Search

The Gram-positive genus Clavibacter is currently divided into seven species ( Clavibacter michiganensis , Clavibacter nebraskensis , Clavibacter capsici , Clavibacter sepedonicus , Clavibacter tessellarius , Clavibacter insidiosus and Clavibacter zhangzhiyongii ) and three subspecies ( C. michiganensis subsp. californiensis , C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli ). Recent studies have indicated that the taxonomic rank of the subspecies must be re-evaluated. In this research, we assessed the taxonomic position of the three C. michiganensis subspecies and clarified the taxonomic nomenclature of other 75 Clavibacter strains. The complete genomes of the type strains of the three Clavibacter subspecies, the type strain of C. tessellarius and C. nebraskensis A6096 were sequenced using PacBio RSII technology. Application of whole-genome-based computational approaches such as average nucleotide identity (ANI), digital DNA–DNA hybridization, multi-locus sequence analysis of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), a phylogenomic tree reconstructed from 1 028 core genes, and ANI-based phylogeny provided sufficient justification for raising C. michiganensis subsp. californiensis to the species level. These results led us to propose the establishment of Clavibacter californiensis sp. nov. as a species with its type strain C55T (=CFBP 8216T=ATCC BAA-2691T). Moreover, the orthologous and in silico dot plot analyses, along with the above described bioinformatic strategies, revealed a high degree of similarity between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli . Based on these analyses, we propose that both subspecies be combined into a single taxon and elevated to the species level as Clavibacter phaseoli sp. nov., with LPPA 982T (= CECT 8144T= LMG 27667T) as the type strain.

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.

Dickeya spp. cause blackleg and soft rot diseases of potato and several other plant species worldwide, resulting in high economic losses. Rapid detection and identification of the pathogen is essential for facilitating efficient disease management. Our aim in this research was to develop a rapid and field-deployable recombinase polymerase amplification (RPA) assay coupled with a lateral flow device (LFD) that will accurately detect Dickeya spp. in infected plant tissues without the need for DNA isolation. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific robust primers and probes for an RPA assay. Assay specificity was validated with 34 representative strains from all Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed when 1 µl of host sap, macerated in Tris-EDTA buffer, was added to each reaction in the sensitivity tests. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Susceptibility of anthurium cultivars to systemic infection by the bacterial blight pathogen, Xanthomonas campestris pv. dieffenbachiae, was examined using a bioengineered bioluminescent strain (V108LRUH1) and compared with susceptibility to foliar infection. Eight cultivars with different levels of susceptibility to foliar infection were evaluated for their susceptibility to systemic infection. Petioles of second youngest leaves cut near the main stem were inoculated with strain V108LRUH1, and subsequent movement of this bacterium into other petioles was monitored by observing bioluminescence from the plants. The actual extent of systemic movement was determined by reisolating V108LRUH1 from dissected segments of the remaining petioles. In susceptible cultivars, the pathogen advanced very rapidly and nearly reached the distal end of petioles. In resistant cultivars, the pathogen was detected in none (or very few) of the petiole segments. However, the susceptibility ranking among the tested cultivars for systemic infection did not always correspond to the ranking determined for foliar infection: i.e., one cultivar that was susceptible to foliar infection was highly resistant to systemic infection, and vice versa. This suggests that cultivar susceptibility of anthuriums to bacterial blight may differ depending on the phase of disease progression, and thus evaluation for both disease phases is essential for complete understanding of cultivar susceptibility.

The first reported outbreak of bacterial heart rot of pineapple (Ananas comosus var. comosus) in Hawaii occurred in December 2003. Of immediate concern was the differentiation of heart rot caused by Erwinia chrysanthemi from a soft rot caused by E. carotovora subsp. carotovora because of regulatory issues. Presumptive identifications of the isolated bacteria were made using bacteriological tests (including reactivity with an Erwinia-specific monoclonal antibody, E2) and compared with identifications obtained by two general methods: carbon source utilization profiling (Biolog) and 16S rDNA sequence analysis. The panel of bacteriological tests consistently differentiated E. chrysanthemi from E. carotovora subsp. carotovora and other nonquarantine organisms. BOX-polymerase chain reaction fingerprint patterns further differentiated the pineapple-isolated E. chrysanthemi strains from those obtained from other plants and irrigation water. Pineapple leaf inoculations revealed that only E. chrysanthemi from pineapple produced watersoaking and rot similar to that observed on the original symptomatic plants, thus identifying these strains as the causal agents of the outbreak. In this situation, where rapid identification of an unknown pathogen was necessary, standard bacteriological tests then available in the laboratory provided reliable differentiation of E. chrysanthemi from E. carotovora subsp. carotovora. Additional strain characterization is needed before the pineapple-isolated E. chrysanthemi strains can be classified into a species of the new genus Dickeya.

Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays.

Pectobacterium species cause serious bacterial soft rot diseases worldwide on economically important fruit and vegetable crops including tomato and potato. Accurate and simple methods are essential for rapid pathogen identification and timely management of the diseases. Recombinase polymerase amplification (RPA) combined with a lateral flow device (LFD) was developed for specific detection of Pectobacterium sp. directly from infected plant materials with no need for DNA isolation. The specificity of RPA-LFD was tested with 26 Pectobacterium sp. strains and 12 non-Pectobacterium species and no false positive or false negative outcomes were observed. RPA primers and probe for host control were also developed to detect the host genome for enhanced reliability and accuracy of the developed assay. The detection limit of 10 fg was obtained with both sensitivity and spiked sensitivity assays. No inhibitory effects were observed on the RPA assay when targets (pathogen and host) were directly detected from infected potato and tomato sap. The developed RPA assay has numerous applications from routine diagnostics at point-of-care, biosecurity, surveillance and disease management to epidemiological studies. In addition, this tool can also be used to discover reservoir hosts for Pectobacterium species.

Bacterial spot (BS), caused by Xanthomonas euvesicatoria, X. vesicatoria, X. gardneri and X. perforans, is an economically important bacterial disease of tomato and pepper. Symptoms produced by all four species are nearly indistinguishable. At present, no point-of-care diagnostics exist for BS. In this research, we examined genomes of X. euvesicatoria, X. vesicatoria, X. gardneri, X. perforans and other species of Xanthomonas; the unique gene recG was chosen to design primers to develop a loop-mediated isothermal amplification (LAMP) assay to rapidly and accurately identify and differentiate X. euvesicatoria from other BS causing Xanthomonas sp. using a field-deployable portable BioRangerTM instrument. Specificity of the developed assay was tested against 39 strains of X. euvesicatoria and 41 strains of other species in inclusivity and exclusivity panels, respectively. The assay detection limit was 100 fg (~18 genome copies) of genomic DNA and 1,000 fg in samples spiked with tomato DNA. The assay unambiguously detected X. euvesicatoria in infected tomato plant samples. Concordant results were obtained when multiple operators performed the test independently. No false positives and false negatives were detected. The developed LAMP assay has numerous applications in diagnostics, biosecurity and disease management.

A loop-mediated amplification (LAMP) assay developed previously to specifically identify Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato, was compared with a commercial lateral flow immunoassay, three PCR assays, and culturing on agar medium for efficacy in detecting the pathogen in pepper and tomato tissues and tomato seeds. The LAMP assay was as effective in identifying C. m. michiganensis-positive tomato seed and tissue samples as the Cmm ImmunoStrip®. The LAMP assay was performed using a handheld real-time fluorescence-monitoring device (Smart-DARTTM) and the iQTM5 Multicolor Real-Time PCR Detection System to compare both systems. Both devices produced equivalent results, thus demonstrating the potential of this LAMP detection system for field detection. A side-by-side comparison of the C. m. michiganensis-LAMP with the Cmm ImmunoStrip® assay, two conventional PCR assays, using Cmm5/6 and PSA8/R primer sets, and one quantitative PCR assay (primers...

Dickeya and Pectobacterium spp. are responsible for soft-rotting diseases of several plant species, some with overlapping host range. On potato, symptoms caused by these pathogens cannot be clearly differentiated. Disease results in the downgrading and rejection of potato seed, thus requiring additional phytosanitary restrictions across Northern Europe and other parts of the world. In an effort to provide a more timely and accurate diagnostic to distinguish these two groups of pathogens, a method for detecting Dickeya spp. using loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay can be used to test crude extracts prepared directly from symptomatic lesions. The entire test can be completed in less than 30 min, making it faster than the current diagnostic standard, the pelADE conventional polymerase chain reaction. Additionally, the LAMP assay was able to detect Dickeya DNA in samples spiked with varying amounts of Pectobacterium DNA, thus demonstrating the highly specific and sensitive nature of the assay, which can be applied on survey samples with mixed soft-rotting bacterial populations.

Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), occurs worldwide. This study focuses on three genes, clvA (clavicidin) and two associated genes (clvF and clvG, encoding a putative ABC-type multidrug transport system ATPase and permease component, respectively). Loop-mediated amplification of clvA and PCR of clvA, clvF and clvG showed that these genes were present only in Cmm and not in other Clavibacter species/subspecies, as well as other genera of plant-associated bacteria. Sequences of clvA, clvF and clvG from 48 geographically diverse strains of Cmm were analyzed phylogenetically to determine sequence variation. Maximum parsimony (MP), neighbour-joining (NJ) and maximum likelihood (ML) analyses placed strains into subgroups irrespective of their geographical origins. Multi-locus sequence analysis (MLSA) of these three genes produced the same results. In sum, all three clv genes are unique and conserved in Cmm, further supporting the use of the clvA gene for identification of Cmm, using either a previously designed LAMP assay or PCR with additional primers developed in this study. The genes investigated here are novel targets for specific detection of Cmm and have numerous molecular diagnostic applications.

Bacterial leaf spot of tomato and pepper (BLS), an economically important bacterial disease caused by four species of Xanthomonas (X. euvesicatoria (Xe), X. vesicatoria (Xv), X. gardneri (Xg), and X. perforans (Xp)), is a global problem and can cause over 50% crop loss under unfavorable conditions. Among the four species, Xe and Xv are prevalent worldwide. Characterization of the pathogens is crucial for disease management and regulatory purposes. In this study, we performed a multilocus sequence analysis (MLSA) with six genes (hrcN, dnaA gyrB, gapA, pdg, and hmbs) on BLS strains. Other Xanthomonas species were included to determine phylogenetic relationships within and among the tested strains. Four BLS species comprising 76 strains from different serological groups and diverse geographical locations were resolved into three major clades. BLS xanthomonads formed distinct clusters in the phylogenetic analyses. Three other xanthomonads, including X. albilineans, X. sacchari, and X. translucens pv. undolusa revealed less than 85%, 88%, and 89% average nucleotide identity (ANI), respectively, with the other species of Xanthomonas included in this study. Both antibody and MLSA data showed that Xv was clearly separated from Xe and that the latter strains were remarkably clonal, even though they originated from distant geographical locations. The Xe strains formed two separate phylogenetic groups, Xe group A1 consisted only of tomato strains, whereas Xe group A2 included strains from pepper and tomato. In contrast, the Xv group showed greater heterogeneity. Some Xv strains from South America were closely related to strains from California, while others grouped closer to a strain from Indiana and more distantly to a strain from Hawaii. Using this information molecular tests can now be devised to track distribution of clonal populations that may be introduced into new geographic areas through seeds and other infected plant materials.

Loop-mediated amplification (LAMP) was used to specifically identify Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato. LAMP primers were developed to detect micA, a chromosomally stable gene that encodes a type II lantibiotic, michiganin A, which inhibits growth of other C. michiganensis subspecies. In all, 409 bacterial strains (351 C. michiganensis subsp. michiganensis and 58 non-C. michiganensis subsp. michiganensis) from a worldwide collection were tested with LAMP to determine its specificity. LAMP results were compared with genetic profiles established using polymerase chain reaction (PCR) amplification of seven genes (dnaA, ppaJ, pat-1, chpC, tomA, ppaA, and ppaC). C. michiganensis subsp. michiganensis strains produced eight distinct profiles. The LAMP reaction identified all C. michiganensis subsp. michiganensis strains and discriminated them from other C. michiganensis subspecies and non-Clavibacter bacteria. LAMP has advantages over immunodiagnostic and other molecular detection methods because of its specificity and isothermal nature, which allows for easy field application. The LAMP reaction is also not affected by as many inhibitors as PCR. This diagnostic tool has potential to provide an easy, one-step test for rapid identification of C. michiganensis subsp. michiganensis.

Bacterial heart rot of pineapple reported in Hawaii in 2003 and reoccurring in 2006 was caused by an undetermined species of Dickeya . Classification of the bacterial strains isolated from infected pineapple to one of the recognized Dickeya species and their phylogenetic relationships with Dickeya were determined by a multilocus sequence analysis (MLSA), based on the partial gene sequences of dnaA, dnaJ, dnaX, gyrB and recN. Individual and concatenated gene phylogenies revealed that the strains form a clade with reference Dickeya sp. isolated from pineapple in Malaysia and are closely related to D. zeae ; however, previous DNA–DNA reassociation values suggest that these strains do not meet the genomic threshold for consideration in D. zeae , and require further taxonomic analysis. An analysis of the markers used in this MLSA determined that recN was the best overall marker for resolution of species within Dickeya . Differential intraspecies resolution was observed with the other markers, suggesting that marker selection is important for defining relationships within a clade. Phylogenies produced with gene sequences from the sequenced genomes of strains D. dadantii Ech586, D. dadantii Ech703 and D. zeae Ech1591 did not place the sequenced strains with members of other well-characterized members of their respective species. The average nucleotide identity (ANI) and tetranucleotide frequencies determined for the sequenced strains corroborated the results of the MLSA that D. dadantii Ech586 and D. dadantii Ech703 should be reclassified as Dickeya zeae Ech586 and Dickeya paradisiaca Ech703, respectively, whereas D. zeae Ech1591 should be reclassified as Dickeya chrysanthemi Ech1591.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

The Goss’s wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss’s wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms.

Ralstonia solanacearum (Rs) race 4 strains cause bacterial wilt of edible ginger (Zingiber officinale). The survival of the pathogen was studied in plant-free soil and potting medium in the presence of plants inoculated by different methods (non-wounded, rhizome-wounded, and stem-wounded) and irrigated on different schedules (alternate and daily). Detection thresholds for Rs were determined for an enzyme-linked immunoabsorbent assay (ELISA), immunostrip assay, and polymerase chain reaction (PCR) using drainage water from soil and potting medium containing known concentrations of Rs. In the absence of a plant or in the presence of non-wounded plants, Rs populations declined rapidly in drainage water from potting medium during the first 9 days and were undetectable after 81 days. When plants were stem- or rhizome-wounded, Rs populations increased by two to three orders of magnitude from the initial population levels for the first 9 to 19 days and then gradually declined and became undetectable after 89 days. Results were similar in experiments with soil except for non-wounded ginger plants, where the initial decline in Rs populations was followed by an abrupt increase after day 11, reaching 7 log cfu/mL on day 21, then declining gradually to non-detectable levels after 137 days. The increase was attributed to natural infection of the plants followed by release of high populations of Rs into the irrigation water when plants wilted. When rhizome-inoculated plants were watered on alternate days, Rs was recovered from 97 to 129 days in soil and potting medium, but when the plants were watered daily, Rs was recovered in soil and potting medium up to 153 days after plant inoculation. ELISA using Ps1a monoclonal antibody detected the pathogen from >95% of the samples from soil and potting medium when viable populations were >5 log cfu/mL. The immunostrip assay (using the same antibody) detected the pathogen from 100% of the samples when viable populations were >3 log cfu/mL. PCR based on the flagellin gene fliC detected the pathogen from >95% of the samples from soil and from >74% of the samples from potting medium when viable populations were >4 log cfu/mL.

A sensitive, selective, and rapid protocol for detecting Ralstonia solanacearum from soil and plant tissues was developed based on the integration of the rapid self-replicating ability of bacteriophages with quantitative PCR (q-PCR). Six bacteriophages were isolated and selected for their ability to specifically infect and lyse R. solanacearum. Sixty-three strains of R. solanacearum and 72 isolates of other bacterial species were tested for their susceptibility to the bacteriophages. Based on the large host range and observed replication speed and reproductive burst sizes in ginger infecting R. solanacearum strain GW-1, phage M_DS1 was selected for the development of the phage-based indirect assay. With primers based on the phage genome, the protocol was used to detect R. solanacearum from a number of substrates. In pure R. solanacearum cultures, the protocol consistently detected approximately 3.3 CFU/ml after an hour's incubation with 5.3 × 102 PFU/ml M_DS1. We used the protocol to confirm the presence of the pathogen in infected potted ginger plants, detecting levels near 102 CFU/g in 0.1 g of leaf tissue and levels near 103 CFU/ml in drainage water from the pots. In soils emended with the bacteria, we observed detection limits down to approximately 102 CFU/g.

Klebsiella spp. are opportunistic pathogens with clinical, veterinary and plant-associated isolates. A previous study showed that bacterial ooze from wetwood of severely declined ironwood trees in Guam contained Ralstonia solanacearum, Klebsiella variicola and K. oxytoca. In this study, Loop-Mediated Isothermal Amplification (LAMP) detected K. variicola and K. oxytoca specifically, using unique primer sets designed individually for each organism. Each LAMP detected its target specifically, while showing negative results for non-target bacteria and negative controls. LAMP detected Klebsiella in inoculated-ironwood stem tissues and bacterial ooze. Due to the presence of plant inhibitors, different sampling protocols were tested. Soaking plant tissue samples to allow diffusion of bacteria into solution, followed by boiling, provided optimum detection of Klebsiella directly from plant samples. False negatives obtained when using crushed plant samples were eliminated by including an enrichment step, involving plating and 12-h incubation. DGGE (denaturing gradient gel electrophoresis) and a colony-blot immunoassay using a Klebsiella-specific antibody also detected Klebsiella in inoculated ironwood. DGGE bands and antibody crossreactions from closely related enterobacters showed the potential for false positive results. The nature of LAMP makes it ideal for point-of-care testing, and when combined with the specificity of the LAMP primers developed in this study, demonstrates its potential as a routine field test for Klebsiella in ironwood in Guam, as well as clinical and veterinary diagnosis of Klebsiella infection. Additionally, the regions targeted for detection in this study have application across all forms of molecular-based diagnostics.

Specificity of a monoclonal antibody (MAb), Cmm1, to geographically diverse strains of the seed-borne tomato pathogen, Clavibacter michiganensis subsp. michiganensis (Cmm), was assessed and the MAb was tested for its usefulness as a tool to separate the pathogen from saprophytes in naturally infested tomato seed. Of the 236 international Cmm strains tested, 99% reacted with MAb Cmm1. MAb Cmm1 was also strongly reactive with an additional 32 strains isolated from seed that were later identified as Cmm by the Biolog MicroLog™ microbial identification system (Biolog, Inc., Hayward, CA) and 16S rDNA sequence analysis. It correctly differentiated these strains from 12 MAb Cmm1-negative seed strains that possessed similar colony morphology but were later identified as other Gram-positive genera and species. The specificity of MAb Cmm1 to the pathogen and the near universality of the MAb Cmm1-reactive antigen among diverse Cmm strains make this antibody a useful detection and identification tool. The finding that a large proportion of the Cmm strains associated with naturally infested tomato seed were putatively hypovirulent or non-virulent indicates that such populations cannot be ignored and points to a need for studies to determine their significance in host-pathogen interactions.

The genus Clavibacter contains one recognized species, Clavibacter michiganensis . Clavibacter michiganensis is subdivided into subspecies based on host specificity and bacteriological characteristics, with Clavibacter michiganensis subsp. michiganensis causing bacterial canker of tomato. Clavibacter michiganensis subsp. michiganensis is often spread through contaminated seed leading to outbreaks of bacterial canker in tomato production areas worldwide. The frequent occurrence of non-pathogenic Clavibacter michiganensis subsp. michiganensis -like bacteria (CMB) is a concern for seed producers because Clavibacter michiganensis subsp. michiganensis is a quarantine organism and detection of a non-pathogenic variant may result in destruction of an otherwise healthy seed lot. A thorough biological and genetic characterization of these seed-associated CMB strains was performed using standard biochemical tests, cell wall analyses, metabolic profiling using Biolog, and single-gene and multilocus sequence analyses. Combined, these tests revealed two distinct populations of seed-associated members of the genus Clavibacter that differed from each other, as well as from all other described subspecies of Clavibacter michiganensis . DNA–DNA hybridization values are 70 % or higher, justifying placement into the single recognized species, C. michiganensis , but other analyses justify separate subspecies designations. Additionally, strains belonging to the genus Clavibacter isolated from pepper also represent a distinct population and warrant separate subspecies designation. On the basis of these data we propose subspecies designations for separate non-pathogenic subpopulations of Clavibacter michiganensis : Clavibacter michiganensis subsp. californiensis subsp. nov. and Clavibacter michiganensis subsp. chilensis subsp. nov. for seed-associated strains represented by C55T ( = ATCC BAA-2691T = CFBP 8216T) and ZUM3936T ( = ATCC BAA-2690T = CFBP 8217T), respectively. Recognition of separate subspecies is essential for improved international seed testing operations.

Two cultivars of Anthurium andraeanum Hort. hybrids, `Paradise Pink' and `Tropic Flame', were transformed by Agrobacterium to contain gene sequences for Shiva-1, a cecropin-based lytic peptide. The antibacterial gene was driven by a 35-35S cauliflower mosaic viral (35-35S CaMV) promoter and the construct included the secretory signal sequence for pathogenesis-related protein 1b (PR1b). Blight tolerance of regenerated plants was tested by inoculation with a virulent strain of Xanthomonas axonopodis (formerly campestris) pv. dieffenbachiae (Xad) that is bioluminescent to allow detection of symptomless infections in Shiva-1 transformants. Primary regenerants for two Shiva-1 transgenic lines of `Paradise Pink' displayed significantly enhanced tolerance to bacterial blight over blight susceptible `Rudolph' and even the blight tolerant `Kalapana'. Two Shiva-1 transgenic lines of `Tropic Flame' showed no improved resistance when compared to the control at the mean percent leaf infection level. One Shiva-1 transgenic line of `Tropic Flame' was unexpectedly more susceptible to blight than the nontransgenic control. Low expression of Shiva-1 observed in this line is hypothesized to be the cause of its increased susceptibility to Xad.

It has been reported that all tested naturally occurring strains of Xanthomonas campestris pv. campestris that are known to be capable of inducing blight symptoms in cabbage react with MAb A11 and hybridize with a 5.4-kb DNA fragment (in plasmid pJC41), cloned from the Xanthomonas campestris species type strain, Xcc528T, whereas all tested naturally occurring strains that do not cause blight react with MAb X21 and do not hybridize to pJC41. The roles of the 5.4-kb DNA in pJC41 and the epitope recognized by MAb A11 in the pathogenicity of X. c. campestris strains that cause blight were examined by mutational analyses. A 4.0-kb deletion of the pJC41 region on the Xcc528T chromosome was created by marker exchange, but the derivatives were evidently not affected in their ability to elicit blight symptoms. Nitrosoguanidine was used to mutagenize two blight strains, Xcc528T and CAM19, and mutants were selected that were not reactive to MAb A11. The MAb A11-negative mutant of Xcc528T was reactive to MAb X21, but was evidently not affected in the ability to elicit blight symptoms. MAb A11-negative mutants of CAM19, however, were not reactive to MAb X21, and showed reduction or loss of virulence, which suggested the requirement for at least one of the two antigens (to MAbs A11 or X21) for pathogenicity. A genomic library of CAM19 was made and screened for genes responsible for the production of the A11 antigen. Cosmid clones were identified that restored MAb A11 reactivity to the mutants. None of these cosmids restored the virulence of the mutant strains that had lost virulence. Therefore, neither the blight-associated 5.4-kb DNA fragment nor the MAb A11 antibody marker were required for blight symptom elicitation or pathogenicity.

Xanthomonas campestris pv. campestris induces two types of symptoms, namely, black rot and blight. Black rot symptoms are V-shaped lesions and black veins on the leaf, and blight symptoms are sudden collapse of interveinal tissues following the lack of veinal necrosis at early stages of infection. These two symptoms can occur simultaneously. However, the tendency to induce either symptom type is strain-dependent. Six strains were evaluated for their rate and pattern of spread in misted seedbeds by using strain-specific monoclonal antibodies and miniplate enrichment/ELISA. Data on pathogen incidence was defined as the presence of the pathogen in or on plants rather than visual symptoms. The results indicated that blight-inducing strains spread to more seedlings than black rot-inducing strains. The high incidences of blight-inducing strains in experimental plots were associated with non-randomness of spatial pattern of pathogen spread, indicating that high incidence is primarily due to the spread from adjacent plants by leaf contact and water splash. Most ELISA-positive seedlings were symptomless, indicating that the sensitivity of the system used in this study was adequate for detection of latent or epiphytic spread.

An adaptation of the most probable number (MPN) method and use of microfluor plates for monitoring a bioluminescent strain (171LIIH-7) of Xanthomonas campestris pv. campestris (Pammel) Dowson in debris-inoculated soil, is described. Microfluor plates with liquid culture medium were inoculated with a dilution series of soil extract previously infested with cabbage debris containing 171LIIH-7. Light produced by the bacteria was detected on X-ray film, and absorbance readings of the images were converted to a binary set of values for estimation of MPN by the computer program MPNES (Woomer et al., 1990). The MPN-microfluor plate system was compared with direct dilution plating on FS medium and MPN-miniplate enrichment ELISA with a pathovar-specific monoclonal antibody. The detection limit of X. c. pv. campestris with FS medium was about 104 cfu g−1 soil. The detection threshold of MPN-microfluor plates (102 bacteria g−1 non-sterile soil) was equivalent to the MPN-miniplate enrichment ELISA and 100 times more sensitive than direct dilution plating. The MPN-microfluor plate system is an easy, accurate, sensitive and inexpensive method to detect and enumerate bioliminescent X. c. pv. campestris in soil, and permits ecological studies in the soil environment that will provide the rationale for changes in crop rotations or other cultural practices.

Summary A total of 70 presumptive non-pathogenic yellow-pigmented strains from different origins were identified to the genus Xanthomonas with the genus-specific monoclonal antibodies (XI and XII), and fatty acid methyl ester (FAME) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein databases. On the basis of FAME profiles and SDS-PAGE protein patterns, these non-pathogenic xanth- omonads formed a heterogeneous population. Fourty-two strains were identified as the same species by both methods, whereas five strains were identified as different species. Only eight strains remained unidentified by both methods, whereas in 15 cases the identification was ambiguous. The identification at pathovar level was usually ambiguous and not concordant, which indicated that the non-pathogenic xanthomonads cannot be classified in the described pathovars. None of the non-pathogenic xanth- omonads was identified as belonging to the pathovars of the plant from which they were isolated.

We used micro- and resonance Raman spectroscopy with 785 nm and 514.5 nm laser excitation, respectively, to characterize a plant pathogenic bacteria, Xanthomonas axonopodis pv. dieffenbachiae D150. The bacterial genus Xathomonas is closely related to bacterial genus Stenotrophomonas that causes an infection in humans. This study has identified for the first time the unique Raman spectra of the carotenoid-like pigment xanthomonadin of the Xanthomonas strain. Xanthomonadin is a brominated aryl-polyene pigment molecule similar to carotenoids. Further studies were conducted using resonance Raman spectroscopy with 514.5 nm laser excitation on several strains of the bacterial genus Xanthomonas isolated from numerous plants from various geographical locations. The current study revealed that the Raman bands representing the vibrations (v1, v2, v3) of the polyene chain of xanthomonadin are 1003-1005 (v3), 1135-1138 (v2), and 1530 (v1). Overtone bands representing xanthomonadin were identified as 2264-2275 (2v2), and combinational bands at 2653-2662 (v1+ v2). The findings from this study validate our previous finding that the Raman fingerprints of xanthomonadin are unique for the genus Xanthomonas. This facilitates rapid identification (~5 minutes) of Xanthomonas spp. from bacterial culture plates. The xanthomonadin marker is different from Raman markers of many other bacterial genus including Agrobacterium, Bacillus, Clavibacter, Enterobacter, Erwinia, Microbacterium, Paenibacillus, and Ralstonia. This study also identified Xanthomonas spp. from bacterial strains isolated from a diseased wheat sample on a culture plate.© (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Two genetically diverse groups of strains were identified among cultures of Xanthomonas campestris pv. vesicatoria isolated from plants with bacterial spot of pepper and tomato. Group A strains do not pit pectate gels or hydrolyze starch, whereas group B strains are strongly positive for these reactions. Group A strains cause a hypersensitive reaction in plants of tomato breeding line Hawaii 7998, but group B strains do not. Other differences between the two groups of strains were discovered in tests for utilization of carbon compounds, serology, fatty acid profiles, silver-stained protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and DNA restriction enzyme digestion profiles. The levels of DNA homology between strains belonging to the same group were more than 74%, but the levels of DNA homology between strains belonging to different groups were less than 46%. The two groups of strains have different genetic backgrounds, but cause essentially the same disease of tomato and pepper.

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants. In this study, both pristine and contaminated soils were sampled as a source of PAH-degrading organisms. Nine strains isolated from these soils were identified as rapidly growing members of the genus Mycobacterium through basic phenotypic characteristics and through sequence similarity of three genes. Because the sequence similarity of the 16S rRNA gene is relatively high among members of this genus, additional conserved genes encoding the beta subunit of RNA polymerase (rpoB) and a heat-shock protein (hsp65) were sequenced. Several analyses were completed to differentiate the strains from one another and to determine their species-level taxonomy, including fatty acid methyl ester analysis, biochemical tests and substrate-utilization profiling. A phylogenetic tree incorporating sequences for all three genes was constructed with the isolates and their close described relatives. Results for biochemical tests, substrate-utilization tests and DNA sequencing were compared with those of the phylogenetically similar organisms to establish the isolated strains as representatives of novel species with characteristics unlike those of previously described species of Mycobacterium. Finally, DNA-DNA hybridization was performed between strains and their close relatives to confirm their position within novel species. Our results demonstrated that the isolates represent five novel species, which were named Mycobacterium crocinum sp. nov. (type strain czh-42(T) =ATCC BAA-1373(T) =CIP 109262(T); reference strains czh-1A =ATCC BAA-1370 =CIP 109266 and czh-3 =ATCC BAA-1371=CIP 109267), Mycobacterium pallens sp. nov. (type strain czh-8(T) =ATCC BAA-1372(T) =CIP 109268(T)), Mycobacterium rutilum sp. nov. (type strain czh-117(T) =ATCC BAA-1375(T) =CIP 109271(T); reference strains czh-107 =ATCC BAA-1374 =CIP 109270 and czh-132 =ATCC BAA-1376 =CIP 109272), Mycobacterium rufum sp. nov. (type strain JS14(T) =ATCC BAA-1377(T) =CIP 109273(T)) and Mycobacterium aromaticivorans sp. nov. (type strain JS19b1(T) =ATCC BAA-1378(T) =CIP 109274(T)).

We report on the engineering of hardware systems and molecular tools to allow the rapid detection and discrimination of sub-populations of Ralstonia solanacearum, a bacterial wilt pathogen affecting a wide host-range of agronomically important plants, which also exhibits a large degree of virulence and environmental fortitude within the species. The hardware is based on a compact disc platform, wherein DNA delivered to a reaction well is replicated at a constant controlled temperature, and detected in real-time through increased fluorescence caused by the displacement of a quencher strand from a fluorescent “molecular zipper” during the DNA polymerization. The primers designed for the reaction were shown to selectively replicate DNA from race 3 biovar 2 populations of the pathogen, which is considered a major threat to potato growers in North America if it were ever introduced, but not DNA from other strains of Ralstonia solanacearum. Selective replication was observed in real time using the molecular zippers. Work is ongoing to demonstrate the molecular replication and detection on the CD media, though results have demonstrated that the temperature control on the CD is effective using non-contact measurements corrected for transmission losses in the reaction well and reflection of ambient radiation. Ultimately, we intend to integrate all of these systems onto a CD where centrifugal control can be used to sequence sample capture, wash, lysis, and DNA extraction followed by the replication and detection on an inexpensive disposable media.

▪ Abstract Disease diagnosis is based on a number of factors, including laboratory tests for pathogen identification. Rapid development of genomic techniques for characterization of bacteria over the past decade has greatly simplified and improved pathogen detection and identification, but DNA-based methods have not yet entirely replaced traditional culture and phenotypic tests in the plant industry. The first section of this review focuses on rapid immunodiagnostic and DNA-based detection methods for known bacterial pathogens in plants or plant products, which often manifest no symptoms of disease. The second section covers the broader topic of disease diagnosis and new methods for identifying and characterizing bacteria.

The hrpX gene is essential for pathogenicity of Xanthomonas species. Loss of hrpX by mutation results in the loss of pathogenicity and a gain in the ability of Xanthomonas to cause the hypersensitive response in their respective host plants, suggesting that hrpX confers a means to evade this host defense response. The function of HrpX protein was predicted by sequencing of hrpXc and hrpXo from X. campestris pv. campestris and X. oryzae, respectively. The predicted amino acid sequences of the protein encoded by these respective genes revealed similarities (45.96%) to the HrpB protein of Burkholderia solanacearum, which has sequence identity to the transcriptional activator VirF of Yersinia enterocolitica and AraC of Escherichia coli. Thus, HrpX may regulate Xanthomonas virulence genes since a putative DNA binding domain present in the carboxyl terminal half of HrpX is highly conserved among HrpB, VirF and AraC and since over-expression of the carboxyl terminal half of HrpX in E. coli is lethal.

We have developed a sensitive and specific assay for Xanthomonas axonopodis pv. citri, the causal agent of citrus bacterial canker. The assay is based on sequential nested amplification by polymerase chain reaction (PCR) of a region of plasmid DNA that is very highly conserved in X. axonopodis pv. citri. Specific amplification products were observed in reactions containing three or fewer target molecules, an improvement of 50- to 100-fold over single-stage PCR, and similar results were observed when beginning with purified DNA or living bacterial cells. Colorimetric detection of amplification products was performed with the DIANA (detection of immobilized amplified nucleic acids) method, which uses labeled primers to allow amplification product capture and detection in a microtiter plate. Predicted amplification products were produced from all strains of X. axonopodis pv. citri and from four of six strains of X. axonopodis pv. aurantifolii but not from other xanthomonads, including citrus epiphytes, except for X. axonopodis pv. vignicola and one strain isolated from Feronia elephantiacum, consistent with previous hybridization results. No amplification products were observed from strains of X. axonopodis pv. citrumelo that incite citrus bacterial spot disease in Florida citrus nurseries. Amplification was completely inhibited by copper hydroxide when present in the reaction mix at 13.6 μg/ml. Concentrated leaf extracts from tangelo and mandarin orange, but not similar extracts from other citrus varieties, also inhibited amplification. Immunomagnetic separation of target bacteria prior to amplification was used to concentrate and recover X. axonopodis pv. citri from samples containing compounds that inhibit amplification (i.e., copper and concentrated citrus extracts). Immunocapture, by concentrating target bacteria from dilute plant extracts, improved the sensitivity of the assay by 100-fold over nested-PCR alone. The combination of sensitivity, specificity, and speed of the assay could make this a widely used assay both in plant quarantine and in areas where X. axonopodis pv. citri is endemic and clean planting stock programs are to be initiated.

Four semiselective media that varied with respect to carbon source, amino acids, and antibiotics were evaluated for the growth of various strains of the rice bacterial blight pathogen, Xanthomonas oryzae pv. oryzae in pure and mixed culture. Plating efficiencies in pure culture ranged from 40 to 90% on these media, and colonies developed in 3-7 days, depending on the strain of X. o. oryzae. In mixtures with Erwinia herbicola and Pseudomonas putida(1:1:1), X. o. oryzae was recovered only on XOS medium. Recovery of a faster growing X. o. oryzae strain (X1-5) was improved by adding 50-100 mg/L of FeEDTA to XOS medium, but recovery of other strains was reduced or unaffected

Monoclonal antibodies (MAbs) specific for strains of Xanthomonas campestris pv. campestris (Xcc) and/or X. campestris pv. armoraciae (Xca) were evaluated to determine their reliability for pathogen identification. Reactivity patterns of 1,023 bacterial isolates from crucifer leaves, seeds, weeds, and alternate crops were compared with original host symptoms; and reactivity patterns of 498 of these isolates were compared with pathogenicity tests. Six MAbs were selected that distinguished strains of Xcc and Xca from nearly all other bacteria tested. However, Xcc and Xca strains were not serologically distinguished as groups from each other, despite numerous attempts to do so. Ninety-nine percent of the Xcc and Xca strains tested reacted with one or more of the Xcc/Xca-specific MAbs. Three MAbs separated all the Xcc (and some Xca) strains into three serological groups. A subset of 12 Xcc and seven Xca strains representing the serological and pathological diversity found in these two pathovars was further evaluated by restriction fragment length polymorphism (RFLP). Strains of both pathovars were heterogeneous by RFLP, and the RFLP patterns of the seven Xca strains overlapped with patterns of the Xcc strains. However, the 12 Xcc strains formed three identifiable groups (C1, C2, and C3), which corresponded to the three serological groups (S1, S2, and S3). The type strain of the species X. campestris (Xcc528 T ) and all members of group S3/C3 caused a distinctive blight symptom that later developed into black rot. Strains in the other two Xcc groups caused typical black rot symptoms but not blight. The panel of antibodies can be used for rapid identification of Xcc and Xca pathogens in Field and seed assays

Xanthomonas campestris pv. vesicatoria strains from a worldwide collection were assigned to the A or B group by testing for amylolytic activity and the presence of the α or β protein band. Cluster analyses of serological, carbon substrate utilization, and fatty acid composition data resulted in dendrograms with interspersed clusters of A and B strains, suggesting that the strain populations within each group are heterogeneous. However, further analysis of the data identified features unique to members of each group. Two monoclonal antibodies reacted only with group A strains, and two reacted only with group B strains. Reactivity patterns further subdivided each group into three serovars [...]

Six monoclonal antibodies (MAbs) were used to group 323 strains of Xanthomonas campestris pv. dieffenbachiae, isolated from 12 genera of aroids and collected from five Hawaiian islands, Florida, California, and Australia over 11 yr. Nine percent of these strains did not react with any of the six MAbs. Of the remaining 91%, 12 major serogroups were identified. Ninety-five percent of the Anthurium strains formed seven groups, four of which contained exclusively Anthurium strains. Thirty-six of 43 strains from Syngonium reacted with the MAbs, including most of those strains previously considered to be «X. vitians» and recently described as a separate pathovar (pv. syngonii) (...)

The heterogeneity of 194 previously uncharacterized strains of Xanthomanas campestris pv. citrumelo isolated from citrus bacterial spot epidemics in four citrus nurseries in central Florida was evaluated by virulence reactions on detached leaves, reaction to a panel of monoclonal antibodies (MAbs), and reaction to a panel of restriction fragment length polymorphism (RFLP) probes. Detached-leaf assays were performed on the 194 strains that had been differentiated into five serological groups based on reactions with six MAbs (...)

A monoclonal antibody (MAb), designated A1, reacted with lipopolysaccharide (LPS) epitopes of all tested strains of Xanthomonas campestris pv. citri isolated from the Asiatic form of citrus bacterial canker (CBC-A), with X. campestris strains pathogenic on ti (Cordyline terminalis), and with some Florida citrus nursery strains associated with citrus bacterial spot (CBS) disease. The A1 MAb did not react with strains associated with other forms of citrus canker (B, C, or D) (...)

A bacterial disease of limes that was detected in Colima, Mexico, and reached epidemic proportions by July 1982, was thought to be citrus canker disease caused by Xanthomonas campestris pv. citri. However, only one of 41 bacterial strains (strain M3) isolated from leaf lesions reacted with anti-Xanthomonas monoclonal antibodies (MCA). MCA were generated to identify reference strains of X. c. pv. citri, types A, B, and C from Japan, Argentina, and Brazil, respectively. The Mexican strain, M3, reacted only with MCA B2, specific for slow-growing type B strains from Argentina and the type C strain (XC70). M3 showed no relationship to faster-growing type B strains, XC64 and XC69 from Argentina. Five additional Mexican strains (S. Rodriguez: T20-T24) reacted with anti-Xanthomonas MCA (X1 and X11), but failed to react with MCA specific for reference strains of X. c. pv. citri. The remaining yellow, oxidative bacteria associated with leaf lesions were separated into 6 groups with a rapid hybridoma technique, and 44 additional freshly-isolated formalin-killed strains were characterized with MCA. The possibility of a mixed etiology involved in Mexican lime bacteriosis is presented.

Inoculation of 3-wk-old and 7-wk-old rice plants with bacteria isolated from lesions confirmed pathogenicity and reproduced symptoms observed in the field. Comparison of the causal organism with Asiatic strains of X.c. oryzae by standard biochemical tests, light and electron microscopy, fatty acid analysis, analysis of restriction fragment length polymorphisms, and reaction to monoclonal antibodies revealed numerous similarities but some consistent differences. Virulence of the U.S. strains on TN-1 was much reduced compared with strains from the Philippines. This is the first report of X.c. oryzae in the United States

Etiologie, symptomatologie des principales pourritures d'entreposage et mesures generales prophylactiques ou curatives de lutte contre les organismes phytopathogenes