gyrB Sequences Research Articles

First Report of a New Leaf Spot Disease Caused by a Xanthomonas sp. on Hamelia patens

HomePlant DiseaseVol. 103, No. 2First Report of a New Leaf Spot Disease Caused by a Xanthomonas sp. on Hamelia patens PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a New Leaf Spot Disease Caused by a Xanthomonas sp. on Hamelia patensC. Conner, C. Marble, A. Khan, and D. J. NormanC. ConnerSearch for more papers by this author, C. MarbleSearch for more papers by this author, A. KhanSearch for more papers by this author, and D. J. Norman†Corresponding author: D. J. Norman; E-mail: E-mail Address: djn@ufl.eduhttp://orcid.org/0000-0002-1373-9416Search for more papers by this authorAffiliationsAuthors and Affiliations C. Conner C. Marble A. Khan D. J. Norman † , UF-IFAS, Mid-Florida Research and Education Center, Apopka, 32703. Published Online:13 Dec 2018https://doi.org/10.1094/PDIS-01-18-0122-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Lime Sizzler firebush (Hamelia patens Grelmsiz) is an ornamental landscape shrub that is cultivated in USDA hardiness zones 8 to 11 (Polanco 2015). It is popular because of its lime-colored foliage and its red-orange flowers. In the summer of 2016, dark necrotic leaf spots were observed on Lime Sizzler plants being produced in Florida. Isolations on nutrient agar from symptomatic tissue consistently yielded smooth, yellow, butyrous colonies of gram negative bacteria. Two strains of the bacterium, X1963 and X1964, were selected for further identification and characterization using fatty acid methyl esters analyses with the MIDI identification system (Sherlock version 6.2B, Microbial ID, Newark, DE), partial 16S rDNA, and gyrB gene sequencing. Using MIDI, the two strains had similarity indexes of 0.870 and 0.812 for Xanthomonas axonopodis pv. citrumelo and X. axonopodis pv. manihotis, respectively. For partial 16S rRNA and gyrB sequencing, we used primers and amplification protocols recommended by Ah-You et al. (2009). For both strains, sequences of 16S rRNA were identical. Similarly, sequences of gyrB gene were also identical for both strains. When using 16S rRNA and gyrB sequences in the National Center of Biotechnology Information (NCBI) BLAST of the nucleotide collection/nonredundant (nt/nr) database, 99% identity with a number of Xanthomonas spp. was found. The 16S rRNA and gyrB sequences were deposited in the NCBI nucleotide database under accession numbers MH591441 and MH681992, respectively. To satisfy Koch’s postulates, the two strains were inoculated on 10 plants each of three Hamelia species: H. patens Grelmsiz (Lime Sizzler firebush), nonnative H. cuprea, dwarf firebush, and native H. patens var. patens (Ahmad et al. 2012). Plants were approximately 15 cm in height with 15 to 25 leaves per plant. Inoculations were done in a greenhouse with temperatures between 21 and 32°C and light levels between 9,688 and 12,917 lm/m2. Suspensions of each strain were sprayed on plants to run-off at a concentration of 1 × 108 CFU/ml. Plants were bagged for 24 h to raise humidity to aid infection. Within 2 weeks, leaf spots developed on all three Hamelia varieties. The same characteristic leaf spots yielded the same type of colony as the one originally inoculated and the identical 16S rRNA sequence. Inoculation experiments were repeated with the same results. To our knowledge, this is the first report of bacterial leaf spot on H. patens caused by Xanthomonas sp.

Development of a New Semi-Selective Lysine-Ornithine-Mannitol-Arginine-Charcoal Medium for the Isolation of Pantoea Species from Environmental Sources in Japan.

Although Pantoea species are widely distributed among plants, water, soils, humans, and animals, due to a lack of efficient isolation methods, the clonality of Pantoea species is poorly characterized. Therefore, we developed a new semi-selective medium designated ‘lysine-ornithine-mannitol-arginine-charcoal’ (LOMAC) to isolate these species. In an inclusive and exclusive study examining 94 bacterial strains, all Pantoea strains exhibited yellow colonies on LOMAC medium. The performance of the medium was assessed using Pantoea-spiked soils. Percent average agreement relative to the Api20E biochemical test was 97%. A total of 24 soil spot samples and 19 plant types were subjected to practical trials. Of the 91 yellow colonies selected on LOMAC medium, 81 were correctly identified as Pantoea species using the biochemical test. The sequencing of 16S rRNA (rrs) and gyrB from these isolates confirmed that Pantoea agglomerans, P. vagans, P. ananatis, and P. deleyi were present in Japanese fields. A phylogenetic analysis using rrs enabled only the limited separation of strains within each Pantoea spp., whereas an analysis using gyrB revealed higher variability and enabled the finer resolution of distinct branches. P. agglomerans isolates were divided into 3 groups, 2 of which were new clades, with the other comprising a large group including biocontrol strains. P. vagans was also in one of the new clades. The present results indicate that LOMAC medium is useful for screening Pantoea species. The use of LOMAC medium will provide new opportunities for identifying the beneficial properties of Japanese Pantoea isolates.

Influence of Environment and Host Plant Genotype on the Structure and Diversity of the Brassica napus Seed Microbiota

Seeds are involved in the transmission of microorganisms from one plant generation to the next and consequently act as reservoirs for the plant microbiota. The driving processes influencing seed microbiota assemblage have not been yet deciphered because of confounding factors related to environmental location, agricultural practices, and host genotype selection. Nine genotypes were chosen among a large panel of genetically diverse Brassica napus accessions. The taxonomic structure of the seed microbiota was monitored by amplification and subsequent high-throughput sequencing of gyrB and internal transcribed spacer 1 markers for two successive years on seed lots collected from self-pollinated plants. Seed germination capacities were compared between all seed lots. Although harvesting year was the main driver of seed microbiota composition, the host genotype also significantly altered the structure of seed microbial assemblages. The core microbiota of B. napus included nine fungal taxa shared between all the genotypes and years, while no bacterial taxa were conserved across all genotypes and years. The harvesting year had the major effect on seed germination but with some differences between genotypes. The study demonstrated the relative contribution of host- and environmental-filtering on the assemblage of the seed microbiota. It suggested some influence of these assemblages on seed germination.

RpoB Targeted Loop-Mediated Isothermal Amplification (LAMP) Assay for Consensus Detection of Mycobacteria Associated With Pulmonary Infections.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid method which has been used to identify mycobacteria including Mycobacterium tuberculosis in clinical microbiology laboratory and point of care settings. Previously published LAMP protocols for detection of mycobacterial species used conventional specific primer and targeted the 16S rRNA, gyrB, and insertion sequence genes. We developed and evaluated a LAMP assay targeting a mycobacterial rpoB gene conserved sequence and incorporating degenerate primers. This assay allowed consensus detection of mycobacterial species from pure culture, clinical respiratory tract samples, and mycobacteria growth indicator tube (MGIT) liquid-based culture medium. A panel of twenty mycobacterial species were successfully detected at detection thresholds of 102 CFU/mL and 103 CFU/mL when respectively performed on pure culture suspension or sputum and MGIT broth. The inclusion of degenerate bases in LAMP primers increased the diversity of mycobacterial species identified by the assay without negatively affecting analytical sensitivity. LAMP-based consensus detection of multiple pathogens can be achieved with degenerate primers therefore allowing the design of rapid multi-disease screening assays. Despite high analytical sensitivity, species specificity and the advantageous operational characteristics of LAMP over PCR, challenges such as potential ambiguity in visual interpretation of results and occasional non-specific amplification precludes the implementation of novel LAMP assay in routine diagnostics both in centralized and point-of-care laboratory.

2009. Misidentification Rate of Acinetobacter baumannii Isolated From Invasive Infections in Children

Background Acinetobacter baumannii (AB) invasive infections are known to have a worse clinical outcome than non-baumannii Acinetobacter infections. However, currently, phenotypic identification by semi-automated commercial identification systems struggle to distinguish Acinetobacter subspecies; especially the four closely related subspecies of the Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex. The purpose of this study was to examine the rate of misidentification of AB isolated from invasive infections in children.MethodsFrom January 2001 to December 2017, patients 18 years old and below who were treated for invasive AB infections at Seoul National University Hospital and Chungnam National University Hospital were included. Acinetobacter baumannii, identified by commercial identification systems, cultured from sterile body fluids of the study participants were prospectively collected. The DNA from the stored bacteria were isolated, and subspecies identification was carried out by PCR and sequencing of the partial gyrB gene. Clinical data were retrospectively reviewed.ResultsDuring the 17-year study period, 113 AB isolates were obtained from patients treated for invasive infections. The median age of the patients was 2 (IQR 0–7) years old and 47 (49.5%) were male. Duplicate isolates were eliminated, and a total 95 isolates underwent further investigation. The isolates were retrieved from the blood (n = 82), peritoneal fluid (n = 8), pleural fluid (n = 2), cerebrospinal fluid (n = 2), and bronchoalveolar fluid (n = 1). Of the AB isolates identified by the commercial identification systems, 55 (57.9%) were AB. Of the non-AB isolates identified by partial gyrB sequencing, 22 (23.2%) were identified as A. nosocomialis, 8 (8.4%) as A. pittii, and 1 (1.1%) as A. calcoaceticus. Non-ACB complex subspecies included A. soli (n = 3), A. seifertii (n = 3), A. iwoffii (n = 1), A. bereziniae (n = 1), and A. junnii (n = 1).ConclusionThere was a high rate of misidentification of the Acinetobacter subspecies causing invasive infections in children. Further studies are needed to analyze the burden that misidentification has on the treatment and outcome of patients with invasive infections.Disclosures H. Lee, Korean Society of Pediatric Infectious Diseases: Member, Research grant.

Ignatzschineria cameli sp. nov., isolated from necrotic foot tissue of dromedaries (Camelus dromedarius) and associated maggots (Wohlfahrtia species) in Dubai.

Five bacterial strains, UAE-HKU57T, UAE-HKU58, UAE-HKU59, UAE-HKU60 and UAE-HKU61, were isolated in Dubai, UAE, from necrotic foot tissue samples of four dromedaries (Camelus dromedarius) and associated maggots (Wohrlfartia species). They were non-sporulating, Gram-negative, non-motile bacilli. They grew well under aerobic conditions at 37 °C, but not anaerobically. The pH range for growth was pH 7.0-9.0 (optimum, pH 7.5-8.0) and the strains could tolerate NaCl concentrations (w/v) up to 2 % (optimum, 0.5 %). They were catalase- and cytochrome oxidase-positive, but caseinase-, gelatinase- and urease-negative. Their phenotypic characters were distinguishable from other closely related species. Phylogenetic analyses of the almost-complete 16S rRNA gene and partial 23S rRNA gene, gyrB, groEL and recA sequences revealed that the five isolates were most closely related to undescribed Ignatzschineria strain F8392 and Ignatzschineria indica, but in most phylogenies clustered separately from these close relatives. Average nucleotide identity analysis showed that genomes of the five isolates (2.47-2.52 Mb, G+C content 41.71-41.86 mol%) were 98.00-99.97% similar to each other, but ≤87.18 % similar to other Ignatzschineriaspecies/strains. Low DNA relatedness between the five isolates to other Ignatzschineriaspecies/strains was also supported by Genome-to-Genome Distance Calculator analysis. The chemotaxonomic traits of the five strains were highly similar. They were non-susceptible (intermediate or resistant) to tetracycline and resistant to trimethoprim/sulphamethoxazole. The name Ignatzschineria cameli sp. nov. is proposed to accommodate these five strains, with strain UAE-HKU57T (=CCOS1165T=NBRC 113042T) as the type strain.

Marinobacter profundi sp. nov., a slightly halophilic bacterium isolated from a deep-sea sediment sample of the New Britain Trench

A piezotolerant, cold-adapted, slightly halophilic bacterium, designated strain PWS21T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. Cells were observed to be Gram-stain negative, rod-shaped, oxidase- and catalase-positive. Growth of the strain was observed at 4-45°C (optimum 37°C), at pH 5.0-9.0 (optimum 7.0) and in 0.5-20% (w/v) NaCl (optimum 3-4%). The optimum pressure for growth was 0.1MPa (megapascal) with tolerance up to 70MPa. 16S rRNA gene sequence analysis showed that strain PWS21T is closely related to Marinobacter guineae M3BT (98.4%) and Marinobacter lipolyticus SM19T (98.2%). Multilocus sequence analysis (MLSA) based on sequences of housekeeping genes gyrB, recA, atpD, rpoB and rpoD indicates that strain PWS21T represents a distinct evolutionary lineage within the genus Marinobacter. Furthermore, strain PWS21T showed low ANI and diDDH values to the closely related species. The principal fatty acids were identified as C12:0, C12:0 3-OH, C16:1ω9c, C16:0 and C18:1ω9c. Ubiquinone-9 was identified as the major respiratory quinone. The polar lipids were identified as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), two unidentified lipids and an unidentified phospholipid (PL). The G + C content of the genomic DNA was determined to be 60.3mol%. On the basis of phenotypic, chemotaxonomic and molecular data, we conclude that strain PWS21T represents a novel species of the genus Marinobacter, for which the name Marinobacter profundi sp. nov. is proposed (type strain PWS21T = KCTC 52990T = MCCC 1K03345T).

Description of a novel species of fast growing mycobacterium: Mycobacterium kyogaense sp. nov., a scotochromogenic strain received as Mycobacterium vaccae.

A polyphasic study was undertaken to determine the taxonomic status of a rapidly growing, scotochromogenic organism that had been received as Mycobacterium vaccaeNCTC 11659T. The organism was found to have chemotaxonomic and cultural properties in accord with its assignment to the genus Mycobacteriumand was distinguished from the type strain of Mycobacterium vaccaeand from other closely related reference strains on the basis of concatenated sequences of 16S rRNA, gyrB, hsp65, recA and rpoB genes. It was also distinguished from M. vaccaestrain DSM 43292T and from the type strain of Mycobacterium obuense, its nearest phylogenetic neighbour, on the basis of chemotaxonomic and phenotypic data and digital DNA -DNA relatedness values of 22.7 and 68.3 %, respectively. These datasets not only indicate that strain NCTC 11659T had been misclassified as M. vaccae but that it merits recognition as representing a novel species of the genus Mycobacterium. It is proposed that the organism be classified as Mycobacteriumkyogaense sp. nov.

Pantoea dispersa causing bulb decay of onion in Taiwan

Onion (Allium cepa L.) bulbs showing symptoms similar to those caused by slippery skin disease were sampled during a market survey in Chiayi city, Taiwan during 2017–2018. Interestingly, a bacterium which phenotypically differed from Burkholderia was isolated along with the slippery skin pathogen from the rotted scales. The purified isolates were initially identified as Pantoea dispersa by the phenotypic and biochemical characterization. Further molecular identification based on the 16S rDNA sequence analysis and multilocus sequence analysis with the atpD, gyrB, and infB sequences confirmed that the isolates are P. dispersa. For pathogenicity assay, surface-sterilized onion bulbs were injected with the isolates, and P. dispersa type strain LMG 2603T was included as a reference strain. Upon artificial inoculation of onion bulbs, all P. dispersa isolates and the strain LMG 2603T induced decays of the internal fleshy scales and yellow to tan discoloration similar to those caused by onion pathogenic Pantoea spp. The same bacterium was consistently re-isolated from the artificially inoculated onion bulbs, fulfilling Koch’s postulates. Moreover, a single injection of a P. dispersa suspension at a concentration of 1 × 104 CFU/mL was sufficient to cause disease. Accordingly, this study indicates, for the first time, that P. dispersa is an onion pathogen causing bulb decay in Taiwan.

Natural occurrence of edwardsiellosis caused by Edwardsiella ictaluri in farmed hybrid red tilapia (Oreochromis sp.) in Southeast Asia

Infections caused by Edwardsiella ictaluri bacteria have mainly affected the catfish aquaculture industry in Asia and America, while few reports reveal E. ictaluri-associated mortality in non-catfish species. Here, we report a natural occurrence of E. ictaluri in hybrid red tilapia juveniles farmed in a floating cage system in Southeast Asia. Cumulative mortality reached 40–50% within the first month after stocking. Diseased fish exhibited the presence of numerous white spots in the swollen spleen and head kidney. Pure cultures of pinpoint bacterial colonies were recovered from internal organs of all naturally diseased fish (n = 10) and subsequently four representative isolates were chosen for further identification and analyses. The bacteria were later confirmed as E. ictaluri based on biochemical characteristics, specific PCR for both genus and species levels, partial 16S rRNA and gyrB sequencing. Artificial infections using Nile tilapia juveniles produced Edwardsiellosis with typical signs of visceral white spots as observed in naturally diseased fish. Discovery of E. ictaluri infection in farmed red tilapia adds to the growing list of emerging pathogens in tilapia aquaculture in the region of which better awareness needs to be made.

Pseudopontivivens aestuariicola gen. nov., sp. nov., a Novel Bacterium of the Class Alphaproteobacteria Isolated from a Tidal Flat

A Gram-stain-negative, non-motile and coccoid, ovoid or rod-shaped bacterial strain, OITF-57T, which was isolated from a tidal flat sediment in South Korea, was characterized taxonomically. Strain OITF-57T grew optimally at 25°C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Strain OITF-57T exhibited the highest 16S rRNA gene sequence similarity value (94.2%) to the type strain of Pontivivens insulae forming a cluster in the neighbour-joining phylogenetic tree. In the maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences and the phylogenetic tress based on gyrB sequences, strain OITF-57T formed evolutionary lineages independent of those of other taxa. Strain OITF-57T contained Q-10 as the predominant ubiquinone and C18:1 ω7c as the major fatty acid. The major polar lipids of strain OITF-57T were phosphatidylcholine and phosphatidylglycerol. The DNA G + C content of strain OITF-57T was 66.0mol%. The chemotaxonomic data and other differential phenotypic properties made it possible to distinguish strain OITF-57T from the genus Pontivivens and other phylogenetically related genera. On the basis of the data presented, strain OITF-57T constitutes a new genus and species within the class Alphaproteobacteria, for which the name Pseudopontivivens aestuariicola gen. nov., sp. nov. is proposed. The type strain is OITF-57T (= KACC 19570T = CGMCC 1.13481T).

Occurrence, virulence factors, and antimicrobial susceptibility patterns of Vibrio spp. isolated from live oyster (Crassostrea gigas) in Korea

AbstractThis study determined the incidence, virulence factors, and antimicrobial susceptibility patterns of Vibrio spp. in live oysters (Crassostrea gigas) marketed in Korea. A total of 41 Vibrio spp. isolates including 23 Vibrio alginolyticus, 11 Vibrio fluvialis and 7 Vibrio antiquarius were isolated and identified. All Vibrio spp. isolates showed DNase, amylase, lipase, phospholipase, and gelatinase activities. For slime production, caseinase and β‐hemolysis isolates were positive in 68%, 78%, and 56%, respectively. PCR‐based assays showed that V. alginolyticus expressed collagenase, tlh, toxR, ctxAB, and VPI virulence genes in 100%, 100%, 78%, 39%, and 48% of the isolates, respectively. All V. fluvialis strains harbored hupO gene while 64% and 55% isolates were positive for toxR and vfh. Besides, V. antiquarius strains were positive for toxR, tlh, ctxAB, and VPI virulence genes in 57%, 86%, 43%, and 43% of the isolates, respectively. Multiple Antibiotic Resistance (MAR) index calculated with disk diffusion test results revealed that 54% of the isolates were having MAR indices >0.2. Our results reveal that consumption of raw oysters may cause potential risk for public health owing to the Vibrio spp. comprising putative virulence properties and antimicrobial resistance.Practical applicationsThe genus Vibrio are most commonly associated with seafood‐borne diseases. Oysters are one of the most preferred seafood in Korea as well as throughout the world. However, it may harbor pathogenic Vibrio spp., can cause foodborne illnesses if consumed as raw or undercooked product. In this study, the prevalence and characteristics of Vibrio spp. isolated from oysters in Korean markets was investigated. Among a total of 41 isolates presumptively identified by biochemical tests as vibrios, the incidence of three Vibrio species was confirmed by gyrB sequencing. Most of the isolates showed positive results for pathogenicity tests and virulence gene tests indicating the putative virulence of Vibrio spp. Susceptibility for 14 antimicrobials were tested using disk diffusion method. All the isolates were resistant to ampicillin while resistance to some other antimicrobials were also observed. According to these results, it is worthwhile to raise consumer awareness that consumption of oysters from markets in Korea may pose a risk of Vibrio infection.

Identification and characterisation of short chain rhamnolipid production in a previously uninvestigated, non-pathogenic marine pseudomonad

This study aimed to identify and characterise biosurfactant compounds produced by bacteria associated with a marine eukaryotic phytoplankton bloom. One strain, designated MCTG214(3b1), was isolated by enrichment with polycyclic aromatic hydrocarbons and based on 16S rDNA, and gyrB sequencing was found to belong to the genus Pseudomonas, however not related to P. aeruginosa. Cell-free supernatant samples of strain MCTG214(3b1) at stationary phase showed significant reductions in surface tension. HPLC-MS and NMR analysis of these samples indicated the presence of five different rhamnolipid (RL) congeners. Di-rhamnolipids accounted for 87% relative abundance and all congeners possessed fatty acid moieties consisting of 8–12 carbons. PCR screening of strain MCTG214(3b1) DNA revealed homologues to the P. aeruginosa RL synthesis genes rhlA and rhlB; however, no rhlC homologue was identified. Using the Galleria mellonella larvae model, strain MCTG214(3b1) was demonstrated to be far less pathogenic than P. aeruginosa. This study identifies for the first time a significantly high level of synthesis of short chain di-rhamnolipids by a non-pathogenic marine Pseudomonas species. We postulate that RL synthesis in Pseudomonas sp. MCTG214(3b1) is carried out by enzymes expressed from rhlA/B homologues similar to those of P. aeruginosa; however, a lack of rhlC potentially indicates the presence of a second novel rhamnosyltransferase responsible for the di-rhamnolipid congeners identified by HPLC-MS.

A Novel Oligonucleotide Pair for Genotyping Members of the Pseudomonas Genus by Single-Round PCR Amplification of the gyrB Gene

Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.

Lelliottia aquatilis sp. nov., isolated from drinking water.

Five beige-pigmented, oxidase-negative bacterial isolates, 6331-17T, 6332-17, 6333-17, 6334-17 and 9827-07, isolated either from a drinking water storage reservoir or drinking water in 2006 and 2017 in Germany, were examined in detail applying by a polyphasic taxonomic approach. Cells of the isolates were rod-shaped and Gram-stain-negative. Comparison of the 16S rRNA gene sequences of these five isolates showed highest sequence similarities to Lelliottia amnigena (99.98 %) and Lelliottia nimipressuralis (99.99 %). Multilocus sequence analyses based on concatenated partial rpoB, gyrB, infB and atpD sequences confirmed the clustering of these isolates with Lelliottia species, but also revealed a clear distinction to the closest related type strains. Analysis of the genome sequences of these isolates indicated >70 % in silico DNA-DNA hybridization and high average nucleotide identities between strains. Nevertheless, they showed only <70 and <95 % similarity to the type strains of these two Lelliottia species. The fatty acid profiles of these isolates were very similar and consisted of the major fatty acids C16:0, C17 : 0cyclo, C15 : 0iso 2-OH/C16 : 1ω7c and C18 : 1ω7c. In addition, physiological/biochemical tests revealed high phenotypic similarity to each other. These cumulative data indicate that these isolates represent a novel Lelliottia species, for which the name Lelliottia aquatilis sp. nov. is proposed, with strain 6331-17T (=CCM 8846T=CIP 111609T=LMG 30560T) as the type strain.

Massilia armeniaca sp. nov., isolated from desert soil.

A Gram-stain-negative, aerobic, motile and rod-shaped bacterium, strain ZMN-3T, was isolated from desert soil sample collected from Ongniod Qi, Inner Mongolia, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZMN-3T was affiliated with the genus Massilia and showed the highest similarity to Massilia humi THG S6-8T (98.9 %) and Massilia buxea A9T (98.2 %). In partial gyrB and lepA sequences, the highest similarity of strain ZMN-3T and M. humi THG S6-8T were 95.9 and 96.8 %, respectively. The DNA-DNA hybridization value between strain ZMN-3T and its closely related type strains were all below 70 %. The major respiratory quinone of strain ZMN-3T was Q-8 and the major cellular fatty acids consisted of summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The predominant polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. The DNA G+C content of strain ZMN-3T was 66.3 mol%. On the basis of this polyphasic taxonomic study, strain ZMN-3T is considered to represent a novel species of the genus Massilia, for which the name Massilia armeniaca sp. nov. is proposed. The type strain is ZMN-3T (=CGMCC 1.16209T=DSM 104676T).

Occurrence, identification, and bactericide sensitivity of Pantoea ananatis causing leaf blight on welsh onion in Taiwan

Leaves of Welsh onion (Allium fistulosum) showing blight and elongated lesions were observed and sampled in central west Taiwan in 2016, from which a bacterium phenotypically similar to Pantoea spp. was isolated. The results of molecular identification based on 16S rDNA and gyrB sequences indicate that the Welsh onion isolates are Pantoea ananatis. Upon artificial inoculation of Welsh onion leaves, P. ananatis isolates induced symptoms indistinguishable from those observed on the naturally infected plants. The bacterium was consistently re-isolated from inoculated leaves, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of P. ananatis causing leaf blight of Welsh onion in Taiwan. The Welsh onion-pathogenic P. ananatis isolates showed copper resistance, implying that copper-based bactericides may not provide effective protection of Welsh onion against P. ananatis.

Description of New and Amended Clades of the Genus Photobacterium.

Phylogenetic relationships between species in the genus Photobacterium have been poorly studied despite pathogenic and ecological relevance of some of its members. This is the first phylogenetic study that includes new species of Photobacterium (validated or not) that have not been included in any of the previously described clades, using 16S rRNA sequences and multilocus sequence analysis (MLSA) in concatenated sequences of gyrB, gapA, topA, ftsZ and mreB housekeeping genes. Sequence analysis has been implemented using Maximum-parsimony (MP), Neighbour-joining (NJ) and Maximum likelihood (ML) treeing methods and the predicted evolutionary relationship between the Photobacterium clades was established on the basis of bootstrap values of >75% for 16S rRNA sequences and MLSA. We have grouped 22 species of the genus Photobacterium into the following 5 clades: Phosphoreum (comprises P. aquimaris, “P. carnosum,” P. iliopiscarium, P. kishitanii, P. phosphoreum, “P. piscicola” and “P. toruni”); clade Profundum (composed of P. aestuarii, P. alginatilyticum, P. frigidiphilum, P. indicum, P. jeanii, P. lipolyticum, “P. marinum,” and P. profundum); clade Damselae (two subspecies of P. damselae, damselae and piscicida); and two new clades: clade Ganghwense (includes P. aphoticum, P. aquae, P. galatheae, P. ganghwense, P. halotolerans, P. panuliri and P. proteolyticum); and clade Leiognathi (composed by P. angustum, P. leiognathi subsp. leiognathi and “P. leiognathi subsp. mandapamensis”). Two additional clades, Rosenbergii and Swingsii, were formed using a phylogenetic method based on 16S rRNA gene, although they are not confirmed by any MLSA methods. Only P. aplysiae could not be included in none of the established clade, constituting an orphan clade.

Serratia marcescens associated with squash leaf chlorosis and necrotic spots in Iran

In autumn 2011, a survey was conducted for the identification of plant pathogenic bacteria affecting Cucurbitaceae grown in southwestern Iran. Squash (Cucurbita pepo var. styriaca) plants with suspected bacterial symptoms, i.e. necrotic spots surrounded by chlorotic haloes, were collected and brought to the laboratory for further analysis. Two Gram-negative, red-pigmented bacterial strains were isolated from squash leaves in Shushtar county, Khuzestan province. The isolates induced hypersensitive reaction (HR) on tobacco plants 24 h post-inoculation. A pathogenicity test on 25-day-old squash plants yielded symptoms consisting of necrotic spots surrounded by chlorotic haloes that appeared 5–10 days post-inoculation. The causal agent was identified as Serratia marcescens based on biochemical and phylogenetic analyses. Phylogenetic analysis of 16S rRNA, gyrB and groE genes sequences showed that the isolates have 99–100% similarity with S. marcescens strains previously described as pathogenic to bell pepper and corn plants. Although non-pigmented strains of S. marcescens have previously been described as the causal agents of cucurbit yellow vine disease, this is the first report of a HR-inducing red-pigmented strain of the bacterium associated with squash leaf chlorosis and necrosis.

Biological conversion of aromatic monolignol compounds by a Pseudomonas isolate from sediments of the Baltic Sea

Bacterial strains were isolated from the sediments of the Baltic Sea using ferulic acid, guaiacol or a lignin-rich softwood waste stream as substrate. In total nine isolates were obtained, five on ferulic acid, two on guaiacol and two on a lignin-rich softwood stream as a carbon source. Three of the isolates were found to be Pseudomonas sp. based on 16S rRNA sequencing. Among them, isolate 9.1, which showed the fastest growth in defined M9 medium, was tentatively identified as a Pseudomonas deceptionensis strain based on the gyrB sequencing. The growth of isolate 9.1 was further examined on six selected lignin model compounds (ferulate, p-coumarate, benzoate, syringate, vanillin and guaiacol) from different upper funneling aromatic pathways and was found able to grow on four out of these six compounds. No growth was detected on syringate and guaiacol. The highest specific growth and uptake rates were observed for benzoate (0.3 h−1 and 4.2 mmol gCDW−1 h−1) whereas the lowest were for the compounds from the coniferyl branch. Interestingly, several pathway intermediates were excreted during batch growth. Vanillyl alcohol was found to be excreted during growth on vanillin. Several other intermediates like cis,cis-muconate, catechol, vanillate and 4-hydroxybenzoate from the known bacterial catabolic pathways were excreted during growth on the model compounds.