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American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3–5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries. [ABSTRACT FROM AUTHOR]

The genus Clavibacter (family Microbacteriaceae) includes gram‐positive actinobacterial species infecting diverse agricultural crops such as alfalfa, maize, pepper, potato, tomato and wheat. Clavibacter tessellarius, causing bacterial mosaic of wheat, was reported for the first time in 1976 in Nebraska (United States). Since then, the pathogen was thought to be restricted to North America (Canada and the United States). While bacterial strains suspected to be C. tessellarius have occasionally been isolated from wheat samples outside North America, occurrence of the pathogen in the Old World has not yet been confirmed by the plant protection authorities. In spring 2020 (January to May) dozens of wheat fields with young plants showing leaf chlorosis and mild mosaic symptoms were surveyed and sampled in southern Iran. Among 192 surveyed wheat fields, gram‐positive, orange‐pigmented actinobacterial strains were isolated from samples of 53 fields. Altogether, 61 bacterial strains were identified as C. tessellarius based on the phenotypic features, PCR‐based molecular tests and pathogenicity assays. Multilocus sequence analysis of five housekeeping genes (atpD, dnaK, gyrB, ppk and rpoB) using 10 representative strains from diverse geographic areas in southern Iran confirmed the phylogenetic status of the strains within C. tessellarius. Our data indicate that the bacterial mosaic pathogen occurs in four southern Iranian provinces including Bushehr, Fars, Kerman and Khuzestan. Thus, occurrence of C. tessellarius outside North America is confirmed. Potential risks associated with the emergence of the pathogen in the Old World, and the need for strict quarantine measures in the area, is further discussed. [ABSTRACT FROM AUTHOR]

Bacterial leaf streak of small-grain cereals is an economically important disease of wheat and barley crops. The disease occurs in many countries across the globe. with particular importance in regions characterized by high precipitation or areas in which sprinkler irrigation is used. Three genetically distinct lineages of the Gram - negative bacterium Xanthomonas trans! ucens (X. transiticens pv. undulosci. X. Trans lucens pv. trans lucens, and X. transhicens py. cerealis) are responsible for most of the bacterial leaf streak infections on wheat and harley crops. Considering the seedborne nature of the pathogens. they are included in the A2 (high-risk) list of quarantine organisms for some European countries; hence, they are under strict quarantine control and zero tolerance. Due to the taxonomic complexities within X. transhicens. the exact geographic distribution of each pathovar has not yet been determined. In this mini review, we provide an updated overview of the detection and diagnosis of the bacterial leaf streak pathogens. First, a shoi·t history of the leaf streak pathogens is provided, followed by the symptomology and host range of the causal agents. Then, the utility of conventional methods and high-throughput molecular approaches in the precise detection and identification of the pathogens is explained. Finally, we highlight the role of quarantine inspections and early detection of the pathogen in combating the risk of bacterial leaf streak in the 21 st century s small-grains cereals-industry. [ABSTRACT FROM AUTHOR]

Goss's bacterial wilt and leaf blight of maize (Zea mays) caused by the gram‐positive coryneform bacterium Clavibacter nebraskensis is an economically important disease in North America. C. nebraskensis is included within the high‐risk list of quarantine pathogens by several plant protection organizations (EPPO code: CORBMI), hence it is under strict quarantine control around the world. The causal agent was reported for the first time on maize in Nebraska (USA) in 1969. After an outbreak during the 1970s, prevalence of the disease decreased in the 1980s to the early 2000s, before the disease resurged causing a serious threat to maize production in North America. The re‐emergence of Goss's wilt in the corn belt of the United States led to several novel achievements in understanding the pathogen biology and disease control. In this review, we provide an updated overview of the pathogen taxonomy, biology, and epidemiology as well as management strategies of Goss's wilt disease. First, a taxonomic history of the pathogen is provided followed by symptomology and host range, genetic diversity, and pathogenicity mechanisms of the bacterium. Then, utility of high‐throughput molecular approaches in the precise detection and identification of the pathogen and the management strategies of the disease are explained. Finally, we highlight the role of integrated pest management strategies to combat the risk of Goss's wilt in the 21st century maize industry. Disease symptoms: Large (2–15 cm) tan to grey elongated oval lesions with wavy, irregular water‐soaked margins on the leaves. The lesions often start at the leaf tip or are associated with wounding caused by hail or wind damage. Small (1 mm in diameter), dark, discontinuous water‐soaked spots, known as "freckles", can be observed in the periphery of lesions. When backlit, the freckles appear translucent. Early infection (prior to growth stage V6) may become systemic and cause seedlings to wilt, wither, and die. Coalescence of lesions results in leaf blighting. Host range: Maize (Zea mays) is the only economic host of the pathogen. A number of Poaceae species are reported to act as secondary hosts for C. nebraskensis. Taxonomic status of the pathogen: Class: Actinobacteria; Order: Micrococcales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter nebraskensis. Synonyms Corynebacterium nebraskense (Schuster, 1970) Vidaver & Mandel 1974; Corynebacterium michiganense pv. nebraskense (Vidaver & Mandel 1974) Dye & Kemp 1977; Corynebacterium michiganense subsp. nebraskense (Vidaver & Mandel 1974) Carlson & Vidaver 1982; Clavibacter michiganense subsp. nebraskense (Vidaver & Mandel 1974) Davis et al. 1984; Clavibacter michiganensis subsp. nebraskensis (Vidaver & Mandel 1974) Davis et al. 1984. Type materials: ATCC 27794T; CFBP 2405T; ICMP 3298T; LMG 3700T; NCPPB 2581T. Microbiological properties: Cells are gram‐positive, orange‐pigmented, pleomorphic club‐ or rod‐shaped, nonspore‐forming, nonmotile, and without flagella, approximately 0.5 × 1–2.0 μm. Distribution: The pathogen is restricted to Canada and the United States. Phytosanitary categorization: EPPO code CORBNE. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa subsp. pauca (Xfp) infects olive trees and other hosts in Southern Apulia (Italy), devastating agriculture and landscape. A containment strategy of the disease requires quick and sensitive detection tools. Therefore, a colorimetric LAMP protocol was developed using as a template a crude alkaline sap obtained from incubation of 50–60 mg of thin slices of olive twigs in a NaOH-containing buffer. This rapid molecular assay can be performed directly in the field, as it needs only a portable isothermal block. Tissues of the same olive trees analysed by this technique were also compared to qPCR (using purified total plant DNA as template) as well as digital droplet PCR (on the same crude alkaline extracts used in cLAMP). A titration of the cLAMP reaction with healthy olive sap, spiked with dilutions of in vitro cultivated Xfp cells and plasmid DNA containing the target sequence, gave positive detection results as low as 102 CFU/mL and up to 169.2 target copies/µL, equivalent to about 0.9 pg of the genomic DNA. A portable, sensitive and target-specific Xfp field test was developed, which has a 40 min sample-to-answer time and does not require any DNA isolation procedure or laboratory equipment. The application of this detection assay could help the monitoring and containment of the disease spread. [ABSTRACT FROM AUTHOR]

Bacterial canker and spot of stone fruits and bacterial blight of walnut, caused by different pathovars of Xanthomonas arboricola, namely X. arboricola pv. pruni and X. arboricola pv. juglandis, respectively, are considered among quarantine or regulated (nonquarantine) agents in different countries. The two pathogens were initially reported in Iran in 2005 and 1977, respectively. However, the precise identity, pathogenicity status and geographic distribution of these pathogens have been questioned. In this study, we have employed a series of comprehensive field surveys and samplings over a course of 5 years (2015–2019) across 16 provinces to reassess and clarify the occurrence and distribution of X. arboricola on fruit and nut trees in Iran. Besides dozens of non‐Xanthomonas and nonpathogenic bacterial strains, 27 X. arboricola strains were isolated from stone fruits (n = 13) and walnut (n = 14). Except for the strains Peh35, Peh78 and Peh79, the stone fruit strains were nonpathogenic on their host of isolation as well as several other plant species, while the walnut strains were pathogenic on their host of isolation. Multilocus sequence analysis (MLSA) of four housekeeping genes, atpD, dnaK, gyrB and rpoD, revealed that among the stone fruit strains only Peh78 and Peh79 could be assigned into X. arboricola pv. pruni, while the walnut strains were identified as X. arboricola pv. juglandis. Based on the results of pathogenicity assays and phylogenetic analyses, we confirm the occurrence of X. arboricola pv. juglandis in Iran, while X. arboricola pv. pruni was absent in the plant samples evaluated in this study. [ABSTRACT FROM AUTHOR]

Bacterial blight and leaf spot of geraniums is a destructive disease of cultivated Pelargonium species around the world. During 2020 to 2021, surveys were conducted in seven geranium-growing provinces of Iran to monitor the status of bacterial blight and leaf spot disease. The disease was observed in six surveyed provinces varying in the extent of occurrence and severity. Twenty-two gram-negative pale-yellow bacterial strains resembling members of Xanthomonas were isolated from symptomatic leaves and stems. Pathogenicity and host range assays showed that the bacterial strains were pathogenic on Pelargonium grandiflorum , P. graveolens , P. peltatum , and P. zonale . All strains were positive for a PCR test using the primer pair XcpM1/XcpM2, which is specific for X. hortorum pv. pelargonii . Phylogenetic analysis using the sequences of gyrB and lepA genes showed that the 22 strains clustered in a clade among the sequences of X. hortorum pv. pelargonii strains retrieved from GenBank but were distinct from the other pathovars of X. hortorum . BOX-PCR-based fingerprinting using BOX-A1R primer revealed that the strains isolated in this study were grouped into two clusters, while no distinct correlation was observed between the host/area of isolation and BOX-PCR fingerprinting. None of the strains obtained in this study nor the reference strain of the pathogen produced bacteriocin against each other. Results obtained in this study shed light on the geographic distribution, taxonomic status, and host range of the bacterial blight and leaf spot pathogen of geraniums in Iran, paving the path for further research on disease management., Competing Interests: The author(s) declare no conflict of interest.

Background: Bacterial ring rot of potato (Solanum tuberosum) caused by the gram‐positive coryneform bacterium Clavibacter sepedonicus is an important quarantine disease threatening the potato industry around the globe. Since its original description in 1906 in Germany, management of ring rot has been a major problem due to the seedborne nature (via seed tubers not true seeds) of the pathogen allowing the bacterium to be transmitted long distances via infected tubers. Disease symptoms: On growing potato plants: interveinal chlorosis on leaflets leading to necrotic areas and systemic wilt. On infected tubers: vascular tissues become yellowish brown with a cheesy texture due to bacterial colonization and decay. Host range: Potato is the main host of the pathogen, but natural infection also occurs on eggplant, tomato, and sugar beet. Taxonomic status of the pathogen: Class: Actinobacteria; Order: Actinomycetales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter sepedonicus (Spieckermann and Kotthoff 1914) Li et al. 2018. Synonyms (nonpreferred scientific names): Aplanobacter sepedonicus; Bacterium sepedonicum; Corynebacterium sepedonicum; Corynebacterium michiganense pv. sepedonicum; Clavibacter michiganensis subsp. sepedonicus. Microbiological properties: Gram‐positive, club‐shaped cells with creamy to yellowish‐cream colonies for which the optimal growth temperature is 20–23°C. Distribution: Asia (China, Japan, Kazakhstan, Nepal, North Korea, Pakistan, South Korea, Uzbekistan, the Asian part of Russia), Europe (Belarus, Bulgaria, Czech Republic, Estonia, Finland, Georgia, Germany, Greece, Hungary, Latvia, Lithuania, Norway, Poland, Romania, European part of Russia, Slovakia, Spain, Sweden, Turkey, Ukraine), and North America (Canada, Mexico, USA). Phytosanitary categorization: CORBSE: EPPO A2 list no. 51. EU; Annex designation I/A2. [ABSTRACT FROM AUTHOR]

One of the most destructive diseases affecting grapevine in Europe is caused by Flavescence Dorée phytoplasma (FDp), which belongs to the 16Sr-V group and is a European Union quarantine pathogen. Although many molecular techniques such as loop-mediated isothermal amplification (LAMP) are widely used for the rapid detection of FDp in infected grapevine plants, there is no developed isothermal amplification assay for FDp detection in the insect vectors that are fundamental for the spread of the disease. For this reason, a simple in-field real-time LAMP protocol was optimized and developed for the specific detection of FDp in the insect vector Scaphoideus titanus. The LAMP assay was optimized to work with crude insect extracts obtained by manually shaking a single insect in a buffer for 5 min. Such a simple, sensitive, specific, economic, and user-friendly LAMP assay allowed the detection of FDp in S. titanus in less than half an hour, directly in the field. The developed insect tissue preparation procedure, combined with the LAMP protocol, promptly revealed the presence of FDp in infected S. titanus directly in the vineyards, allowing for monitoring of the spread of the pathogen in the field and to apply timely strategies required for the mandatory control of this pathogen. [ABSTRACT FROM AUTHOR]

Seeds are important microbial vectors, and seed-associated pathogens can be introduced into a country through trade, resulting in yield and quality losses in agriculture. The aim of this study was to characterize the microbial communities associated with barley seeds, and based on which, to develop technical approaches to trace their geographical origins, and to inspect and identify quarantine pathogens. Our analysis defined the core microbiota of barley seed and revealed significant differences in the barley seed-associated microbial communities among different continents, suggesting a strong geographic specificity of the barley seed microbiota. By implementing a machine learning model, we achieved over 95% accuracy in tracing the origin of barley seeds. Furthermore, the analysis of co-occurrence and exclusion patterns provided important insights into the identification of candidate biocontrol agents or microbial inoculants that could be useful in improving barley yield and quality. A core pathogen database was developed, and a procedure for inspecting potential quarantine species associated with barley seed was established. These approaches proved effective in detecting four fungal and three bacterial quarantine species for the first time in the port of China. This study not only characterized the core microbiota of barley seeds but also provided practical approaches for tracing the regional origin of barley and identifying potential quarantine pathogens. [Display omitted] • We characterized the barley-seed associated bacterial and fungal communities of Oceania, North America, and Europe. • Our analysis defined the core microbiota of barley seed and revealed significant differences among different continents, suggesting a strong geographic specificity of the barley seed microbiota. • We could accurately predict the original countries of imported barley grains within the RF prediction model, with an accuracy greater than 95%. • We constructed a barley-related quarantine pathogens database. Based on this database, some important pathogens were intercepted through metabarcoding sequencing. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa subsp. pauca (Xfp) infects olive trees and other hosts in Southern Apulia (Italy), devastating agriculture and landscape. A containment strategy of the disease requires quick and sensitive detection tools. Therefore, a colorimetric LAMP protocol was developed using as a template a crude alkaline sap obtained from incubation of 50–60 mg of thin slices of olive twigs in a NaOH-containing buffer. This rapid molecular assay can be performed directly in the field, as it needs only a portable isothermal block. Tissues of the same olive trees analysed by this technique were also compared to qPCR (using purified total plant DNA as template) as well as digital droplet PCR (on the same crude alkaline extracts used in cLAMP). A titration of the cLAMP reaction with healthy olive sap, spiked with dilutions of in vitro cultivated Xfp cells and plasmid DNA containing the target sequence, gave positive detection results as low as 102 CFU/mL and up to 169.2 target copies/µL, equivalent to about 0.9 pg of the genomic DNA. A portable, sensitive and target-specific Xfp field test was developed, which has a 40 min sample-to-answer time and does not require any DNA isolation procedure or laboratory equipment. The application of this detection assay could help the monitoring and containment of the disease spread.

Citrus vein enation virus (CVEV), which was regulated as a quarantine virus in Korea, was firstly found on Jeju Island in 2017. In February 2018, a survey was carried out to determine the distribution of CVEV in the main commercial areas growing Citrus spp. and Poncirus trifoliata. The survey was performed at 203 groves in the southern Korean Peninsula and Jeju Island. CVEV infection was determined by reverse transcription polymerase chain reaction detection and sequencing. The coat protein (CP) gene sequences obtained from the CVEV-infected samples showed high similarities (more than 98%) to the previously reported CVEV CP sequences. In summary, CVEV was detected in 136 groves (67%), in which 85.4% of Citrus junos and 77.8% of Citrus unshiu were infected by CVEV. In Jeju Island, the infection rate of CVEV was relatively higher (90.6%). Our result revealed that CVEV has spread widely in Citrus and Poncirus in Korea. Based on the result, the Korean quarantine agency decide to exclude CVEV from quarantine in Korea.

In recent years, there has been an apparent Europe-wide emerging presence of brown spot needle blight, a disease of pine species caused by the fungus Lecanosticta acicola. In this study, we report the first well-documented occurrences of the pathogen in Slovakia, identified molecularly using species-specific primers and based on sequencing of the ITS region and TEF1 gene. Among the material collected from 84 locations within the country, L. acicola was present in 17 samples from 13 different locations, mainly distributed in urban environments. Four pine species were identified as hosts, among which, Pinus nigra and P. mugo were found to be the most frequently infected. Analysis of the mating type genes of 24 isolates obtained from two localities revealed the presence of a single mating type in Slovakia. All analyzed ITS sequences of the Slovak isolates were found to be uniform. However, although analysis of the TEF1 gene indicated that all Slovak isolates could be grouped into a single lineage, we detected nucleotide polymorphisms suggestive of a certain degree of genetic diversification within central European populations of the fungus. [ABSTRACT FROM AUTHOR]

Curtobacterium flaccumfaciens (Microbacteriaceae), a plant-pathogenic coryneform species includes five pathovars with valid names and a number of proposed – but unvalidated – new members. In this study, phenotypic features and DNA similarity indexes were investigated among all C. flaccumfaciens members. Results showed that the C. flaccumfaciens pv. poinsettiae strains causing bacterial canker of Euphorbia pulcherrima in the USA as well as the orange-/red-pigmented strains of C. flaccumfaciens pv. flaccumfaciens pathogenic on dry beans in Iran are too distinct from each other and from the type strain of the species to be considered members of C. flaccumfaciens. Hence, the latter two groups were elevated at the species level as C. poinsettiae sp. nov. (ATCC 9682T = CFBP 2403T = ICMP 2566T = LMG 3715T = NCPPB 854T as type strain), and C. aurantiacum sp. nov. (50RT = CFBP 8819T = ICMP 22071T as type strain). Within the emended species C. flaccumfaciens comb. nov., yellow-pigmented strains causing bacterial wilt of dry beans and those causing bacterial canker of Euphorbia pulcherrima in Europe were retained as C. flaccumfaciens pv. flaccumfaciens and C. flaccumfaciens pv. poinsettiae, respectively; while taxonomic position of the sugar beet pathogen C. flaccumfaciens pv. beticola ATCC BAA144PT was confirmed. The newly described onion pathogen C. allii was also reclassified as C. flaccumfaciens pv. allii with the pathotype strain LMG 32517PT. Furthermore, C. flaccumfaciens pv. basellae causing bacterial leaf spot of malabar spinach (Basella rubra) was transferred to C. citreum pv. basellae with ATCC BAA143PT as pathotype. [ABSTRACT FROM AUTHOR]

One of the most destructive diseases affecting grapevine in Europe is caused by Flavescence Dorée phytoplasma (FDp), which belongs to the 16Sr-V group and is a European Union quarantine pathogen. Although many molecular techniques such as loop-mediated isothermal amplification (LAMP) are widely used for the rapid detection of FDp in infected grapevine plants, there is no developed isothermal amplification assay for FDp detection in the insect vectors that are fundamental for the spread of the disease. For this reason, a simple in-field real-time LAMP protocol was optimized and developed for the specific detection of FDp in the insect vector Scaphoideus titanus. The LAMP assay was optimized to work with crude insect extracts obtained by manually shaking a single insect in a buffer for 5 min. Such a simple, sensitive, specific, economic, and user-friendly LAMP assay allowed the detection of FDp in S. titanus in less than half an hour, directly in the field. The developed insect tissue preparation procedure, combined with the LAMP protocol, promptly revealed the presence of FDp in infected S. titanus directly in the vineyards, allowing for monitoring of the spread of the pathogen in the field and to apply timely strategies required for the mandatory control of this pathogen.

Bacterial wilt of dry beans (family Fabaceae) caused by the actino-bacterial agent Curtobacterium flaccumfaciens pv. flaccumfaciens is one of the most important diseases threatening edible legume production around the globe. Despite the economic losses due to the bacterial wilt disease, the pathogen has not so far been investigated for its genomic features, pathogenicity determinants, and virulence strategies. Here we present the first complete genome sequence of a highly virulent bacteriocin-producing C. flaccumfaciens pv. flaccumfaciens strain P990. The bacterium has a circular chromosome consisting of 3,736 kbp with the G+C% content of 71.0%. Furthermore, a 147-kbp circular plasmid (pCffl) with 66.1% G+C% content as well as two circular plasmid-like DNAs with sizes of 25 and 22 kbp were detected within the genomic contents of C. flaccumfaciens pv. flaccumfaciens. Phylogenetic analyses revealed that only a few number of Curtobacterium sp. strains deposited in the public databases could be classified within the species C. flaccumfaciens. Comparative genomics of C. flaccumfaciens pv. flaccumfaciens using the genome sequences of actinobacterial plant pathogens revealed the presence of a set of unique low G+C% content genomic islands in the C. flaccumfaciens pv. flaccumfaciens genome. Homologs of pathogenicity-determinant loci capable of producing 1,4-beta-xylanase (xysA), pectate lyase (pelAl and pelA2), serine protease (chpC, chpG, and pat-1), and sortase (srtA) were detected in C. flaccumfaciens pv. flaccumfaciens genome. The genomic data presented here extend our understanding of the C. flaccumfaciens pv. flaccumfaciens genomic features and pave the ways of research on functional and interaction genetics to combat the risk of bacterial wilt disease in the 21st century's dry bean industry. [ABSTRACT FROM AUTHOR]

Bacterial wilt and tan spot of dry beans (family Fabaceae), caused by Curtobacterium flaccumfaciens pv. flaccumfaciens, is an important emerging disease threatening the edible legume industry around the globe. The management of bacterial wilt has been a major problem since its original description in 1922. This is in part due to the seedborne nature of the pathogen allowing the bacterium to be transmitted long distances via infected seeds, as well as a lack of detailed molecular information concerning the pathogenicity repertoires and virulence determinates of the pathogen. Identification can also be difficult owing to the presence of five different colony colour variants (i.e., yellow, orange, pink, purple, and red) on culture media. In this review, we provide an overview of the aetiology, epidemiology, and management strategies of bacterial wilt disease. First, a comprehensive and comparative symptomology of the disease on different dry bean species is described. Then, the taxonomic history of the causal agent and utility of high‐throughput sequencing‐based approaches in the precise characterization of the pathogen is explained. Furthermore, we provide an updated outline on the global distribution of the pathogen, highlighting expansion of the causal agent into the areas with no history of the disease until the beginning of the current century. Finally, because there are limited options for use of conventional pesticides against the pathogen, we highlight the use of integrated pest management strategies, for example quarantine inspections, resistant cultivars, and crop sanitation, to combat the risk of bacterial wilt disease in the dry bean industry. Disease symptoms: Interveinal chlorosis on leaflets leading to necrotic areas and systemic wilt. Seed discolouration to yellow, orange, pink, or purple is seen in white‐seeded cultivars. Host range: Causes bacterial wilt and tan spot disease on edible dry beans in the Fabaceae family, including common bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), mungbean (Vigna radiata), soybean (Glycine max), as well as a number of weed species. Taxonomic status of the pathogen: Bacteria; phylum Actinobacteria; order Actinomycetales; suborder: Micrococcineae; family Microbacteriaceae; genus Curtobacterium; species Curtobacterium flaccumfaciens. Synonyms: Corynebacterium flaccumfaciens subsp. flaccumfaciens; Corynebacterium flaccumfaciens pv. flaccumfaciens, Corynebacterium flaccumfaciens, Phytomonas flaccumfaciens, Bacterium flaccumfaciens. Microbiological properties: Multicoloured (yellow, orange, pink, purple, and red), gram‐positive, aerobic, curved rod, nonspore‐forming, polar flagellated, motile cells. Distribution: Widespread in America (Brazil, Canada, and the USA), Australia, and Iran. Restricted occurrence in Africa and Europe. Phytosanitary categorization: EPPO A2 list no. 48, EU Annex II⁄B. [ABSTRACT FROM AUTHOR]

Pantoea stewartii subsp. stewartii (Pnss) and P. stewartii subsp. indologenes (Pnsi) are closely related plant pathogens that differ in their host specificities. Pnss is the causal agent of Stewart's wilt of corn, whereas Pnsi causes disease on millets but not corn. Comparative genomics is a valuable method for characterizing the differences between genomes, but there are few studies on this important quarantine pathogen. Here, we compared publicly available genomes of seven strains of Pnss and three strains of Pnsi. Pan- and core-genome analyses showed that strains isolated from close geographical regions are more similar in their genome structures. Gene content and collinearity analyses further revealed numerous strain-specific genes. In particular, the Pnss type strain DC283 contained over 1200 additional genes compared with other strains. Importantly, we also identified eleven genes that are only present in Pnsi genomes and thus may be useful to distinguish between Pnss and Pnsi strains. Overall, this study characterized the common and distinct genomic features of Pnss and Pnsi, which lay the foundation for future development of molecular methods to detect the Stewart's wilt pathogen in maize for quarantine regulations and distinguish its two subspecies. [ABSTRACT FROM AUTHOR]

Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B. cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum), 510-bp (B. cactivora), 313-bp (P. nicotinae), and 447-bp (P. cactorum). The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti.

Tobacco ringspot virus (TRSV) is a quarantine pathogen in Europe. During an official inspection in November 2011, Impatiens walleriana plants showing symptoms were found in a nursery in the Czech Republic. The causal agent of the disease was detected as Nepovirus group A by RT-PCR using specific primers of the Nepovirus group. Sequence analysis of PCR fragments confirms that the detected virus is TRSV. TRSV detection in these plants was further confirmed by ELISA and one-step RT-PCR using specific primers. The coat protein (CP) gene of the Czech TRSV isolate was sequenced, and the sequence analysis showed high identity of both nucleotide (99.28%) and amino acid (99.96%) levels compared with other known TRSV isolates from GenBank. Two amino acid motifs characteristic of nepoviruses, FDDY (FDAY) and FWGR (FYGR), were found equally at positions 80 and 497 of the TRSV CP genes, respectively, including the sequences described in this study.

A 2-year comprehensive field survey was conducted across major tomato-growing areas of Iran. Two hundred and thirty-four tomato fields and six tomato-producing greenhouses were surveyed for the potential presence of bacterial spot disease. Five hundred and ninety-six tomato samples with and without symptoms were analysed. While Xanthomonas spp. were found in association with tomato plants both with and without symptoms from five surveyed counties, the bacterial spot disease was observed only in plants from three of them. Only strains isolated from plants with symptoms induced disease symptoms on tomato, while those isolated from symptomless plants caused symptoms only on cabbage and common bean. None of the isolates caused disease symptoms on pepper and eggplant. Phylogenetic analysis showed that X. perforans is the causal agent of tomato bacterial spot in Iran, although X. campestris and X. axonopodis were also associated with symptomless tomato plants. All X. perforans isolates in this study were sensitive to streptomycin, copper sulphate and copper oxychloride at concentrations of 50 mg L−1, 200 mg L−1 and 0.8 g L−1, respectively. Unlike the type strain of X. perforans, isolates in this study did not produce bacteriocin against other Xanthomonas spp., nor were they detected using the usual species-specific primer pair Bs-XpF/Bs-XpR. This suggests an atypical nature of X. perforans strains in Iran, which leads to the hypothesis that X. perforans strains in Iran may have a separate origin to those causing disease epidemics elsewhere. The aggregated dispersal pattern of the diseased tomato fields signifies the seedborne introduction of the pathogen into the country. [ABSTRACT FROM AUTHOR]

In April 2015, reddish banding at last year's needles of Pinus jeffreyi Balf. (Jeffrey pine) and Pinus ponderosa Douglas ex C. Lawson (Ponderosa Pine) was found in an Arboretum in the federal state of Brandenburg (Germany). These symptoms were observed in systematic controls at Pinus attenuata Lemmon (Knobcone pine) and Pinus thunbergii Parl. (Japanese black pine) later too. Based on micromorphological investigations and laborative tests, the suspected infection by the quarantine fungus Dothistroma septosporum (DOROGIN) M. Morelet (causative agent of Dothistroma needle blight) was confirmed. Only younger trees in an area with persistently high air humidity were diseased. The worldwide occurring pathogen infects mainly pine species (Pinus spp.). It caused serious damage especially in the southern hemisphere until now. Lately however Dothistroma septosporum was remarkably often detected in several European countries. The increased presence of the fungus north of the Equator is supposed to originate in the recognizable climate change of the last few decades. The pathogen is capable of damaging the native to Europe Black pine (Pinus nigra J.F. Arnold) considerably. Therefore forestry risks result. In addition, it is unclear, what intensity an infestation of pure stands of Scots pine (Pinus sylvestris L.) would reach. [ABSTRACT FROM AUTHOR]

Bacterial leaf streak disease caused by Xanthomonas translucens pv. undulosa is an economically important disease threatening wheat and barley crops around the globe. Thus far, specific PCR-based detection and identification tests for X. translucens pathovars are not available. In this study, we used comparative genomics approach to design a pathovar-specific primer pair for detection of X. translucens pv. undulosa in naturally infected seeds and its differentiation from other pathovars of the species. For this aim, complete genome sequences of strains of different X. translucens pathovars were compared and the specific PCR primer pair XtuF/XtuR was designed. These primers were strictly specific to X. translucens pv. undulosa because the expected 229-bp DNA fragment was not amplified in the closely related pathovars or in other xanthomonads, wheat-pathogenic bacteria, and other plant-pathogenic bacteria. High sensitivity of the primer pair XtuF/XtuR allowed detection of pure DNA of the pathogen in a concentration as low as 4.5 pg/μl. The pathogen was also detected in water suspension at a concentration of 8.6 × 10 2 CFU/ml. The PCR test was capable of detecting the pathogen in extracts of naturally infected wheat seeds at a concentration of 3.5 × 10 4 CFU/g while a culture-plate method was able to detect the pathogen at a concentration of 50 × 10 5 CFU/g of the same seeds. The PCR test developed in this study is a step forward for precise detection and identification of X. translucens pv. undulosa to prevent outbreaks of the bacterial leaf streak disease., Competing Interests: The author(s) declare no conflict of interest.

[Display omitted] • We characterized the soybean-grain associated bacterial and fungal communities of world's largest soybean-producing countries (China, United States of America, Brazil, and Argentina). • Our results showed that the soybean-associated microbiota from different continents significantly separated, presenting strong geographic variations. The core microbial taxa and geographically specified taxa were defined. • We build the Random Forest (RF) model to predict the source of imported soybean grains. We could accurately predict the original countries of imported soybean grains within the RF prediction model, with an accuracy greater than 95%. • We constructed a soybean-related quarantine pathogens database using full-length sequences of fungal ITS region and bacterial 16S rDNA. Based on this database, two phytopathogenic fungi, Diaporthe caulivora , and Cladosporium cucumerinum , listed in the China quarantine catalog, were intercepted through metabarcoding sequencing. Soybean and derived products are among the most important food for both humans and animals. China is the world's largest importer of soybeans, with more than 100 million tons of annual imports, mainly from the United States of America (US), Brazil, and Argentina. However, there have been limited studies on the microbiota associated with imported soybean grains. Here, we reveal the soybean microbiota using amplicon sequencing based on samples from four countries on three continents of North America (US), South America (Argentina, Brazil), and Asia (China). Our results showed that the soybean-associated microbiota from different continents significantly separated, presenting strong geographic variations. The core microbial taxa and geographically specified taxa were defined, with Alternaria , Enterobacter , Plectosphaerella , Stenotrophomanas, and Xeromyces defined as the core microbiota for soybean from Asia; Amanita , Aspergillus , Fusarium , Nigrospora , Herbiconiux , Pseudomonas , Saccharopolyspora , and Schumannella from North America; and Bradyrhizobium , Colletotrichum , Filobasidium , Phialosimplex , Mycosphaerella , Septoria , Sphingomonas , and Weissalla , from South America. In addition, we build the Random Forest (RF) model to predict the source of imported soybean grains. We could accurately predict the original countries of imported soybean grains within the RF prediction models, with accuracies greater than 95 %. We constructed a database of soybean-related quarantine pathogens using full-length sequences of fungal ITS region and bacterial 16S rDNA region. Two phytopathogenic fungi, Diaporthe caulivora and Cladosporium cucumerinum, listed in the Chinese quarantine catalog, were intercepted through metabarcoding sequencing. The former was further confirmed using an available national standard protocol of qPCR diagnosis. In summary, our NGS-based approach revealed the microbiota associated with soybeans. It could provide comprehensive information and valuable method on the trace the origin of soybean and detection of quarantine pathogens at Customs and departments of inspection and quarantine. [ABSTRACT FROM AUTHOR]

The phytopathogen Ralstonia solanacearum is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the R. solanacearum species complex strains have been reclassified into three genospecies: R. solanacearum , Ralstonia pseudosolanacearum and Ralstonia syzygii . An unidentified R. solanacearum strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). Currently, sequevars of R. solanacearum species complex strains can only be determined by phylogenetic analysis of a partial endoglucanase ( egl ) sequence of approximately 700-bp in length. Such analysis, however, requires expert knowledge to properly trim the sequence, to include the correct reference strains, and to interpret the results. By comparing GenBank egl sequences of representative R. solanacearum species-complex strains, we identified genospecies- and sequevar 1 and 2-specific single nucleotide polymorphisms (SNPs). We also designed primers to amplify a shorter, 526-bp, egl fragment from R. solanacearum species complex strains for easy sequencing of the amplicon, and to facilitate direct and specific amplification of egl from R. solanacearum -infected plant samples without the need of bacterial isolation. We wrote a computer program ( Ralstonia solanacearum typing program) that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. The program correctly typed all 371 tested egl sequences with known sequevars, obtained either from GenBank or through personal communication. Additionally, the program successfully typed 25 R. solanacearum strains in our collection with no prior sequevar information, as well as 4 strains in infected plant samples, using their partial egl sequences amplified and sequenced with primers designed in this study. The Ralstonia solanacearum typing program does not require expertise or specific knowledge to use, gives results in seconds, and provides data interpretation for the user. The program and primers can help expert or non-expert users to quickly type an unknown R. solanacearum species-complex strain and determine whether it is a highly regulated R. solanacearum strain. The program can also serve as a confirmation method, since it is the only method that can easily and directly determine whether the strain in question is a sequevar 1 or 2 strain of R. solanacearum . [ABSTRACT FROM AUTHOR]

Tobacco ringspot virus (TRSV) is a quarantine pathogen in Europe. During an official inspection in November 2011, Impatiens walleriana plants showing symptoms were found in a nursery in the Czech Republic. The causal agent of the disease was detected as Nepovirus group A by RT-PCR using specific primers of the Nepovirus group. Sequence analysis of PCR fragments confirms that the detected virus is TRSV. TRSV detection in these plants was further confirmed by ELISA and one-step RT-PCR using specific primers. The coat protein (CP) gene of the Czech TRSV isolate was sequenced, and the sequence analysis showed high identity of both nucleotide (99.28%) and amino acid (99.96%) levels compared with other known TRSV isolates from GenBank. Two amino acid motifs characteristic of nepoviruses, FDDY (FDAY) and FWGR (FYGR), were found equally at positions 80 and 497 of the TRSV CP genes, respectively, including the sequences described in this study. [ABSTRACT FROM AUTHOR]

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers. [ABSTRACT FROM AUTHOR]

Severe decline of olive trees was observed in the Lecce province, Apulia (Italy), and received the name 'complesso del disseccamento rapido dell'olivo' (olive rapid decline complex). Affected plants showed leaf scorch symptoms and dieback of twigs, branches and even of the whole plant. Similar symptoms, unusual for the area, have also been observed in other Apulian localities (Cerignola, Foggia, Canosa di Puglia, and Andria). Three fungal species were associated with the symptoms: Phaeoacremonium aleophilum, Neofusicoccum parvum, and Pleurostomophora richardsiae. The latter is the first report of this fungal species infecting olives. In the Lecce province, the bacterium Xylella fastidiosa also was detected from affected olive trees. Xylella fastidiosa is a quarantine agent in Europe that had been previously reported in the Mediterranean region, but did not spread probably because of the lack of a vector. Present findings suggest that this fundamental condition has now been met. [ABSTRACT FROM AUTHOR]

Melampsora medusae is a quarantine fungus in the European Union (EU) that causes a damaging leaf rust disease on poplars. Two formae speciales of the pathogen can be distinguished, M. medusae f. sp. deltoidae and M. medusae f. sp. tremuloidae, but the EU plant health directive 2000/29/EC currently in force does not make the distinction between them. EU countries must have the ability to detect and identify rapidly the introduction of these quarantine fungi and to conduct extensive surveys in case of outbreaks. Efficient detection tools are thus needed. In this study, a sensitive real-time PCR assay was developed to detect the presence of M. medusae in poplar leaf samples. A unique primer/hydrolysis probe combination targeting both formae speciales was designed using species-specific polymorphisms observed within the internal transcribed spacer region. An additional primer/hydrolysis probe combination was designed from a region of the 28S rDNA that is highly conserved in the genus Melampsora and used in a separate real-time PCR assay in order to check the quality of the DNA extracted from Melampsora urediniospores. The test developed demonstrated a high sensitivity since it enables the reproducible detection of two M. medusae urediniospore in a mixture of 2 mg of urediniospores (ca 800 000 urediniospores) of other Melampsora species. This new real-time PCR tool should be useful for laboratories in charge of official analyses since it has many advantages over the techniques currently used to monitor this quarantine pathogen in Europe. [ABSTRACT FROM AUTHOR]

Abstract: Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe. [Copyright &y& Elsevier]

This study investigated survival of the pathogens Phytophthora ramorum, P. alni and P. kernoviae as zoospores or sporangia in response to an important water quality parameter, electrical conductivity (EC), at its range in irrigation water reservoirs and irrigated cropping systems. Experiments with different strengths of Hoagland's solution showed that all three pathogens survived at a broad range of EC levels for at least 3 days and were stimulated to grow and sporulate at ECs > 1·89 dS m−1. Recovery of initial populations after a 14-day exposure was over 20% for P. alni subsp. alni and P. kernoviae, and 61·3% and 130% for zoospores and sporangia of P. ramorum, respectively. Zoospore survival of these pathogens at ECs < 0·41 dS m−1 was poor, barely beyond 3 days in pure water; only 0·3% ( P. alni), 2·9% ( P. kernoviae) and 15·1% ( P. ramorum) of the initial population survived after 14 days at EC = 0·21 dS m−1. The variation in rates of survival at different EC levels suggests that these pathogens survive better in cropping systems than in irrigation water. Containment of run-off and reduction in EC levels may therefore be non-chemical control options to reduce the risk of pathogen spread through natural waterways and irrigation systems. [ABSTRACT FROM AUTHOR]

Ash decline induced by Hymenoscyphus pseudoalbidus is an emerging disease that severely affects Fraxinus excelsior stands in Europe. There has been an invasive spread of the disease from east to west in Europe over the last decade. Wood discoloration on infected trunks has been reported, but few data are available on the involvement of H. pseudoalbidus in such symptoms. Transport and trade of ash logs could introduce the pathogen into disease-free areas and therefore accelerate its dissemination. The aim of this study was to assess the prevalence and severity of H. pseudoalbidus in ash logs in infested areas located in the northeast of France and to clarify the role of secondary pathogens in ash decline. The results showed that prevalence of H. pseudoalbidus on collar lesions was high in the study area. The pathogen was able to produce conidia from infected wood. Thus, export of ash logs could represent a potential risk for spreading the disease. Involvement of Armillaria spp. in the decline process was confirmed, while no Phytophthora-induced collar lesions were found. Studying both disease prevalence and the age of callus tissues surrounding collar lesions in 60 ash stands enabled the origin of the disease in the study area to be determined. [ABSTRACT FROM AUTHOR]

The genetic diversity of Spanish and French field populations of Monilinia fructicola, a quarantine fungal pathogen in Europe, was compared with that of Californian, Uruguayan, and New Zealand M. fructicola populations using inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers. Unweighted pair-group method with arithmetic average (UPGMA) cluster analysis and principal component analysis (PCA) of the ISSR data set revealed that the Spanish and French M. fructicola isolates were more closely related between themselves than to the non-European isolates. The levels of genetic diversity in the Spanish and French isolates are lower than those of the non-European isolates, indicating that M. fructicola is a recently introduced pathogen. UPGMA cluster analysis and PCA of the combined ISSR + RAPD data set of the European M. fructicola populations revealed that the Spanish isolates were more closely related among themselves than with the French isolates. Analysis of molecular variance partitioned the genetic variance to among the two regions (Spain and France) (20%), among the regional populations (35%), and within the populations in each region (45%) suggesting restricted gene flow between the three European populations. The observed index of association (I) in each European M. fructicola populations indicates that the French and Spanish populations of M. fructicola are mainly asexually reproducing, with the Sudanell population potentially having a teleomorphic stage. The present finding of low genetic diversity in the Spanish and French M. fructicola populations is probably due to founder effects and genetic drift. [ABSTRACT FROM AUTHOR]

Abstract: Fire blight is an invasive disease caused by Erwinia amylovora that threatens pome fruit production globally. Effective implementation of phytosanitary control measures depends upon rapid, reliable pathogen detection and disease diagnosis. We developed a lateral-flow immunoassay specific for E. amylovora with a detection limit of log 5.7CFU/ml, typical of pathogen concentrations in symptomatic plant material. The simple assay had comparable sensitivity to standard culture plating, serum agglutination and nested PCR when validated for application in a phytosanitary laboratory as a confirmatory test of cultured isolates and for first-line diagnosis of phytosanitary samples that represent the full range of commercial, ornamental and forestry host species. On-site validation in ring-trials with local plant inspectors demonstrated robust and reliable detection (compared to subsequent plating and PCR analysis). The simplicity, inspector acceptance and facilitation of expedited diagnosis (from 2days for laboratory submitted samples to 15min with the immunoassay), offers a valuable tool for improved phytosanitary control of fire blight. [Copyright &y& Elsevier]

Abstract: A PCR-based method was developed for the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni (Xap), which provides rapid, sensitive and specific in planta detection and isolate identification. Primers specific for Xap were identified using random amplified polymorphic DNA (RAPD). Simplex PCR with these primers had a limit of detection per PCR reaction of approximately 10CFU for isolate cultures and 50CFU for plant material when used on tenfold dilutions of isolate culture or genomic DNA extracted from spiked samples, respectively. The primers were adapted as a high-throughput single-step screening based on a digoxigenin-labeled DNA probe assay with a detection limit of 4×102 CFU from isolate cultures. A duplex-PCR method was designed that includes the pathovar-level with species-level primers based on species-specific regions of the quinate metabolic gene qumA, increasing diagnostic confidence and offering the first molecular test for all X. arboricola pathovars. [Copyright &y& Elsevier]

Bacterial wilt of dry beans (family Fabaceae) caused by the actinobacterial agent Curtobacterium flaccumfaciens pv. flaccumfaciens is one of the most important diseases threatening edible legume production around the globe. Despite the economic losses due to the bacterial wilt disease, the pathogen has not so far been investigated for its genomic features, pathogenicity determinants, and virulence strategies. Here we present the first complete genome sequence of a highly virulent bacteriocin-producing C. flaccumfaciens pv. flaccumfaciens strain P990. The bacterium has a circular chromosome consisting of 3,736 kbp with the G+C% content of 71.0%. Furthermore, a 147-kbp circular plasmid (pCff1) with 66.1% G+C% content as well as two circular plasmid-like DNAs with sizes of 25 and 22 kbp were detected within the genomic contents of C. flaccumfaciens pv. flaccumfaciens . Phylogenetic analyses revealed that only a few number of Curtobacterium sp. strains deposited in the public databases could be classified within the species C. flaccumfaciens . Comparative genomics of C. flaccumfaciens pv. flaccumfaciens using the genome sequences of actinobacterial plant pathogens revealed the presence of a set of unique low G+C% content genomic islands in the C. flaccumfaciens pv. flaccumfaciens genome. Homologs of pathogenicity-determinant loci capable of producing 1,4-beta-xylanase ( xysA ), pectate lyase ( pelA1 and pelA2 ), serine protease ( chpC , chpG , and pat-1 ), and sortase ( srtA ) were detected in C. flaccumfaciens pv. flaccumfaciens genome. The genomic data presented here extend our understanding of the C. flaccumfaciens pv. flaccumfaciens genomic features and pave the ways of research on functional and interaction genetics to combat the risk of bacterial wilt disease in the 21st century's dry bean industry.

Members of the genus Clavibacter are economically important bacterial plant pathogens infecting a set of diverse agricultural crops (e.g., alfalfa, corn, potato, tomato, and wheat). Tomato-associated Clavibacter sp. strains account for a great portion of the genetic diversity of the genus, and C. michiganensis sensu stricto (formerly C. michiganensis subsp. michiganensis ), causing bacterial canker disease, is considered one of the most destructive seed-borne agents for the crop worldwide. However, current taxonomic descriptions of the genus do not reflect the existing diversity of the strains, resulting in unsatisfactory results in quarantine surveys for the pathogens. In this study, we used all the available genome sequences of Clavibacter sp. strains, including the type strains of newly described subspecies, to provide precise insight into the diversity of tomato-associated members of the genus and further clarify the taxonomic status of the strains using genotypic and phenotypic features. The results of phylogenetic analyses revealed the existence of nine hypothetical new species among the investigated strains. None of the three new subspecies (i.e., C. michiganensis subsp. californiensis , C. michiganensis subsp. chilensis , and C. michiganensis subsp. phaseoli ) is included within the tomato-pathogenic C. michiganensis sensu stricto lineage. Although comparative genomics revealed the lack of chp and tomA pathogenicity determinant gene clusters in the nonpathogenic strains, a number of pathogenicity-related genes were noted to be present in all the strains regardless of their pathogenicity characteristics. Altogether, our results indicate a need for a formal taxonomic reconsideration of tomato-associated Clavibacter sp. strains to facilitate differentiation of the lineages in quarantine inspections. IMPORTANCE Clavibacter spp. are economically important bacterial plant pathogens infecting a set of diverse agricultural crops, such as alfalfa, corn, pepper, potato, tomato, and wheat. A number of plant-pathogenic members of the genus (e.g., C. michiganensis sensu stricto and C. sepedonicus , infecting tomato and potato plants, respectively) are included in the A2 (high-risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Although tomato-associated members of Clavibacter spp. account for a significant portion of the genetic diversity in the genus, only the strains belonging to C. michiganensis sensu stricto (formerly C. michiganensis subsp. michiganensis ) cause bacterial canker disease of tomato and are subjected to the quarantine inspections. Hence, discrimination between the pathogenic and nonpathogenic Clavibacter sp. strains associated with tomato seeds and transplants plays a pivotal role in the accurate detection and cost-efficient management of the disease. On the other hand, detailed information on the genetic contents of different lineages of the genus would lead to the development of genome-informed specific detection techniques. In this study, we have provided an overview of the phylogenetic and genomic differences between the pathogenic and nonpathogenic tomato-associated Clavibacter sp. strains. We also noted that the taxonomic status of newly introduced subspecies of C. michiganensis (i.e., C. michiganensis subsp. californiensis , C. michiganensis subsp. chilensis , and C. michiganensis subsp. phaseoli ) should be reconsidered., (Copyright © 2020 American Society for Microbiology.)