Search

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae), causing soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera, and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with 10-fold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18–20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Ironwood (Casuarina equisetifoliasubsp.equisetifolia) trees on Guam have been in decline since 2002. This study applied proportional odds logistic multiple regression modeling to a set of biological variables in order to find significant decline predictors as a first step towards identifying pathogenic contributors. Based on the analysis of a set of 77 medium and large trees, the bacteriumRalstonia solanacearumand the fungusGanoderma australespecies complex were found to be significant predictors, withpvalues of R. solanacearumbyG. australeinteraction was not a strong effect, indicating additive rather than multiplicative behavior of these effects on decline severity (pvalue>0.087). When nine covariates were applied univariately to a data subset of 30 tree cross-sections, the significance ofR. solanacearumwas strongly upheld while that ofG. australewas moderately reduced. Also significant were percent cross-sectional area with bacterial wetwood and the formation of ooze within 24 h. Wetwood bacteria ofKlebsiellaspp. andK. oxytocawere found across all levels of decline and were not significant predictors. Other enteric bacteria identified includedKosakonia,Enterobacter, Pantoea, Erwinia,andCitrobacter.

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.

Ironwood (Casuarina equisetifolia subsp. equisetifolia) is a nitrogen-fixing tree of considerable social, economic and environmental importance that commonly occurs in tropical/subtropical zones of Asia, the Pacific, Africa, and Central America. Ironwood decline was first noticed on Guam in 2002 and is now affecting thousands of trees and impacting the ecosystem. In 2012, a survey showed that Ralstonia solanacearum and Klebsiella spp. were associated with wetwood symptoms of declining trees. R. solanacearum strains isolated from diseased ironwood in Guam were similar to R. solanacearum strain GMI1000, having similar BOX-PCR profiles and belonging to phylotype I and biovar 3. Two Klebsiella species (K. variicola and K. oxytoca) were recovered, with K. variicola being the more prevalent species. Pathogenicity tests revealed that R. solanacearum caused wilt in tomato and ironwood seedlings, whereas neither Klebsiella spp. produced symptoms. There were no differences in virulence between Guam R. solanacearum and control strains following inoculation into tomato and ironwood from Hawaii. Additionally, no observable differences in ironwood susceptibility to Ralstonia strains from Guam or Hawaii, were observed, suggesting that the association of Guam R. solanacearum with Guam ironwood is not specific. Co-inoculation studies with both R. solanacearum and Klebsiella variicola and K. oxytoca revealed that Klebsiella sp. did not affect symptoms produced by R. solanacearum alone. In planta studies were feasible only on seedlings and young trees in Hawaii; thus, possible interactions between R. solanacearum and Klebsiealla sp. in adult trees remain to be investigated. A new in-field survey of declining ironwood is needed to better understand the role of Klebsiella and Ralstonia in ironwood tree decline in Guam.

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.

New rapid diagnostic methods are urgently needed to discriminate the quarantine pathogen Ralstonia solanacearum (Rs) race 3 biovar 2 (R3B2) from other populations of Rs that lack the adaptation to cause bacterial wilt disease in temperate regions. We used an in silico bioinformatic approach to identify several genome sequences potentially specific to R3B2 strains. Primer sets were designed to PCR-amplify sequences in these regions, and four sets were ultimately shown to be >99% accurate for detection of R3B2 strains. On the basis of these results, several primers were designed to enable development of a loop-mediated isothermal amplification assay that was rapid, technologically simple, and essentially 100% accurate for identification of R3B2 when applied to a comprehensive collection of geographically diverse Rs strains. We fortuitously found that a sequence in one of the “R3B2-specific” regions has ~90% identity to a sequence present in strains of the blood disease bacterium (BDB), a member of the Rs species complex that infects banana. Alignments of these sequences allowed design of a second PCR primer set that proved 100% accurate for identification of BDB strains when tested on the 22 BDB strains available to us. These results demonstrate the power of in silico genomic subtraction for rapid identification of population-specific DNA sequences and for the development of simple, reliable detection methods for Rs subpopulations.

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 survival of the bacterial pathogen, Xanthomonas campestris pv. campestris (Xcc), was studied in plant debris-infested soil with different matric potentials (0, −10, −30, −50, −100, −200, and −900 kPa), and on the phylloplane of crops used for rotation with cabbage. Populations of cellulolytic and proteolytic microorganisms were studied in relation to soil matric potential and Xcc. The survival of Xcc was negatively correlated (r=−0.710; P=0.06) with soil matric potential and with the abundance of cellulolytic microorganisms (P=0.05). In saturated soil, Xcc survived for only 19–28 days, while at −900 kPa there was no significant change in the bacterial population within this period. Survival of Xcc on the phylloplane of cabbage (Brassica oleracea var. capitata) (host) was similar to that on mustard and lettuce (48 days), whereas the pathogen was detected for only 9 days on rice. It appears that high matric potentials reduced populations of Xcc in soil, whereas epiphytic survival of this pathogen depended on the plant species.

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.