Survival and Growth of Wild-Type and rpoS-Deficient Salmonella Newport Strains in Soil Extracts Prepared with Heat-Treated Poultry Pellets

ABSTRACT Manure runoff can transfer pathogens to farmlands or to water sources, leading to subsequent contamination of produce. Untreated biological soil amendments, like manure, can be contaminated with foodborne pathogens, such as Salmonella Newport, which may lead to transfer of the pathogen to fruits or vegetables. Studies have reported the occurrence and survival of Salmonella in manure or manure slurries. However, data on the survival and growth of Salmonella Newport is lacking in matrices simulating runoff. We quantified the survival and growth of wild-type (WT) Salmonella Newport and rpoS-deficient (ΔrpoS) strains in sterile and nonsterile soil extracts prepared with (amended) or without (unamended) heat-treated poultry pellets at 25°C. Salmonella Newport WT and ΔrpoS populations reached a maximum cell density of 6 to 8 log CFU/mL in 24 to 30 h in amended and unamended soil extracts and remained in stationary phase for up to 4 days. Salmonella Newport in amended soil extracts exhibited a decreased lag phase (λ, 2.87 ± 1.01 h) and greater maximum cell densities (Nmax, 6.84 ± 1.25 CFU/mL) compared with λ (20.10 ± 9.53 h) and Nmax (5.22 ± 0.82 CFU/mL) in unamended soil extracts. In amended soil extract, the ΔrpoS strain had no measurable λ, similar growth rates (μmax) compared with WT, and a lower Nmax compared with the WT strain. Unamended, nonsterile soil extracts did not support the growth of Salmonella Newport WT and led to a decline in populations for the ΔrpoS strain. Salmonella Newport had lower cell densities in nonsterile soil extracts (5.94 ± 0.95 CFU/mL) than it did in sterile soil extracts (6.66 ± 1.50 CFU/mL), potentially indicating competition for nutrients between indigenous microbes and Salmonella Newport. The most favorable growth conditions were provided by amended sterile and nonsterile soil extracts, followed by sterile, unamended soil extracts for both Salmonella Newport strains. Salmonella Newport may grow to greater densities in amended extracts, providing a route for increased Salmonella levels in the growing environments of produce.

Evaluation of Bacillus Strains for Plant Growth Promotion and Predictability of Efficacy by In Vitro Physiological Traits

Bacilli are commonly used as plant growth-promoting agents but can be limited in effectiveness to certain crop and soil environments. The objectives of this study were to (1) identify Bacillus strains that can be consistent in promoting the growth of corn, wheat, and soybean and (2) determine whether physiological traits expressed in vitro can be predictive of growth promotion efficacy/consistency and be used for selecting effective strains. Twelve Bacillus strains isolated from wheat rhizospheres were evaluated in greenhouse pot tests with nonsterile soil for their effects on the growth of corn, soybean, and wheat. The strains also were assessed in vitro for multiple physiological traits. All 12 strains increased corn growth significantly compared to the controls. The four most efficacious strains on corn—Bacillus megaterium R181, B. safensis R173, B. simplex R180, and Paenibacillus graminis R200—also increased the growth of soybean and wheat. No set of traits was a predictor of growth promotion efficacy. The number of traits expressed by a strain also was not an indicator of efficacy as strain R200 that was positive for only one trait showed high growth promotion efficacy. Effective strains can be identified through pot tests on multiple crop plants, but in vitro physiological assays are unreliable for strain selection.

Interactions between Glyphosate,FusariumInfection of Common Waterhemp (Amaranthus rudis), and Soil Microbial Abundance and Diversity in Soil Collections from Missouri

Greenhouse and laboratory experiments were conducted on common waterhemp and soil collected from 131 soybean fields in Missouri that contained late-season common waterhemp escapes. The objectives of these experiments were to determine the effects of soil sterilization on glyphosate-resistant (GR) and -susceptible (GS) common waterhemp survival, to determine the effects of soil sterilization and glyphosate treatment on infection of GR and GS common waterhemp biotypes byFusariumspp., and to determine the soil microbial abundance and diversity in soils collected from soybean fields with differences in common waterhemp biotypes and herbicide and crop rotation histories. Common waterhemp biotypes were treated with 1.7 kg glyphosate ae ha−1or left untreated once plants reached approximately 15 cm in height. Common waterhemp survival was visually assessed at 21 d after glyphosate treatment (21 DAT). To determineFusariuminfection frequency, a single intact common waterhemp root was harvested from each treatment at 0, 3, 7, 14, and 21 DAT and surface sterilized, and 10 to 15–mm common waterhemp root sections were plated on Komada culture medium. After 14 d incubation, fungal colonies were selected from colonized roots and maintained on potato dextrose agar medium amended with antibiotics before identification. Speciation ofFusariumisolates was conducted through microscopic examination of fungal characters and confirmed by sequencing and analysis of ribosomal DNA. Soil samples from 131 different collections were subjected to phospholipid fatty acid (PLFA) analysis and were conducted utilizing gas chromatography to determine the soil microbial community abundance and structure. Common waterhemp plants grown in sterile soils had the highest common waterhemp survival, regardless of biotype. After treatment with glyphosate, survival of GS common waterhemp grown in nonsterile soil was only 29% 21 DAT, whereas survival of GS common waterhemp grown in nonsterile soil was only 10%. Similarly, GR common waterhemp survival was reduced from 83 to 61% following treatment with glyphosate when grown in nonsterile compared to sterile soil.Fusariumspp. were recovered from only 12% of the assayed roots (223 treatments withFusariumout of a total 1,920 treatments). The greatest occurrence ofFusariumroot infection in both GR and GS common waterhemp occurred in nonsterile soils following a glyphosate treatment. Few differences in total PLFA were observed in field soil collected from locations with either GR or GS common waterhemp, and regardless of herbicide or crop history. This research supports previous findings that plant species are more sensitive to glyphosate in nonsterile than sterile soils and indicates glyphosate may predispose plants to soil-borne phytopathogens. This research also suggests that continuous use of glyphosate does not significantly affect soil microbial abundance or diversity.

Sexual Reproduction in Aspergillus flavus Sclerotia Naturally Produced in Corn

Aspergillus flavus is the major producer of carcinogenic aflatoxins worldwide in crops. Populations of A. flavus are characterized by high genetic variation and the source of this variation is likely sexual reproduction. The fungus is heterothallic and laboratory crosses produce ascospore-bearing ascocarps embedded within sclerotia. However, the capacity for sexual reproduction in sclerotia naturally formed in crops has not been examined. Corn was grown for 3 years under different levels of drought stress at Shellman, GA, and sclerotia were recovered from 146 ears (0.6% of ears). Sclerotia of A. flavus L strain were dominant in 2010 and 2011 and sclerotia of A. flavus S strain were dominant in 2012. The incidence of S strain sclerotia in corn ears increased with decreasing water availability. Ascocarps were not detected in sclerotia at harvest but incubation of sclerotia on the surface of nonsterile soil in the laboratory resulted in the formation of viable ascospores in A. flavus L and S strains and in homothallic A. alliaceus. Ascospores were produced by section Flavi species in 6.1% of the 6,022 sclerotia (18 of 84 ears) in 2010, 0.1% of the 2,846 sclerotia (3 of 36 ears) in 2011, and 0.5% of the 3,106 sclerotia (5 of 26 ears) in 2012. For sexual reproduction to occur under field conditions, sclerotia may require an additional incubation period on soil following dispersal at crop harvest.

The effect of selected fungicides on survival of Agrobacterium tumefaciens in various kinds of soils

The effect of 10 fungicides on survival of A. tumefaciens in various types of soils was studied. In fertile, nonsterile soil Dithane M-45 (mancozeb), Euparen 50 WP (tolyfluanid), Kaptan 50 WP (captan) and Ridomil Gold 80 WP (metalaxyl) at concentration of 1000 ppm showed the highest antibacterial activity. Similar trends in activity of these fungicides occurred in fertile, sterile soil, however a little lower in case of Kaptan and Euparen. In most of investigated soils Befran 25 SL (imimnoctadyne), Syllit 65 WP (dodine) and Thiram Granulfo 80 WG (thiram) increased bacteria number. In sandy acidic soil (pH 3,5) all tested fungicides totally eliminated bacteria. On the other hand in sandy neutral soil only Dithane, Euparen, Kaptan and Ridomil showed such activity. Ten fold decrease of fungicides concentration generally did not influence Kaptan and Ridomil effectiveness but it decreased the activity of Dithane and Euparen.

Use of Green Fluorescent Protein and Image Analysis to Quantify Proliferation of Trichoderma harzianum in Nonsterile Soil

One drawback of traditional methods for fungal biomass measurement is the inability to distinguish biomass of an introduced fungus from that of the indigenous microbial community in nonsterile soil. We quantified biomass of a specific fungal biological control agent in nonsterile soil using epifluorescence microscopy and image analysis of green fluorescent protein (GFP)-expressing Trichoderma harzianum (ThzID1-M3). Numbers of colony forming units on a semiselective medium were compared with biomass estimates from image analysis, after ThzID1-M3 was incubated in soil that either remained moist (-0.05 MPa) for 14 to 21 days or remained moist for approximately 5 days and then was allowed to dry to <-3.0 MPa. Recovery of significant numbers of ThzID1-M3 propagules lagged approximately 3 days behind initiation of hyphal growth. Reductions in both colony counts and biomass were observed over time when soil was allowed to dry. However, in soil that remained moist, colony counts increased over a 14- to 21-day period even though biomass declined after approximately 3 to 5 days. Our results confirm that use of GFP, along with epifluorescence microscopy, is a useful tool to distinguish active hyphal biomass, the form of the fungus that is functional for biological control, from inactive propagules such as conidia or chlamydospores that are enumerated by plate counts.

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

ABSTRACT The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g−1) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ∼2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.

Inactivation of the Regulatory Gene algU or gacA Can Affect the Ability of Biocontrol Pseudomonas fluorescens CHA0 To Persist as Culturable Cells in Nonsterile Soil

ABSTRACT Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor σE) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil.