Diogo Neves Proença

Diversity of Bacteria Associated with Bursaphelenchus xylophilus and Other Nematodes Isolated from Pinus pinaster Trees with Pine Wilt Disease

The pinewood nematode (PWN), Bursaphelenchus xylophilus, has been thought to be the only causal agent of pine wilt disease (PWD), however, since bacteria have been suggested to play a role in PWD, it is important to know the diversity of the microbial community associated to it. This study aimed to assess the microbial community associated with B. xylophilus and with other nematodes isolated from pine trees, Pinus pinaster, with PWD from three different affected forest areas in Portugal. One hundred and twenty three bacteria strains were isolated from PWN and other nematodes collected from 14 P. pinaster. The bacteria strains were identified by comparative analysis of the 16S rRNA gene partial sequence. All except one Gram-positive strain (Actinobacteria) belonged to the Gram-negative Beta and Gammaproteobacteria. Most isolates belonged to the genus Pseudomonas, Burkholderia and to the family Enterobacteriaceae. Species isolated in higher percentage were Pseudomonas lutea, Yersinia intermedia and Burkholderia tuberum. The major bacterial population associated to the nematodes differed according to the forest area and none of the isolated bacterial species was found in all different forest areas. For each of the sampled areas, 60 to 100% of the isolates produced siderophores and at least 40% produced lipases. The ability to produce siderophores and lipases by most isolates enables these bacteria to have a role in plant physiological response. This research showed a high diversity of the microbial community associated with B. xylophilus and other nematodes isolated from P. pinaster with PWD.

Chitinophaga costaii sp. nov., an endophyte of Pinus pinaster, and emended description of Chitinophaga niabensis.

Bacterial strain A37T2(T) was isolated from the endophytic microbial community of a Pinus pinaster tree trunk and characterized. Strain A37T2(T) was Gram-stain-negative, formed rod-shaped cells, and grew optimally at 26-30 °C and at pH 5.5-7.5. The G+C content of the DNA was 46.6 mol%. The major respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids were C16 : 1ω5c and iso-C15 : 0, representing 61.7 % of the total fatty acids. The polar lipids consisted of phosphatidylethanolamine, four unidentified aminophospholipids, one unidentified phospholipid, two unidentified aminolipids and three unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain A37T2(T) belonged to the family Chitinophagaceae, forming a distinct branch with Chitinophaga niabensis JS13-10(T) within the genus Chitinophaga. Strain A37T2(T) shared between 92.7 and 95.1 % 16S rRNA gene sequence similarity with the type strains of species of the genus Chitinophaga. The phylogenetic, phenotypic and chemotaxonomic data presented indicate that strain A37T2(T) represents a novel species of the genus Chitinophaga, for which the name Chitinophaga costaii sp. nov. is proposed. The type strain is A37T2(T) ( = CIP 110584(T) = LMG 27458(T)). An emended description of Chitinophaga niabensis JS13-10(T) is also proposed.

Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion.

Draft Genome Sequence of Serratia sp. Strain M24T3, Isolated from Pinewood Disease Nematode Bursaphelenchus xylophilus

Here we report the draft genome sequence of Serratia sp. strain M24T3, which is associated with pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. Serratia sp. strain M24T3 has been identified as a bionematocide for B. xylophilus in vitro, and multiple genes potentially involved in virulence and nematotoxity were identified.

Mucilaginibacter pineti sp. nov., isolated from Pinus pinaster wood from a mixed grove of pines trees

Bacterial strain M47C3B(T) was isolated from the endophytic microbial community of a Pinus pinaster tree branch from a mixed grove of pines. Phylogenetic analysis of 16S rRNA gene sequences showed that this organism represented one distinct branch within the family Sphingobacteriaceae, most closely related to the genus Mucilaginibacter. Strain M47C3B(T) formed a distinct lineage, closely related to Mucilaginibacter dorajii KACC 14556(T), with which it shared 97.2% 16S rRNA gene sequence similarity. The other members of the genus Mucilaginibacter included in the same clade were Mucilaginibacter lappiensis ATCC BAA-1855(T) sharing 97.0% similarity and Mucilaginibacter composti TR6-03(T) that had a lower similarity (95.7%). The novel strain was Gram-staining-negative, formed rod-shaped cells, grew optimally at 26 °C and at pH 7, and was able to grow with up to 0.3% (w/v) NaCl. The respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids of the strain were summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 3-OH, representing 73.5% of the total fatty acids. The major components of the polar lipid profile of strain M47C3B(T) consisted of phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. The G+C content of the DNA was 40.6 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics we propose the name Mucilaginibacter pineti sp. nov. for the novel species represented by strain M47C3B(T) ( = CIP 110632(T) = LMG 28160(T)).

Draft Genome Sequence of Pseudomonas sp. Strain M47T1, Carried by Bursaphelenchus xylophilus Isolated from Pinus pinaster

The draft genome sequence of Pseudomonas sp. strain M47T1, carried by the Bursaphelenchus xylophilus pinewood nematode, the causative agent of pine wilt disease, is presented. In Pseudomonas sp. strain M47T1, genes that make this a plant growth-promoting bacterium, as well as genes potentially involved in nematotoxicity, were identified.

Understanding pine wilt disease: roles of the pine endophytic bacteria and of the bacteria carried by the disease‐causing pinewood nematode

Pine wilt disease (PWD) is one of the most destructive diseases in trees of the genus Pinus and is responsible for environmental and economic losses around the world. The only known causal agent of the disease is the pinewood nematode (PWN) Bursaphelenchus xylophilus. Despite that, bacteria belonging to several different genera have been found associated with PWN and their roles in the development of PWD have been suggested. Molecular methodologies and the new era of genomics have revealed different perspectives to the problem, recognizing the manifold interactions between different organisms involved in the disease. Here, we reviewed the possible roles of nematode‐carried bacteria in PWD, what could be the definition of this group of microorganisms and questioned their origin as possible endophytes, discussing their relation within the endophytic community of pine trees. The diversity of the nematode‐carried bacteria and the diversity of pine tree endophytes, reported until now, is revised in detail in this review. What could signify a synergetic effect with PWN harming the plant, or what could equip bacteria with functions to control the presence of nematodes inside the tree, is outlined as two possible roles of the microbial community in the etiology of this disease. An emphasis is put on the potential revealed by the genomic data of isolated organisms in their potential activities as effective tools in PWD management.

Draft Genome Sequence of Bacillus anthracis BF-1, Isolated from Bavarian Cattle

Bacillus anthracis BF-1 was isolated from a cow in Bavaria (Germany) that had succumbed to anthrax. Here, we report the draft genome sequence of this strain, which belongs to the European B2 subclade of B. anthracis. The closest phylogenetic neighbor of strain BF-1 is a strain isolated from cattle in France.

Diversity of Bacteria Carried by Pinewood Nematode in USA and Phylogenetic Comparison with Isolates from Other Countries

Pine wilt disease (PWD) is native to North America and has spread to Asia and Europe. Lately, mutualistic relationship has been suggested between the pinewood nematode (PWN), Bursaphelenchus xylophilus the causal nematode agent of PWD, and bacteria. In countries where PWN occurs, nematodes from diseased trees were reported to carry bacteria from several genera. However no data exists for the United States. The objective of this study was to evaluate the diversity of the bacterial community carried by B. xylophilus, isolated from different Pinus spp. with PWD in Nebraska, United States. The bacteria carried by PWN belonged to Gammaproteobacteria (79.9%), Betaproteobacteria (11.7%), Bacilli (5.0%), Alphaproteobacteria (1.7%) and Flavobacteriia (1.7%). Strains from the genera Chryseobacterium and Pigmentiphaga were found associated with the nematode for the first time. These results were compared to results from similar studies conducted from other countries of three continents in order to assess the diversity of bacteria with associated with PWN. The isolates from the United States, Portugal and China belonged to 25 different genera and only strains from the genus Pseudomonas were found in nematodes from all countries. The strains from China were closely related to P. fluorescens and the strains isolated from Portugal and USA were phylogenetically related to P. mohnii and P. lutea. Nematodes from the different countries are associated with bacteria of different species, not supporting a relationship between PWN with a particular bacterial species. Moreover, the diversity of the bacteria carried by the pinewood nematode seems to be related to the geographic area and the Pinus species. The roles these bacteria play within the pine trees or when associated with the nematodes, might be independent of the presence of the nematode in the tree and only related on the bacterias relationship with the tree.

Virulence factors and infection ability of Pseudomonas aeruginosa isolates from a hydropathic facility and respiratory infections.

To compare the virulence pool and acute infection ability of Pseudomonas aeruginosa isolates from a hydropathic facility, used to treat respiratory conditions by inhalation of untreated natural mineral water, with clinical isolates from respiratory infections.