Seed treatment with prodigiosin controls damping-off of cucumber caused by Pythium ultimum.

Ethanol extract of cell mass of Serratia marcescens strain N4-5, when applied as a treatment to cucumber seed, has been shown to provide control of the oomycete soil-borne plant pathogen Pythium ultimum equivalent to that provided by a seed-treatment chemical pesticide in some soils. Two dominant compounds in this extract, prodigiosin and the serratamolide serrawetin W1, were identified based on mass and collision induced dissociation mass fragmentation spectra. An additional four compounds with M+H+ masses (487, 541, 543, and 571) consistent with serratamolides reported in the literature were also detected. Several other compounds with M+H+ masses of 488, 536, 684, 834, 906, and 908 m/z were detected in this ethanol extract inconsistently over multiple liquid chromatography coupled with tandem mass spectrometry (LC/MS-MS) runs. A purified preparation of prodigiosin provided control of damping-off of cucumber caused by P. ultimum when applied as a seed treatment while ethanol extract of cell mass of strain Tn246, a transposon-mutant-derivative of strain N4-5, did not. Strain Tn246 contained a mini-Tn5 Km insertion in a prodigiosin biosynthetic gene and was deficient in production of prodigiosin. All other compounds detected in N4-5 extract were detected in the Tn246 extract. This is the first report demonstrating that prodigiosin can control a plant disease. Other compounds in ethanol extract of strain N4-5 may contribute to disease control.

Deciphering the Role of Trehalose in Tripartite Symbiosis Among Rhizobia, Arbuscular Mycorrhizal Fungi, and Legumes for Enhancing Abiotic Stress Tolerance in Crop Plants.

Drought is a critical factor limiting the productivity of legumes worldwide. Legumes can enter into a unique tripartite symbiotic relationship with root-nodulating bacteria of genera Rhizobium, Bradyrhizobium, or Sinorhizobium and colonization by arbuscular mycorrhizal fungi (AMF). Rhizobial symbiosis provides nitrogen necessary for growth. AMF symbiosis enhances uptake of diffusion-limited nutrients such as P, Zn, Cu, etc., and also water from the soil via plant-associated fungal hyphae. Rhizobial and AMF symbioses can act synergistically in promoting plant growth and fitness, resulting in overall yield benefits under drought stress. One of the approaches that rhizobia use to survive under stress is the accumulation of compatible solutes, or osmolytes, such as trehalose. Trehalose is a non-reducing disaccharide and an osmolyte reported to accumulate in a range of organisms. High accumulation of trehalose in bacteroids during nodulation protects cells and proteins from osmotic shock, desiccation, and heat under drought stress. Manipulation of trehalose cell concentrations has been directly correlated with stress response in plants and other organisms, including AMF. However, the role of this compound in the tripartite symbiotic relationship is not fully explored. This review describes the biological importance and the role of trehalose in the tripartite symbiosis between plants, rhizobia, and AMF. In particular, we review the physiological functions and the molecular investigations of trehalose carried out using omics-based approaches. This review will pave the way for future studies investigating possible metabolic engineering of this biomolecule for enhancing abiotic stress tolerance in plants.

Soil Microbial Communities in Diverse Agroecosystems Exposed to the Herbicide Glyphosate.

Despite glyphosate's wide use for weed control in agriculture, questions remain about the herbicide's effect on soil microbial communities. The existing scientific literature contains conflicting results, from no observable effect of glyphosate to the enrichment of agricultural pathogens such as Fusarium spp. We conducted a comprehensive field-based study to compare the microbial communities on the roots of plants that received a foliar application of glyphosate to adjacent plants that did not. The 2-year study was conducted in Beltsville, MD, and Stoneville, MS, with corn and soybean crops grown in a variety of organic and conventional farming systems. By sequencing environmental metabarcode amplicons, the prokaryotic and fungal communities were described, along with chemical and physical properties of the soil. Sections of corn and soybean roots were plated to screen for the presence of plant pathogens. Geography, farming system, and season were significant factors determining the composition of fungal and prokaryotic communities. Plots treated with glyphosate did not differ from untreated plots in overall microbial community composition after controlling for other factors. We did not detect an effect of glyphosate treatment on the relative abundance of organisms such as Fusarium spp.IMPORTANCE Increasing the efficiency of food production systems while reducing negative environmental effects remains a key societal challenge to successfully meet the needs of a growing global population. The herbicide glyphosate has become a nearly ubiquitous component of agricultural production across the globe, enabling an increasing adoption of no-till agriculture. Despite this widespread use, there remains considerable debate on the consequences of glyphosate exposure. In this paper, we examine the effect of glyphosate on soil microbial communities associated with the roots of glyphosate-resistant crops. Using metabarcoding techniques, we evaluated prokaryotic and fungal communities from agricultural soil samples (n = 768). No effects of glyphosate were found on soil microbial communities associated with glyphosate-resistant corn and soybean varieties across diverse farming systems.

Comparative measurements of arbuscular mycorrhizal fungal responses to agricultural management practices.

Arbuscular mycorrhizal (AM) fungi are considered to be a key group of soil organisms for assessments of soil biological properties and developing relationships among crop production management practices, soil properties, crop performance, and ecosystem services. In a field study of cover crop treatments established during the transition from small grains to corn (Zea mays L), we assessed multiple measures of AM fungal responses to the management treatments: soil propagule numbers, biomass via lipid biomarkers, and root colonization extent. Our objectives were to determine response variables that reliably distinguished cover crop treatments and formed consistent relationships with grain yield, plant biomass, and mineral nutrient concentrations of the following corn crop. The number of soil AM fungal propagules and amount of the NLFA biomarker C16:1cis11 measured on fall-collected soils most consistently and significantly responded to fall cover crop treatments. Neither of these measures of soil inoculum potential was strongly related to measures of crop performance. The PLFA biomarker C16:1cis11 was marginally responsive to cover crop but did not strongly relate to crop performance parameters. Corn root colonization by AM fungi was not significantly affected by cover crop treatment, but significant negative relationships were found between root colonization and grain N concentration and plant biomass at maturity. In contrast, a significant positive relationship between root colonization and plant N concentration at the 6-leaf stage was found. Understanding the relative effectiveness and limitations of AM fungal response variables will inform their application in field studies of agricultural management practices.

Characterization of soil nematode communities in three cropping systems through morphological and DNA metabarcoding approaches.

We used complementary morphological and DNA metabarcoding approaches to characterize soil nematode communities in three cropping systems, conventional till (CT), no-till (NT) and organic (ORG), from a long-term field experiment. We hypothesized that organic inputs to the ORG system would promote a more abundant nematode community, and that the NT system would show a more structured trophic system (higher Bongers MI) than CT due to decreased soil disturbance. The abundance of Tylenchidae and Cephalobidae both showed positive correlations to soil organic carbon and nitrogen, which were highest in the ORG system. The density of omnivore-predator and bacterial-feeding nematodes was reduced in NT soils compared to CT, while some plant-parasitic taxa increased. NT soils had similar Bongers MI values to CT, suggesting they contained nematode communities associated with soils experiencing comparable levels of disturbance. Metabarcoding revealed within-family differences in nematode diversity. Shannon and Simpson's index values for the Tylenchidae and Rhabditidae were higher in the ORG system than CT. Compared to morphological analysis, metabarcoding over- or underestimated the prevalence of several nematode families and detected some families not observed based on morphology. Discrepancies between the techniques require further investigation to establish the accuracy of metabarcoding for characterization of soil nematode communities.

Microbial community biomass and structure in saline and non-saline soils associated with salt- and boron-tolerant poplar clones grown for the phytoremediation of selenium.

Poplar trees (Populus spp.) are often used in bioremediation strategies because of their ability to phytoextract potential toxic ions, e.g., selenium (Se) from poor quality soils. Soil microorganisms may play a vital role in sustaining health of soil and/or tolerance of these trees grown in poor quality soils by contributing to nutrient cycling, soil structure, overall soil quality, and plant survival. The effect of naturally occurring salts boron (B) and Se on soil microbial community composition associated with poplar trees is not known for bioremediation strategies. In this study, three Populus clones 13-366, 345-1, and 347-14 were grown in spring 2006 under highly saline, B, and Se clay-like soils in the west side of the San Joaquin Valley (SJV) of CA, as well as in non-saline sandy loam soils located in the east side of the SJV. After 7 years of growing in the respective soils of different qualities, soil samples were collected from poplar clones grown in saline and non-saline soils to examine and compare soil quality effects on soil microbial community biomass and composition. The phospholipid fatty acid (PLFA) analysis was used to characterize microbial community composition in soils from trees grown at both locations. This study showed that microbial biomass and the amount and proportion of arbuscular mycorrhizal fungal (AMF) community were lower in all three poplar clones grown in saline soil compared to non-saline soil. Amounts of Gram + bacterial and actinomycetes PLFAs were significantly lower in poplar clone 13-366 grown in saline soil compared to non-saline soil; however, they did not differ significantly in poplar clones 347-14 and 345-1. Additionally, amounts of saprophytic fungal, Gram - bacterial and eukaryotic PLFA remained similar at saline and non-saline sites under poplar clones 347-14, 345-1, and 13-366. Therefore, this study suggested that salinity and B do have an impact on microbial biomass and AMF; however, these poplar clones still recycled sufficient amount of nutrients to support and protect saprophytic fungal and bacterial communities from the effects of poor quality soils.

Belowground competition among invading detritivores.

The factors regulating soil animal communities are poorly understood. Current theory favors niche complementarity and facilitation over competition as the primary forms of non-trophic interspecific interaction in soil fauna; however, competition has frequently been suggested as an important community-structuring factor in earthworms, ecosystem engineers that influence belowground processes. To date, direct evidence of competition in earthworms is lacking due to the difficulty inherent in identifying a limiting resource for saprophagous animals. In the present study, we offer the first direct evidence of interspecific competition for food in this dominant soil detritivore group by combining field observations with laboratory mesocosm experiments using 13C and 15N double-enriched leaf litter to track consumption patterns. In our experiments, the Asian invasive species Amynthas hilgendorfi was a dominant competitor for leaf litter against two European species currently invading the temperate deciduous forests in North America. This competitive advantage may account for recent invasion success of A. hilgendorfi in forests with established populations of European species, and we hypothesize that specific phenological differences play an important role in determining the outcome of the belowground competition. In contrast, Eisenoides lonnbergi, a common native species in the Eastern United States, occupied a unique trophic position with limited interactions with other species, which may contribute to its persistence in habitats dominated by invasive species. Furthermore, our results supported neither the hypothesis that facilitation occurs between species of different functional groups nor the hypothesis that species in the same group exhibit functional equivalency in C and N translocation in the soil. We propose that species identity is a more powerful approach to understand earthworm invasion and its impacts on belowground processes.

Yeasts associated with plums and their potential for controlling brown rot after harvest.

Bacterial and yeast antagonists isolated from fruit surfaces have been effective in controlling various post-harvest diseases, and several microbial antagonists have been developed into commercial products. Our knowledge of the fruit microbial community, with the exception of grapes, apples and some citrus fruit, is rudimentary and the potential of the resident yeasts for biocontrol remains largely unknown. We determined the occurrence of yeasts on plum surfaces during fruit development from the pre-hardening stage until harvest for 2 years. A total of 16 species from 13 genera were isolated. Species from three genera, basidiomycetes Rhodotorula (29.5%) and Sporidiobolus (24.7%) and the dimorphic ascomycete genus Aureobasidium (24.7%), constituted 78.7% of all isolations and were recovered throughout fruit development, while Cryptococcus spp. constituted only 6.2% of the total plum isolates. The yeast community in the final sampling was significantly different from the first three samplings, reflecting a rapidly changing fruit habitat during the maturation of fruit. For example, Hanseniaspora, Pichia, Zygosaccharomyces and Wickerhamomyces occurred only on the most mature fruit. Screening of the yeasts for antagonistic activity against Monilinia fructicola, a fungus that causes brown rot, revealed a range of biocontrol activities. Several isolates provided complete control of the decay on plums, challenged with a pathogen suspension of 10(3) conidia/ml and > 90% of control on fruit inoculated with the pathogen at a concentration 10 times higher. Some of the best antagonists included A. pullulans and R. phylloplana. Populations of both of these antagonists increased rapidly by several orders of magnitude in wounds of plums incubated at 24ºC and 4ºC. Our results indicate that plum surfaces harbour several yeast species, with excellent potential for use in biological control of brown rot of stone fruits.

Genes expressed by the biological control bacterium Pseudomonas protegens Pf-5 on seed surfaces under the control of the global regulators GacA and RpoS.

Gene expression profiles of the biological control strain Pseudomonas protegens Pf-5 inhabiting pea seed surfaces were revealed using a whole-genome oligonucleotide microarray. We identified genes expressed by Pf-5 under the control of two global regulators (GacA and RpoS) known to influence biological control and secondary metabolism. Transcript levels of 897 genes, including many with unknown functions as well as those for biofilm formation, cyclic diguanylate (c-di-GMP) signalling, iron homeostasis and secondary metabolism, were influenced by one or both regulators, providing evidence for expression of these genes by Pf-5 on seed surfaces. Comparison of the GacA and RpoS transcriptomes defined for Pf-5 grown on seed versus in broth culture overlapped, but most genes were regulated by GacA or RpoS under only one condition, likely due to differing levels of expression in the two conditions. We quantified secondary metabolites produced by Pf-5 and gacA and rpoS mutants on seed and in culture, and found that production profiles corresponded generally with biosynthetic gene expression profiles. Future studies evaluating biological control mechanisms can now focus on genes expressed by Pf-5 on seed surfaces, the habitat where the bacterium interacts with seed-infecting pathogens to suppress seedling diseases.

Endophytic fungi from plums (Prunus domestica) and their antifungal activity against Monilinia fructicola.

Enophytic fungi were isolated from plum (Prunus domestica) leaves, identified with ITS1 and ITS4 primers, and their antagonistic activity was tested against Monilinia fructicola, which causes brown rot, blossom blight, and twig blight of stone fruits, and Colletotrichum gloeosporioides, which causes anthracnose on a variety of fruit crops. The production of antifungal compounds was determined in agar-diffusion and volatile inverted-plate tests. A total of 163 fungi were recovered from 30 plum trees, representing 22 cultivars. Twenty-nine morphotypes were detected, but only 14 species were identified genetically. The most frequently isolated species was Phaeosphaeria nodorum, constituting 86.5% of the total isolates. Four isolates produced inhibitory volatiles to M. fructicola; however, no isolate produced volatiles inhibitory to C. gloeosporioides. The volatiles produced by these fungi were identified as ethyl acetate, 3-methyl-1-butanol, acetic acid, 2-propyn-1-ol, and 2-propenenitrile. The fungal volatiles inhibited growth and reduced width of the hyphae, and caused disintegration of the hyphal content. This is the first study describing fungal endophytes in stone fruits. The P. nodorum strains producing inhibitory volatiles could play a significant role in reduction of M. fructicola expansion in plum tissues. Potential of these strains for biological control of this pathogen on stone fruits warrants further investigation.

Formulations of the endophytic bacterium Bacillus subtilis Tu-100 suppress Sclerotinia sclerotiorum on oilseed rape and improve plant vigor in field trials conducted at separate locations.

Sclerotinia sclerotiorum causes serious yield losses in crops in the People's Republic of China. Two formulations of oilseed rape seed containing the bacterium Bacillus subtilis Tu-100 were evaluated for suppression of this pathogen in field trials conducted at two independent locations. The pellet formulation significantly reduced disease (incidence and disease index) and increased plant dry mass, while the wrap formulation significantly reduced disease incidence and significantly increased plant dry mass at both field locations. Mean seed yield per 120 plants with both formulations of isolate Tu-100 was significantly greater than the appropriate controls, but at only one of the locations. Both formulations provided stable B. subtilis Tu-100 biomass (≥10(5) CFU·g(-1)) and seed germination (≥85%) over a 6 month period at room temperature. Polymerase chain reaction and DNA sequence analysis identified ituC and ituD, and bacAB and bacD in the genome of isolate Tu-100. These genes are involved in the biosynthesis of iturin and bacilysin. Iturin was detected in culture filtrates from isolate Tu-100, with thin layer chromatography. Detection of bacilysin was not attempted. Experiments reported here indicate the commercial viability of B. subtilis Tu-100 for suppression of S. sclerotiorum on oilseed rape.

Culturable bacterial microflora associated with nectarine fruit and their potential for control of brown rot.

Microflora of fruit surfaces have been the best source of antagonists against fungi causing postharvest decay of fruit. However, there is little information on microflora colonizing surfaces of fruits other than grape, apple, and citrus. We characterized bacterial microflora on nectarine fruit surfaces from the early stage of development until harvest. Identification of bacterial strains was made using MIDI (fatty acid methyl ester analysis) and Biolog systems. Biolog identified 35% and MIDI 53% of the strains. Thus results from MIDI were used to determine the frequency of occurrence of genera and species. The most frequently occurring genera were Curtobacterium (21.31%), followed by Pseudomonas (19.99%), Microbacterium (13.57%), Clavibacter (9.69%), Pantoea (6.59%), and Enterobacter (4.26%). The frequency of isolations of some bacteria - for example, the major pseudomonads (Pseudomonas syringae, Pseudomonas putida, and Pseudomonas savastanoi) or Pantoea agglomerans - tended to decline as fruit developed. As Pseudomonas declined, Curtobacterium became more dominant. Time of isolation was a significant factor in the frequency of occurrence of different bacteria, indicating succession of the genera. Throughput screening of the bacterial strains against Monilinia fructicola on nectarine fruit resulted in the detection of strains able to control brown rot. The 10 best-performing antagonistic strains were subjected to secondary screening. Four strains reduced decay severity by more than 50% (51.7%-91.4% reduction) at the high pathogen inoculum concentration of 105 conidia/mL.

Isolation and partial characterization of Bacillus subtilis ME488 for suppression of soilborne pathogens of cucumber and pepper.

Environmentally friendly control measures are needed for suppression of soilborne pathogens of vegetable crops in the Republic of Korea. In vitro challenge assays were used to screen approximately 500 bacterial isolates from 20 Korean greenhouse soils for inhibition of diverse plant pathogens. One isolate, Bacillus subtilis ME488, suppressed the growth of 39 of 42 plant pathogens tested. Isolate ME488 also suppressed the disease caused by Fusarium oxysporum f. sp. cucumerinum on cucumber and Phytophthora capsici on pepper in pot assays. Polymerase chain reaction was used to screen isolate ME488 for genes involved in biosynthesis of 11 antibiotics produced by various isolates of B. subtilis. Amplicons of the expected sizes were detected for bacD and bacAB, ituC and ituD, and mrsA and mrsM involved in the biosynthesis of bacilysin, iturin, and mersacidin, respectively. The identity of these genes was confirmed by DNA sequence analysis of the amplicons. Bacilysin and iturin were detected in culture filtrates from isolate ME488 by gas chromatography coupled with mass spectroscopy and by thin layer chromatography, respectively. Detection of mersacidin in ME488 culture filtrates was not attempted. Experiments reported here indicate that B. subtilis ME488 has potential for biological control of pathogens of cucumber and pepper possibly due to the production of antibiotics.

Role of sdhA and pfkA and catabolism of reduced carbon during colonization of cucumber roots by Enterobacter cloacae.

We have been using a mutational approach to determine how plant-beneficial bacteria such as Enterobacter cloacae 501R3 obtain carbon and energy for colonization of subterranean portions of cucumber and other plants. Reduced carbon detected in cucumber root exudate consisted of 73.3 % amino acids, 22.2 % organic acids and 4.4 % carbohydrate. Ent. cloacae M2, a mini-Tn5 Km transposon mutant of strain 501R3, was severely reduced in in vitro growth relative to strain 501R3 on the mixture of amino acids and organic acids detected in cucumber root exudate when these compounds were supplied as the sole source of carbon and energy, but was similar in growth on the mixture of carbohydrates detected in this exudate. Molecular and biochemical characterization of Ent. cloacae M2 indicated that the transposon was inserted in sdhA, which encodes a subunit of succinate dehydrogenase. Ent. cloacae A-11, a mutant of strain 501R3 with a mini-Tn5 Km insertion in pfkA, was severely reduced in in vitro growth relative to strain 501R3 on the mixture of carbohydrates detected in cucumber root exudate, but similar in growth on the mixture of amino acids and organic acids. When strains A-11 and M2 were coapplied with strain 501R3 to cucumber seeds above carrying capacity in competitive root colonization assays, populations of strains A-11 and M2 were roughly one order of magnitude lower than those of strain 501R3 in cucumber rhizosphere, while populations of strains A-11 and M2 were similar to one other when coapplied to cucumber seeds. When Ent. cloacae strains were coapplied to cucumber seeds below carrying capacity, populations of A-11 and M2 were roughly two to three orders of magnitude lower than those of 501R3 in cucumber rhizosphere, and populations of A-11 were significantly lower than those of M2 when these two strains were coapplied to cucumber seed. The experiments reported here indicate an important role for pfkA and sdhA and the catabolism of carbohydrates, and of amino acids and organic acids, respectively, in the colonization of cucumber roots by Ent. cloacae. The results reported here also indicate that catabolism of carbohydrates by this bacterium is more important than catabolism of amino acids and organic acids at lower population densities, despite the much higher relative quantities of amino acids and organic acids detected in cucumber root exudate.

Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities.

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E(var)) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E(var)). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.

Rapid and sensitive FAME analysis of bacteria by cold trap injection gas chromatography.

Whole cell fatty acid analysis is commonly used to identify bacteria. A cold trap, using a commercially available device that directs a stream of compressed air across a section of the GC column, is used to focus peaks at the head of the column. When combined with a rapid sample processing method that uses smaller volumes of solvents, it becomes possible to correctly identify bacteria from 1 to 2 mg of biomass.

A mutation in an exbD gene reduces tagetitoxin production by Pseudomonas syringae pv. tagetis.

A mutant of Pseudomonas syringae pv. tagetis EB037 with limited ability to produce tagetitoxin was isolated after transposon mutagenesis and the mutation was characterized. The mutation occurred in a gene with a high degree of sequence identity to exbD. exbD is contiguous with tonB and exbB upstream and with a gene for a TonB-dependent receptor downstream. Using reverse transcription - polymerase chain reaction with RNA from the wild-type and exbD mutant strains, we demonstrated that the mutation in exbD did not have a polar affect on the expression of downstream genes. The exbD mutant was able to grow well in conditions where iron is not freely available. Siderophore production by the exbD mutant was similar to that of the wild-type strain. We conclude that the mutation in exbD disrupts tagetitoxin production without compromising iron metabolism. The results indicate that tagetitoxin export by P. syringae pv. tagetis involves an efflux pump that requires a functional TonB system that is not essential for normal iron metabolism.

Phylum- and class-specific PCR primers for general microbial community analysis.

Amplification of a particular DNA fragment from a mixture of organisms by PCR is a common first step in methods of examining microbial community structure. The use of group-specific primers in community DNA profiling applications can provide enhanced sensitivity and phylogenetic detail compared to domain-specific primers. Other uses for group-specific primers include quantitative PCR and library screening. The purpose of the present study was to develop several primer sets targeting commonly occurring and important groups. Primers specific for the 16S ribosomal sequences of Alphaproteobacteria, Betaproteobacteria, Bacilli, Actinobacteria, and Planctomycetes and for parts of both the 18S ribosomal sequence and the internal transcribed spacer region of Basidiomycota were examined. Primers were tested by comparison to sequences in the ARB 2003 database, and chosen primers were further tested by cloning and sequencing from soil community DNA. Eighty-five to 100% of the sequences obtained from clone libraries were found to be placed with the groups intended as targets, demonstrating the specificity of the primers under field conditions. It will be important to reevaluate primers over time because of the continual growth of sequence databases and revision of microbial taxonomy.

Endophytic bacteria in Coffea arabica L.

Eighty-seven culturable endophytic bacterial isolates in 19 genera were obtained from coffee plants collected in Colombia (n = 67), Hawaii (n = 17), and Mexico (n = 3). Both Gram positive and Gram negative bacteria were isolated, with a greater percentage (68%) being Gram negative. Tissues yielding bacterial endophytes included adult plant leaves, various parts of the berry (e.g., crown, pulp, peduncle and seed), and leaves, stems, and roots of seedlings. Some of the bacteria also occurred as epiphytes. The highest number of bacteria among the berry tissues sampled was isolated from the seed, and includes Bacillus , Burkholderia , Clavibacter , Curtobacterium , Escherichia , Micrococcus , Pantoea , Pseudomonas , Serratia , and Stenotrophomonas . This is the first survey of the endophytic bacteria diversity in various coffee tissues, and the first study reporting endophytic bacteria in coffee seeds. The possible role for these bacteria in the biology of the coffee plant remains unknown.