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。     

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.     

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。     

Influence of Ring Nematode Infestation and Calcium, Nitrogen, and Indoleacetic Acid Applications on Peach Susceptibility to Pseudomonas syringae pv. syringae

ABSTRACT Two field experiments were conducted to study the effects of added nitrogen, calcium, and indoleacetic acid, in the presence or absence of ring nematodes (Mesocriconema xenoplax), on susceptibility of peach to bacterial canker. When noninfested soil was inoculated with ring nematodes, peach tree susceptibility to bacterial canker infection caused by Pseudomonas syringae pv. syringae was dramatically increased after a period of 2 years. However, no evidence was found that ring nematode infestation increased tree water stress or, in turn, altered plant calcium uptake. Soil fumigation with methyl bromide prior to planting in a commercial orchard significantly reduced both nematode populations and peach tree susceptibility to bacterial canker infection when compared with nonfumigated treatments. In both experiments, tree susceptibility, as measured by canker length following inoculation of stems with P. syringae pv. syringae, was negatively correlated with plant tissue nitrogen content and positively correlated with tissue calcium content. A principal components analysis showed that tissue nitrogen and calcium levels were negatively correlated, and that high-nitrogen, low-calcium tissues were less susceptible to bacterial canker than low-nitrogen, high-calcium tissues. These results indicate that the increased susceptibility of peach to P. syringae pv. syringae under nematode infestation conditions is mediated by both nutritional effects (primarily nitrogen) and nutritional-independent effects, but do not support previous reports of beneficial effects of calcium for reducing bacterial canker.     

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.     

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。     

Interaction Between Nitrogen-Fertilized Peach Trees and Expression of syrB, a Gene Involved in Syringomycin Production in Pseudomonas syringae pv. syringae

ABSTRACT The in vitro expression of the syrB gene that controls the synthesis of syringomycin, a non-host-specific phytotoxin produced by Pseudomonas syringae pv. syringae van Hall, was studied using aqueous extracts derived from bark tissues collected from nitrogen-fertilized and nonfertilized peach trees. Expression of the syrB gene was quantified as beta- galactosidase activity expressed by P. syringae pv. syringae B3AR-132 containing a syrB::lacZ fusion. Gene expression was significantly less in three of four paired comparisons using extracts derived from fertilized versus nonfertilized trees; however, canker lengths were significantly different in only one of four comparisons. Expression was negatively correlated with plant tissue nitrogen content and positively correlated with a plant carbon/nitrogen ratio. Bark tissue from ring nematodeinfested trees had significantly higher concentrations of total soluble phenolic compounds and carbon/nitrogen ratios than bark samples from trees without nematodes, and canker size was significantly greater in trees growing in ring nematode-infested soil compared with noninfested soil. Nitrogen fertilization significantly decreased the plant carbon/nitrogen ratio, which was positively correlated with the concentration of total soluble phenolic compounds. Canker size developing after bacterial inoculation was positively correlated with higher plant carbon/nitrogen ratios and total soluble phenolic compounds. These results support the hypothesis that one reason why nitrogen fertilization decreases host susceptibility to bacterial canker is by either reducing the amount of plant metabolites that can induce syrB gene expression, or producing or increasing the concentration of compounds that antagonize syrB inducing compounds.     

贵州六盘水市猕猴桃病害调查及病原鉴定

针对2014年以来六盘水市猕猴桃病害日益严重的问题,分别于2015年10月、2016年4月及8月对六盘水市共12个乡镇26个代表性猕猴桃栽培园区病害进行了调查。采集了大量典型病害样本,综合生物学特性、分子鉴定及致病力测试对分离得到的病原菌进行鉴定。结果表明,六盘水市猕猴桃夏季和秋冬季病害主要为细菌性溃疡病和真菌性软腐病、灰斑病、褐斑病,其次是炭疽病和黑斑病,春季部分园区有细菌性溃疡病,真菌病害相对较轻。本文研究结果为六盘水市猕猴桃病害的预测预报及综合防治提供了理论依据。     

不同猕猴桃品种RAPD分析及其与抗溃疡病的关系

对不同猕猴桃品种的分子生物学试验表明：猕猴桃的DNA浓度在920 μg/mL符合RAPD分析的要求。通过60个随机引物的PCR扩增,报道了6个不同品种和类型猕猴桃种质资源的RAPD多态性,计算了它们之间的遗传距离,构建了聚类图,并讨论了其亲缘关系。聚类分析图反映出来源于安徽省主要猕猴桃产区的6个样品可以分为3组,其中抗病与感病的相对较为集中,由此可推断出现这种聚类的原因可能是由于它们基因组中有相同的DNA片段。抗病品系都有一条1 458 bp DNA片段,而感病品系均没有该带。故该片段可能与猕猴桃植株抗溃疡病相关。RAPD多态性从分子水平上反映出了猕猴桃种质资源不同品种及不同类型间复杂的遗传背景,为抗病育种的亲本选配提供了依据,也为合成猕猴桃抗溃疡病探针并用于检测猕猴桃抗溃疡病种质和分子标记辅助育种奠定了基础。     

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.     

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。     

Influence of rootstock on the susceptibility of sweet cherry scions to bacterial canker, caused by Pseudomonas syringae pvs morsprunorum and syringae

In a field trial to determine whether the rootstock influenced the susceptibility of cherry cultivars to bacterial canker three cultivars (Napoleon, Roundel and JI 14039), each grafted on two rootstocks (F 12/1 and Colt), were subjected to natural infectionand to inoculation with three bacterial canker pathogens (Pseudomonas syringae pv. morsprunorum races 1 and 2 and P. syringae pv. syringae). Inoculations were made through leaf scars and through wounds. The high susceptibility of Napoleon and high resistance of JI 14039 were confirmed. Napoleon was more susceptible to inoculation through branches when on F12/1 than when on Colt but the reverse was true for leaf scar inoculations. JI14039 was more susceptible to race 1 inoculated through leaf scars when grown on F12/1 than when on Colt. No rootstock/scion interaction was detected with Roundel.
The complexity of the relationships between the pseudomonad pathogens and their cherry hosts is briefly discussed.     

猕猴桃园区坡度和晴雨天对猕猴桃溃疡病传播的影响

猕猴桃溃疡病是由丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv. actinidiae,Psa)引起的细菌性病害,严重影响猕猴桃产业发展。明确猕猴桃溃疡病在田间的传播对其防治具有重要意义。本研究于2020年4月16-22日和2021年3月24-30日,在湖北省赤壁市调查了半岛南猕猴桃种植园区内猕猴桃溃疡病的发病情况,通过孢子捕捉器在猕猴桃园区的不同地势(坡度为10°、30°和45°的区域)和不同天气环境下进行24 h连续病原捕集,并据猕猴桃叶片上溃疡病显症所需时长反映不同地势和天气环境对病害传播的影响。结果表明,在坡度10°区域进行病原捕集的猕猴桃叶片显症所需时间比坡度30°和45°区域晚1~2 d。雨天在孢子捕捉器内的叶片上显症所需时长仅需7 d,而晴天显症时长则需10~12 d。明确了露地栽培猕猴桃园区坡度和雨天显著促进了猕猴桃溃疡病的传播。对猕猴桃溃疡病防治技术制订和实施有一定意义。     

Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae

Several factors affecting the severity of bacterial canker of pear were studied. In the orchard, infection of shoots by Pseudomonas syringae pv. syringae occurred only when the inoculum dose exceeded 10⁶ colony-forming units/shoot. However, under favourable conditions in a growth chamber, cankers formed on detached shoots inoculated with 5 cfu/shoot. A second-order polynomial relationship was established between log₁₀ transformed canker length and log₁₀ transformed inoculum dose. In orchard and growth chamber experiments, shoots were susceptible from the time of bud swell until after fruit harvest. The severity of Pseudomonas canker of detached shoots increased if they were frozen at – 10°C for 24 h before inoculation. Shoots were most susceptible when inoculated immediately after wounding, and no cankers developed in the orchard when 3-day-old wounds were inoculated. Additionally, no cankers resulted from inoculation of leaf scars at leaf drop. Actively growing, current-season shoots were more susceptible than shoots that had set a terminal bud. The practical implications of these results are discussed as a basis for control of bacterial canker of pear.     

Influence of stem diameter, water content, and freezing-thawing on bacterial canker development in excised stems of dormant stone fruit

ABSTRACT In excised dormant stems of peach (Prunus persica), prune (Prunus domestica), and almond (Prunus dulcis), stem diameter, stem hydration, and freezing-thawing influenced the extent of infection caused by Pseudomonas syringae pv. syringae. Bacterial lesion length increased with increasing stem diameter, demonstrating the need to account for the effects of stem diameter when lesion length data are analyzed. Lesion length increased or decreased with stem hydration or dehydration, respectively. However, tissue water content was not a good indicator of tissue susceptibility to infection by P. syringae pv. syringae, as larger diameter stems had larger lesions and lower water content than did smaller diameter stems. After freezing at -5 degrees C for 12 to 24 h, inoculations made during the thawing process produced significantly larger lesions than did inoculations performed before freezing or after thawing. These results support the hypothesis that the increased susceptibility to bacterial canker that is associated with noninjurious freezing is a result of the increased passive spread of bacteria through water redistribution when inoculation is performed during the thawing process. Plant tissue water relationship characteristics that can influence water movement during freezing and thawing may be an important component of bacterial canker development in stone fruit trees.     

不同猕猴桃品种对细菌性溃疡病的抗病性及其聚类分析

通过田间定点抗病性调查,结合前人的研究结果得出:不同猕猴桃品种对细菌性溃疡病的抗性存在明显差异。金魁和两种雄株中华软、美味硬最为抗病,早鲜次之,魁蜜稍抗。华美2号、海沃德中感。秦美、金丰最感病。用最长距离法对安徽省9个猕猴桃品种进行抗细菌性溃疡病系统聚类,可以将9个品种划分为5类。在谱系图上,抗病性相同的品种大致划分在同一个类中,第1类中华软、美味硬(8、9)为免疫品种;第2类早鲜、金魁(6、7)为高抗品种;第3类魁蜜(5)为中抗品种;第4类为中感品种,华美2号(4)为偏抗病,海沃德(3)为感病品种;第5类则为感病品种,金丰(1)为高感品种,秦美(2)为感病品种。其结果与实际抗性调查情况基本相符。     

Infection of horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi and its detection by quantitative real-time PCR

Pseudomonas syringae pv. aesculi is a pathogenic bacterium causing bleeding canker disease of horse chestnut ( Aesculus hippocastanum ). This is a serious disease which has been affecting horse chestnut in several European countries over the last five years; however, very little is known about the biology of the causal agent. One of the obstacles to studying this pathogen is the lengthy procedure associated with confirming its presence on the host. In this study, P. syringae pv. aesculi was isolated from lesions on different parts of horse chestnut and its pathogenicity confirmed on horse chestnut saplings using two inoculation techniques. Real-time PCR primers were developed based on gyrase B gene sequence data for the specific detection of P. syringae pv. aesculi . Primer specificity was tested on isolates of the target pathogen as well as on a broad range of related non-target bacteria and other bacterial spp. which inhabit horse chestnut. The real-time primers reliably amplified P. syringae pv. aesculi down to 1 pg of extracted DNA, with and without the presence of host DNA, and also amplified unextracted DNA in whole cells of the bacterium down to at least 160 colony forming units. Detection and quantification of the target pathogen in phloem and xylem of both naturally infected and inoculated horse chestnut tissues was also demonstrated. This quantitative real-time PCR assay provides the facility to study several important aspects of the biology of P. syringae pv. aesculi on horse chestnut including its potential for dissemination in different substrates.     

猕猴桃品种中糖分及木质素含量 与抗溃疡病的关系

以安徽省猕猴桃主栽品种金魁、早鲜、魁蜜和金丰为研究对象，于展叶孕蕾期分别取发病的枝条、叶片，以未发病的健株相应组织为对照，采用生理生化的方法，分析枝条、叶片中木质素和可溶性糖的含量变化以及与抗溃疡病的关系。结果表明：抗病品种金魁健株枝条、叶片中可溶性糖及木质素含量显著高于感病品种金丰。自然发病后，抗感病品种枝条、叶片中可溶性糖含量都降低，感病品种降低更多，金魁叶片可溶性糖含量下降4．20％，金丰叶片可溶性糖含量下降55.35％；木质素含量都升高，且抗病品种金魁叶片中的木质素含量比感病品种金丰高得多，其变化率分别为7．17％、3．01％，枝条中的木质素含量变化率分别为110．39％、68．98％，其差异达到显著水平。相关分析表明，枝条中木质素、可溶性糖含量与品种发病率也呈负相关，γ分别为-0．9583和-0．9282；叶片中木质素、可溶性糖含量与品种发病率呈负相关，γ分别为-0．8099和-0、8266。从而说明枝条、叶片中可溶性糖及木质素含量与品种抗性呈正相关。抗感品种淀粉含量无明显规律性变化，与品种抗性关系不大。     

猕猴桃溃疡病调查研究

对陕西西安(周至、户县、长安)地区12个村60个猕猴桃园的猕猴桃溃疡病发病特点调查分析,发现品种、树龄与猕猴桃溃疡病的发生直接相关,4个主栽品种(红阳、亚特、秦美和海沃德)中红阳最感病,亚特和海沃德次之,秦美发病最轻;且随着树龄的增大溃疡病危害相应加重(3~13 a生)。地区之间猕猴桃溃疡病严重程度存在着不同程度的差异,除地区间小气候外,管理水平也是影响溃疡病发生的重要因素之一。     

Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa.