Quantitative Amplicon Sequencing Is Necessary to Identify Differential Taxa and Correlated Taxa Where Population Sizes Differ.

High-throughput, multiplexed-amplicon sequencing has become a core tool for understanding environmental microbiomes. As researchers have widely adopted sequencing, many open-source analysis pipelines have been developed to compare microbiomes using compositional analysis frameworks. However, there is increasing evidence that compositional analyses do not provide the information necessary to accurately interpret many community assembly processes. This is especially true when there are large gradients that drive distinct community assembly processes. Recently, sequencing has been combined with Q-PCR (among other sources of total quantitation) to generate "Quantitative Sequencing" (QSeq) data. QSeq more accurately estimates the true abundance of taxa, is a more reliable basis for inferring correlation, and, ultimately, can be more reliably related to environmental data to infer community assembly processes. In this paper, we use a combination of published data sets, synthesis, and empirical modeling to offer guidance for which contexts QSeq is advantageous. As little as 5% variation in total abundance among experimental groups resulted in more accurate inference by QSeq than compositional methods. Compositional methods for differential abundance and correlation unreliably detected patterns in abundance and covariance when there was greater than 20% variation in total abundance among experimental groups. Whether QSeq performs better for beta diversity analysis depends on the question being asked, and the analytic strategy (e.g., what distance metric is being used); for many questions and methods, QSeq and compositional analysis are equivalent for beta diversity analysis. QSeq is especially useful for taxon-specific analysis; QSeq transformation and analysis should be the default for answering taxon-specific questions of amplicon sequence data. Publicly available bioinformatics pipelines should incorporate support for QSeq transformation and analysis.

Cultivation and Genome Sequencing of Bacteria Isolated From the Coffee Berry Borer (Hypothenemus hampei), With Emphasis on the Role of Caffeine Degradation.

The coffee berry borer, the most economically important insect pest of coffee worldwide, is the only insect capable of feeding and reproducing solely on the coffee seed, a food source containing the purine alkaloid caffeine. Twenty-one bacterial species associated with coffee berry borers from Hawai'i, Mexico, or a laboratory colony in Maryland (Acinetobacter sp. S40, S54, S55, Bacillus aryabhattai, Delftia lacustris, Erwinia sp. S38, S43, S63, Klebsiella oxytoca, Ochrobactrum sp. S45, S46, Pantoea sp. S61, Pseudomonas aeruginosa, P. parafulva, and Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75) were found to have at least one of five caffeine N-demethylation genes (ndmA, ndmB, ndmC, ndmD, ndmE), with Pseudomonas spp. S31, S32, S37, S60 and P. parafulva having the full complement of these genes. Some of the bacteria carrying the ndm genes were detected in eggs, suggesting possible vertical transmission, while presence of caffeine-degrading bacteria in frass, e.g., P. parafulva (ndmABCDE) and Bacillus aryabhattai (ndmA) could result in horizontal transmission to all insect life stages. Thirty-five bacterial species associated with the insect (Acinetobacter sp. S40, S54, S55, B. aryabhattai, B. cereus group, Bacillus sp. S29, S70, S71, S72, S73, D. lacustris, Erwinia sp. S38, S43, S59, S63, K. oxytoca, Kosakonia cowanii, Ochrobactrum sp. S45, S46, Paenibacillus sp. S28, Pantoea sp. S61, S62, P. aeruginosa, P. parafulva, Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75, Stenotrophomonas sp. S39, S41, S48, S49) might contribute to caffeine breakdown using the C-8 oxidation pathway, based on presence of genes required for this pathway. It is possible that caffeine-degrading bacteria associated with the coffee berry borer originated as epiphytes and endophytes in the coffee plant microbiota.

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.

Complete genome sequence of the biocontrol agent Serratia marcescens strain N4-5 uncovers an assembly artefact.

Serratia marcescens are gram-negative bacteria found in several environmental niches, including the plant rhizosphere and patients in hospitals. Here, we present the genome of Serratia marcescens strain N4-5 (=NRRL B-65519), which has a size of 5,074,473 bp (664-fold coverage) and contains 4840 protein coding genes, 21 RNA genes, and an average G + C content of 59.7%. N4-5 harbours a plasmid of 11,089 bp and 43.5% G + C content that encodes six unique CDS repeated 2.5× times totalling 13 CDS. Our genome assembly and manual curation uncovered the insertion of two extra copies of the 5S rRNA gene in the assembled sequence, which was confirmed by PCR and Sanger sequencing to be a misassembly. This artefact was subsequently removed from the final assembly. The occurrence of extra copies of the 5S rRNA gene was also observed in most complete genomes of Serratia spp. deposited in public databases in our comparative analysis. These elements, which also occur naturally, can easily be confused with true genetic variation. Efforts to discover and correct assembly artefacts should be made in order to generate genome sequences that represent the biological truth underlying the studied organism. We present the genome of N4-5 and discuss genes potentially involved in biological control activity against plant pathogens and also the possible mechanisms responsible for the artefact we observed in our initial assembly. This report raises awareness about the extra copies of the 5S rRNA gene in sequenced bacterial genomes as they may represent misassemblies and therefore should be verified experimentally.

Dark septate endophyte improves salt tolerance of native and invasive lineages of Phragmites australis.

Fungal endophytes can improve plant tolerance to abiotic stress. However, the role of these plant-fungal interactions in invasive species ecology and their management implications remain unclear. This study characterized the fungal endophyte communities of native and invasive lineages of Phragmites australis and assessed the role of dark septate endophytes (DSE) in salt tolerance of this species. We used Illumina sequencing to characterize root fungal endophytes of contiguous stands of native and invasive P. australis along a salinity gradient. DSE colonization was assessed throughout the growing season in the field, and effects of fungal inoculation on salinity tolerance were investigated using laboratory and greenhouse studies. Native and invasive lineages had distinct fungal endophyte communities that shifted across the salinity gradient. DSE colonization was greater in the invasive lineage and increased with salinity. Laboratory studies showed that DSE inoculation increased P. australis seedling survival under salt stress; and a greenhouse assay revealed that the invasive lineage had higher aboveground biomass under mesohaline conditions when inoculated with a DSE. We observed that P. australis can establish mutualistic associations with DSE when subjected to salt stress. This type of plant-fungal association merits further investigation in integrated management strategies of invasive species and restoration of native Phragmites.

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.

Glyphosate Resistance Technology Has Minimal or No Effect on Maize Mineral Content and Yield.

Controversy continues to exist regarding whether the transgene for glyphosate resistance (GR) and/or glyphosate applied to GR crops adversely affect plant mineral content. Field studies were conducted in 2013 and 2014 in Stoneville, MS and Urbana, IL to examine this issue in maize. At each location, the experiment was conducted in fields with no history of glyphosate application and fields with several years of glyphosate use preceding the study. Neither glyphosate nor the GR transgene affected yield or mineral content of leaves or seed, except for occasional (<5%) significant effects that were inconsistent across minerals, treatments, and environments. Glyphosate and AMPA (aminomethylphosphonic acid), a main degradation product of glyphosate, were found in leaves from treated plants, but little or no glyphosate and no AMPA was found in maize seeds. These results show that the GR transgene and glyphosate application, whether used for a single year or several years, have no deleterious effect on mineral nutrition or yield of GR maize.

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.

Lack of transgene and glyphosate effects on yield, and mineral and amino acid content of glyphosate-resistant soybean.

BACKGROUND: There has been controversy as to whether the glyphosate resistance gene and/or glyphosate applied to glyphosate-resistant (GR) soybean affect the content of cationic minerals (especially Mg, Mn and Fe), yield and amino acid content of GR soybean. A two-year field study (2013 and 2014) examined these questions at sites in Mississippi, USA. RESULTS: There were no effects of glyphosate, the GR transgene or field crop history (for a field with both no history of glyphosate use versus one with a long history of glyphosate use) on grain yield. Furthermore, these factors had no consistent effects on measured mineral (Al, As, Ba, Cd, Ca, Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Ni, Pb, Rb, Se, Sr, Tl, U, V, Zn) content of leaves or harvested seed. Effects on minerals were small and inconsistent between years, treatments and mineral, and appeared to be random false positives. No notable effects on free or protein amino acids of the seed were measured, although glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), were found in the seed in concentrations consistent with previous studies. CONCLUSIONS: Neither glyphosate nor the GR transgene affect the content of the minerals measured in leaves and seed, harvested seed amino acid composition, or yield of GR soybean. Furthermore, soils with a legacy of GR crops have no effects on these parameters in soybean. © 2017 Society of Chemical Industry.

Characterization of mechanisms underlying degradation of sclerotia of Sclerotinia sclerotiorum by Aspergillus aculeatus Asp-4 using a combined qRT-PCR and proteomic approach.

BACKGROUND: The biological control agent Aspergillus aculeatus Asp-4 colonizes and degrades sclerotia of Sclerotinia sclerotiorum resulting in reduced germination and disease caused by this important plant pathogen. Molecular mechanisms of mycoparasites underlying colonization, degradation, and reduction of germination of sclerotia of this and other important plant pathogens remain poorly understood. RESULTS: An RNA-Seq screen of Asp-4 growing on autoclaved, ground sclerotia of S. sclerotiorum for 48 h identified 997 up-regulated and 777 down-regulated genes relative to this mycoparasite growing on potato dextrose agar (PDA) for 48 h. qRT-PCR time course experiments characterized expression dynamics of select genes encoding enzymes functioning in degradation of sclerotial components and management of environmental conditions, including environmental stress. This analysis suggested co-temporal up-regulation of genes functioning in these two processes. Proteomic analysis of Asp-4 growing on this sclerotial material for 48 h identified 26 up-regulated and 6 down-regulated proteins relative to the PDA control. Certain proteins with increased abundance had putative functions in degradation of polymeric components of sclerotia and the mitigation of environmental stress. CONCLUSIONS: Our results suggest co-temporal up-regulation of genes involved in degradation of sclerotial compounds and mitigation of environmental stress. This study furthers the analysis of mycoparasitism of sclerotial pathogens by providing the basis for molecular characterization of a previously uncharacterized mycoparasite-sclerotial interaction.

Managing the plant microbiome for biocontrol fungi: examples from Hypocreales.

Feeding an increasing global population requires continued improvements in agricultural efficiency and productivity. Meeting estimated future production levels requires the adoption of practices that increase output without environmental degradation associated with external inputs to supplement nutrition or control pests. Enriching the community of microbes associated with plants in agricultural systems for those providing ecosystem services such as pest control is one possible component towards achieving sustainable productivity increases. In this review we explore the current state of knowledge for Hypocreales fungi used in biological control. Advances in understanding the field ecology, diversity and genetic determinants of host range and virulence of hypocrealean fungi provide the means to improve their efficacy.

A Novel Approach to Estimating Nitrous Oxide Emissions during Wetting Events from Single-Timepoint Flux Measurements.

Precipitation and irrigation induce pulses of NO emissions in agricultural soils, but the magnitude, duration, and timing of these pulses remain uncertain. This uncertainty makes it difficult to accurately extrapolate emissions from unmeasured time periods between chamber sampling events. Therefore, we developed a modeling protocol to predict NO emissions from data collected daily for 7 d after wetting events. Within a cover crop-based corn ( L.) production system in Beltsville, MD, we conducted the 7-d time series during four time periods representing a range of corn growth stages in 2013 and 2014. Treatments included mixtures and monocultures of grass and legume cover crops that were fertilized with pelletized poultry litter or urea-ammonium nitrate solution (9-276 kg N ha). Most fluxes did not exhibit the expected exponential decay over time (82%); therefore, cumulative emissions were calculated using trapezoidal integration over 7 d after the wetting event. Cumulative 7-d emissions were well correlated with single point gas fluxes on the second day after a wetting event using a generalized linear mixed model (ln[emissions] = 0.809∙ln[flux] + 2.47). Soil chemical covariates before or after a wetting event were weakly associated with cumulative emissions. The ratio of dissolved organic C to total inorganic N was negatively correlated with cumulative emissions ( = 0.23-0.29), whereas nitrate was positively correlated with cumulative emissions ( = 0.23-0.33). Our model is an innovative approach that is calibrated using site-specific time series data, which may then be used to estimate short-term NO emissions after wetting events using only a single flux measurement.

Seed Treatment with Ethanol Extract of Serratia marcescens is Compatible with Trichoderma Isolates for Control of Damping-off of Cucumber Caused by Pythium ultimum.

Environmentally friendly control measures for soilborne plant pathogens are needed that are effective in different soils when applied alone or as components of an integrated disease control strategy. An ethanol extract of Serratia marcescens N4-5, when applied as a cucumber seed treatment, effectively suppressed damping-off caused by Pythium ultimum in potting mix and in a sandy loam soil. Plant stand associated with this treatment was similar to that of seed treated with the chemical pesticide Thiram in the sandy loam soil. The N4-5 ethanol extract did not consistently provide significant disease control in a loam soil. The N4-5 ethanol extract was compatible with two Trichoderma isolates, not affecting in vitro or in situ colonization of cucumber by these biological control fungi. Control of damping-off of cucumber was never diminished when this ethanol extract was applied as a seed treatment in combination with in-furrow application of the Trichoderma isolates, and disease control was improved in certain instances with these combinations in the loam soil. Data presented here indicate that the N4-5 ethanol extract is compatible with certain beneficial fungi, suggesting that this extract can be used as a component of integrated disease control strategies featuring biological control fungi.

Components of a Rice-Oilseed Rape Production System Augmented with Trichoderma sp. Tri-1 Control Sclerotinia sclerotiorum on Oilseed Rape.

Sclerotinia sclerotiorum causes serious yield losses on many crops throughout the world. A multicomponent treatment that consisted of the residual rice straw remaining after rice harvest and Trichoderma sp. Tri-1 (Tri-1) formulated with the oilseed rape seedcake fertilizer was used in field soil infested with S. sclerotiorum. This treatment resulted in oilseed rape seed yield that was significantly greater than the nontreated control or when the fungicide carbendizem was used in the presence of this pathogen in field trials. Yield data suggested that the rice straw, oilseed rape seedcake, and Tri-1 components of this treatment all contributed incrementally. Similar treatment results were obtained regarding reduction in disease incidence. Slight improvements in yield and disease incidence were detected when this multicomponent treatment was combined with a fungicide spray. Inhibition of sclerotial germination by this multicomponent treatment trended greater than the nontreated control at 90, 120, and 150 days in field studies but was not significantly different from this control. This multicomponent treatment resulted in increased yield relative to the nontreated control in the absence of pathogen in a greenhouse pot study, while the straw alone and the straw plus oilseed rape seedcake treatments did not; suggesting that Tri-1 was capable of promoting growth. Experiments reported here indicate that a treatment containing components of a rice-oilseed rape production system augmented with Tri-1 can control S. sclerotiorum on oilseed rape, be used in integrated strategies containing fungicide sprays for control of this pathogen, and promote plant growth.

Microbial rRNA:rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils.

We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communities. 'Activity ratios' were calculated for bacteria and archaea in soil sampled from a tropical rainforest and temperate agricultural field and incubated for one year at two levels of moisture availability and with and without carbon additions. Prior to calculating activity ratios, we corrected the relative abundances of OTUs to account for multiple copies of the 16S gene per genome. Although necessary to ensure accurate activity ratios, this correction did not change our interpretation of differences in microbial community composition across treatments. Activity ratios in this study were lower than those previously published (0.0003-210, logarithmic mean=0.24), suggesting significant extracellular DNA preservation. After controlling for the influence of individual incubation jars, significant differences in activity ratios between all members of each phylum were observed. Planctomycetes and Firmicutes had the highest activity ratios and Crenarchaeota had the lowest activity overall. Our results suggest that greater caution should be taken in interpreting soil microbial community data derived from extracted DNA. Indirect extraction methods may be useful in ensuring that microbes identified from extracellular DNA are not erroneously interpreted as components of an active microbial community.

Community composition and population genetics of insect pathogenic fungi in the genus Metarhizium from soils of a long-term agricultural research system.

Fungi in the genus Metarhizium are insect pathogens able to function in other niches, including soil and plant rhizosphere habitats. In agroecosystems, cropping and tillage practices influence soil fungal communities with unknown effects on the distribution of Metarhizium, whose presence can reduce populations of crop pests. We report results from a selective media survey of Metarhizium in soils sampled from a long-term experimental farming project in the mid-Atlantic region. Field plots under soybean cultivation produced higher numbers of Metarhizium colony-forming units (cfu) than corn or alfalfa. Plots managed organically and via chisel-till harboured higher numbers of Metarhizium cfu than no-till plots. Sequence typing of Metarhizium isolates revealed four species, with M. robertsii and M. brunneum predominating. The M. brunneum population was essentially fixed for a single clone as determined by multilocus microsatellite genotyping. In contrast, M. robertsii was found to contain significant diversity, with the majority of isolates distributed between two principal clades. Evidence for recombination was observed only in the most abundant clade. These findings illuminate multiple levels of Metarhizium diversity that can be used to inform strategies by which soil Metarhizium populations may be manipulated to exert downward pressure on pest insects and promote plant health.

Development of a biologically based fertilizer, incorporating Bacillus megaterium A6, for improved phosphorus nutrition of oilseed rape.

Sustainable methods with diminished impact on the environment need to be developed for the production of oilseed rape in China and other regions of the world. A biological fertilizer consisting of Bacillus megaterium A6 cultured on oilseed rape meal improved oilseed rape seed yield (P < 0.0001) relative to the nontreated control in 2 greenhouse pot experiments using natural soil. This treatment resulted in slightly greater yield than oilseed rape meal without strain A6 in 1 of 2 experiments, suggesting a role for strain A6 in improving yield. Strain A6 was capable of solubilizing phosphorus from rock phosphate in liquid culture and produced enzymes capable of mineralizing organic phosphorus (acid phosphatase, phytase) in liquid culture and in the biological fertilizer. The biologically based fertilizer, containing strain A6, improved plant phosphorus nutrition in greenhouse pot experiments resulting in significantly greater available phosphorus in natural soil and in significantly greater plant phosphorus content relative to the nontreated control. Seed yield and available phosphorus in natural soil were significantly greater with a synthetic chemical fertilizer treatment, reduced in phosphorus content, than the biological fertilizer treatment, but a treatment containing the biological fertilizer combined with the synthetic fertilizer provided the significantly greatest seed yield, available phosphorus in natural soil, and plant phosphorus content. These results suggest that the biological fertilizer was capable of improving oilseed rape seed yield, at least in part, through the phosphorus-solubilizing activity of B. megaterium A6.

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.

Selection of genetically diverse Trichoderma spp. isolates for suppression of Phytophthora capsici on bell pepper.

Environmentally compatible control measures are needed for suppression of Phytophthora capsici on pepper. Twenty-three isolates of Trichoderma were screened for suppression of a mixture of 4 genetically distinct isolates of this pathogen on bell pepper (Capsicum anuum) in greenhouse pot assays. Of these 23 isolates, GL12, GL13, and Th23 provided significant suppression of P. capsici in at least 2 assays. These isolates were then compared with Trichoderma virens isolates GL3 and GL21 for suppression of this disease in the presence and absence of the harpin-based natural product Messenger. Isolates GL3 and Th23 provided significant disease suppression (P ≤ 0.05) in 3 of 4 assays, while GL12, GL13, and GL21 provided significant suppression in 2 of 4 assays. There was no apparent benefit from the application of Messenger. Phylogenetic analysis of these 5 isolates (based on the ITS1 region of the nuclear rDNA cluster and tef1), and an additional 9 isolates that suppressed P. capsici in at least 1 assay, separated isolates into 2 clades, with 1 clade containing GL3, GL12, GL13, and GL21. There were also 2 more distantly related isolates, one of which was Th23. We report here the identification of genetically distinct Trichoderma isolates for potential use in disease management strategies employing isolate combinations directed at suppression of P. capsici on pepper.