16S rDNA与23S rDNA之间的间隔区域(Inter Transcript Space, ITS)的HaeIII酶切片段类型,可将所获得的假单胞菌群分为19种不同的基因型,其中合成PCA的菌群可分为16个不同的基因型。从不同地点基因型的不同分布我们可以看出基因型A5广泛分布于各个不同的采样点,同时每个采样点都有彼此不相同的特异基因型分布。从顾庄和马桥两地分离得到的合成PCA的菌株分别占了PCA合成菌株总数的31.1%和26.7%。在6个采样点中顾庄和马桥两地的甜椒受真菌感染程度最轻,这也从侧面暗示了合成PCA的菌株作为潜在生防菌群起到了一定的生防作用。而且在真菌病害发生较少的顾庄与马桥两地发现了更多的隶属于基因型A5的菌株,这也在一定程度上验证了基因型A5在PCA合成菌群中的主导地位以及作为今后进一步筛选分离潜在生防菌株的分子遗传标记。随机选取每种基因型中的一个菌株为代表,通过对该区段进行扩增、测序以及NCBI数据库中的已有序列比对,我们发现PCA产生菌群中占主导地位的菌群主要为荧光假单胞菌(Pseudomonas fluorescence),
Phenazine and pyoluteorin producing pseudomonads prove to be effective biocontrol agents based on previous reports of our laboratory. After sampling in six different green pepper rhizosphere located in Shanghai and Jiangsu provice, we design molecular detecting probes according to the conserved gene fragments of phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) gene clusters in Pseudomonas spp. M18. Finally we’ve got altogether 48 PCA and Plt producing isolates with colony hybridization followed by fermentation confirmation, which is 0.4% of total colonies selected by King’s B medium (KMB) approximately. 94% of the total isolates (45) belong to widely distributed PCA producing group, while 6% (3) for both PCA and Plt producing group. None of them can produce Plt alone. After analyzing the 16S rDNA plus ITS (Inter Transcript Space) between 16S and 23S rDNA region of these isolates with PCR-RFLP (Restriction Fragment Length Polymorphism) method, we’ve got altogether 19 genotypes in these isolates. 16 of them belong to PCA producing isolates. What’s more, genotype A5 was widely distributed in six sampling locations. Except for genotype A5, different genotypes could be found in each sampling location. The isolates obtained from Guzhuang and Maqiao occupied 31.1% and 26.7% of the total PCA producing isolates respectively. It is found that green peppers grown in these two regions suffer from less plant fungi disease infection than that in other four sampling sites, providing a complementary proof for biocontrol of plant fungi pathogens by these antibiotic-producing isolates. Moreover, more isolates belonging to genotype A5 have been found in these two
Phenazine and pyoluteorin producing pseudomonads prove to be effective biocontrol agents based on previous reports of our laboratory. After sampling in six different green pepper rhizosphere located in Shanghai and Jiangsu provice, we design molecular detecting probes according to the conserved gene fragments of phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) gene clusters in Pseudomonas spp. M18. Finally we’ve got altogether 48 PCA and Plt producing isolates with colony hybridization followed by fermentation confirmation, which is 0.4% of total colonies selected by King’s B medium (KMB) approximately. 94% of the total isolates (45) belong to widely distributed PCA producing group, while 6% (3) for both PCA and Plt producing group. None of them can produce Plt alone. After analyzing the 16S rDNA plus ITS (Inter Transcript Space) between 16S and 23S rDNA region of these isolates with PCR-RFLP (Restriction Fragment Length Polymorphism) method, we’ve got altogether 19 genotypes in these isolates. 16 of them belong to PCA producing isolates. What’s more, genotype A5 was widely distributed in six sampling locations. Except for genotype A5, different genotypes could be found in each sampling location. The isolates obtained from Guzhuang and Maqiao occupied 31.1% and 26.7% of the total PCA producing isolates respectively. It is found that green peppers grown in these two regions suffer from less plant fungi disease infection than that in other four sampling sites, providing a complementary proof for biocontrol of plant fungi pathogens by these antibiotic-producing isolates. Moreover, more isolates belonging to genotype A5 have been found in these two
引文
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