Consequences of the taxonomic revision of Xanthomonas axonopodis pv. dieffenbachiae and of the analysis of its associated pathogenicity for the recommendation of regulation (EPPO A2 List)

Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of bacterial blight of Araceae (aroids), is a regulated pest in several countries and is included in the EPPO A2 List. Reference strains of Xanthomonas axonopodis pv. dieffenbachiae have recently been reclassified into the species Xanthomonas phaseoli, Xanthomonas citri and Xanthomonas euvesicatoria on the basis of different features, including multilocus sequence analysis, average nucleotide identity and homology in DNA–DNA hybridization analyses. Based on pathogenicity tests, Constantin et al. (2017) proposed naming the pathogens on aroids as X. phaseoli pv. dieffenbachiae, X. phaseoli pv. syngonii and X. citri pv. aracearum. Recommendations are made on how to deal with these changes for the group of pathogenic bacteria for Araceae. The name Xanthomonas axonopodis pv. dieffenbachiae on the EPPO List should be adjusted to the names proposed in the taxonomic study by Constantin et al. (2016). The current EPPO Diagnostic Standard is directed at strains pathogenic on Anthurium. They mainly belong to X. phaseoli pv. dieffenbachiae, but some also to X. citri pv. aracearum that are not detected by the EPPO Diagnostic Standard. Xanthomonas phaseoli pv. syngonii strains are also aggressive, but with a host range restricted to Syngonium. The pathogenicity specific to aroids of the bacterial isolates reclassified as Xanthomonas euvesicatoria was not confirmed and no pathovar epithet has been retained for these strains.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.     

红掌拮抗内生菌Y-54的分离、鉴定及其生防作用

为筛选具有广谱拮抗作用的内生菌,采用平板对峙法从健康红掌组织中分离和筛选对多种病原菌有拮抗作用的内生菌株,选择拮抗作用较好的菌株进行抑菌谱和拮抗作用测定,通过形态学特征、生理生化特性和分子生物学特征对其进行鉴定,利用盆栽试验测定其对红掌的促生作用和对红掌细菌性疫病的防效。结果显示,在健康红掌中共分离得到237株内生细菌,其中菌株Y-54的拮抗作用最强,其50倍发酵液对红掌细菌性疫病菌Xanthomonas axonopodis pv. dieffenbachiae的抑制作用最强,抑制率达37.78%;同时对多种病原真菌具有较强的抑制作用,其中对番茄叶霉病菌Cladosporium fulvum的抑制率达86.42%。结合形态学特征、生理生化特性和分子生物学特征将菌株Y-54鉴定为暹罗芽胞杆菌Bacillus siamensis。菌株Y-54的50倍发酵液可显著提高红掌叶长、叶宽和叶绿素相对含量(soil and plant analyzer development,SPAD)值,施药14 d和28 d后对红掌细菌性疫病的防效分别为62.94%和59.56%,与对照药剂的防效相当,表明菌...     

Pathogenicity and virulence gene content of Xanthomonas strains infecting Araceae,formerly known as Xanthomonas axonopodis pv. dieffenbachiae

Bacterial leaf blight of aroids is caused by a heterogeneous group of xanthomonads listed as Xanthomonas axonopodis pv. dieffenbachiae (Xad) on the EPPO A2 quarantine list. Recently, Xad strains were shown not to belong to X. axonopodis but to the species X. citri, X. phaseoli and X. euvesicatoria. Here, to verify the pathovar designation, 11 representative strains were tested for pathogenicity on six aroid genera. They had overlapping host ranges and only the strain isolated from Syngonium showed host specificity. The X. citri strains, isolated from various hosts, showed dissimilarity in virulence to the tested aroid genera. The X. phaseoli strains, isolated from Anthurium and Syngonium, were generally more virulent and, additionally, induced systemic infections. The X. euvesicatoria strains, isolated from Philodendron, were scored as not pathogenic on the tested aroids. Four representative strains were genome sequenced and showed a variable virulence‐associated gene content. Pathogenicity to aroids was correlated with the presence of three specific T3 effector genes and with a T6SS gene sequence. Together, the phylogenetic and pathogenic differentiation among Xad strains justifies the installation of three pathovar epithets for the pathogens on aroids: X. phaseoli pv. dieffenbachiae comb. nov. for the strains isolated from Anthurium; X. phaseoli pv. syngonii comb. nov. for the strain isolated from Syngonium; and X. citri pv. aracearum comb. nov. for the strains isolated from Aglaonema, Xanthosoma and Dieffenbachia. It is proposed that phytosanitary regulations for xanthomonads on aroids are restricted to these three pathovars.     

Leaf blight and defoliation caused by two new pathovars of Xanthomonas axonopodis on Schinus terebinthifolius and Mabea fistulifera

Schinus terebinthifolius and Mabea fistulifera have been used for forest repositioning and urban forestry in Brazil. In October 2012, in a routine inspection at the research nursery of the Forestry Department of the Universidade Federal de Viçosa, in Minas Gerais, Brazil, a mortality of approximately 40% of the seedlings was observed as a result of diseases characterized by leaf blight and intense defoliation, which culminated in the death of the plants. Microscopy observations revealed oozing from the infected tissue and isolations revealed a bacterial aetiology for both diseases. Bacterial cells that formed bright yellow mucoid colonies with round edges were routinely isolated from lesion margins. Inoculation of isolated strains into healthy seedlings reproduced the symptoms observed under natural conditions. Bacterial cells showing the same morphological, biochemical and molecular characteristics as those originally isolated from naturally infected plants were reisolated from inoculated plants. Morphological, physiological and biochemical tests as well as 16S rDNA sequencing and multilocus sequence analysis using four housekeeping genes, dnaK, fyuA, gyrB and rpoD, confirmed the newly isolated strains belong to Xanthomonas axonopodis. Plant cross‐inoculations showed the strains did not belong to any known phylogenetically related pathovar. Pathovars X. axonopodis pv. schini pv. nov. and X. axonopodis pv. mabeae pv. nov. are proposed as the causal agents of bacterial leaf blight on S. terebinthifolius and M. fistulifera, respectively.     

Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N‐PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N‐PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90·6, 88·7 and 47·2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92·5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N‐PCR assay. The N‐PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N‐PCR had undeniable advantages compared to the reference method (less labour‐intensive and less time‐consuming). In addition, post‐test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N‐PCR assay has since been included in a revised version of the EPPO detection protocol.     

Survival of the Anthurium Blight Pathogen, Xanthomonas axonopodis pv. Dieffenbachiae, in Field Crop Residues

Bacterial blight, caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad), is a major threat to the anthurium cut flower industry worldwide. Two field trials in Hawaii evaluated the long-term persistence of Xad in artificially-infested crop residues. Xad survived in leaf, petiole, and root residues for as long as 4 months when tissues were left on the surface or buried 15cm deep. Survival was considerably shorter (approximately 20 days) outside of residues. Xad that was recovered from residues over a period of 4 months retained pathogenicity. Xad was isolated from living roots of naturally-infected plants which further suggests that roots left in the field after culling may be particularly important, but overlooked, inoculum source. This information is key to determining minimum fallow periods before replanting devastated fields.     

Bacterial blight of rice and its control in Nepal

Research on Xanthomonas oryzae pv. oryzae, the bacterial blight of rice pathogen, was initiated at the Institute of Agriculture and Animal Science (IAAS) with the main objective of assessing the population structure of X. o. pv. oryzae through the use of both conventional and molecular markers in combination with virulence typing. A high DNA polymorphism was detected in the pathogen populations using different DNA probes and rep-PCR primers. Most strains were avirulent to cultivars containing the bacterial blight resistance gene Xa-21, which suggested the strategy that targets gene deployment is feasible in Nepal.     

Development of a specific molecular tool for detecting Xanthomonas campestris pv. musacearum

A specific and rapid diagnostic tool has been developed to detect Xanthomonas campestris pv. musacearum, the causal agent of bacterial wilt of banana. PCR primers were developed from intergenic regions of X. campestris pv. musacearum following its partial sequence. A total of 48 primers were tested for specificity to X. campestris pv. musacearum strains collected from various regions in Uganda. These were also tested for specificity against related Xanthomonas species from the vasicola group, Xanthomonas species pathogenic to other crops, and against those existing saprophytically on banana plants. Seven primer sets (Xcm12, Xcm35, Xcm36, Xcm38, Xcm44, Xcm47 and Xcm48) were found to be very specific to X. campestris pv. musacearum. These primer sets directed the amplification of the expected product for all 52 strains of X. campestris pv. musacearum collected from different locations in Uganda. No amplification products were obtained with unrelated phytopathogenic bacteria or endophytic/epiphytic bacteria from banana. A detection limit of 10³ CFU mL^?1 corresponding to about four cells per PCR reaction was observed when X. campestris pv. musacearum cells were used for all the seven primer sets. The DNA samples from symptomless plant tissues also tested positive with primer set Xcm38. The specific PCR method described here is a valuable diagnostic tool which can be used to detect the pathogen at early stages of infection and monitor disease.     

实时荧光定量PCR法检测十字花科细菌性黑斑病菌

为有效防控我国的检疫性有害生物十字花科细菌性黑斑病菌Pseudomonas syringae pv.maculicola在国内的传播与蔓延,通过设计1对特异性引物3539,利用132株靶标和非靶标菌为模板进行PCR扩增,建立了实时荧光定量PCR法,并进行了模拟种子带菌试验。结果显示,引物3539为只针对十字花科细菌性黑斑病菌扩增出的特异性产物;在模拟种子带菌检测中,常规PCR对菌悬液的检测限为10~5CFU/m L,实时荧光定量PCR的检测限为10~3CFU/m L,其中10~8CFU/m L菌液的Ct值最低,为22.90,10~3CFU/m L菌液的Ct值最高,为35.73,且不同浓度菌液间的Ct值均有显著差异;不同带菌率模拟种子的检测结果表明,常规PCR和实时荧光定量PCR能检测到的带菌率分别为0.5%和0.1%。研究表明,实时荧光定量PCR法不仅可用于病种的检测,也可用于病害的早期诊断。     

PCR-mediated Detection of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>by Amplification of the 16S–23S rDNA Spacer Region Sequence

A detection method specific for Xanthomonas oryzae pv. oryzae, the pathogen responsible for bacterial blight of rice, was based on the polymerase chain reaction (PCR) and designed by amplifying the 16S–23S rDNA spacer region from this bacterium. The nucleotide sequence of the spacer region between the 16S and 23S rDNA, consisting of approximately 580-bp, from X. oryzae pv. oryzae, X. campestris pv. alfalfae, X. campestris pv. campestris, X. campestris pv. cannabis, X. campestris pv. citri, X. campestris pv. cucurbitae, X. campestris pv. pisi, X. campestris pv. pruni and X. campestris pv. vitians, was determined. The determined sequences had more than 95% identity. Therefore, a pair of primers, XOR-F (5′-GCATGACGTCATCGTCCTGT-3′) and XOR-R2 (5′-CTCGGAGCTATATGCCGTGC-3′) was designed and found to specifically amplify a 470-bp fragment from all strains of X. oryzae pv. oryzae isolated from diverse regions in Japan. No PCR product was amplified from X. campestris pathovars alfalfae, campestris, cannabis, carotae, cucurbitae, dieffenbachiae, glycines, pisi, pruni, vitians or zantedeschiae, except for pathovars citri, incanae and zinniae. The method could also detect the pathogen in infected rice leaves within 3 hr, at a detection limit of 4×10¹ cfu/ml. Received 17 December 1999/ Accepted in revised form 10 April 2000     

PCR-based detection of Xanthomonas campestris pathovars in Brassica seed

Xanthomonas campestris is a seedborne bacterium that causes black rot of crucifers. Substantial crop losses may result from the rapid spread of the bacteria under favourable conditions, especially those occurring during seedling production. A PCR-based method has been developed for the rapid and sensitive detection of the pathovars of X. campestris that affect crucifers. Primers were designed to specifically amplify a 619 bp fragment of the hrpF gene from X. campestris . Amplification products were not detected from other Xanthomonas species, or from other pathogenic or epiphytic bacteria occurring on these plants. To avoid false-negative results arising from the presence of amplification inhibitors in plant extracts, primers targeting a 360 bp section of the internal transcribed spacer (ITS) region from Brassica spp. were included in a multiplex PCR. The assay readily detected X. campestris pv. campestris infections in diseased plants and from bacterial colonies isolated on growth media, and was more sensitive and specific than traditional plating methods and a commercially available ELISA. A seed-washing protocol was optimized to allow the detection of a single artificially infected seed among 10 000 healthy seeds using the multiplex PCR.     

PM 7/23 (2): Xanthomonas axonopodis pv. dieffenbachiae

Specific scope

This standard describes a diagnostic protocol for Xanthomonas axonopodis pv. dieffenbachiae ¹ 1 Use of brand names of chemicals or equipment in these EPPO Standards implies no approval of them to the exclusion of others that may also be suitable.
.

Specific approval and amendment

First approved in 2003–09. Revised in 2009–09.

     

红掌细菌性疫病病原菌的PCR特异性检测

 红掌细菌性疫病(Xanthomonas axonopodis pv. dieffenbachiae,简称Xad)是红掌等天南星科花卉毁灭性病害,该病通过带菌种苗调运不断在我国扩散蔓延,国内尚未有检测方法。通过筛选和重新设计引物,建立了Xad 的PCR 检测方法,结果表明,利用引物Xad-F / Xad-R 进行PCR,能扩增出检测Xad 的特异性DNA 片断,其灵敏度可达1 × 102 CFU / mL,DNA 的最低检出限为0. 44 ng / μL,可用于红掌苗的带菌检测和红掌细菌病害的鉴定。     

Rep-PCR技术对中国水稻条斑病菌的遗传多样性初析

 采用Rep-PCR技术,对30个水稻细菌性条斑病菌株(Xanthomonas oryzae pv.oryzicola)进行遗传多样性分析,同时对李氏禾条斑病菌等其它10个参试菌株也进行了比较。Rep-PCR是利用一些基于细菌的短的重复序列引物(ERIC和BOX)的DNA扩增特性,2种引物组合的电泳图谱结合并分析,以水稻细菌性条斑病菌各自的指纹谱型在相似率80%时可分为6簇,初步表明我国水稻细菌性条斑病菌群体的遗传分化明显;发现自然界存在的弱或无毒性菌株与毒性菌株的Rep-PCR指纹图谱差异很大;毒性菌株的遗传分簇与其致病性具有一定的相关性。用ERIC扩增水稻条斑病菌基因组DNA的指纹比BOX更为多样,两者对菌株的分辨率不同。因此,Rep-PCR技术可有效地用于监测水稻细菌性条斑病菌的遗传变异,还可应用于菌株的鉴定和分类学研究。     

Detection of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis>, the causal agent of cassava bacterial blight diseases in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) in Ghana by polymerase chain reaction

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of cassava bacterial blight (CBB) disease. CBB is a major constraint to cassava cultivation in Ghana. In this study, a survey was conducted in eight regions of Ghana to assess the presence of CBB disease. Out of the eight regions visited, CBB, though at different prevalence, was observed in five regions. Cassava plants samples showing suspected bacterial blight symptoms were collected for analysis by Polymerase Chain Reaction (PCR). The results of the analysis showed that Ashanti region had the highest prevalence in percentage of CBB, which recorded (70%), followed by Volta region (60%); Brong Ahafo region (40%); Eastern region (40%) and Greater Accra region (20%). Morphological examination of the putative pathogen was carried out on Cefazolin trehalose agar (CTA) and Nutrient agar (NA) media. The isolates were subjected to conventional PCR using Xanthomonas genus specific primer, RST2/RST3, Xam specific Variable Number Tandem Repeat (VNTRs) loci, XaG1_67F/R and X-gumD primers, which produced 840, 446 and 402 bp, respectively. The isolates also tested positive with SYBR Green fluorescent dye, using Real-time PCR. The resulting PCR products were sequenced and analyzed using a BLASTn program, which revealed homology between 93 and 100% with several other Xam strains retrieved from GenBank nucleotide database. The pathogenicity test of the isolates on the susceptible Esam cassava variety produced symptoms typical of Xam and the pathogen was consistently re-isolated from the inoculated cassava plants and thereby satisfying the Koch’s postulates.     

Persistence of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">vignicola</Emphasis> in weeds and crop debris and identification of <Emphasis Type="Italic">Sphenostylis stenocarpa</Emphasis> as a potential new host

The survival of Xanthomonas axonopodis pv. vignicola, incitant of cowpea bacterial blight and pustule, in residues of infested cowpea leaves was studied in the field in the forest savanna transition zone of South Benin and under variable controlled conditions. The pathogen survived for up to 60 days when placed on the soil surface, and up to 45 days buried at depths of 10 and 20 cm. In the glasshouse, bacteria survived in residue mixed with soil for at least 2 months in dry soil and less than 2 months in moist soil. The pathogen survived at least 30 days in the field after spray-inoculation on the weed species Euphorbia heterophylla, Digitaria horizontalis and Synedrella nodiflora; 20 days on Panicum subalbidum; 10 days on Euphorbia hirta; and 5 days on Talinum triangulare. After leaf-infiltration under glasshouse conditions, the pathogen was detected after 90 days in D. horizontalis; 75 days in T. triangulare, P. subalbidum and S. nodiflora; 60 days in E. hirta, and 30 days in E. heterophylla. Among 12 legume species tested as alternative hosts of X. axonopodis pv. vignicola, only Sphenostylis stenocarpa (African yam bean) showed typical symptoms of cowpea bacterial blight in a glasshouse experiment following artificial inoculation. This is the first time this legume species has been identified as a potential host of X. axonopodis pv.vignicola. Crop residue and weeds are likely sources of primary inoculum when planting two consecutive cowpea crops per year and they probably play a role in dissemination of the pathogen during the cropping season. The alternate host may form a bridge for primary inoculum between cropping seasons.     

Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

Molecular diagnostic procedures for production of pathogen-free propagation material

Production of disease-free propagation material is a major means of controlling most bacterial diseases of plants, particularly when neither resistant clones nor effective chemical treatments are available. For this purpose sensitive, specific and rapid detection methods are required. The advent of molecular biology and, in particular, the polymerase chain reaction (PCR) has opened new ways for the characterization and identification of plant pathogens and the development of disease-management strategies. PCR-based detection methods rely on the development of primers for the specific detection of the pathogen. The use of pathogenicity genes as targets for primer design is the preferred procedure for obtaining specific primers but other procedures may also be useful for this purpose. In the present review we describe four examples of procedures for detecting four important bacterial pathogens in Israel: Erwinia herbicola pv gypsophilae in gypsophila, Xanthomonas campestris pv pelargonii in geranium, Agrobacterium tumefaciens in asters and roses, and Xanthomonas campestris pv campestris in crucifers. Procedures for constructing specific PCR primers for each bacterium are illustrated and discussed as well as the combination of PCR with other methods.