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首页> 外文期刊>European Journal of Plant Pathology >Enhanced detection and isolation of the walnut pathogen Brenneria rubrifaciens, causal agent of deep bark canker
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Enhanced detection and isolation of the walnut pathogen Brenneria rubrifaciens, causal agent of deep bark canker

机译：深层树皮溃疡病原体病原体核桃病原体Brenneria rubrifaciens的增强检测和分离

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摘要

Deep bark canker (DBC) of walnut is caused by the bacterium Brenneria rubrifaciens which produces the red pigment rubrifacine. This disease of English walnut trees, is characterized by deep vertical cankers which exude sap laden with B. rubrifaciens. Although DBC is not observed on young trees, it is hypothesized that B. rubrifaciens is present in host tissue years before symptom development. Therefore, a sensitive technique would be useful in detecting B. rubrifaciens in asymptomatic trees. Tn5 mutants deficient in rubrifacine production (pig ) were generated and DNA sequences from pig mutants were used to design two primer sets; GSP1F-GSP1R and GSP2F-GSP2R. A third primer pair, BR1-BR3 was designed from the 16S rRNA gene. The three primer pairs did not amplify the diagnostic bands from members of the following bacterial genera: Agrobacterium, Erwinia, Pseudomonas, Ralstonia, and Rhizobium. In addition, no amplification was observed using DNA from the following Brenneria species, alni, nigrifluens, quercina, or salicis. All three DNA primer sets detected B. rubrifaciens in spiked greenhouse soil and infiltrated walnut leaf tissue. PCR detection limits for BR, GSP1, and GSP2 primer pairs were 254, 254, and 2.54 x 10t colony forming units (CFU) respectively. Real-time PCR detection limit for BR primers was 8 CFU. The differential medium, yeast extract dextrose calcium carbonate agar (YDCA) was amended with novobiocin, and bacitracin, to enhance isolation from environmental samples. The improved detection and isolation methods described here will facilitate examination of B. rubrifaciens ecology under both nursery and orchard conditions.
机译：核桃的深树皮溃疡病（DBC）是由产生红色色素Rubrifacine的Br​​enneria rubrifaciens细菌引起的。这种英国胡桃树病的特征是深深的垂直溃疡，渗出液汁中充满了B. rubrifaciens。尽管在幼树上未观察到DBC，但据推测在症状发展之前的几年中，宿主组织中就存在红景天芽孢杆菌。因此，一种灵敏的技术将可用于检测无症状树中的红景天杆菌。产生了缺乏红霉素的猪Tn5突变体，猪突变体的DNA序列用于设计两个引物对。 GSP1F-GSP1R和GSP2F-GSP2R。从16S rRNA基因设计了第三对引物BR1-BR3。这三个引物对未扩增以下细菌属成员的诊断条带：农杆菌属，欧文氏菌属，假单胞菌属，罗氏菌属和根瘤菌属。此外，使用以下布雷氏菌属物种（链球菌，黑气菌，槲皮或唾液酸）的DNA均未观察到扩增。所有这三个DNA引物组在加标的温室土壤和浸润的核桃叶组织中均检测到红芽孢杆菌。 BR，GSP1和GSP2引物对的PCR检测限分别为254、254和2.54 x 10t集落形成单位（CFU）。 BR引物的实时PCR检测极限为8 CFU。将差异培养基酵母提取物右旋糖碳酸钙琼脂（YDCA）用新霉素和杆菌肽进行了改良，以增强与环境样品的分离。此处描述的改进的检测和隔离方法将有助于在苗圃和果园条件下检查红景天杆菌的生态。

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