猕猴桃溃疡病菌biovar 3群体MLVA分型技术的建立与应用

 丁香假单胞菌猕猴桃致病变种生物型3(Pseudomonas syringae pv. actinidiae biovar 3,Psa3)是猕猴桃溃疡病菌的世界流行群体,但仅在中国存在复杂的遗传多样性。开发适于Psa3群体分型的MLVA(multilocus variable-number tandem-repeat analysis)技术是探索中国Psa3起源与流行学特性的基础。本研究对7个Psa3菌株进行了全基因组测序,结合已公布的86个全基因组数据,进行比较分析发现,中国Psa3至少存在7个亚群;在各亚群间存在多态性的24个串联重复序列中,其中10个可以通过琼脂糖凝胶电泳区分开且变异指数合适,据此建立了适于Psa3的MLVA技术。采用该技术对分别来自贵州和陕西的62和9个Psa3菌株进行群体分型,分型结果与全基因组分析高度一致,证明该MLVA体系分型准确。MLVA分型结果表明:贵州主产区修文县Psa3有3个MLVA群体,其中亚群4的组内分化明显,代表最早发生的群体;而亚群 1和3的结构单一,且多在新果园发现,是新传入群体。总之,本研究建立了一套可用于Psa3群体分型的MLVA技术,将有助于解析中国各猕猴桃产区Psa3群体结构,以及探索中国Psa3的起源、传播和流行学特征。     

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。     

猕猴桃溃疡病菌在中国的适生性分析

通过分析猕猴桃溃疡病菌在中国的适生性,为科学制定有效的检疫监管措施,防范其入侵和扩散,确保猕猴桃产业健康发展提供理论依据。本研究根据前人研究结果,采用模糊数学综合评判的原理和方法,定量分析猕猴桃细菌性溃疡病菌(Pseudomonas syringae pv.actinidiae)在我国各个地区的适生性。猕猴桃溃疡病菌在我国最适宜的省份主要分布在四川、云南、贵州、福建、安徽、湖南、湖北、河南、江西、陕西、浙江、重庆、西藏。鉴于该病具有发生发展迅速,危害性强,防治难度大等特点,应当加强猕猴桃种苗等繁殖材料的检疫,加强对果园的管理和病害监测,积极采取有效的防治措施并加强抗病育种方面的研究。     

软枣猕猴桃种质资源溃疡病抗性鉴定方法的建立与评价

以51份软枣猕猴桃Actinidia arguta种质资源为材料,以中华猕猴桃Actinidia chinensis Planch'红阳'、美味猕猴桃Actinidia chinensis var.deliciosa'徐香'为对照,利用丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv.actin...     

四种杀菌剂防治猕猴桃溃疡病的效果及田间应用技术

为探究猕猴桃溃疡病的化学防治技术,采用离体枝条室内生测及田间试验的方法,对72%农用链霉素SPX、46%氢氧化铜WG、20%叶枯唑WP及1.5%噻霉酮EW的预防和治疗效果以及施药的时期和方法进行了研究。结果显示,供试杀菌剂对猕猴桃溃疡病均有显著的预防效果和一定的治疗效果,在浓度为4.0 mg/m L和2.0 mg/m L时,对病害病斑15 d的预防效果分别达到100%和68.0%以上,较相同浓度的治疗效果高2~3倍,预防和治疗效果均达到90%时的杀菌剂浓度分别为3.07~3.35 mg/m L和8.32~10.81 mg/m L,药效持效期约为11~14 d。田间全树喷雾和树干涂药试验表明,农用链霉素和氢氧化铜预防效果较好,超过60%;秋冬季枝干发病前施药4~5次预防效果可达70%左右;枝干发病显症初期采用病斑划道后涂药治疗效果较好,但对大于15 cm的中大型病斑治疗效果较差,而枝干发病后采取药剂注射及药剂涂抹法治疗病斑效果均较差。表明秋冬季是预防猕猴桃溃疡病的关键期,而发病初期为治疗最佳时期。     

猕猴桃品种对细菌性溃疡病的抗性机制

研究了安徽省猕猴桃主栽品种金魁(高抗)、早鲜(中抗)、魁蜜(抗病)、华美2号(感病)、秦美(中感)和金丰(高感)对细菌性溃疡病的抗性机制.结果表明:形态结构抗性方面,感病品种的皮孔密度和长度及气孔密度、长度和宽度都明显高于抗病品种,抗、感品种间皮孔、气孔的排列方式也存在一定差异.相关分析表明,皮孔密度和长度及气孔密度、长度和宽度与品种发病率都有较高的相关性,尤其以皮孔长度和气孔长度相关性最高,r分别为0.9278和0.9794;生理生化抗性方面,品种未感染溃疡病菌前,芽中的POD酶活性与品种抗性存在规律性不强,一年生枝条、叶片中的POD酶活性与品种抗性有密切关系.品种感染溃疡病菌后,POD酶活性均升高,但抗病品种中酶活提高倍数高于感病品种.一年生枝条上病斑越大,POD酶活性越高.POD同工酶谱带表现出与酶活相一致的规律.     

四川省猕猴桃溃疡病发生现状及绿色防控技术体系的建立

通过调查,明确了猕猴桃溃疡病在四川的发生分布情况,以及猕猴桃品种抗性、园区管理情况,建立了猕猴桃溃疡病绿色防控体系。     

猕猴桃溃疡病菌不致病菌株G230的鉴定及形成原因分析

为探索田间猕猴桃溃疡病菌Pseudomonas syringae pv. actinidiae(Psa)致病力丧失的分子机制,针对从猕猴桃果园中分离获得的1株不致病菌株G230,通过特异性引物检测和多基因序列分析明确其分类地位,并设计引物检测其是否由已知遗传变异引起,通过比较基因组学、基因表达、超敏反应和荧光素酶报告菌株检测确定引起菌株G230致病力丧失的原因。结果表明,不致病菌株G230为Psa生物型3(Psa3),其致病缺陷不是由已报道的遗传变异引起;基于基因组比较分析发现菌株G230中的hrpS基因被转座子ISPsy36插入破坏,导致Ⅲ型分泌系统（type Ⅲ secretion system,T3SS）不能正常表达;而在不致病菌株G230中表达hrpS基因后能恢复其T3SS功能,使其具备致病能力及激发非寄主超敏反应的能力。表明转座子ISPsy36插入hrpS基因内部可以破坏Psa的T3SS功能进而使其丧失致病力,这是自然条件下Psa3丧失致病力的一种新型机制。     

猕猴桃溃疡病菌Ⅲ型效应蛋白HopAZ1功能研究与互作蛋白鉴定

细菌Ⅲ型分泌系统可向寄主植物细胞分泌多种效应蛋白,在丁香假单胞菌猕猴桃致病变种(Psa)侵染猕猴桃致病中发挥关键作用.但是,尚不清楚Psa如何利用这些效应蛋白与寄主互作.Psa的5个生物型群体具有不同种类的效应子,但其中共有的14个效应子可能是参与病菌与寄主亲和互作的关键因子.本研究系统调查了其中HopAZ1的功能.结...     

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。     

Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.

Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die‐back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.     

Comparative genomics‐informed design of two LAMP assays for detection of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae and discrimination of isolates belonging to the pandemic biovar 3

The aim of this study was to develop a rapid, sensitive and reliable field‐based assay for detection of the quarantine pathogen Pseudomonas syringae pv. actinidiae (Psa), the causal agent of the most destructive and economically important bacterial disease of kiwifruit. A comparative genomic approach was used on the publicly available Psa genomic data to select unique target regions for the development of two loop‐mediated isothermal amplification (LAMP) assays able to detect Psa and to discriminate strains belonging to the highly virulent and globally spreading Psa biovar 3. Both LAMP assays showed specificity in accordance with their target and were able to detect reliably 125 CFU per reaction in less than 30 min. The developed assays were able to detect the presence of Psa in naturally infected kiwifruit material with and without symptoms, thus increasing the potential of the LAMP assays for phytosanitary use.     

Frost promotes the pathogenicity of Pseudomonas syringae pv. actinidiae in Actinidia chinensis and A. deliciosa plants

Frost occurs in all major areas of cultivation, presenting a threat for the production of kiwifruit crops worldwide. A series of experiments were performed on 1‐year‐old, potted plants or excised twigs of Actinidia chinensis and A. deliciosa to verify whether strict relationships exist between bacterial canker outbreaks from Pseudomonas syringae pv. actinidiae (Psa) attacks and the occurrence of autumn and winter frost events. The association between the occurrence of autumn frost and the sudden outbreak of bacterial canker in A. chinensis in central Italy has been confirmed. Both autumn and winter frosts promote Psa multiplication in the inoculated twigs of both species. The day after the frost, reddish exudates oozing from the inoculation sites were consistently observed in both species, and Psa was re‐isolated in some cases. During the thawing of both A. deliciosa and A. chinensis twigs, the 2‐cm upward and downward migration of Psa from the inoculation site was observed within 3 min, and the leaves were consistently colonized with the pathogen. A consistent brown discoloration, accompanied with a sour‐sap odour, was observed throughout the length of the excised twigs of both Actinidia species after Psa inoculation and winter frost. Psa inoculation induced a remarkably higher necrosis in excised twigs that were not frozen compared with P. s. pv. syringae inoculation. Antifreeze protection using irrigation sprinklers did not influence the short‐term period of Psa and P. s. pv. syringae multiplication in both A. deliciosa and A. chinensis twigs. Thus, the damage from frost, freeze thawing and the accumulation of Psa in Actinidia twigs promotes the migration of the pathogen within and between the orchards. Taken together, the results obtained in this study confirmed that A. deliciosa is more frost tolerant than A. chinensis, autumn frosts are more dangerous to these crops than winter frosts, and in the absence of Psa, young kiwifruit plants remain sensitive to frost.     

Evaluation of the wild Actinidia germplasm for resistance to Pseudomonas syringae pv. actinidiae

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is a catastrophic disease that threatens the global kiwifruit industry. As yet, no cure has been developed. Planting resistant cultivars is considered as one of the most effective ways to control Psa. However, most existing cultivars lack Psa-resistance genes. Wild Actinidia resources contain rich genetic diversity and may have powerful disease-resistance genes under long-term natural selection, but lack of knowledge about the resistance to Psa for most Actinidia species results in some excellent wild resistant genotypes being underutilized. In this study, the response to Psa of 104 wild genotypes of 30 Actinidia species (including 37 taxa) was tested with an in vitro bioassay, and a considerable number of individuals from different species with tolerance or high resistance to Psa were identified. The results showed high consistency between years. This is the first large-scale evaluation of diverse Actinidia species with resistance to Psa through an in vitro bioassay. The resistant genotypes of A. chinensis identified could be used in future kiwifruit improvement programmes. The findings should help provide an understanding of the resistance to Psa.     

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。     

湘西地区猕猴桃细菌性溃疡病抗性资源筛选及其抗性机理研究

由Pseudomonas syringae pv.actinidiae(Psa)引起的溃疡病是猕猴桃生产中威胁最大的细菌性病害。种植抗病品种是防治猕猴桃溃疡病最有效途径,猕猴桃抗源是抗病育种的物质基础。本试验通过离体接种,在室内评价了4个不同种的7个猕猴桃资源及品种对溃疡病的抗性。结果表明:供试材料离体叶片、枝条接种溃疡病菌后,其发病时间、病斑大小、发病率差异明显。按病斑大小排序,其抗性强弱依次为毛花猕猴桃‘M9808’>对萼猕猴桃‘S9801’>美味猕猴桃‘M-06’>美味猕猴桃‘米良1号’>中华猕猴桃‘Z-12’>中华猕猴桃‘东红’>中华猕猴桃‘红阳’(CK)。其中,毛花猕猴桃‘M9808’抗病性最强,表现为发病最晚,离体叶片、枝条分别于接种后10 d、20 d发病,比对照推迟了8 d、10 d;病斑最小,为对照的1/25、1/11;发病率最低,为对照的1/28、1/10。抗病性相关酶活性比对发现,不同材料的PAL、CAT、POD酶活性差异较大,抗性材料均高于感病材料,且峰值出现时间早于感病材料。其中,毛花猕猴桃‘M9808’的PAL峰值最高,接种后5 d出现,比对照早10 d;CAT、POD峰值接种后10 d出现,比对照早5 d。说明毛花猕猴桃‘M9808’对溃疡病抗性最强,可作为今后猕猴桃抗病育种或抗性砧木的理想材料。这为猕猴桃抗病育种提供了理论依据,为猕猴桃溃疡病的防控提供了参考。