拮抗根癌土壤杆菌的桃内生细菌的筛选鉴定

Screening and Identification of Peach Endophytic Bacteria with Antagonism Against Agrobacterium tumefaciens

【目的】从对根癌病具有获得抗性(SAR)的桃单株"西北13-1"的枝条内分离鉴定内生细菌,分析其种群组成并从中筛选拮抗根癌土壤杆菌的高效生防菌,为桃根癌病的生物防治探索新方向并提供新的生防菌资源。【方法】首先,将根癌土壤杆菌(Agrobacterium tumefaciens)接种到"西北13-1"的桃树枝条上,并以无菌水接种作为对照;然后,分别于接种前、接种后10 d和接种后60 d采集接种处理和对照处理的"西北13-1"枝条。表面消毒桃树枝条样品后采用涂布平板法分离内生细菌;并结合16S r DNA序列鉴定,比对初步鉴定菌株,分析内生细菌的数量及组成差异。采用平板拮抗法、温室防病效果测定筛选拮抗根癌土壤杆菌的活性菌株,通过生理生化测定和分子鉴定,明确其分类地位。为揭示拮抗菌可能具有的其他拮抗作用机制,通过电击转化将含有GFP基因的p BBR1MCS-2质粒导入拮抗菌株中,然后进行GFP标记菌株对根癌土壤杆菌的抑菌试验、生长动力学分析、稳定性测试。采用灌根法提前接种标记菌株菌悬液,第2天接种根癌土壤杆菌菌悬液,应用激光扫描共聚焦显微镜和平板稀释法研究标记菌株在番茄根部的定殖情况。【结果】从"西北13-1"桃枝条中共分离到108株内生细菌,基于16S r DNA序列,这些菌株分别属于伽马变形菌类(Gammaproteobacteria)、阿尔法变形菌类(Alpharoteobacteria)、放线菌类(Actinobacteria)、厚壁菌类(Firmicutes)、拟杆菌(Bacteroidetes),共计5个细菌类群17个属,显示了桃树枝条中内生细菌丰富的种群多样性。其中,伽马变形菌类的肠杆菌属(Enterobacter)、泛菌属(Pantoea)和根瘤菌属(Rhizobium)最多,其数量在接种根癌土壤杆菌后显著上升,包含了14株产抑菌圈的拮抗菌中的10株细菌。经鉴定,筛选出的两株抑菌活性较高、具有生防潜力的拮抗菌10DM4-1和10DI2-2分别为泛菌(Pantoea deleyi)和肠杆菌(Enterobacter cowanii),它们可显著抑制向日葵根癌瘤的形成,防治效果分别为86.08%和89.87%。标记菌株10DM4-1-gfp和10DI2-2-gfp灌根番茄后,可在根部细胞间隙观察到目标菌落,标记菌株的数量在0—10 d时呈现急剧下降趋势,最后保持相对稳定的较低水平(104CFU/g),说明了它们在番茄根组织内具有良好的生存和定殖能力。【结论】桃"西北13-1"的内生菌中存在着能够拮抗根癌土壤杆菌的细菌,这些具有拮抗活性的菌株与桃"西北13-1"具有根癌病抗性有关。分离得到的泛菌10DM4-1和肠杆菌10DI2-2可能通过产拮抗物质、占据根部有利生态位拮抗根癌土壤杆菌。

[Objective]The objectives of this study are to identify the population composition of cultivable endophytes in peach twigs of ‘Xibei 13-1＇ cultivar, and to screen the new antagonists for biological control of crown gall disease. [Method] Peach twigs of ‘Xibei 13-1＇ cultivar were inoculated with Agrobacterium tumefaciens, and mock inoculation of the control was performed in a similar manner, but sterilized distilled water was used instead of the bacterial suspension. Twigs with different treatments were collected at different time points（before inoculation, 10 and 60 days after inoculation, respectively）. Surface sterilization, endophytes isolation and counts, and 16 S r DNA sequencing, were conducted to analyze the quantity and diversity of endophytic bacteria among samples. Their antagonisms against the causal agent of crown gall disease, A. tumefaciens, were tested by pair co-culturing method, and the efficacy of the antagonists in suppressing crown gall disease was further evaluated in greenhouse using sunflowers as a susceptible plant. The taxonomic status was clarified by physiological and biochemical and molecular methods. In order to reveal the antagonistic mechanism, the plasmid of p BBR1 MCS-2 that contains green fluorescent protein gene（GFP） was transformed into the antagonistic strains by electroporation. Antibacterial activities, dynamic analysis and stability of the GFP-labeled strain were tested. The labeled strain suspension was inoculated using root irrigation method, then the A. tumefaciens suspension was inoculated at the 2 nd day. Their re-colonization on the tomato roots was examined by fluorescence microscope and plate dilution method. [Result]A total of 108 endophytic bacterial isolates were obtained from peach twigs of ‘Xibei 13-1＇ cultivar. All isolates were identified as 17 genera of the 5 bacterial groups-phylogenetically based on 16 S r DNA. The 5 groups are Gammaproteobacteria, Alpharoteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. This result showed the biodiversity of endophytic bacteria from peach twigs of ‘Xibei 13-1＇ cultivar. Among them, genera Enterobacter, Pantoea, and Rhizobium were the most abundant and significantly increased following inoculation of A. tumefaciens. In addition, most of the antagonists belong to these three genera as well（10/14）. In vivo, strains 10 DM4-1 and 10 DI2-2 showed good performances in disease control with the efficacy of 86.08% and 89.87%, respectively. Strains 10 DM4-1 and 10 DI2-2 were then identified as Pantoea deleyi and Enterobacter cowanii by their 16 S r DNA sequences in combination with their biochemical and physiological characteristics, respectively. The population of transformants 10 DM4-1-gfp and 10 DI2-2-gfp on tomato roots decreased sharply in the first 10 days, then declined slowly after 10 th day and remained constant at lower level（104 CFU/g）, indicating that they could stably colonize and survive in the intercellular of tomato roots. [Conclusion]The resistance of peach ‘Xibei 13-1＇ cultivar against crown gall disease may be related to its endogenous Enterobacter, Pantoea and Rhizobium. P. deleyi 10 DM4-1 and E. cowanii 10 DI2-2 can effectively suppress crown gall disease caused by A. tumefaciens, produce antagonistic substances, colonize the favorable ecological niche and has high potential application value for biological control of crown gall disease.