Identification and characterization of bacteria associated with decline of ironwood (Casuarina equisetifolia) in Guam

Ayin, Caleb; Schlub, Robert L.; Yasuhara-Bell, Jarred; Alvarez, Anne M.

doi:10.1007/s13313-014-0341-4

Cited by 19 publications

(40 citation statements)

References 20 publications

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“…Plant inoculations were performed according to a previously established protocol [13,14]. Briefly, bacterial suspensions at 10 8 CFU/ ml were prepared from 48 hr cultures of test strains from TZC plates.…”

Section: Plant Inoculationmentioning

confidence: 99%

“…Two plants were co-inoculated with Ralstonia solanacearum and Klebsiella variicola, but one was an older, 10-month old ironwood (Plant 1) that was used to try to produce bacterial ooze. The ironwood seedlings were then grown under the same conditions as described previously [14].…”

Section: Plant Inoculationmentioning

confidence: 99%

“…Three complimentary approaches were used to isolate and identify the pathogen: growth on modified Rs-semiselective medium (mSMSA), Rs-specific immunostrip assays, and confirmation with a Rs-specific loop-mediated isothermal amplification (LAMP) reaction [11,12]. The identity of isolates was determined in Hawai'i and the two predominant types of bacteria associated with ironwood decline were characterized [13,14]. Bacteriological tests, along with multiplex PCR, BOX-PCR, and phylogenetic analysis of the dnaA RIF marker [15], were performed to further characterize and evaluate the genetic diversity among the Rs isolates [13,14].…”

Section: Introductionmentioning

confidence: 99%

“…The identity of isolates was determined in Hawai'i and the two predominant types of bacteria associated with ironwood decline were characterized [13,14]. Bacteriological tests, along with multiplex PCR, BOX-PCR, and phylogenetic analysis of the dnaA RIF marker [15], were performed to further characterize and evaluate the genetic diversity among the Rs isolates [13,14]. Non-Rs isolates were presumptively identified as Klebsiella spp.…”

Section: Introductionmentioning

confidence: 99%

“…Non-Rs isolates were presumptively identified as Klebsiella spp. using bacteriological tests [13,14,16,17]. Most strains showed closest similarity to K. variicola following sequence analysis of the 16S rRNA gene, while one showed closest match to K. oxytoca.…”

Section: Introductionmentioning

confidence: 99%

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Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

Yasuhara-Bell¹,

Ayin²,

Hatada³

et al. 2015

J Plant Pathol Microbiol

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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.

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Section: Plant Inoculationmentioning

confidence: 99%

Section: Plant Inoculationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

Yasuhara-Bell¹,

Ayin²,

Hatada³

et al. 2015

J Plant Pathol Microbiol

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Molecular markers reveal low genetic diversity in Casuarina equisetifolia clonal plantations in South China

Zhang

et al. 2019

New Forests

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Ralstonia solanacearum, Ganoderma australe, and bacterial wetwood as predictors of ironwood tree (Casuarina equisetifolia) decline in Guam

Ayin¹,

Alvarez²,

Awana³

et al. 2019

Australasian Plant Pathol.

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Ironwood (Casuarina equisetifolia subsp. 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 bacterium Ralstonia solanacearum and the fungus Ganoderma australe species complex were found to be significant predictors, with p values of <0.001 and < 0.008, respectively. Their respective values for symptomless trees were 18% and 0%, compared to 80% and 35% for those nearly dead trees of which 30% had both organisms and 15% had neither. More complex models were also fit, that included interactions. The R. solanacearum by G. australe interaction was not a strong effect, indicating additive rather than multiplicative behavior of these effects on decline severity (p value>0.087). When nine covariates were applied univariately to a data subset of 30 tree cross-sections, the significance of R. solanacearum was strongly upheld while that of G. australe was moderately reduced. Also significant were percent cross-sectional area with bacterial wetwood and the formation of ooze within 24 h. Wetwood bacteria of Klebsiella spp. and K. oxytoca were found across all levels of decline and were not significant predictors. Other enteric bacteria identified included Kosakonia, Enterobacter, Pantoea, Erwinia, and Citrobacter.

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Identification and characterization of bacteria associated with decline of ironwood (Casuarina equisetifolia) in Guam

Cited by 19 publications

References 20 publications

Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

Molecular markers reveal low genetic diversity in Casuarina equisetifolia clonal plantations in South China

Ralstonia solanacearum, Ganoderma australe, and bacterial wetwood as predictors of ironwood tree (Casuarina equisetifolia) decline in Guam

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