Carrie L. Brady

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.

Pantoea vagans sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyi sp. nov. and Pantoea anthophila sp. nov.

Bacteria isolated from eucalyptus leaves and shoots showing symptoms of blight and die-back collected in Uganda, Uruguay and Argentina and from maize displaying brown stalk rot symptoms in South Africa were tentatively placed in the genus Pantoea on the basis of phenotypic and biochemical tests. These isolates, together with two strains (LMG 2558 and LMG 2560) previously assigned to Pantoea agglomerans based on protein electrophoregrams but later excluded from this species, were further investigated using molecular techniques. 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) revealed that the strains were phylogenetically closely related to Pantoea agglomerans, Pantoea stewartii and Pantoea ananatis. MLSA and amplified fragment length polymorphism analysis placed the strains into four separate clusters, not containing any of the type strains of species of the genus Pantoea. DNA-DNA hybridization confirmed the classification of the isolates into four novel species, for which the names Pantoea vagans sp. nov. (type strain R-21566T=LMG 24199T=BCC 105T=BD 765T), Pantoea eucalypti sp. nov. (type strain R-25678T=LMG 24197T=BCC 076T=BD 769T), Pantoea deleyi sp. nov. (type strain R-31523T=LMG 24200T=BCC 109T=BD 767T) and Pantoea anthophila sp. nov. (type strain LMG 2558T=BD 871T=NCPPB 1682T) are proposed.

Brenneria goodwinii sp. nov., associated with acute oak decline in the UK

A group of nine Gram-negative staining, facultatively anaerobic bacterial strains isolated from native oak trees displaying symptoms of acute oak decline (AOD) in the UK were investigated using a polyphasic approach. 16S rRNA gene sequencing and phylogenetic analysis revealed that these isolates form a distinct lineage within the genus Brenneria, family Enterobacteriaceae, and are most closely related to Brenneria rubrifaciens (97.6 % sequence similarity to the type strain). Multilocus sequence analysis based on four housekeeping genes (gyrB, rpoB, infB and atpD) confirmed their position within the genus Brenneria, while DNA-DNA hybridization indicated that the isolates belong to a single taxon. The isolates can be differentiated phenotypically from their closest phylogenetic neighbours. The phylogenetic and phenotypic data demonstrate that these isolates from oak with symptoms of AOD represent a novel species in the genus Brenneria, for which the name Brenneria goodwinii sp. nov. (type strain FRB 141(T)  = R-43656(T)  = BCC 845(T)  = LMG 26270(T)  = NCPPB 4484(T)) is proposed.

Erwinia oleae sp. nov., isolated from olive knots caused by Pseudomonas savastanoi pv. savastanoi

Three endophytic bacterial isolates were obtained in Italy from olive knots caused by Pseudomonas savastanoi pv. savastanoi. Phenotypic tests in combination with 16S rRNA gene sequence analysis indicated a phylogenetic position for these isolates in the genera Erwinia or Pantoea, and revealed two other strains with highly similar 16S rRNA gene sequences (>99 %), CECT 5262 and CECT 5264, obtained in Spain from olive knots. Rep-PCR DNA fingerprinting of the five strains from olive knots with BOX, ERIC and REP primers revealed three groups of profiles that were highly similar to each other. Multilocus sequence analysis (MLSA) based on concatenated partial atpD, gyrB, infB and rpoB gene sequences indicated that the strains constituted a single novel species in the genus Erwinia. The strains showed general phenotypic characteristics typical of the genus Erwinia and whole genome DNA-DNA hybridization data confirmed that they represented a single novel species of the genus Erwinia. The strains showed DNA G+C contents ranging from 54.7 to 54.9 mol%. They could be discriminated from phylogenetically related species of the genus Erwinia by their ability to utilize potassium gluconate, l-rhamnose and d-arabitol, but not glycerol, inositol or d-sorbitol. The name Erwinia oleae sp. nov. (type strain DAPP-PG 531(T)= LMG 25322(T) = DSM 23398(T)) is proposed for this novel taxon.

Pantoea rodasii sp. nov., Pantoea rwandensis sp. nov. and Pantoea wallisii sp. nov., isolated from Eucalyptus

Several Gram-negative-staining, facultatively anaerobic bacterial isolates were obtained from Eucalyptus seedlings showing symptoms of bacterial blight and dieback in Colombia, Rwanda and South Africa. Partial 16S rRNA gene sequencing, together with partial gyrB sequencing, placed the isolates in the genus Pantoea and indicated that they constituted three novel species. Multilocus sequence analysis (MLSA) based on partial sequences of gyrB, rpoB, infB and atpD revealed Pantoea dispersa, Pantoea eucrina and Pantoea cypripedii as their closest phylogenetic relatives. DNA-DNA hybridization studies confirmed the classification of the new isolates as three novel species and phenotypic tests allowed them to be differentiated from their closest phylogenetic neighbours. The names Pantoea rodasii sp. nov. [type strain LMG 26273(T)=BD 943(T) (deposited with the Plant Pathogenic and Plant Protecting Bacteria Collection, South Africa)=BCC 581(T) (deposited with the Bacterial Culture Collection, Forestry and Agricultural Institute, South Africa)], Pantoea rwandensis sp. nov. (type strain LMG 26275(T)=BD 944(T)=BCC 571(T)) and Pantoea wallisii sp. nov. (type strain LMG 26277(T)=BD 946(T)=BCC 682(T)) are proposed.

Pantoea allii sp. nov., isolated from onion plants and seed

Eight yellow-pigmented, Gram-negative, rod-shaped, oxidase-negative, motile, facultatively anaerobic bacteria were isolated from onion seed in South Africa and from an onion plant exhibiting centre rot symptoms in the USA. The isolates were assigned to the genus Pantoea on the basis of phenotypic and biochemical tests. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), based on gyrB, rpoB, infB and atpD sequences, confirmed the allocation of the isolates to the genus Pantoea. MLSA further indicated that the isolates represented a novel species, which was phylogenetically most closely related to Pantoea ananatis and Pantoea stewartii. Amplified fragment length polymorphism analysis also placed the isolates into a cluster separate from P. ananatis and P. stewartii. Compared with type strains of species of the genus Pantoea that showed >97 % 16S rRNA gene sequence similarity with strain BD 390(T), the isolates exhibited 11-55 % whole-genome DNA-DNA relatedness, which confirmed the classification of the isolates in a novel species. The most useful phenotypic characteristics for the differentiation of the isolates from their closest phylogenetic neighbours are production of acid from amygdalin and utilization of adonitol and sorbitol. A novel species, Pantoea allii sp. nov., is proposed, with type strain BD 390(T) ( = LMG 24248(T)).

Pectinolytic bacteria associated with potato soft rot and blackleg in South Africa and Zimbabwe

Soft rot and blackleg can cause severe economic losses in potato production in South Africa and Zimbabwe depending on climatic conditions. The aim of the study was to identify the predominant bacteria causing potato soft rot and blackleg in these countries. Samples, comprising of stems and tubers from potato plants with blackleg and soft rot symptoms were collected from 2006–2009 from potato production areas where disease outbreaks occurred. The isolates from these plants and tubers yielded Gram negative, pectinolytic bacteria on crystal violet pectate and inoculated tubers. Identification was based on biochemical and phenotypic characteristics, rep-PCR, Amplified Fragment Length Polymorphisms and sequences of gyrB and recA genes. Isolates from Zimbabwe were identified as Pectobacterium carotovorum subsp. brasiliensis (Pcb) (21 isolates), Dickeya dadantii subsp. dadantii (Dd) (20 isolates), P. c. subsp. carotovorum (Pcc) (16 isolates) and P. atrosepticum (Pa) (4 isolates). Pcb, Pcc and Dd subsp. dadantii were isolated from samples collected from all the regions, while Pa was isolated from Nyanga the coolest region in Zimbabwe. In South Africa, however, Pcb was the most common causal agent of soft rot and blackleg. P. atrosepticum was the only pathogen isolated from samples collected in Nyanga, Zimbabwe, and was not isolated from any South African samples. AFLP analysis separated the Pcb strains into 12 clusters, reflecting subdivision in terms of geographic origin, and Pcc isolates were clearly differentiated from Pcb isolates. A large degree of DNA polymorphism was evident among these 12 clusters. The study identified all the pathogens associated with the blackleg/soft rot disease complex.

Isolation of Enterobacter cowanii from Eucalyptus showing symptoms of bacterial blight and dieback in Uruguay

Aims:  This study was performed to identify bacterial strains isolated simultaneously with Pantoea species from Eucalyptus trees showing symptoms of bacterial blight and dieback in Uruguay.