Bacillus swezeyi sp. nov. and Bacillus haynesii sp. nov., isolated from desert soil.

Two isolates of Gram-reaction-positive, facultatively anaerobic, motile, rod-shaped, endospore-forming bacteria were identified during a survey of the diversity of strains belonging to the genus Bacillus deposited in the Agriculture Research Service Culture Collection. These strains were originally isolated from soil in Evolution Canyon III (Israel) in a survey of ecological diversification. Phylogenetic analysis of the 16S rRNA gene of strains NRRL B-41294T and NRRL B-41327T determined they were closely related to members of the Bacillus licheniformis clade. The genome of each strain was sequenced, and further analysis indicated that the strains represented unique species based on in silico DNA-DNA hybridization analyses. A phylogenomic analysis revealed that NRRL B-41294T and NRRL B-41327T were closely related to the group that includes B. licheniformis. In phenotypic characterization, both NRRL B-41294T and NRRL B-41327T were found to grow at temperatures of between 15 and 60 °C and tolerated up to 12 % NaCl (w/v). The predominant cellular fatty acids were anteiso-C15 : 0 and iso-C15 : 0, and peptidoglycan from cell walls contained meso-diaminopimelic acid. The DNA G+C content was 45.7 and 44.3 mol% for NRRL B-41327T and NRRL B-41294T, respectively. Furthermore, each strain had a unique carbon utilization pattern that distinguished it from its nearest phylogenetic neighbours. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that these strains represent two novel species within the genus Bacillus, for which the name Bacillus swezeyi sp. nov. is proposed, with type strain NRRL B-41294T (=CCUG 70177T), and the name Bacillus haynesii sp. nov. is proposed, with type strain NRRL B-41327T (=CCUG 70178T).

Genome analysis shows Bacillus axarquiensis is not a later heterotypic synonym of Bacillus mojavensis; reclassification of Bacillus malacitensis and Brevibacterium halotolerans as heterotypic synonyms of Bacillus axarquiensis.

Bacillus axarquiensis and Bacillus malacitensis were previously reported to be later heterotypic synonyms of Bacillus mojavensis, based primarily on DNA-DNA relatedness values. We have sequenced draft genomes of Bacillus axarquiensis NRRL B-41617T and Bacillus malacitensis NRRL B-41618T. Comparative genomics and DNA-DNA relatedness calculations showed that while Bacillus axarquiensis and Bacillus malacitensis are synonymous with each other, they are not synonymous with Bacillus mojavensis. In addition, a draft genome was completed for Brevibacterium halotolerans, a strain long suspected of being a Bacillus subtilis group member based on 16S rRNA similarities (99.8 % with Bacillus mojavensis). Comparative genomics and DNA-DNA relatedness calculations showed that Brevibacterium halotolerans is synonymous with Bacillus axarquiensis and Bacillus malacitensis. The pairwise in silico DNA-DNA hybridization values calculated in comparisons between the three conspecific strains were all greater than 92 %, which is well above the standard species threshold of 70 %. While the pairwise in silico DNA-DNA hybridization values calculated in comparisons of the three conspecific strains with Bacillus mojavensis were all less than 65 %. The combined results of our genotype and phenotype studies showed that Bacillus axarquiensis, Bacillus malacitensis and Brevibacterium halotolerans are conspecific and distinct from Bacillus mojavensis. Because the valid publication of the name Bacillus axarquiensis predates the publication of the name Bacillus malacitensis, we propose that Bacillus malacitensis be reclassified as a synonym of Bacillus axarquiensis. In addition, we propose to reclassify Brevibacterium halotolerans as a synonym of Bacillus axarquiensis. An amended description of Bacillus axarquiensis is provided.

Bacillus nakamurai sp. nov., a black-pigment-producing strain.

Two isolates of a Gram-stain-positive, strictly aerobic, motile, rod-shaped, endospore-forming bacterium were identified during a survey of the Bacillus diversity of the Agriculture Research Service Culture Collection. These strains were originally isolated from soil and have a phenotype of producing a dark pigment on tryptic soy agar. Phylogenetic analysis of the 16S rRNA gene indicated that these strains were related most closely to Bacillus subtilis subsp. inaquosorum (99.7 % similarity) and Bacillus axarquiensis (99.7 %). In phenotypic characterization, the novel strains were found to grow between 17 and 50 °C and can tolerate up to 9 % (w/v) NaCl. Furthermore, the strains grew in media of pH 5.5-10 (optimal growth at pH 7.0-8.0). The predominant cellular fatty acids were anteiso-C15 : 0 (34.8 %) and iso-C15 : 0 (21.9 %). The cell-wall peptidoglycan contained meso-diaminopimelic acid. A draft genome of both strains was completed. The DNA G+C content was 43.8 mol%. A phylogenomic analysis on the core genome of these two new strains and all members of the Bacillus subtilis group revealed these two strains formed a distinct monophyletic clade with the nearest neighbour Bacillus amyloliquefaciens. DNA-DNA relatedness studies using in silico DNA-DNA hybridizations showed the two strains were conspecific (93.8 %), while values with all other species (<31.5 %) were well below the species threshold of 70 %. Based on the consensus of phylogenetic and phenotypic analyses, these strains are considered to represent a novel species within the genus Bacillus, for which the name Bacillus nakamurai sp. nov. is proposed, with type strain NRRL B-41091T (=CCUG 68786T).

Evaluation of economically feasible, natural plant extract-based microbiological media for producing biomass of the dry rot biocontrol strain Pseudomonas fluorescens P22Y05 in liquid culture.

The production of microbial biomass in liquid media often represents an indispensable step in the research and development of bacterial and fungal strains. Costs of commercially prepared nutrient media or purified media components, however, can represent a significant hurdle to conducting research in locations where obtaining these products is difficult. A less expensive option for providing components essential to microbial growth in liquid culture is the use of extracts of fresh or dried plant products obtained by using hot water extraction techniques. A total of 13 plant extract-based media were prepared from a variety of plant fruits, pods or seeds of plant species including Allium cepa (red onion bulb), Phaseolus vulgaris (green bean pods), and Lens culinaris (lentil seeds). In shake flask tests, cell production by potato dry rot antagonist Pseudomonas fluorescens P22Y05 in plant extract-based media was generally statistically indistinguishable from that in commercially produced tryptic soy broth and nutrient broth as measured by optical density and colony forming units/ml produced (P ≤ 0.05, Fisher's protected LSD). The efficacy of biomass produced in the best plant extract-based media or commercial media was equivalent in reducing Fusarium dry rot by 50-96% compared to controls. In studies using a high-throughput microbioreactor, logarithmic growth of P22Y05 in plant extract-based media initiated in 3-5 h in most cases but specific growth rate and the time of maximum OD varied as did the maximum pH obtained in media. Nutrient analysis of selected media before and after cell growth indicated that nitrogen in the form of NH4 accumulated in culture supernatants, possibly due to unbalanced growth conditions brought on by a scarcity of simple sugars in the media tested. The potential of plant extract-based media to economically produce biomass of microbes active in reducing plant disease is considerable and deserves further research.

Selection of Biocontrol Agents of Pink Rot Based on Efficacy and Growth Kinetics Index Rankings.

The microbiota of 84 different agricultural soils were transferred to separate samples of a γ irradiation-sterilized field soil enriched with potato periderm, and the resulting soils were assayed for biological suppressiveness to Phytophthora erythroseptica and their effect on zoospore production. The 13 most suppressive soil samples, which reduced zoospore production by 14 to 93% and disease severity on tubers by 6 to 21%, were used to isolate 279 organisms. Fourteen strains that reduce pink rot infections in preliminary tests were selected for further study. Six bacterial strains that reduced the severity of disease (P ≤ 0.05, Fischer's protected least significant difference) in subsequent tests were identified as Bacillus simplex (three strains), Pantoea agglomerans, Pseudomonas koreensis, and P. lini. Relative performance indices (RPIs) for biocontrol efficacy and for each of four kinetic parameters, including total colony-forming units (CFUmax), biomass production values (DWmax), cell production after 8 h (OD8), and time of recovery from oxygen depletion (DT) were calculated for each strain. Overall RPIEff,Kin values for each strain then were calculated using strain RPI values for both efficacy (RPIEff) and kinetics (RPIKin). Strains with the highest RPIEff,Kin possess the best biocontrol efficacy of the strains tested and liquid culture growth characteristics that suggest commercial development potential.

Carbon-to-Nitrogen Ratio and Carbon Loading of Production Media Influence Freeze-Drying Survival and Biocontrol Efficacy of Cryptococcus nodaensis OH 182.9.

ABSTRACT Fusarium head blight (FHB), caused by Gibberella zeae, is a devastating disease of wheat worldwide. Cryptococcus nodaensis OH 182.9 is an effective biocontrol agent for this disease. Development of a dried product of OH 182.9 would have potential advantages of ease of handling, favorable economics, and acceptance by end users. Isolate OH 182.9 was grown for 48 and 72 h in semi-defined complete liquid (SDCL) medium with carbon-to-nitrogen (C/N) ratios of 6.5:1, 9:1, 11:1, 15:1, and 30:1, and in SDCL C/N 30:1 media with varied carbon loadings of 7, 14, 21, and 28 g/liter. Total biomass production and cell survival at 15 days after freeze-drying were evaluated. Biomass production of OH 182.9 (CFU per milliliter) was not different for all cultivation time by medium C/N or carbon loading combinations. In general, cells harvested at 48 h survived freeze-drying better than those harvested at 72 h. Survival of freeze-dried cells was greatest for cells grown for 48 h in C/N30:1 medium. Cells produced in C/N 6.5:1 medium generally exhibited the poorest survival. For the C/N 30:1 media, cells from 7 g/liter carbon loading medium harvested after 48 h had the best survival after freeze-drying. The difference in freeze-dried cell populations between superior and inferior treatments was typically 1 to 2 log units at 15 days after freeze-drying. The biomass of OH 182.9 produced in SDCL with varied C/N ratios and in SDCL C/N 30:1 media with differing carbon loadings was tested for biocontrol efficacy against FHB in greenhouse studies. The biomass harvested from SDCL C/N 9:1, 11:1, and 15:1 media after 48 h significantly reduced symptoms of FHB. None of the treatments with cells harvested at 72 h consistently reduced FHB severity (P

Liquid culture production of microsclerotia and submerged conidia by Trichoderma harzianum active against damping-off disease caused by Rhizoctonia solani.

Media and culturing protocols were identified that supported the formation of submerged conidia and microsclerotia (MS) by Trichoderma harzianum Rifai strain T-22 using liquid culture fermentation. Liquid media with a higher carbon concentration (36 g L(-1)) promoted MS formation at all C:N ratios tested. Hyphae aggregated to form MS after 2 d growth and after 7 d MS were fully melanized. This is the first report of MS formation by T. harzianum or any species of Trichoderma. Furthermore, submerged conidia formation was induced by liquid culture media, but yields, desiccation tolerance, and storage stability varied with C:N ratio and carbon rate. Air-dried MS granules (<4% moisture) retained excellent shelf life under cool and unrefrigerated storage conditions with no loss in conidial production. A low-cost complex nitrogen source based on cottonseed flour effectively supported high MS yields. Amending potting mix with dried MS formulations reduced or eliminated damping-off of melon seedlings caused by Rhizoctonia solani. Together, the results provide insights into the liquid culture production, stabilization process, and bioefficacy of the hitherto unreported MS of T. harzianum as a potential biofungicide for use in integrated management programs against soilborne diseases.

Biological control of Fusarium head blight of wheat and deoxynivalenol levels in grain via use of microbial antagonists.

Efforts to reduce mycotoxin contamination in food logically start with minimizing plant infection by mycotoxin producing pathogens. Fusarium graminearum (perfect state, Gibberella zeae) infects wheat heads at flowering, causing the disease Fusarium head blight (FHB) and losses of over 2.6 billion dollars in the U.S. during the last 10 years. The pathogen often produces deoxynivalenol (DON) resulting in grain size and quality reduction. Highly resistant wheat cultivars currently are not available for reducing FHB, and labeled fungicides are not consistently effective. The feasibility of biologically controlling FHB is currently being evaluated. Microbial isolates obtained from wheat anthers were screened for their ability to utilize tartaric acid, a compound that is poorly utilized by F. graminearum and could be utilized in formulations of biological control agents. Four strains that utilized tartaric acid and three that did not were effective in reducing FHB disease severity by up to 95% in greenhouse and 56% in field trials. Additional research programs around the globe have identified other antagonist strains with potential for biologically controlling FHB. Though a considerable body of research remains to be completed, strategies and microorganisms for biologically controlling FHB have reached an advanced stage of development and offer the promise of being an effective tool that could soon contribute to the reduction of FHB severity and DON contamination of grain in commercial agriculture.

Characterization of the surface properties of wheat spikelet components grown under different regimens and the biocontrol yeast Cryptococcus flavescens.

Surface properties play an important role in plant-microbe interactions and determine if microbial propagules adhere to the surface of a plant. Fusarium head blight is an important disease of wheat that is initiated by the pathogen colonizing the wheat head. To better understand how surface properties of wheat may affect disease development and spray applications, the surface properties of wheat (Triticum aestivum L.) spikelet components were characterized under different environmental growing regimes. In addition, the surface properties of the biocontrol yeast Cryptococcus flavescens OH 182.9, which has been shown to effective in managing Fusarium head blight, were characterized. Wheat samples grown in a greenhouse environment were compared with samples produced in the field for two wheat cultivars. The results show changes occurring in the surface energy parameters and estimates of roughness during this period between the two cultivars. In general, the greenhouse samples were more hydrophobic than those grown in the field. The surface properties of the biocontrol yeast C. flavescens OH 182.9 were determined from contact angles on microbial lawns and revealed the cells were hydrophobic with a free energy of aggregation of -86.3 mJ/m(2) in water.

Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight.

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.

Osmotic shock tolerance and membrane fluidity of cold-adapted Cryptococcus flavescens OH 182.9, previously reported as C. nodaensis, a biocontrol agent of Fusarium head blight.

Cryptococcus flavescens (previously reported as C. nodaensis), a biological control agent of Fusarium head blight, has been previously shown to have improved desiccation tolerance after cold adaptation. The goal of the current study was to determine the effect of cold adaptation on the physicochemical properties of C. flavescens that may be responsible for its improved desiccation tolerance. The results show that cold adaptation improves liquid hyperosmotic shock tolerance and alters the temperature dependence of osmotic shock tolerance. Fluorescence anisotropy was used to characterize differences in the membrane fluidity of C. flavescens with and without cold adaptation. Force curves from atomic force microscopy showed a significant increase in the cell wall spring constant after cold adaptation. Cold adaptation of C. flavescens during culturing was shown to produce smaller cells and produced a trend towards higher CFU yields. These results suggest that cold adaptation significantly alters the membrane properties of C. flavescens and may be an effective method of improving the desiccation tolerance of microorganisms. In addition, we provide information on the correct naming of the isolate as C. flavescens.

Discovery and development of biological agents to control crop pests / Descoberta e desenvolvimento de agentes de controle biológico de pragas de culturas

Thousands of potential microbial biocontrol agents have been isolated from agricultural fields and crops during research over the last 80 years, yet only a few are in commercial use. Recently, public health and safety concerns about the environmental impact of chemical pesticides have led to consideration of biological control as a natural approach to maintaining crop health. Despite environmental incentives and strong research efforts, commercialization of biocontrol agents has been slow to evolve. The momentum of the chemical industry is difficult to shift, and fermentation processes tend to be more expensive to operate than synthetic chemical processes.Yet there is a demand for biological control products, especially in agricultural niche markets, where there is no chemical competitor. However, given this market demand, the fundamental methods of economical large-scale production and application of biological control agents are lacking. Many aspects of biocontrol agent production and development represent untrodden territory in the progression of industrial fermentation technology beyond its well-established food and pharmaceuticals niche. Distinguishing them from traditional fermentation products, biocontrol agents must not only be produced in high yield but must also meet the following quality criteria: high (near 100 percent) retention of cell viability with maintenance of crop compatibility and consistent bioefficacy during several months of storage. Research examples will be reviewed to illustrate the challenges and strategies of developing processes to manufacture and deliver biological agents for insect, weed, and plant disease control.

Milhares de agentes de controle microbiano potenciais têm sido isolados a partir de campos cultivados ou de culturas pelo esforço da pesquisa nos últimos 80 anos. Entretanto, apenas alguns microrganismos têm uso comercial. Recentemente, medidas de saúde pública e de segurança sobre o impacto ambiental de pesticidas químicos têm levado a se considerar o controle biológico como uma medida promissora para proteção das culturas. Apesar do incentivo ambiental e esforços de pesquisa, a evolução da comercialização dos agentes de biocontrole tem sido lenta. O "momentum" da indústria química é difícil de mudar, e processos fermentativos tendem a ser mais caros e custosos do que os processos químicos sintéticos. Mesmo assim, há demanda para produtos de controle biológico, especialmente em nichos de mercados agrícolas onde não existe um competidor químico. Entretanto, os fundamentos dos métodos econômicos de produção e aplicação dos agentes de biocontrole em larga escala não existem. Muitos aspectos do desenvolvimento e da produção de agentes de controle biológico representam uma área diferenciada na tecnologia de produção industrial por fermentação, já bem estabelecida no nicho farmacêutico e alimentar. Ao se diferenciar dos produtos fermentados tradicionais, os agentes de biocontrole necessitam atingir os critérios de alta (ca. 100 por cento) retenção da viabilidade celular e compatibilidade com a cultura e bioeficiência durante vários meses de armazenagem. Exemplos da pesquisa são discutidos exemplificando-se os desafios e as estratégias dos processo de desenvolvimento para produzir e viabilizar agentes de biocontrole de insetos, ervas-daninhas e doenças.