Identification of biochemical features of defective Coffea arabica L. beans.

Coffee organoleptic properties are based in part on the quality and chemical composition of coffee beans. The presence of defective beans during processing and roasting contribute to off flavors and reduce overall cup quality. A multipronged approach was undertaken to identify specific biochemical markers for defective beans. To this end, beans were split into defective and non-defective fractions and biochemically profiled in both green and roasted states. A set of 17 compounds in green beans, including organic acids, amino acids and reducing sugars; and 35 compounds in roasted beans, dominated by volatile compounds, organic acids, sugars and sugar alcohols, were sufficient to separate the defective and non-defective fractions. Unsorted coffee was examined for the presence of the biochemical markers to test their utility in detecting defective beans. Although the green coffee marker compounds were found in all fractions, three of the roasted coffee marker compounds (1-methylpyrrole, 5-methyl- 2-furfurylfuran, and 2-methylfuran) were uniquely present in defective fractions.

What's Inside That Seed We Brew? A New Approach To Mining the Coffee Microbiome.

Coffee is a critically important agricultural commodity for many tropical states and is a beverage enjoyed by millions of people worldwide. Recent concerns over the sustainability of coffee production have prompted investigations of the coffee microbiome as a tool to improve crop health and bean quality. This review synthesizes literature informing our knowledge of the coffee microbiome, with an emphasis on applications of fruit- and seed-associated microbes in coffee production and processing. A comprehensive inventory of microbial species cited in association with coffee fruits and seeds is presented as reference tool for researchers investigating coffee-microbe associations. It concludes with a discussion of the approaches and techniques that provide a path forward to improve our understanding of the coffee microbiome and its utility, as a whole and as individual components, to help ensure the future sustainability of coffee production.

Soil Conditions That Can Alter Natural Suppression of Escherichia coli O157:H7 in Ohio Specialty Crop Soils.

Food-borne pathogen persistence in soil fundamentally affects the production of safe vegetables and small fruits. Interventions that reduce pathogen survival in soil would have positive impacts on food safety by minimizing preharvest contamination entering the food chain. Laboratory-controlled studies determined the effects of soil pH, moisture content, and soil organic matter (SOM) on the survivability of this pathogen through the creation of single-parameter gradients. Longitudinal field-based studies were conducted in Ohio to quantify the extent to which field soils suppressed Escherichia coli O157:H7 survival. In all experiments, heat-sensitive microorganisms were responsible for the suppression of E. coli O157 in soil regardless of the chemical composition of the soil. In laboratory-based studies, soil pH and moisture content were primary drivers of E. coli O157 survival, with increases in pH after 48 h (P = 0.02) and decreases in moisture content after 48 h (P = 0.007) significantly increasing the log reduction of E. coli O157 numbers. In field-based experiments, E. coli O157 counts from both heated and unheated samples were sensitive to both season (P = 0.004 for heated samples and P = 0.001 for unheated samples) and region (P = 0.002 for heated samples and P = 0.001 for unheated samples). SOM was observed to be a more significant driver of pathogen suppression than the other two factors after 48 h at both planting and harvest (P = 0.002 at planting and P = 0.058 at harvest). This research reinforces the need for both laboratory-controlled experiments and longitudinal field-based experiments to unravel the complex relationships controlling the survival of introduced organisms in soil.

Association of shifting populations in the root zone microbiome of millet with enhanced crop productivity in the Sahel region (Africa).

This study characterized specific changes in the millet root zone microbiome stimulated by long-term woody-shrub intercropping at different sites in Senegal. At the two study sites, intercropping with woody shrubs and shrub residue resulted in a significant increase in millet [Pennisetum glaucum (L.) R. Br.] yield (P < 0.05) and associated patterns of increased diversity in both bacterial and fungal communities in the root zone of the crop. Across four experiments, operational taxonomic units (OTUs) belonging to Chitinophaga were consistently significantly (P < 0.001) enriched in the intercropped samples, and "Candidatus Koribacter" was consistently significantly enriched in samples where millet was grown alone. Those OTUs belonging to Chitinophaga were enriched more than 30-fold in residue-amended samples and formed a distinct subgroup from all OTUs detected in the genus. Additionally, OTUs belonging to 8 fungal genera (Aspergillus, Coniella, Epicoccum, Fusarium, Gibberella, Lasiodiplodia, Penicillium, and Phoma) were significantly (P < 0.005) enriched in all experiments at all sites in intercropped samples. The OTUs of four genera (Epicoccum, Fusarium, Gibberella, and Haematonectria) were consistently enriched at sites where millet was grown alone. Those enriched OTUs in intercropped samples showed consistently large-magnitude differences, ranging from 30- to 1,000-fold increases in abundance. Consistently enriched OTUs in intercropped samples in the genera Aspergillus, Fusarium, and Penicillium also formed phylogenetically distinct subgroups. These results suggest that the intercropping system used here can influence the recruitment of potentially beneficial microorganisms to the root zone of millet and aid subsistence farmers in producing higher-yielding crops.

Draft Genome Sequence of Cryptococcus flavescens Strain OH182.9_3C, a Biocontrol Agent against Fusarium Head Blight of Wheat.

Cryptococcus flavescens strain OH182.9_3C (3C) is a novel biopesticidal agent that can be used to control fusarium head blight of wheat. Here we present the draft genome sequence for 3C, the first for the species C. flavescens. Additionally, several genes that may contribute to the biocontrol activities of 3C were identified in silico.

Different roles of glycine-rich RNA-binding protein7 in plant defense against Pectobacterium carotovorum, Botrytis cinerea, and tobacco mosaic viruses.

Glycine-rich RNA-binding protein7 (AtGRP7) has previously been demonstrated to confer plant defense against Pseudomonas syringae DC3000. Here, we show that AtGRP7 can play different roles in plant defense against diverse pathogens. AtGRP7 enhances resistance against a necrotrophic bacterium Pectobacterium carotovorum SCC1 or a biotrophic virus tobacco mosaic virus. By contrast, AtGRP7 plays a negative role in defense against a necrotrophic fungus Botrytis cinerea. These results provide evidence that AtGRP7 is a potent regulator in plant defense response to diverse pathogens, and suggest that the regulation of RNA metabolism by RNA-binding proteins is important for plant innate immunity.

Draft genome sequence of Pantoea ananatis B1-9, a nonpathogenic plant growth-promoting bacterium.

Pantoea ananatis B1-9 is an endophytic Gram-negative rhizobacterium that was isolated for its ability to promote plant growth and improve crop yield in the field. Here we report the draft genome sequence of P. ananatis B1-9. Comparison of this sequence to the sequenced genome of a plant-pathogenic P. ananatis strain, LMG20103, indicated that the pathogenesis-related genes were absent, but a subset of gene functions that may be related to its plant growth promotion were present.

Draft genome sequences of the Pseudomonas fluorescens biocontrol strains Wayne1R and Wood1R.

Pseudomonas fluorescens strains Wayne1R and Wood1R have proven capacities to improve plant health. Here we report the draft genome sequences and automatic annotations of both strains. Genome comparisons reveal similarities with P. fluorescens strain Pf-5, reveal the novelty of Wood1R, and indicate some genes that may be related to biocontrol.

Draft genome sequences of the biocontrol bacterium Mitsuaria sp. strain H24L5A.

Mitsuaria sp. strain H24L5A is a plant-associated bacterium with proven capacities to suppress plant pathogens. Here, we report the draft genome sequences and automatic annotation of H24L5A. Comparative genomic analysis indicates H24L5A's similarity to the Leptothrix and Methylibium species, as well as several genes potentially contributing to its biocontrol activities.

Draft genome sequence of the biocontrol bacterium Pseudomonas putida B001, an oligotrophic bacterium that induces systemic resistance to plant diseases.

Pseudomonas putida B001 is a rhizobacterium that was isolated on the basis of its abilities to grow under low-nutrient conditions and induce systemic resistance against bacterial, fungal, and viral diseases of plants. Here we report the draft genome sequence and automatic annotation of strain B001. Comparison of this sequence to the sequenced genome of P. putida KT2440 points to a subset of gene functions that may be related to the defense-inducing functions of B001.

Draft genome sequence of the biocontrol bacterium Chromobacterium sp. strain C-61.

Chromobacterium sp. strain C-61 is a plant-associated bacterium with proven capacities to suppress plant diseases. Here, we report the draft genome sequence and automatic annotation of strain C-61. A comparison of this sequence to the sequenced genome of Chromobacterium violaceum ATCC 12472 indicates the novelty of C-61 and a subset of gene functions that may be related to its biocontrol activities.

The multifactorial basis for plant health promotion by plant-associated bacteria.

On plants, microbial populations interact with each other and their host through the actions of secreted metabolites. However, the combined action of diverse organisms and their different metabolites on plant health has yet to be fully appreciated. Here, the multifactorial nature of these interactions, at the organismal and molecular level, leading to the biological control of plant diseases is reviewed. To do so, we describe in detail the ecological significance of three different classes of secondary metabolites and discuss how they might contribute to biological control. Specifically, the roles of auxin, acetoin, and phenazines are considered, because they represent very different but important types of secondary metabolites. We also describe how studies of the global regulation of bacterial secondary metabolism have led to the discovery of new genes and phenotypes related to plant health promotion. In conclusion, we describe three avenues for future research that will help to integrate these complex and diverse observations into a more coherent synthesis of bacterially mediated biocontrol of plant diseases.

General suppression of Escherichia coli O157:H7 in sand-based dairy livestock bedding.

Sand bedding material is frequently used in dairy operations to reduce the occurrence of mastitis and enhance cow comfort. One objective of this work was to determine if sand-based bedding also supported the microbiologically based suppression of an introduced bacterial pathogen. Bedding samples were collected in summer, fall, and winter from various locations within a dairy operation and tested for their ability to suppress introduced populations of Escherichia coli O157:H7. All sources of bedding displayed a heat-sensitive suppressiveness to the pathogen. Differences in suppressiveness were also noted between different samples at room temperature. At just 1 day postinoculation (dpi), the recycled sand bedding catalyzed up to a 1,000-fold reduction in E. coli counts, typically 10-fold greater than the reduction achieved with other substrates, depending on the sampling date. All bedding substrates were able to reduce E. coli populations by over 10,000-fold within 7 to 15 dpi, regardless of sampling date. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to identify bacterial populations potentially associated with the noted suppression of E. coli O157:H7 in sand bedding. Eleven terminal restriction fragments (TRFs) were overrepresented in paired comparisons of suppressive and nonsuppressive specimens at multiple sampling points, indicating that they may represent environmentally stable populations of pathogen-suppressing bacteria. Cloning and sequencing of these TRFs indicated that they represent a diverse subset of bacteria, belonging to the Cytophaga-Flexibacter-Bacteroidetes, Gammaproteobacteria, and Firmicutes, only a few of which have previously been identified in livestock manure. Such data indicate that microbial suppression may be harnessed to develop new options for mitigating the risk and dispersal of zoonotic bacterial pathogens on dairy farms.

Seed treatment with 2,4-diacetylphloroglucinol-producing pseudomonads improves crop health in low-pH soils by altering patterns of nutrient uptake.

Seed treatment with a 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strain ameliorated abiotic stress disorder in corn caused by growth in a low-pH soil. In two consecutive growing seasons, Wood1R-treated seed gave rise to plants that grew taller (P

Distinct amino acids of the Phytophthora infestans effector AVR3a condition activation of R3a hypersensitivity and suppression of cell death.

The AVR3a protein of Phytophthora infestans is a polymorphic member of the RXLR class of cytoplasmic effectors with dual functions. AVR3a(KI) but not AVR3a(EM) activates innate immunity triggered by the potato resistance protein R3a and is a strong suppressor of the cell-death response induced by INF1 elicitin, a secreted P. infestans protein that has features of pathogen-associated molecular patterns. To gain insights into the molecular basis of AVR3a activities, we performed structure-function analyses of both AVR3a forms. We utilized saturated high-throughput mutant screens to identify amino acids important for R3a activation. Of 6,500 AVR3a(EM) clones tested, we identified 136 AVR3a(EM) mutant clones that gained the ability to induce R3a hypersensitivity. Fifteen amino-acid sites were affected in this set of mutant clones. Most of these mutants did not suppress cell death at a level similar to that of AVR3a(KI). A similar loss-of-function screen of 4,500 AVR3a(KI) clones identified only 13 mutants with altered activity. These results point to models in which AVR3a functions by interacting with one or more host proteins and are not consistent with the recognition of AVR3a through an enzymatic activity. The identification of mutants that gain R3a activation but not cell-death suppression activity suggests that distinct amino acids condition the two AVR3a effector activities.

Evaluation of an antibiotic-producing strain of Pseudomonas fluorescens for suppression of plant-parasitic nematodes.

The antibiotic 2,4-diacetylphloroglucinol (DAPG), produced by some strains of Pseudomonas spp., is involved in suppression of several fungal root pathogens as well as plant-parasitic nematodes. The primary objective of this study was to determine whether Wood1R, a D-genotype strain of DAPG-producing P. fluorescens, suppresses numbers of both sedentary and migratory plant-parasitic nematodes. An experiment was conducted in steam-heated soil and included two seed treatments (with Wood1R and a control without the bacterium) and six plant-nematode combinations which were Meloidogyne incognita on cotton, corn, and soybean; M. arenaria on peanut; Heterodera glycines on soybean; and Paratrichodorus minor on corn. Wood 1R had no effect on final numbers of M. arenaria, P. minor, or H. glycines; however, final numbers of M. incognita were lower when seeds were treated with Wood1R than left untreated, and this reduction was consistent among host plants. Population densities of Wood1R were greater on the roots of corn than on the other crops, and the bacterium was most effective in suppressing M. incognita on corn, with an average reduction of 41%. Despite high population densities of Wood1R on corn, the bacterium was not able to suppress numbers of P. minor. When comparing the suppression of M. incognita on corn in natural and steam-heated soil, egg production by the nematode was suppressed in natural compared to steamed soil, but the presence of Wood1R did not result in additional suppression of the nematodes in the natural soil. These data indicate that P. fluorescens strain Wood1R has the capacity to inhibit some populations of plant-parasitic nematodes. However, consistent suppression of nematodes in natural soils seems unlikely.

Diversity and Ecology of Biocontrol Pseudomonas spp. in Agricultural Systems.

ABSTRACT Diverse Pseudomonas spp. may act as biological controls of plant pathogens, but the ecology of those natural populations is not well understood. And, while biocontrol potential has been identified in multiple pseudomonad strains, the linkages between genotype and phenotype have yet to be fully delineated. However, intensive studies of one class of biocontrol strains, i.e., those that can produce 2,4-diacetylphloroglucionl (DAPG), have provided new insights into the diversity, distribution, and interactions of biocontrol pseudomonads. Those studies also laid the foundation for future research and development of pseudomonad-based biocontrol strategies. Over the past several years, numerous studies have also revealed that biocontrol pseudomonads are widely distributed in agricultural soils, and that multiple crop and soil factors can affect their abundance and activities. Recent work has shown that a variety of farm management practices that reduce soilborne disease pressure can also alter the rhizosphere abundance of DAPG producers in complex ways. Such studies provide support for the hypothesis of an ecological feedback mechanism whereby a native biocontrol population increase and subsequently reduce root disease severity following infection. It is well established that complex biological interactions can take place among bio-control pseudomonads, plant pathogens, their hosts, and other members of the microbial community. The net result of such interactions likely dilutes biocontrol efficacy at the field scale. Nonetheless, inoculation can be effective, and several successful applications of biocontrol pseudomonads have been developed. Future applications of microbial ecology research will hopefully improve the consistency and efficacy of bio-control mediated by Pseudomonas spp. Current applications and future opportunities for improving pseudomonad-based biological control are discussed.

Identification and Characterization of Novel Genetic Markers Associated with Biological Control Activities in Bacillus subtilis.

ABSTRACT Suppressive subtractive hybridization (SSH) was used to identify genetic markers associated with biological control of plant pathogens by Bacillus subtilis. The genomes of two commercialized strains, GB03 and QST713, were compared with that of strain 168, which has no defined biocontrol capacities, to obtain a pool of DNA fragments unique to the two biocontrol strains. The sequences of 149 subtracted fragments were determined and compared with those present in GenBank, but only 80 were found to correspond to known Bacillus genes. Of these, 65 were similar to genes with a wide range of metabolic functions, including the biosynthesis of cell wall components, sporulation, and antibiotic biosynthesis. Sixteen subtracted fragments shared a high degree of similarity to sequences found in multiple B. subtilis strains with proven biocontrol capacities. Oligonucleotide primers specific to nine of these genes were developed. The targeted genes included five genes involved in antibiotic synthesis (bmyB, fenD, ituC,srfAA, and srfAB) and four additional genes (yndJ, yngG, bioA, and a hypothetical open reading frame) not previously associated with biological control. All nine markers were amplified from the commercialized B. subtilis strains GB03, QST713, and MBI600, with the exception of ituC, which was not detected in GB03. The markers also were amplified from four other B. subtilis isolates, but they were not amplified from other related Bacillus strains, including the plant growth-promoting rhizobacteria IN937a and IN937b. Sequencing of the amplified markers revealed that all seven of the isolates that scored positive for multiple markers were genotypically distinct strains. Interestingly, strains scored positive for the amplifiable markers generally were more effective at inhibiting the growth of Rhizoctonia solani and Pythium ultimum than other Bacillus isolates that lacked the markers. The potential utility of the defined genetic markers to further define the diversity, ecology, and biocontrol activities of B. subtilis are discussed.

Distribution and Biocontrol Potential of phlD(+) Pseudomonads in Corn and Soybean Fields.

ABSTRACT The abundance and diversity of phlD(+) Pseudomonas spp. colonizing the rhizospheres of young, field-grown corn and soybean plants were assayed over a 3-year period. Populations of these bacteria were detected on the large majority of plants sampled in the state of Ohio, but colonization was greater on corn. Although significant variation in the incidence of rhizosphere colonization was observed from site to site and year to year on both crops, the magnitude of the variation was greatest for soybean. The D genotype was detected on plants collected from all 15 counties examined, and it represented the most abundant subpopulation on both crops. Additionally, six other genotypes (A, C, F, I, R, and S) were found to predominate in the rhizosphere of some plants. The most frequently observed of these were the A genotype and a newly discovered S genotype, both of which were found on corn and soybean roots obtained from multiple locations. Multiple isolates of the most abundant genotypes were recovered and characterized. The S genotype was found to be phylogenetically and phenotypically similar to the D genotype. In addition, the novel R genotype was found to be most similar to the A genotype. All of the isolates displayed significant capacities to inhibit the growth of an oomycete pathogen in vitro, but such phenotypes were highly dependent on media used. When tested against multiple oomycete pathogens isolated from soybean, the A genotype was significantly more inhibitory than the D genotype when incubated on 1/10x tryptic soy agar and 1/5x corn meal agar. Seed inoculation with different isolates of the A, D, and S genotypes indicated that significant root colonization, generally in excess of log 5 cells per gram of root, could be attained on both crops. Field trials of the A genotype isolate Wayne1R indicated the capacity of inoculant populations to supplement the activities of native populations so as to increase soybean stands and yields. The relevance of these findings to natural and augmentative biocontrol of root pathogens by these bacteria is discussed.