Identification and characterization of bacteria associated with decline ...

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Dec 25, 2014 - ... (Casuarina equisetifolia) in Guam. Authors; Authors and affiliations. C. M. Ayin; R. L. Schlub; J. Yasuhara-Bell; A. M. AlvarezEmail author.

Australasian Plant Pathol. (2015) 44:225–234 DOI 10.1007/s13313-014-0341-4


Identification and characterization of bacteria associated with decline of ironwood (Casuarina equisetifolia) in Guam C. M. Ayin & R. L. Schlub & J. Yasuhara-Bell & A. M. Alvarez

Received: 14 August 2014 / Accepted: 11 December 2014 / Published online: 25 December 2014 # Australasian Plant Pathology Society Inc. 2014

Abstract 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 BOXPCR 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. CoC. M. Ayin : J. Yasuhara-Bell Department of Molecular Biosciences and Bioengineering, University of Hawaii at Mānoa, 3190 Maile Way, St. John Room 315, Honolulu, HI 96822, Hawaii R. L. Schlub Cooperative Extension Service, University of Guam, Agriculture and Life Sciences, Building Room 105E, Mangilao, Guam 96923 A. M. Alvarez (*) Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Mānoa, 3190 Maile Way, St. John Room 315, Honolulu, HI 96822, Hawaii e-mail: [email protected]

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. Keywords Casuarina equisetifolia . Ironwood . Decline . Ralstonia solanacaerum . Klebsiella

Introduction Ironwood (Casuarina equisetifolia subsp. equisetifolia) has a wide natural distribution, comprising most of the Central Indo-Pacific coastline. Due to the tree’s versatility, it has been widely planted outside it natural range and is now a common feature of most tropical and warm subtropical countries. It is very fast-growing, salt tolerant, readily colonizes rocky coasts, dunes, sandbars, islets and islands, and is considered a valuable fuel in parts of Asia and Africa (Morton 1980). It is a member of Guam’s natural ecosystem (Fosberg et al. 1979; Stone 1970) and dating of soil pollen samples indicated that ironwood has grown on Guam for thousands of years (Athens and Ward 2004). In 2002, a farmer in Guam noticed that 20 of his 10-year old ironwood windbreaker trees were dead or dying (Mersha et al. 2009; Mersha et al. 2010; Schlub 2010; Schlub, Mersha et al. 2010). Five trees exhibited symptoms of wilt and 15 of decline. The wilt progressed rapidly from the base of the tree, turning branchlets yellow and giving the tips of branches a burnt appearance. Trees usually were dead within 6 months. In


C.M. Ayin et al.

contrast, trees in decline exhibited twig thinning, starting in the crown and progressing downwards, with tree death occurring after several years (Mersha et al. 2010; Schlub, Mersha et al. 2010). Inspections of other tree stands in 2002 revealed a low prevalence of wilt, whereas decline occurred at dozens of locations. By 2005, thousands of ironwood trees across Guam exhibited symptoms of decline, which preceded the death of hundreds of trees (Fig. 1a) (Mersha et al. 2009; Mersha et al. 2010; Schlub 2010; Schlub, Mersha et al. 2010). To determine potential causes of ironwood decline in Guam, various modeling techniques were applied to data collected from a set of 1,427 surveyed trees, beginning in 2009 (Schlub 2010; Schlub, Mersha et al. 2010). Factors associated with decline in 2010 were presence of the wood rotting fungus Ganoderma australe complex, termites, and poor tree practices. Numerous diseased ironwood trees showed obvious termite damage (Mersha et al. 2009); however, termites were not usually associated with the death of healthy trees, are rarely observed on young trees, and termite damage was not always observed on diseased ironwood trees. Conks of the fungus Ganoderma australe were visible on trees exhibiting decline; however, conks are not always present on diseased ironwood trees, and Ganoderma was not observed in cross sections. In addition, Ganoderma is not normally associated with the death of young trees, while many young trees in Guam are in decline. Therefore, in an attempt to improve modeling for early prediction of decline onset, unequivocal evidence for the causal agent (s) was sought. The bacterial wilt pathogen Ralstonia solanacearum was earlier disregarded as a causal agent based largely on

Fig. 1 Symptoms of ironwood tree decline in Guam. a Defoliation and dying branches. b Wetwood symptom in transverse section of trunk showing white bacterial ooze. Ralstonia solanacaerum-specific Immunostrips were positive and both R. solanacearum and Klebsiella were cultured from the ooze. c Cream-colored ooze typical of R. solanacearum infections in China (Photographed at Huian Chihu State Protection Forest Farm, Fujian, China, by He Xue-You of the Fujian Academy of Forestry)


dissimilarities between cross-sections of trees from Guam (Fig. 1b) and China (Fig. 1c); however, the bacterial pathogen regained attention when Melody Putnam discovered that ooze and tissue samples from declining trees tested positive using Ralstonia solanacearum-specific Immunostrips (Agdia, Inc.) (Schlub et al. 2011a, b). Bacterial wilt and dieback of ironwood have been reported in China (Ali et al. 1991), India (Ali et al. 1991), and Mauritius (Orian 1961). In India, young inoculated plants display yellowing that proceeded upward, eventually killing the seedling. Stem and roots of the wilted seedlings displayed bacterial ooze and browning of the vascular tissues (Ali et al. 1991). In Mauritius, young trees infected with R. solanacearum also display yellowing of twigs, followed by desiccation and plant death. However, in tall trees on Mauritius, loss of needles and branches is reported to begin at the base and proceed upward until the tree finally dies. Additionally, in Mauritius, infected young trees may be asymptomatic for many months, yet bacterial ooze consisting of R. solanacearum emerges from the woody tissues (Orian 1961). Based on strong evidence demonstrating an association of R. solanacearum with poor tree health (Ali et al. 1991; Orian 1961), an additional survey of declining ironwood in Guam was conducted in 2012 using additional diagnostic tools (Schlub et al. 2013). All bacterial ooze samples obtained from diseased ironwood trees during this survey tested positive with R. solanacearum-specific Immunostrips. The identity of bacteria was confirmed using recently developed loopmediated isothermal amplification (LAMP) primers for R. solanacearum (Kubota et al. 2008). Bacterial colonies were



Identification and characterization of bacteria associated

cultured on modified semi-selective medium of South Africa (mSMSA) (Engelbrecht 1994), and following transfer to a differential medium containing tetrazolium chloride (modified TZC) (Norman and Alvarez 1989), colonies showed typical Ralstonia morphology and pigmentation. In addition to Ralstonia, a second bacterium consistently appeared on TZC medium and overgrew the Ralstonia-like colonies in all original isolations from trees exhibiting wetwood. The second bacterium did not grow on mSMSA and was consistently isolated from tissues exhibiting wetwood symptoms. Wetwood is defined as a “bacterial condition where the heartwood, or center of a tree, is discolored and appears wet” (Hartley et al. 1961). It is not considered deleterious, unless it spreads into the sapwood, which commonly occurs in Guam’s decling trees (Fig. 1b). The objective of this study was to confirm the identity of R. solanacearum isolated from ironwood samples, and to identify any other bacteria isolated from diseased ironwood trees, through thorough characterization using complimentary approaches, including bacteriological, immunological and DNAbased tests, genetic analysis and plant pathogenicity studies.

Materials and methods

227 Table 1

Description of strains used in this study



Host and geographical regionb


Host symptomsd






Solanum lycopersicon1 Heliconia2

S-3 S-4 S-5 S-6 S-7

A6125 A6126 A6116 A6117 A6118

C. equisetifolia3 C. equisetifolia3 C. equisetifolia3 C. equisetifolia3 C. equisetifolia3


S-8 S-11 S-15 S-18 S-19 S-20 S-25

A6127 A6119 A6120 A6121 A6128 A6122 A6123

C. equisetifolia3 C. equisetifolia3 C. equisetifolia3 C. equisetifolia3 C. equisetifolia3 C. equisetifolia3 C. equisetifolia3


wilt, stem discoloration wilt, rhizome discoloration Decline, WW Decline, WW Decline, WW Decline, WW wilt and yellowing of foliage Decline, WW Decline, WW Decline, WW Decline, WW Decline, WW Decline, WW Decline, WW



PBC=Pacific Bacterial Collection; Acquisition number


1=Trinidad and Tobago, 2=Hawaii, U.S.A, 3=Guam, U.S.A


Bacterial isolation and cultural characteristics

S=stem, R=rhizome, BO=bacterial ooze from cross section of trunk, SD=saw dust, MOS=maceration of stem, MWPT=macerated wood pieces of trunk


Samples were obtained from diseased ironwood trees on Guam in December 2012 (Table 1). Bacterial isolation was performed based on colony appearance on a modified Kelman's tetrazolium chloride medium (TZC) (Norman and Alvarez 1989) following incubation for 24–48 h at 28 °C. A modified semi-selective medium developed in South Africa (mSMSA) (Engelbrecht 1994) was used to help isolate R. solanacearum colonies when TZC plates were overgrown by saprophytes. Purified bacterial colonies were transferred to sterile distilled water for long-term storage at 25 °C, and a set of cultures was stored at −80 °C in 1 %w/v tryptone, 0.5 %w/v yeast extract containing 15 %v/v glycerol. Purified strains were Gram stained according to the manufacturer’s instructions (Fisher Diagnostics, Middletown, VA). Oxidativefermentative tests were performed in 13 cm tubes containing 1 % glucose, according to a previously established protocol (Hugh and Leifson 1953). Immunostrip assays Purified bacterial strains were grown on modified TZC medium for 24–48 h at 28 °C. A full loop of an individual colony was placed into 1.5 ml Eppendorf tubes containing 300 μl of BEB1 (bacterial extraction buffer 1) buffer (R. solanacearum immunostrip test buffer) and tested according to manufacturer’s instructions (Agdia Inc., Elkhart, IN).



six-month old ironwood seedling grown for the study; inoculated with bacterial ooze from an infected tree

f isolated from tomato inoculated with bacterial ooze from which A6121 was isolated

DNA extraction and LAMP Strains were cultured overnight in 3 ml of yeast extract broth (PY) medium (0.8 %w/v peptone and 0.01 %w/v yeast extract). Broth cultures were centrifuged at 14,000×g for 10 min to pellet cells. Genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega corp., Madison, WI), following the manufacturer’s instructions. DNA was quantified with a Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Rockland, DE). Real time LAMP reactions for R. solanacearum, as well as R. solanacearum Race3Biovar2, were performed according to a previously established method (Kubota et al. 2011). BIOLOG and API identification assays Bacterial identification using the BIOLOG and API systems was performed according to the manufacturer’s instructions. Identification of strains with BIOLOG was obtained using the


BIOLOG Gen III plate, MicroStation reader and database (BIOLOG, Hayward, CA, U.S.A). Identification obtained using API 20E required access to the API online identification website (API Web, Biovar determination Purified bacterial cultures were grown on modified TZC medium for 24–48 h at 28 °C. Biovar determination, which tests for oxidation of disaccharides and utilization of hexose alcohols (Hayward 1964; He et al. 1983), was performed according to the Laboratory Guide for Identification of Plant Pathogenic Bacteria (Schaad et al. 2001). PCR and DNA sequencing Phylotype multiplex PCR reactions were performed according to methods established previously (Fegan and Prior 2005), with modifications in a total volume of 25 μl containing 1× GoTaq reaction buffer (Promega Corp., Madison, WI), 1.75 mM MgCl2, 0.2 mM of each dNTP, 100 ng Extreme Thermostable Single-Stranded DNA Binding Protein (ET SSB) (Biolabs Inc., Ipswich, MA), 0.875 U Taq polymerase (Promega Corp.), 6 pmol of primers Nmult21:1 F, Nmult21:2 F, Nmult22: InF, and Nmult22:RR, 18 pmol of Nmult23:AF, and 4 pmol of the R. solanacearum-specific primer pair 759/760 (Opina et al. 1997). Reactions were as follows: an initial denaturing at 95 °C for 5 min, followed by 30 cycles of denaturing at 95 °C for 15 s, annealing at 53 °C for 30 s, and elongation at 72 °C for 30 s, with a final elongation step of 72 °C for 10 min. PCR products were resolved using electrophoresis on a 2 % agarose gel. BOX-PCR occurred in a total volume of 26 μl, containing 5× Gitschier buffer (Kogan et al. 1987), 10 %v/v DMSO, 4 μg BSA, 1.25 μM of each dNTP, 44.4 μM BOXA1R primer (5′CTA CGG CAA GGC GAC GCT GAC G-3′), 2 U Taq polymerase, and 2 ng DNA template. BOX-PCR reaction conditions were as follows: an initial denaturation at 95 °C for 7 min, followed by 30 cycles of 94 °C for 3 min, 92 °C for 30 s, 50 °C for 1 min, and 65 °C for 8 min, with a final extension step of 65 °C for 8 min. PCR products were resolved on 1.5 % agarose. 16S rDNA PCR was carried out in a 10 μl reaction containing 5 μM of forward primer (5′-AGA GTT TGA TCC TGG CTC AG-3′), 5 μM reverse primer (5′-ACG GCT ACC TTG TTA CGA CTT-3′) and 1× Jumpstart REDTaq Ready Mix (Sigma-Aldrich, St. Louis, MO) and 1 μL of sample. PCR amplification reaction conditions were as follows: an initial denaturing at 94 °C for 7 min, followed by 30 cycles of denaturing at 94 °C for 30 s, annealing at 55 °C for 1 min, and elongation at 72 °C for 2 min, with a final elongation step of 72 °C for 5 min. PCR products were examined by 1.5 % agarose gel electrophoresis.

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RIF marker (dnaA) amplification was performed according to a previously established protocol (Schneider et al. 2011), in 50 μl reaction containing 0.05 μM of each R. solanacearum-specific primer, 25 μl RedTaq Ready Mix (Sigma-Aldrich, St. Louis, MO, U.S.A), 5 μl DNA template (1 ng/μl), and 15 μl ultrapure water. PCR amplification reaction conditions were as follows: an initial denaturing at 94 °C for 10 min, followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at 61 °C for one min, and elongation at 72 °C for 30 s, with a final extension step at 72 °C for 10 min. All 16S and dnaA PCR products were resolved by 1.5 % agarose gel electrophoresis. For DNA sequencing, excess primers and nucleotides were removed using ExoSap-It (Affymetrix Inc., Santa Clara, CA), following manufacturer’s instructions. Sequencing was performed at the Biotech Core facility, University of Hawaii at Mānoa. Nucleotide accession numbers The 16S rDNA sequences have been deposited into the GenBank Database with accession numbers KM275652 and KM275663-KM275666, for Ralstonia solanacearum strain A6121 and Klebsiella strains A6125-A6128, respectively. The dnaA sequences have been deposited into the GenBank Database with accession numbers KM275653-KM275662. For Ralstonia solanacearum strains China-IW, A3450, A6116-A6123, respectively. Sequence analysis 16S rDNA sequence assembly was performed using DNA Baser (Heracle BioSoft S.R.L., Romania). Assembled contigs were queried in the National Center for Biotechnology Information (NCBI) database using the Basic Alignment Search Tool (BLAST) (Altschul et al. 1990). RIF sequences were assembled using DNA BASER v3.5.4 (Heracle BioSoft SRL Romania, Molecular genetic analysis software (MEGA) (Tamura et al. 2011) was used to trim and align RIF sequences to those published previously (Schneider et al. 2011). A Neighbor-joining tree with bootstrap scores representing 2000 replicates was constructed in MEGA, using nucleotide substitutions and number of differences to indicate branch length. A maximum parsimony (MP) tree was constructed using the nucleotide substitution model at an MP search level of 5. Trees were rooted using an orthologous sequence from Burkholderia mallei strain ATCC 23344 (Accession: CP000010.1). Hypersensitivity (HR) assay Purified cultures of all strains isolated from samples in Guam (Table 1) were grown on TZC medium for 24–48 h at 28 °C. Bacterial suspensions of ~108 CFU/ml (OD600nm =0.1) were

Identification and characterization of bacteria associated

prepared in 30 ml of de-ionized water. Bacterial suspensions were injected directly into the intercellular spaces of tobacco leaves with a syringe. Plants were checked for HR at 24 and 48 h, following infiltration. Pathogenicity and co-inoculation studies in tomato Bacterial strains were cultured on TZC medium for 24– 48 h. Bacterial suspensions were prepared as described previously. Tomato seedlings were grown in potting mix (Sunshine Mix 4; Sun Gro Horticulture Canada Ltd, British Columbia, Canada) for approximately 2–3 weeks, until the 2–3 leaf stage. Plants were then uprooted and roots soaked in the 30 ml bacterial suspension for 1 h. The seedlings were then transplanted into individual pots containing fresh Sunshine potting mix and placed under growth lamps (Phillips F40 Agro and Sylvania GROLUX F40), with a 12 h light/dark cycle and at approximately 28 °C. Bacterial suspensions for co-inoculation studies inoculum was prepared by mixing equal volumes of suspensions containing 2 × 108 CFU/ml ( OD600nm = 0.2) of each test strain. Hawaii and Guam ironwood pathogenicity tests Bacterial suspensions (108 CFU/ml) were prepared from 48-h TZC plate cultures. Seeds of C. equisetifolia trees, collected from the University of Hawaii at Mānoa campus, were germinated on damp filter paper, in the dark, and then transplanted to community pots. Established seedlings were transplanted to 6.7 cm pots containing artificial soil (Sunshine Mix 4). Roots of 10-week old seedlings were wounded by drawing a sterile scalpel through the soil at four sides of the pot, within the root zone. Ten milliliters of the bacterial suspension was pipetted into the soil following the wounding process. Older ironwood seedlings (~4 months old), were inoculated with 50 ml of bacterial suspension, in the same manner. Ironwood seedlings were grown under the same conditions as described for tomato plants. Pathogenicity studies were also conducted on 5-month old ironwood seedlings from Guam following the same protocol. Re-isolation of bacteria from inoculated plants Stem sections of plants were surface-sterilized in 10 % Clorox (final concentration: 0.8 % sodium hypochlorite) for 30 s and then rinsed three times in sterile deionized water. Cleaned stem sections were macerated in ~200 μl of sterile deionized water and the subsequent suspension was streaked onto TZC. Presumptive identification of the reisolated bacteria was by appearance on TZC, and identity was confirmed by R. solanacearum-specific Immunostrips and LAMP.


Results Bacterial identification Two prevalent bacterial types were isolated from Guam ironwood. The first type was identified as a Gram-negative rod. Colonies were considered to be Guam R. solanacearum strains, as they were runny and white, with pink or light red centers, and opaque, irregular margins on TZC, just like both reference R. solanacearum strains (A3450, A3908), and were often only visible after 48 h. The second type of colony contained larger coccobacilli. These colonies were raised and white, with red to crimson centers and transparent margins, and smelly. Colonies were visible within 12 h and following purification were identified as Klebsiella spp. (A6125, A6126, A6127, A6123). Suspect R. solanacearum strains grew on modified SMSA and strains utilized glucose aerobically (oxidative), while suspect Klebsiella strains did not grow on SMSA and were facultative anaerobes, utilizing glucose both aerobically and anaerobically. All presumptive R. solanacearum strains gave positive reactions with R. solanacearum-specific Immunostrips, as well as the R. solanacearum-specific LAMP; however they did not react positively with the Race3Biovar2 LAMP primers. Results of carbon source utilization tests identified all Guam R. solanacearum strains as Biovar 3. Strains A3450 and A3908 are known to be Biovar 1 (Alvarez et al. 1993; Cook et al. 1989; Gabriel et al. 2006). Reference strain GMI1000 is Biovar 3 (Salanoubat et al. 2002). All suspect Klebsiella spp. strains gave negative results for both the R. solanacearum Immunostrip and LAMP. BIOLOG identified the typical Guam R. solanacearum strain (A6116) as Ralstonia solanacearum, with a 100 % probability and 72.6 % similarity. BIOLOG failed to positively identify R. solanacearum strain A3450 (tomato), but correctly identified A3908 (heliconia) and GMI1000 (tomato) as Ralstonia solanacearum. BIOLOG identified Klebsiella spp. strains A6125, A6126, A6127, and A6123 as Klebsiella oxytoca, with Klebsiella pneumoniae identification as a close second. API 20E strips identified strain A6125 as K. oxytoca with a 97.6 % identification score. The remaining Klebsiella strains (A6126, A6127, and A6123) were identified as K. pneumoniae, with a 97.4 % identification score. API 20E identification scores were achieved after confirmation with an independent urease test. Phylotype multiplex PCR showed that all Guam R. solanacearum strains, along with the R. solanacearum reference strain (GMI1000), had bands identical to phylotype I (Asia), and all tested R. solanacearum strains contained the 280 bp amplicon, which is specific to the R. solanacearum species complex. Strains A3450 (tomato) and A3908 (heliconia) were phylotype II (Americas). BOX-PCR profiles allowed visual comparisons of Guam R. solanacearum strains


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and showed little variability amongst Guam ironwood strains (Fig. 2). R. solanacearum reference strain A3450 and A3908 showed banding patterns that differed from each other, as well as the Guam ironwood R. solanacearum strains (Fig. 2, Lanes 1 and 2), while Guam ironwood R. solanacearum strains were similar to GMI1000 (Fig. 2, Lanes 3–11). Klebsiella strains from Guam ironwood displayed two distinct banding patterns that differed from the K. pneumoniae control (Fig. 3). One pattern was unique to strain A6125 (Klebsiella oxytoca) (Fig. 3, Lane 1), while the remaining strains (A6126, A6127, A6123) (Klebsiella variicola) displayed nearly identical banding patterns (Fig. 3, Lane 2–4). As BOX-PCR profiles showed that Guam R. solanacearum strains were highly similar, 16S rDNA analysis was performed on one representative strain. NCBI BLAST queries of 16S rDNA sequences obtained from Guam R. solanacearum strain A6121 resulted in the highest matches with R. solanacearum strain GMI1000 16S rDNA complete sequence (Accession: NR_074551.1) (100 % identity), R. solanacearum strain LMG 2,299 16S rDNA partial sequence (Accession: EF016361.1) (99 % identity), and R.syzygii strain ATCC

Kb 10.0 -




















Kb 10.0 -

2.0 -

1.5 -

1.2 1.0 -

0.5 -

0.4 3.0 -

2.0 -

1.5 -

Fig. 3 BOX-PCR profiles on agarose gel of Klebsiella spp. strains using the BOXA1R primer. Lane M=TriDye 2-log DNA ladder, Lane 1= A6125 (K. oxytoca), Lane 2–4=K. variicola (A6126, A6127, A6123, respectively) Lane 5=K. pneumoniae strain ATCC 13,883

1.2 1.0 -

0.5 0.4 -

Fig. 2 BOX-PCR patterns of Ralstonia solanacearum strains on gel agarose using the BOX A1R primer. Lane M=TriDye 2-log DNA ladder, Lane 1=A3450 - tomato strain; Lane 2=A3908 - heliconia strain, Lane 3 = GMI 1,000 - tomato strain, Lanes 4–11 = Guam ironwood strains

49,543 16S rDNA complete sequence (Accession: NR_040803.1) (99 % identity). The 16S rDNA sequence from Klebsiella strain A6125 resulted in the highest matches with K. oxytoca strain KCTC 1686 16S rRNA complete sequence (Accession: NR_102982.1) (99 % identity) and K. oxytoca strain ATCC 13,182 16S rDNA partial sequence (Accession: NR_119277.1) (99 % identity). The remaining Klebsiella strains (A6126, A6127 and A6123) had identical BLAST results, with highest matches to K. variicola strain F2R9 16S rDNA partial sequence (Accession: NR_025635.1) (99 % identity), K. variicola strain At-22 16S rDNA complete sequence (Accession: NR_074729.1) (99 % identity), and K. pneumoniae strain DSM 30,104 16S rDNA complete sequence (Accession: NR_117686.1) (99 % identity).

Identification and characterization of bacteria associated

Phylogenetic analysis The R. solanacearum RIF marker was successfully amplified and sequenced from all Guam ironwood R. solanacearum strains, the reference control strain (A3450), and a sample of R. solanacearum-infected ironwood from China (strain China-IW). RIF marker analysis (Fig. 4) grouped Guam R. solanacearum strains, reference strain GMI1000, and the China ironwood strain, into the same clade. R. solanacearum reference strains A3450 and A3908 clustered in a separate clade. Neighbor-joining and maximum parsimony analysis produced trees of congruent topology (data not shown).

Hypersensitivity and pathogenicity All R. solanacearum strains from Guam ironwood, except A6117, caused necrosis characteristic of HR in tobacco leaves within 24 h. In contrast, infiltration of tobacco leaves with Klebsiella spp. strains produced only mild yellowing of the Fig. 4 Maximum parsimony cladogram for Ralstonia solanacaerum using RIF marker sequences (Schneider et al. 2011). Bootstrap values shown at the node are expressed as a percentage of 2,000 replicates. Identical sequences are represented by a single strain. The outgroup Burkholderia mallei strain ATCC 23,344 (Accession: CP000010.1) was used to root the cladogram. R. solanacearum reference strains, along with R. solanacearum strains from ironwood, are boxed. Strain A6116 represents all R. solanacearum strains isolated from Ironwood in Guam


leaf panels, in only about half of the trials. In the remaining trials, infiltrated sections of tobacco leaves gave no response. Guam R. solanacearum strains, with the exception of strain A6117, as well as R. solanacearum strain A3450, wilted 2-3weeks old tomato plants at 3 dpi (days post inoculation) and eventually caused plant collapse within 7–10 day. Bacteria were successfully re-isolated from infected tomato seedlings from three separate 10 mm sections, taken at the base, middle portion, and the tip of the tomato stem, respectively. Reisolated colonies showed typical R. solanacearum morphology on TZC medium and gave positive reactions with Immunostrips and LAMP. Tomato seedlings inoculated with Klebsiella remained asymptomatic at 30 dpi. Co-inoculation of Klebsiella and R. solanacearum into tomato plants yielded the same results as compared with plants inoculated with R. solanacearum alone, suggesting that R. solanacearum alone is sufficient to cause wilt in tomatoes. R. solanacearum strains produced wilt in 10-week and 4-month old (Fig. 5b and c) ironwood seedlings. Bacteria were successfully re-isolated from diseased ironwood stems and identified as


R. solanacearum using the tests previously described. The majority of inoculated seedlings remained asymptomatic 30– 60 dpi, though stem macerations prepared from the asymptomatic ironwood seedlings 30 dpi tested positive for R. solanacearum using Immunostrips and LAMP, indicating R. solanacearum was present in the plant tissues. Attempts to re-isolate R. solanacearum from the macerated tissue obtained from asymptomatic trees proved unsuccessful, using both TZC and 0.8 % peptone 0.1 % yeast extract agar (PYEAM). However, motile cells were observed under 400× magnification of liquid from macerated tissues of asymptomatic plants, indicating the presence of live bacterial cells. All ironwood seedlings inoculated with Klebsiella strains were asymptomatic (Fig. 5e). Furthermore, re-isolation of Klebsiella from inoculated ironwood seedlings was achieved in only 2 out of 64 plants. Co-inoculation of Klebsiella and R. solanacearum into ironwood (Fig. 5d) yielded the same results as compared with plants inoculated with R. solanacearum alone, suggesting that R. solanacearum alone is sufficient to produce wilt in ironwood, and thus the role of Klebsiella remains unknown. In the case that there might be a significant difference in the susceptibility of Guam ironwood to these bacteria, in comparison to ironwood from Hawaii, pathogenicity tests were reproduced in ironwood seedlings sent from Guam. Bacterial colonies typical of R. solanacearum on TZC were obtained from 6 out of 15 Guam ironwood seedlings following inoculation with R. solanacearum strain A6116. Re-isolation attempts were unsuccessful from the remaining 9 plants, however macerated stem tissue obtained from these 9 plants tested positive with Immunostrips at sites 2 and 6 cm above the inoculation point. Macerated stems from seedlings inoculated with Klebsiella strain A6127 produced no bacterial growth on agar plates containing either TZC or PYEAM media. Results indicate that R. solanacearum alone is sufficient to produce

Fig. 5 Casuarina equisetifolia seedlings approximately 4- months old. Plants symptoms were recorded 23 day post inoculation with a dH20, b and c Ralstonia solanacearum from Guam (strain A6116), d R. solanacearum and Klebsiella (strains A6116 and A6127, respectively) and e Klebsiella (strain A6126)

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wilt in ironwood, Guam ironwood seedlings were not more susceptible to diesease than Hawaiian seedlings, and the association with Klebsiella during co-infection remains unknown.

Discussion Causative agents of ironwood decline in Guam have not been fully determined, with scientists attributing disease to various factors such as termite damage and fungal pathogens (Schlub, Mersha et al. 2010). The possibility that a bacterial pathogen contributed to Guam ironwood decline had not been considered a likely possibility based on symptoms, although there have been previous reports of bacterial wilt of ironwood (Ali et al. 1991; Orian 1961). In a 2012 survey of declining trees in Guam (Schlub et al. 2013) demonstrated an association of R. solanacearum with diseased ironwood trees. This was the first report to identify and characterize R. solanacearum and Klebsiella spp. associated with ironwood decline in Guam. Ralstonia solanacearum strains isolated in that survey were highly similar to GMI1000, based on BOX-PCR, phylotyping and biovar determination. Two different Klebsiella spp. were isolated from diseased ironwood, based on BOX-PCR and 16S rRNA analysis, with 3 of the 4 strains being K. variicola, a recently described species of Klebsiella comprised of clinical and plant-associated isolates (Rosenblueth et al. 2004), and the remaining isolate being K. oxytoca, a known pathogen causing septicemia, pneumonia and urinary tract infection in humans and sepsis, infections of the urinary and respiratory tracts, and mastitis in animals. The genus Klebsiella comprises opportunistic pathogens that are frequently isolated from humans and animals (Podschun and Ullmann 1998), so it is interesting to find this pathogen associated with wetwood in Guam ironwood. Ralstonia solanacearum strains isolated from diseased ironwood trees in Guam were capable of causing wilt and death of both tomato and ironwood seedlings. There were no significant differences in virulence observed between the Guam Ralstonia isolates and the control Ralstonia strains. Additionally, there were no differences in susceptibility between ironwood seedlings sent from Guam and those grown in Hawaii, suggesting that the association between Guam Ralstonia isolates and Guam ironwood is not specific. Although Ralstonia strains isolated from Guam ironwood caused wilt and death in ironwood seedlings, not all seedlings produced symptoms following inoculation. Nevertheless, numerous asymptomatic plants tested positive with R. solanacearum-specific Immunostrips and LAMP (the detection limit of both assays is ~105 cells/ml), and motile bacteria were observed in plant tissue macerates by light microscopy. Taken together, these findings give rise to the

Identification and characterization of bacteria associated

possibility that R. solanacearum resides within the infected plant in a viable, but non-culturable (non-reproductive) state. The viable but non-culturable (VBNC) state of bacteria occurs when bacteria are deemed viable, due to metabolic activity (Ghezzi and Steck 1999; Oliver 2000) but are unable to reproduce and form visible colonies on media that facilitate their growth, and yet, upon “resuscitation,” become culturable (Oliver 2000). This VBNC state of bacteria (Xu et al. 1982) has been shown in a number of different bacteria (Oliver 2005), and while poorly understood, is thought to be a survival mechanism of bacterial cells under stress. Both Klebsiella pneumoniae (Oliver 2005) and R. solanacearum (Grey and Steck 2001) have been observed to enter this VBNC state. The VBNC state of R. solanacearum may explain the slow progression of wilt and latent infections of C. equisetifolia by R. solanacearum that have been observed in this study and in Mauritius (Orian 1961). Klebsiella produced no symptoms on either tomato or ironwood seedlings, following inoculation through stems or roots, and co-inoculation with Klebsiella and R. solanacearum on plants of the same age showed neither an increase nor decrease in the rate of wilt symptom production. It is yet unknown how these two bacteria will behave individually or in concert in older ironwood trees. Klebsiella as the first invader may cause wetwood symptoms that allow Ralstonia to colonize mature woody tissues. Conversely, Ralstonia infection may favor establishment of Klebsiella in mature trees. The observation that many Ralstonia-infected ironwood trees in Guam also exhibited wetwood associated with Klebsiella highlights the need for a larger survey of declining ironwood and further in-depth studies of the bacterial interactions. Other enteric bacteria also were present in wetwood samples, though Klebsiella was clearly dominant in Guam. Rapid assays for specific detection of Klebsiella are needed to elucidate the significance of the association between Klebsiella and Ralstonia-infected ironwood trees in Guam. Studies are currently underway to develop and evaluate such diagnostics. Acknowledgments This work was supported in part by USDA National Institute for Food and Agriculture, Project HAW00987-H, administered by the College of Tropical Agriculture and Human Resources, University of Hawaii at Mānoa. This work was also supported in part by WPDN-201303063-01 and in part by the Guam Cooperative Extension, University of Guam, Mangilao Guam. The authors thank Zhong Chonglu at the Research Institute of Tropical Forestry, Longdong, Guangzhou, China, for providing the diseased China ironwood sample.

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