Antifungal and sprout regulatory bioactivities of phenylacetic acid, indole-3-acetic acid, and tyrosol isolated from the potato dry rot suppressive bacterium Enterobacter cloacae S11:T:07.

Enterobacter cloacae S11: T:07 (NRRL B-21050) is a promising biological control agent that has significantly reduced both fungal dry rot disease and sprouting in laboratory and pilot potato storages. The metabolites phenylacetic acid (PAA), indole-3-acetic acid (IAA), and tyrosol (TSL) were isolated from S11:T:07 liquid cultures provided with three different growth media. The bioactivities of these metabolites were investigated via thin-layer chromatography bioautography of antifungal activity, wounded potato assays of dry rot suppressiveness, and cored potato eye assays of sprout inhibition. Relative accumulations of PAA, IAA, and TSL in cultures were nutrient dependent. For the first time, IAA, TSL, and PAA were shown to have antifungal activity against the dry rot causative pathogen Gibberella pulicaris, and to suppress dry rot infection of wounded potatoes. Disease suppression was optimal when all three metabolites were applied in combination. Dosages of IAA that resulted in disease suppression also resulted in sprout inhibition. These results suggest the potential for designing culture production and formulation conditions to achieve a dual purpose biological control agent able to suppress both dry rot and sprouting of stored potatoes.

Formulation of Bacillus spp. for Biological Control of Plant Diseases.

ABSTRACT Maximizing the potential for successfully developing and deploying a biocontrol product begins with a carefully crafted microbial screening procedure, proceeds with developing mass production protocols that optimize product quantity and quality, and ends with devising a product formulation that preserves shelf-life, aids product delivery, and enhances bioactivity. Microbial selection procedures that require prospective bio-control agents to possess both efficacy and amenability to production in liquid culture increase the likelihood of selecting agents with enhanced commercial development potential. Scale-up of biomass production procedures must optimize product quantity without compromise of product efficacy or amenability to stabilization and formulation. Formulation of Bacillus spp. for use against plant pathogens is an enormous topic in general terms but limited in published specifics regarding formulations used in commercially available products. Types of formulations include dry products such as wettable powders, dusts, and granules, and liquid products including cell suspensions in water, oils, and emulsions. Cells can also be microencapsulated. Considerations critical to designing successful formulations of microbial biomass are many fold and include preserving biomass viability during stabilization, drying, and rehydration; aiding biomass delivery, target coverage, and target adhesion; and enhancing biomass survival and efficacy after delivery to the target. Solutions to these formulation considerations will not necessarily be compatible. Data from several biocontrol systems including the use of B. subtilis OH 131.1 (NRRL B-30212) to reduce Fusarium head blight of wheat are used to illustrate many of these issues. Using our recently described assay for efficiently evaluating biomass production and formulation protocols, we demonstrate the effectiveness, in vitro, of UV protectant compounds lignin (PC 1307) and Blankophor BBH in reducing OH 131.1 morbidity when cells were exposed to UV light from artificial sunlight.

Greenhouse and Field Evaluation of Biological Control of Fusarium Head Blight on Durum Wheat.

Fusarium head blight (FHB) is a devastating disease that causes extensive yield and quality losses to wheat and barley. In durum wheat, the pathogen-produced toxin deoxynivalenol (DON) is retained in semolina at ˜50%, and the causal agent of FHB, Gibberella zeae, has a strong adverse effect on pasta color. Two bacteria and two yeast strains with known efficacy against G. zeae on hexaploid wheats were produced in liquid culture and assayed on two cultivars of durum wheat in greenhouse bioassays. All antagonists reduced FHB severity on cultivar Renville, and three of the four reduced severity on cultivar Ben, with Bacillus subtilis strain AS 43.3 decreasing FHB severity by as much as 90%. In separate greenhouse bioassays, the car-bon:nitrogen ratio of the medium used to produce antagonists did not consistently influence antagonist efficacy. All antagonist/production medium combinations but one were effective in reducing disease on both durum cultivars. Of six antagonists tested at field sites, Cryptococcus sp. OH 71.4 and C. nodaensis OH 182.9 reduced disease severity by as much as 57% in Peoria, IL, while Cryptococcus sp. OH 181.1 reduced disease severity by as much as 59% in a trial at Langdon, ND. Antagonists did not influence the DON content of grain in the Peoria trial. Relative performance indices for four antagonists calculated from greenhouse and field results on the two durum cultivars demonstrated that the bioassay location, but not the cultivar of durum, influenced the relative performance of antagonists. Yeast antagonists OH 71.4, OH 181.1, and OH 182.9 appear to have the highest potential for contributing to the reduction of FHB on du-rum wheat in the field.

Selection and Evaluation of Microorganisms for Biocontrol of Fusarium Head Blight of Wheat Incited by Gibberella zeae.

Gibberella zeae incites Fusarium head blight (FHB), a devastating disease that causes extensive yield and quality losses to wheat and barley. Of over 700 microbial strains obtained from wheat anthers, 54 were able to utilize tartaric acid as a carbon source when the compound was supplied as choline bitartrate in liquid culture. Four tartaric acid-utilizing and three nonutilizing strains reduced FHB in initial tests and were selected for further assays. Antagonists were effective against three different isolates of G. zeae when single wheat florets were inoculated with pathogen and antagonist inoculum. All seven antagonists increased 100-kernel weight when applied simultaneously with G. zeae isolate Z3639 (P ≤ 0.05). Bacillus strains AS 43.3 and AS 43.4 and Cryptococcus strain OH 182.9 reduced disease severity by >77, 93, and 56%, respectively. Five antagonists increased 100-kernel weight of plants inoculated with G. zeae isolate DAOM 180378. All antagonists except one increased 100-kernel weight, and four of seven antagonists reduced disease severity (P ≤ 0.05) when tested against G. zeae isolate Fg-9-96. In spray-inoculation experiments, Bacillus strains AS 43.3 and AS 43.4 and Cryptococcus strains OH 71.4 and OH 182.9 reduced disease severity, regardless of the sequence, timing, and concentration of inoculum application (P ≤ 0.05), though 100-kernel weight did not always increase when antagonists were applied 4 h after inoculum of G. zeae. Overall, 4 of 54 isolates that utilized tartaric acid in vitro were effective against G. zeae versus only 3 of 170 isolates tested that did not utilize tartaric acid (P ≤ 0.05, χ-square test of goodness of fit), demonstrating the potential benefit of prescreening candidate antagonists of FHB for their ability to utilize tartaric acid. Biological control shows promise as part of an integrated pest management program for managing FHB.

Isolation, identification, and accumulation of 2-acetamidophenol in liquid cultures of the wheat take-all biocontrol agent Pseudomonas fluorescens 2-79.

Pseudomonas fluorescens strain 2-79 (NRRL B-15132) is a classic biological control agent known to produce phenazine-l-carboxylic acid (PCA) as its primary means of suppressing take-all disease of wheat. In addition to PCA, an unknown metabolite was discovered in a liquid culture used to produce the biocontrol agent. The objective of the current study was to isolate, identify, and evaluate the accumulation of this compound in production cultures. Upon centrifugal fractionation of a production culture, thin-layer chromatography (TLC) analyses of extracts of the cells and cell-free supernatant indicated the compound to be primarily in the supernatant. Purified compound was obtained by extraction of culture supernatant, followed by flash chromatography of the extract and preparative TLC. The 'H and 13C nuclear magnetic resonance and electron impact mass spectra indicated the compound to be 2-acetamidophenol (AAP). Measured by reversed-phase HPLC, the accumulations of AAP and PCA in cultures of strain 2-79 reached 0.05 g/l and 1 g/l, respectively. The accumulations of AAP and PCA in liquid cultures were linearly correlated (P < 0.001), as shown by studies of cultures stimulated to yield varying levels of PCA by controlling levels of oxygen transfer, pH, and growth medium composition. In this study, oxygen limitation, a defined amino-acid-free medium, and neutral pH stimulated maximal production of both AAP and PCA. Furthermore, a transposon mutant of 2-79 [2A40 2-79 (phz-)] unable to produce PCA did not accumulate AAP. These findings indicate that AAP and PCA are likely to share a common segment of biosynthetic pathway. This is the first report of AAP production by a strain of P. fluorescens. Possible routes of AAP production are discussed relative to current knowledge of the phenazine biosynthetic pathway of strain 2-79. The pertinence of AAP to the design of commercial seed inoculants of phenazine-producing bacteria for controlling wheat take-all is also considered.

Effects of antagonist cell concentration and two-strain mixtures on biological control of fusarium dry rot of potatoes.

ABSTRACT Eighteen bacterial strains were individually assayed against Gibberella pulicaris (5 x 10(5) conidia per ml) by coinoculating antagonist and pathogen in wounds in cv. Russet Burbank potatoes. All antagonist concentrations (10(6), 10(7), and 10(8) CFU/ml) decreased disease (38 to 76% versus control, P < 0.05). When four strains were assayed at 11 concentrations (range 10(5) to 10(8) CFU/ml) against G. pulicaris, linear regression of the log-dose, log-response data was significant for all four strains (P < 0.001 to 0.01, R(2) = 0.50 to 0.74). Challenging G. pulicaris with all possible antagonist pairings within 2 sets of 10 antagonist strains (5 x 10(5) CFU of each strain per ml) resulted in 16 of 90 pairs controlling disease better than predicted based on averaging the performance of the individual strains making up the pair (P < 0.10). Successful pairs reduced disease by ~70% versus controls, a level of control comparable to that obtained with 100 times the inoculum dose of a single antagonist strain. Neither strain genus nor soil of origin were useful in predicting successful antagonist pairs. Factors potentially influencing dose-response relationships and the effectiveness of antagonist pairs in controlling disease are discussed.

Selection of Microorganisms for Biological Control of Silver Scurf (Helminthosporium solani) of Potato Tubers.

Few management strategies exist for silver scurf, an important postharvest disease of potatoes. In this study, the microbiota of 47 agricultural soils and 7 tuber samples was screened for biological control agents of silver scurf. Soil or periderm samples were transferred to separate samples of γ irradiation-sterilized field soil enriched with potato periderm. After incubation, the samples were assayed for biological suppressiveness to silver scurf using a whole-tuber/infested soil assay. Over 430 isolates of bacteria, yeasts, and actinomycetes were recovered from tubers and soil associated with the 12 most suppressive soil samples. Thirteen strains were selected for further study on three different strains of Helminthosporium solani, including one that was resistant to thiabendazole. Microbial strains that significantly inhibited H. solani (P ≤ 0.05) in at least one experiment were identified as Pseudomonas putida (PM1), Nocardia globerula (S244), and Xanthomonas campestris (P76). Colonization studies with rifampicin-resistant strains of putative biological control agents revealed that long-term colonization of the tuber surface was not necessary to reduce disease symptoms. Highly variable levels of conidiophore production prevented selection of the single most suppressive strain. Possible sources of variability in biological control are discussed, including physiological age of the tuber, tuber infection in the field, and uneven free moisture in the storage.

Pyrrolnitrin Production by Biological Control Agent Pseudomonas cepacia B37w in Culture and in Colonized Wounds of Potatoes.

Bacterial strain B37w (= NRRL B-14858), an isolate noteworthy because it inhibits the growth of the bioherbicide fungus Colletotrichum truncatum, was selected for further studies of bacterial antifungal properties. This isolate was identified as a Pseudomonas cepacia strain by performing carbohydrate utilization and fatty acid profile analyses, as well as other biochemical and physiological tests. Petri plate assays revealed that strain B37w exhibited antifungal activity against the potato dry rot fungus Fusarium sambucinum. Using bioautography, we correlated antifungal activity with production of a specific compound. Isolation from strain B37w and identification of the antifungal antibiotic pyrrolnitrin are described. A whole-potato assay revealed B37w's ability to colonize potato wounds. Wounded potatoes were inoculated with B37w, and pyrrolnitrin was detected in these potatoes by thin-layer chromatography-bioautography at a concentration on the order of nanograms per wound. We performed an assay in which we examined efficacy against F. sambucinum-incited potato dry rot and found that B37w inhibited disease development. This is the first report of P. cepacia or pyrrolnitrin activity against the economically important potato pathogen F. sambucinum.

Production of Colletotrichum truncatum for use as a mycoherbicide: effects of culture, drying and storage on recovery and efficacy.

Colletotrichum truncatum (Schwein.) Andrus and Moore NRRL 13737 (= NRRL 18434) is a fungal plant pathogen which shows promise as a bioherbicide against the troublesome weed Sesbania exaltata (Raf.) Rydb. ex A. W. Hill. Previous studies showed similar amounts of spores were produced/ml of medium in liquid and solid-state fermentations. In this study, Colletotrichum truncatum spores were produced in liquid (LC), solid/vermiculite (SV), and solid/perlite-cornmeal-agar (SP). After drying at room temperature with flowing air, SV and SP retained the most viability. Each product was then stored at 4 degrees, 15 degrees, and 25 degrees C. All three products stored at 4 degrees C and SP stored at 15 degrees C retained highest viability. Efficacy based upon assays utilizing equal numbers of viable spores showed SV and SP spores incited more severe disease symptoms than LC spores.

The Composition and Attributes of Colletotrichum truncatum Spores Are Altered by the Nutritional Environment.

Previous sporulation studies with Colletotrichum truncatum NRRL 13737, a fungal pathogen of the noxious weed Sesbania exaltata, showed that the carbon-to-nitrogen (CN) ratio of the conidiation medium influenced spore yield, morphology, and efficacy in inciting disease in S. exaltata. Spores produced in a medium with a CN ratio of 10:1 were more effective than were spores produced in a 30:1 or 80:1 ratio in causing disease in S. exaltata. With a basal salts medium supplemented with glucose and Casamino Acids, substrate utilization, spore production, biomass accumulation, and biomass and spore composition were compared in submerged cultures of C. truncatum grown in media with CN ratios of 80:1, 30:1, and 10:1. All cultures were sporulating by day 2, and spore concentrations in 5-day-old cultures were significantly different: 30:1 > 10:1 > 80:1. Amino acid and glucose utilization was balanced in cultures grown in media with a CN ratio of 10:1, whereas cultures grown in media with a CN ratio of 30:1 or 80:1 depleted amino acids prior to glucose. Conidia produced in media with a CN ratio of 10:1 contained significantly more protein (32% of dry weight) and less lipid (17% of dry weight) than conidia produced in media with a CN ratio of either 30:1 (15% protein, 33% lipid) or 80:1 (12% protein, 37% lipid). The higher lipid content of spores produced in media with a CN ratio of 30:1 or 80:1 was associated with the presence of increased numbers of lipid droplets. Optimization studies on conidia produced in media with CN ratios between 30:1 and 10:1 which compared yield, attributes, and efficacy in inciting disease in S. exaltata suggest that media with a CN ratio of 15:1 to 20:1 may be optimal for conidium production.