<Emphasis Type="Italic">Bacillus berkeleyi</Emphasis> sp. nov., isolated from the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

A bacterial strain, designated KMM 6244^T, was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244^T was Bacillus decolorationis LMG 19507^T with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA–DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507^T. Strain KMM 6244^T grew at 4–45°C and with 0–12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C_15:0, iso-C_15:0, anteiso-C_17:0, C_15:0, iso-C_16:0 and iso-C_14:0. The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244^T represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244^T (KCTC 12718^T = LMG 26357^T).     

Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.     

Pathogenicity and biotechnological applications of the genus <Emphasis Type="Italic">Burkholderia</Emphasis>

Bacteria belonging to the genus Burkholderia are well known for their adaptability to habitats as diverse as freshwater sediments, lungs of cystic fibrosis patients and plant tissues. This genus includes also plant, animal and human pathogenic species, such as Burkholderia glumae, Burkholderia pseudomallei and the Burkholderia cepacia complex. Over the past few years, several newly discovered non-pathogenic plant associated Burkholderia species have raised particular interest for their potential use in plant growth promotion, biocontrol of plant pathogens, phytoremediation and xenobiotics degradation. Highlights from recent studies on the taxonomy, ecology and pathogenicity of different species of the Burkholderia genus are presented with the aim to evaluate their potential use in biotechnology.     

Common Features of Environmental and Potentially Beneficial Plant-Associated <Emphasis Type="Italic">Burkholderia</Emphasis>

The genus Burkholderia comprises more than 60 species isolated from a wide range of niches. Although they have been shown to be diverse and ubiquitously distributed, most studies have thus far focused on the pathogenic species due to their clinical importance. However, the increasing number of recently described Burkholderia species associated with plants or with the environment has highlighted the division of the genus into two main clusters, as suggested by phylogenetical analyses. The first cluster includes human, animal, and plant pathogens, such as Burkholderia glumae, Burkholderia pseudomallei, and Burkholderia mallei, as well as the 17 defined species of the Burkholderia cepacia complex, while the other, more recently established cluster comprises more than 30 non-pathogenic species, which in most cases have been found to be associated with plants, and thus might be considered to be potentially beneficial. Several species from the latter group share characteristics that are of use when associating with plants, such as a quorum sensing system, the presence of nitrogen fixation and/or nodulation genes, and the ability to degrade aromatic compounds. This review examines the commonalities in this growing subgroup of Burkholderia species and discusses their prospective biotechnological applications.     

Effects of the Inoculation of <Emphasis Type="Italic">Burkholderia vietnamensis</Emphasis> and Related Endophytic Diazotrophic Bacteria on Grain Yield of Rice

During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some “endophytes” were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using ¹⁵N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.     

High-resolution mapping of <Emphasis Type="Italic">Rsn1</Emphasis>, a locus controlling sensitivity of rice to a necrosis-inducing phytotoxin from <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG1-IA

Rhizoctonia solani is a necrotrophic fungal pathogen that causes disease on many crop-plant species. Anastomosis group 1-IA is the causal agent of sheath blight of rice (Oryza sativa L.), one of the most important rice diseases worldwide. R. solani AG1-IA produces a necrosis-inducing phytotoxin and rice cultivar’s sensitivity to the toxin correlates with disease susceptibility. Unlike genetic analyses of sheath blight resistance where resistance loci have been reported as quantitative trait loci, phytotoxin sensitivity is inherited as a Mendelian trait that permits high-resolution mapping of the sensitivity genes. An F₂ mapping population derived from parent cultivars ‘Cypress’ (toxin sensitive) and ‘Jasmine 85’ (toxin insensitive) was used to map Rsn1, the necrosis-inducing locus. Initial mapping based on 176 F₂ progeny and 69 simple sequence repeat (SSR) markers located Rsn1 on the long arm of chromosome 7, with tight linkage to SSR marker RM418. A high-resolution genetic map of the region was subsequently developed using a total of 1,043 F₂ progeny, and Rsn1 was mapped to a 0.7 cM interval flanked by markers NM590 and RM418. Analysis of the corresponding 29 Kb genomic sequences from reference cultivars ‘Nipponbare’ and ‘93-11’ revealed the presence of four putative genes within the interval. Two are expressed cytokinin-O-glucosyltransferases, which fit an apoptotic pathway model of toxin activity, and are individually being investigated further as potential candidates for Rsn1.     

Colonization of <Emphasis Type="Italic">Morus alba</Emphasis> L. by the plant-growth-promoting and antagonistic bacterium <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> strain Lu10-1

Background  

Anthracnose, caused by Colletotrichum dematium, is a serious threat to the production and quality of mulberry leaves in susceptible varieties. Control of the disease has been a major problem in mulberry cultivation. Some strains of Burkholderia cepacia were reported to be useful antagonists of plant pests and could increase the yields of several crop plants. Although B. cepacia Lu10-1 is an endophytic bacterium obtained from mulberry leaves, it has not been deployed to control C. dematium infection in mulberry nor its colonization patterns in mulberry have been studied using GFP reporter or other reporters. The present study sought to evaluate the antifungal and plant-growth-promoting properties of strain Lu10-1, to clarify its specific localization within a mulberry plant, and to better understand its potential as a biocontrol and growth-promoting agent.     

Biological control of rice sheath blight using hyphae-associated bacteria: development of an <Emphasis Type="Italic">in planta</Emphasis> screening assay to predict biological control agent performance under field conditions

A pathogenicity assay for in planta screening of biological control agents (BCAs) towards sheath blight in rice was developed, and used to evaluate a panel of Rhizoctonia solani Kühn hyphae-associated bacteria for their ability to control sheath blight under screenhouse conditions. The best performing BCA, Burkholderia sp. strain A-7.3, was selected for field trials. Disease incidence and disease severity were significantly reduced leading to a significant grain yield increase of 7%. Furthermore, R. solani sclerotia formation and sclerotia viability were significantly reduced, thus lowering the reservoir for primary infections in the next crop cycle. The paper presents a reliable pathogenicity assay that can be used to test BCA performance under different scenarios e.g. plant variety and soil conditions, and help predict translation of results obtained under screenhouse conditions to field performance. Furthermore, it supports the hypothesis that hyphae-associated bacteria are a promising source of niche-specific BCAs towards fungal pathogens.     

<Emphasis Type="Italic">Pedobacter panacis</Emphasis> sp. nov., isolated from <Emphasis Type="Italic">Panax ginseng</Emphasis> soil

A novel strain, DCY108^T was isolated from soil of a Panax ginseng field, Yeoncheon province (38°04′N 126°57′E), Republic of Korea. Strain DCY108^T is Gram-negative, non-motile, non-flagellate, rod-shaped, and aerobic. The bacterium grows optimally at 25–30 °C, pH 6.5–7.0 and 1 % NaCl. Phylogenetically, strain DCY108^T is closely related to Pedobacter jejuensis JCM 18824^T, Pedobacter aquatilis JCM 13454^T, Pedobacter kyungheensis LMG 26577^T and the type strain of the genus Pedobacter heparinus DSM 2366^T. The DNA–DNA relatedness values between strain DCY108^T and its close phylogenetic neighbors were below 30.0 %. The DNA G+C content of strain DCY108^T was determined to be 45.1 mol%. The predominant quinone was menaquinone 7 (MK-7). The major polar lipids were identified as phosphatidylethanolamine and three unidentified aminolipids AL1, AL13 and AL17. Iso-C_15:00, iso-C_17:03OH and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) were identified as the major fatty acids present in strain DCY108^T. The results of physiological and biochemical tests allowed strain DCY108^T to be differentiated phenotypically from other recognized species belonging to the genus Pedobacter. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Pedobacter panacis sp. nov is proposed with the type strain designated as DCY108^T (=CCTCCAB 2015196^T = KCTC 42748^T).     

<Emphasis Type="Italic">Achromobacter panacis</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Panax ginseng</Emphasis>

A novel strain DCY105^T was isolated from soil collected from the rhizosphere of ginseng (Panax ginseng), in Gochang, Republic of Korea. Strain DCY105^T is Gram-reaction-negative, white, non-motile, non-flagellate, rod-shaped and aerobic. The bacteria grow optimally at 30°C, pH 6.5–7.0 and in the absence of NaCl. Phylogenetically, strain DCY105^T is most closely related to Achromobacter marplatensis LMG 26219^T (96.81%). The DNA G+C content of strain DCY105^T was 64.4 mol%. Ubiquinone 8 was the major respiratory quinone, and phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were amongst the major polar lipids. C_16:00, C_8:03OH and iso-C_17:03OH were identified as the major fatty acids present in DCY105^T. The results of physiological and biochemical tests allowed strain DCY105^T to be differentiated phenotypically from other recognized species belonging to the genus Achromobacter. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Achromobacter panacis sp. nov. is proposed with the type strain designated as DCY105^T (=CCTCCAB 2015193^T =KCTC 42751^T).     

Studies on Endophytic Colonization Ability of Two Upland Rice Endophytes, <Emphasis Type="Italic">Rhizobium</Emphasis> sp. and <Emphasis Type="Italic">Burkholderia</Emphasis> sp., Using Green Fluorescent Protein Reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized roots of upland cultivated rice viz., Rhizobium sp. and Burkholderia sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp/gusA). Confocal laser scanning microscopy (CLSM) of gnotobiotically grown seedlings of Narendradhan 97, inoculated with gfp/gusA-tagged endophytes, revealed that both Rhizobium sp. and Burkholderia sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp/gusA-tagged Rhizobium sp. was severely inhibited when co-inoculated with an equal number (10⁶ cfu ml⁻¹) of wild type Burkholderia sp. Burkholderia sp. was a more aggressive endophytic colonizer of rice than Rhizobium sp. The potential of using gfp/gusA reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is demonstrated in this study.     

Diverse mechanisms adopted by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> PGC2 during the inhibition of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Rhizoctonia solani and Phytophthora capsici are two of the most destructive phytopathogens occurring worldwide and are only partly being managed by traditional control strategies. Fluorescent Pseudomonas isolates PGC1 and PGC2 were checked for the antifungal potential against R. solani and P. capsici. Both the isolates were screened for the ability to produce a range of antifungal compounds. The results of this study indicated the role of chitinase and β-1,3-glucanase in the inhibition of R. solani, however, antifungal metabolites of a non-enzymatic nature were responsible for inhibition of P. capsici. The study confirmed that multiple and diverse mechanisms are adopted by the same antagonist to suppress different phytopathogens, as evidenced in case of R. solani and P. capsici.     

Diversity of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> Complex from the Moso Bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>) Rhizhosphere Soil

The purpose of this study was to determine the existence of Burkholderia cepacia complex (Bcc) at species level and the predominant species in the environment of moso bamboo plantations in Hangzhou, China. A total of 423 isolates were recovered from moso bamboo rhizhosphere soil samples of three sites on the selective medium during 2007–2008. Isolates were identified by Bcc-specific PCR assays, followed by recA-restriction fragment length polymorphism assays, species-specific PCR analysis, recA gene sequencing, multilocus sequence typing (MLST) scheme, and BOX-PCR fingerprinting for genomic diversity. Out of 423 isolates, 278 isolates were assigned to the following Bcc species, eight B. stabilis, 26 B. anthina, 193 B. pyrrocinia, and 51 B. arboris, which indicated B. pyrrocinia as the most dominant species followed by B. arboris. Moreover, false positives were observed in certain isolates of B. arboris while performing species-specific PCR test. Furthermore, the results of recA gene sequence similarity and MLST data demonstrated that nine isolates formed a single discrete cluster but were PCR negative to species-specific primers representing novel species may exist within the Bcc. In addition, BOX-PCR fingerprinting for all the Bcc isolates also showed the strain diversity. It is the first report of the existence of B. arboris and predominance of B. pyrrocinia in the moso bamboo environment.     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Cell-free culture medium of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> improves seed germination and seedling growth in maize (<Emphasis Type="Italic">Zea mays</Emphasis>) and rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

The saprophytic bacterium Burkholderia cepacia has been shown to play an active role as plant growth promoting bacteria (PGPB). In this study, the ability of cell-free culture medium (CFCM) of B. cepacia to improve early developmental stages of plants has been assessed on two agronomically important crops, maize (Zea mays) and rice (Oryza sativa). Treating maize and rice seeds for 45 min before germination significantly improved seed germination and consequent seedling growth. The effect of CFCM was confirmed by the increased biomass of the shoot and, mainly, the root systems of treated seedlings. Chromatographic characterization of the CFCM revealed that the spent culture medium of B. cepacia is a complex mix of different classes of metabolites including, among others, salicylic acid, indole-3-acetic acid (IAA) and several unidentified phenolic compounds. Fractionation of the CFCM components revealed that the impressive development of the root system of CFCM-treated seedlings is due to the synergistic action of several groups of components rather than IAA alone. The data presented here suggest that a CFCM of B. cepacia can be used to improve crop germination.     

<Emphasis Type="Italic">Salinarchaeum laminariae</Emphasis> gen. nov., sp. nov.: a new member of the family <Emphasis Type="Italic">Halobacteriaceae</Emphasis> isolated from salted brown alga <Emphasis Type="Italic">Laminaria</Emphasis>

Halophilic archaeal strains R26^T and R22 were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells from the two strains were pleomorphic rods and Gram negative, and colonies were red pigmented. Strains R26^T and R22 were able to grow at 20–50°C (optimum 37°C) in 1.4–5.1 M NaCl (optimum 3.1–4.3 M) at pH 5.5–9.5 (optimum pH 8.0–8.5) and neither strain required Mg²⁺ for growth. Cells lyse in distilled water and the minimum NaCl concentration required to prevent cell lysis was 8% (w/v) for strain R26^T and 12% (w/v) for strain R22. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and minor phosphatidylglycerol sulfate; glycolipids were not detected. Phylogenetic analyses based on 16S rRNA genes and rpoB′ genes revealed that strains R26^T and R22 formed a distinct clade with the closest relative, Natronoarchaeum mannanilyticum. The DNA G+C content of strains R26^T and R22 was 65.8 and 66.4 mol%, respectively. The DNA–DNA hybridization value between strains R26^T and R22 was 89%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strains R26^T and R22 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarchaeum laminariae gen. nov., sp. nov. is proposed. The type strain is R26^T (type strain R26^T = CGMCC 1.10590^T = JCM 17267^T, reference strain R22 = CGMCC 1.10589).     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.