猕猴桃溃疡病菌的分子检测技术研究

 猕猴桃溃疡病是猕猴桃生产上的主要病害,为建立该病的快速诊断技术,本实验通过RAPD分析获得一条1 300 bp左右的致病菌的特异片段,对该片段进行克隆测序,在测序的基础上设计并合成一对特异引物F7/R7,优化特异引物扩增条件,并验证引物的特异性和灵敏性。利用该特异引物对包括猕猴桃溃疡病菌在内的14个菌株基因组DNA进行PCR扩增表明,只有猕猴桃溃疡病菌能扩增出1条约为950 bp的特异条带,其他菌株及对照均未扩增出特异条带。对采自果园的染病枝干组织和接种致病菌的枝干组织的检测表明,该特异引物能特异性地检测到猕猴桃溃疡病菌的存在,其在组织中的检测灵敏度为100 fg/μL。因此,利用设计合成的特异引物F7/R7,参考优化的体系和程序,结合简单的试剂盒法提取猕猴桃溃疡病菌或植物组织DNA,可以在短时间内完成对该病原菌的分子检测。     

利用EMAqPCR建立快速检测猕猴桃溃疡病菌活菌的方法

利用叠氮溴乙锭(ethidium monoazide bromide,EMA)与实时荧光定量PCR技术相结合(EMA-qPCR),建立了一种有效快速检测猕猴桃溃疡病菌活菌的方法。以猕猴桃溃疡病菌ITS序列为检测靶标,菌体经EMA渗透处理,再进行qPCR特异性扩增。结果显示,qPCR检测灵敏度为2cfu;当EMA的浓度为2.0μg/mL时,能有效抑制1.0×10~7 cfu/mL经高温灭活的死菌的扩增,对活菌的扩增没有影响。当活菌数在1.0×10~1~1.0×10~5 cfu范围内,每个qPCR反应体系中活菌数与Ct值呈线性相关(R~2=0.988)。不同温度处理活菌菌悬液后用EMA-qPCR检测猕猴桃溃疡病菌的存活情况并与平板计数法进行比较,结果表明待检样品可在4℃和20℃短期保存。对疑似带病猕猴桃材料进行EMA-qPCR检测,结果表明能减少猕猴桃溃疡病菌PCR的假阳性结果。本研究建立的EMAqPCR方法是一种有效检测猕猴桃溃疡病菌活菌的方法,能有效避免PCR检测实际样品可能造成的假阳性结果。     

气候变化情景下四川省猕猴桃溃疡病菌潜在地理分布模拟

近年来,猕猴桃溃疡病在四川各猕猴桃主产区严重发生,造成严重经济损失。本研究采用MaxEnt模型分析四川省猕猴桃溃疡病菌潜在分布,并预测2030年代、2050年代、2070年代和2080年代的RCP2.6、RCP4.5和RCP8.5等3种气候变化情景下适生区变化。预测结果运用ROC曲线评价模拟准确性。结果表明:所建立13个模型的训练数据和测试数据AUC (areas under curve)值均高于0.9,达到极高的精度。当前气候条件下,猕猴桃溃疡病菌在四川的高适生区主要位于成都市、德阳市、绵阳市、广元市、巴中市、达州市和雅安市,中适生区在四川21地市州均有分布。2030年代-2080年代,气候变化情景下,与当前情景相比,高适生区和低适生区区域均显著增加,中适生区区域先增加后减少,不同适生区几何中心位置和迁移规律均有所不同但总体上均向北移动。     

防治猕猴桃溃疡病涂抹剂的研制及其应用

由丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv. actinidiae (Psa)引起的猕猴桃溃疡病是猕猴桃产业上的毁灭性病害，生产中常施用水基型制剂进行防治，但因其附着性低且不易成膜保护树体，防治效果并不理想。为研制可有效防治猕猴桃溃疡病的新技术措施，本研究通过对不同填料和增稠剂进行筛选，并将两者混合后得到成膜助剂，再加入优化后的渗透剂制备得到基础涂抹剂(涂抹剂基^#)，对其成膜性、在树体上的附着性及耐雨水冲刷能力等性能指标进行了评价；向涂抹剂基^#中分别加入复配杀菌剂组合1^#(四霉素与戊唑醇质量比2:1)和组合2^#(四霉素与噻霉酮质量比5:1)，制得涂抹剂1^#与涂抹剂2^#；采用滤纸片法测定了涂抹剂基^#、涂抹剂1^#、涂抹剂2^#及所对应的杀菌剂组合对猕猴桃溃疡病菌的室内抑制活性，并通过田间试验验证了其对猕猴桃溃疡病的防治效果。结果表明：配方筛选所得最适填料为纳米膨润土...     

苹果壳色单隔孢溃疡病菌

苹果壳色单隔孢溃疡病菌(Botryosphaeria stevensii Shoemaker)是我国新修订的进出境动植物检疫性有害生物名录之一,目前在我国无分布。本文主要从地理分布、寄主范围、症状及危害、形态特征、生物学习性、检疫鉴定方法、防治措施等方面对该菌进行了综述,同时对其传入中国的风险进行评估,评估结果R值为2.17,传入总体风险高,口岸应加大检疫力度,严防该菌的传入。     

红心猕猴桃溃疡病及其防治技术

湖北省恩施自治州属亚热带季风性山地湿润气候,适宜红心猕猴桃生长。多年来,由于溃疡病频繁发生,直接影响红心猕猴桃产量和品质,制约了恩施州猕猴桃产业高质量发展。本文阐述了红心猕猴桃溃疡病的发病条件、危害症状及发病原因,并提出了避雨栽培、植物检疫、农业防治及化学防治等综合防控措施,旨在为山区红心猕猴桃溃疡病的防控提供指导服务。     

植物内生放线菌活性物质防治猕猴桃溃疡病

以供试内生放线菌发酵液的皿内抑菌活性为指标,得到对猕猴桃细菌性溃疡病菌有抑制活性的菌株76株,其中35株的发酵液对猕猴桃溃疡菌的抑菌圈直径≥20mm,5株对猕猴桃溃疡病菌均具有显著的抑制活性.其中gCLA4菌株发酵液抑菌谱最广,不仅对4种靶标细菌具有抑制活性,还对10种植物病原真菌有较强的抑制活性.通过发酵液制备及大孔吸附树脂D101处理得到gCLA4菌粗提活性物质.田间防治结果表明,gCLA4菌株粗提活性物质100倍稀释液对猕猴桃溃疡病28d的相对防效达64.9%,明显优于化学药剂施那宁.     

中国猕猴桃细菌性花腐病菌的鉴定

 从福建、湖南和湖北猕猴桃病花上分离到能引起花腐病的32个细菌菌株,经细菌学和BiologGN测试板测定,可以看出中国的猕猴桃细菌性花腐病菌与新西兰的猕猴桃花腐病菌、丁香假单胞菌丁香致病变种Pseudomonas syringae pv.syringae和绿黄假单胞菌P.viridiflava相似,与萨氏假单胞菌P.savastanoi和猕猴桃溃疡病菌P.syringae pv.actinidiae有更多的不同,但是DNA/DNA同源性测定结果却显示出中国的菌株可分为2个类型:第1个类型与新西兰猕猴桃花腐病菌和萨氏假单胞菌有很高的同源性,第2类型与绿黄假单胞菌有很高的同源性,说明中国菌株分别属于这2个种。第1类型来自于福建和湖北,第2类型来自于湖南。     

修文县猕猴桃溃疡病综合防控措施

通过对修文县猕猴桃溃疡病的调查研究,掌握猕猴桃溃疡病的发生特点、发病因素和田间消长规律,并提出综合防治措施。     

猕猴桃常见病虫害种类及综合防治

猕猴桃产业的发展受到病虫害的严重困扰,该文介绍果实熟腐病、根腐痛、蒂腐病、溃疡病、褐斑病、花腐病、叶蝉、吸果夜蛾、蝙蝠蛾和根结线虫病等猕猴桃常见病虫害的为害症状,并提出了相应的防治措施。     

Phylogenetic Relationships Among Global Populations of Pseudomonas syringae pv. actinidiae

ABSTRACT Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit (Actinidia spp.) vines, was first detected in Japan in 1984, followed by detections in Korea and Italy in the early 1990s. Isolates causing more severe disease symptoms have recently been detected in several countries with a wide global distribution, including Italy, New Zealand, and China. In order to characterize P. syringae pv. actinidiae populations globally, a representative set of 40 isolates from New Zealand, Italy, Japan, South Korea, Australia, and Chile were selected for extensive genetic analysis. Multilocus sequence analysis (MLSA) of housekeeping, type III effector and phytotoxin genes was used to elucidate the phylogenetic relationships between P. syringae pv. actinidiae isolates worldwide. Four additional isolates, including one from China, for which shotgun sequence of the whole genome was available, were included in phylogenetic analyses. It is shown that at least four P. syringae pv. actinidiae MLSA groups are present globally, and that marker sets with differing evolutionary trajectories (conserved housekeeping and rapidly evolving effector genes) readily differentiate all four groups. The MLSA group designated here as Psa3 is the strain causing secondary symptoms such as formation of cankers, production of exudates, and cane and shoot dieback on some kiwifruit orchards in Italy and New Zealand. It is shown that isolates from Chile also belong to this MLSA group. MLSA group Psa4, detected in isolates collected in New Zealand and Australia, has not been previously described. P. syringae pv. actinidiae has an extensive global distribution yet the isolates causing widespread losses to the kiwifruit industry can all be traced to a single MLSA group, Psa3.     

Bacterial canker on kiwifruit in Italy: anatomical changes in the wood and in the primary infection sites

The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae is a severe threat to kiwifruit production worldwide. Many aspects of P. syringae pv. actinidiae biology and epidemiology still require in-depth investigation. The infection by and spread of P. syringae pv. actinidiae in xylem and phloem was investigated by carrying out artificial inoculation experiments with histological and dendrochronological analyses of naturally diseased plants in Italy. We found that the bacterium can infect host plants by entering natural openings and lesions. In naturally infected kiwifruit plants, P. syringae pv. actinidiae is present in the lenticels as well as in the dead phloem tissue beneath the lenticels, surrounded by a lesion in the periderm which appears to indicate the importance of lenticels to kiwifruit infection. Biofilm formation was observed outside and inside plants. In cases of advanced stages of P. syringae pv. actinidiae infection, neuroses of the phloem occur, which are followed by cambial dieback and most likely by infection of the xylem. Anatomical changes in wood such as reduced ring width, a drastic reduction in vessel size, and the presence of tyloses were observed within several infected sites. In the field, these changes occur only a year after the first leaf symptoms are observed suggesting a significant time lapse between primary and secondary symptoms. It was possible to study the temporal development of P. syringae pv. actinidiae-induced cambial dieback by applying dendrochronology methods which revealed that cambial dieback occurs only during the growing season.     

Genomic and phenotypic characterization of the bacterium causing blight of kiwifruit in New Zealand

Strains of the pathogen causing bacterial blight of kiwifruit in New Zealand, previously identified as Pseudomonas viridiflava, were examined using phenotypic and genotypic methods. Percentage DNA–DNA reassociation values for strains of the pathogen, with the type strains representing P. viridiflava and P. savastanoi, and representative strains within P. syringae , were obtained using the S1 endonuclease method. Strains of the pathogen were most similar to the type strain of P. savastanoi. This similarity was supported by examination of the Δ T _m between representative strains. It is concluded that the pathogen can be considered as a member of the P. savastanoi genomic species. The pathogen from kiwifruit in New Zealand was also differentiated in genomic terms from P. syringae pv. actinidiae . Strains of the kiwifruit pathogen compared using the Biomerieux API Biotype 100 system exhibited consistent determinative tests which distinguished the pathogen from P. viridiflava and P. syringae pv. actinidiae . The origins of the pathogen in New Zealand are discussed.     

Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy

Pseudomonas syringae pv. actinidiae has been isolated from kiwifruit plants for the first time in Italy. Biochemical tests were consistent with those characterizing the type-strain; pathogenicity tests yielded severe blights in the inoculated kiwifruit plants and no symptoms on lilac, pear and peach. Nutritional tests as well as whole-cell protein profiles revealed slight differences between the strains isolated in Japan and those of the present study. The main symptoms observed in the field are a red-rusty exudation covering the bark of twigs and trunks, blight of young canes and plants, angular leaf spots surrounded by chlorotic haloes and tiny cankers along the twigs.     

猕猴桃品种酚类物质及可溶性蛋白含量与抗溃疡病的关系

以安徽省猕猴桃主栽品种金魁、早鲜、魁蜜、华美2号、秦美、金丰为研究对象,于展叶孕蕾期分别取发病的枝条、叶片,以未发病健株的相应组织为对照,分析枝条、叶片中酚类物质和可溶性蛋白的含量变化。结果表明：抗病品种健株枝条、叶片中可溶性蛋白含量显著高于易感病品种,说明枝条中可溶性蛋白含量与品种抗性成正相关。自然发病后,感病品种枝条中可溶性蛋白含量增加,抗病品种可溶性蛋白含量降低。抗病品种健枝条、叶片中酚类物质含量高于易感病品种的健枝、叶,发病后抗感品种酚类物质含量都增加。     

First report of bacterial canker of kiwifruit in Iran

Symptoms of bacterial canker of kiwifruit consisting of darkening and shrivelling along the bark were observed in kiwifruit orchards in Tonkabon, Iran. Longidutinal sections showed darkening of cortical and woody tissues. A fluorescent pseudomonad was consistently isolated from canker tissues. According to biochemical, physiological and pathogenicity tests the causal agent was identified as Pseudomonas syringae pv. syringae. This is the first report of the disease in Iran.     

Expression analysis of defense-related genes in wild kiwifruit (Actinidia rufa) tolerant to bacterial canker

Journal of General Plant Pathology - Kiwifruit bacterial canker, which is caused by Pseudomonas syringae pv. actinidiae biovar 3 (Psa3), is found throughout kiwifruit-growing countries. Here, we...     

Loop-mediated isothermal amplification of bacterial effector genes to detect Pseudomonas syringae pv. actinidiae biovars 1 and 3

Journal of General Plant Pathology - Loop-mediated isothermal amplification (LAMP) was designed to rapidly detect biovars 1 and 3 of Pseudomonas syringae pv. actinidiae (Psa), a serious, global...