Population dynamics of the Fusarium head blight biocontrol agent Cryptococcus flavescens OH 182.9 on wheat anthers and heads

Biological Control 70 (2014) 17–27

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Population dynamics of the Fusarium head blight biocontrol agent Cryptococcus flavescens OH 182.9 on wheat anthers and heads David A. Schisler a,⇑, Amanda B. Core b, Michael J. Boehm b, Leona Horst c, Charles Krause c, Christopher A. Dunlap a, Alejandro P. Rooney a a b c

National Center for Agricultural Utilization Research (NCAUR), United States Department of Agriculture–Agricultural Research Service (USDA-ARS), Peoria, IL 61604, USA Department of Plant Pathology, Ohio State University (OSU), Columbus, OH 43210, USA Application Technology Research Unit, USDA-ARS, Wooster, OH 44691, USA

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 C. flavescens OH 182.9 colonized

anthers inside wheat florets prior to flowering.  Populations of OH 182.9 on anthers often increased irrespective of inoculation time.  Higher OH 182.9 population on spikelets when inoculate at flowering versus split boot.  SEM found OH 182.9 often on abaxial surfaces of glume and lemma and apex of palea.

a r t i c l e

i n f o

Article history: Received 19 March 2013 Accepted 29 November 2013 Available online 5 December 2013 Keywords: Colonization Spikelet Floret Anther Fusarium head blight Biocontrol

a b s t r a c t Cryptococcus flavescens OH 182.9 (NRRL Y-30216) reduces Fusarium head blight (FHB) incited by Fusarium graminearum and deoxynivalenol (DON) contamination of grain. Yet little is known about the population dynamics of OH 182.9 on wheat heads and anthers. Biomass of OH 182.9 was produced in liquid culture and applied to greenhouse and field grown wheat prior to and during early anthesis. In greenhouse studies, populations of OH 182.9 were similar on anthers for heads inoculated before (Feekes 10.5) or early in flowering (Feekes 10.5.1) but were 1–3 log units lower in Feekes 10.5 inoculated wheat after 8–10 days. In greenhouse and field studies, OH 182.9 colonized anthers inside florets prior to anthesis. In the field, populations of OH 182.9 on anthers increased or, less frequently, remained stable through 12 days, regardless of application time and peaked at 1–2 log units higher than in the greenhouse. Strain OH 182.9 reduced FHB severity (P < 0.05, FPLSD) but not other disease parameters in the same field study. Application of OH 182.9 at split boot (Feekes 10.1) or Feekes 10.5.1 resulted in higher populations on spikelets treated at flowering on a CFU/g fresh weight tissue basis and as a percentage of the total recoverable microbial population in one of two field studies. Scanning electron microscopy revealed cells of OH 182.9 in microcolonies, groups of several cells and as individual cells, most frequently on the abaxial surfaces of glume and lemma tissues and near the apex of palea tissues. The survival of yeast OH 182.9 on anthers and wheat heads for 12 days and more suggests the strain has the potential to reduce late kernel infections by F. graminearum that can increase DON. Published by Elsevier Inc.

1. Introduction Abbreviations: DPI, days post inoculation; TSBA/5, one fifth strength Tryptic soy broth agar; DON, deoxynivalenol. ⇑ Corresponding author. Address: 1815 N. University Street, NCAUR, USDA-ARS, Peoria, IL 61604, USA E-mail address: [email protected] (D.A. Schisler). 1049-9644/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.biocontrol.2013.11.011

Fusarium head blight (FHB), also known as scab of wheat, is caused by Fusarium graminearum Schwabe (teleomorph of Gibberella zeae (Schwein.) Petch). FHB is a devastating disease of wheat

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and barley worldwide (Bai and Shaner, 1994; McMullen et al., 2012). The disease was observed first in North America in 1884 (Arthur, 1891) and in 1909 G. zeae was identified as the causal agent of FHB on wheat (Selby and Manns, 1909). Although FHB has been recognized for more than 100 years, it has reemerged as a major and chronic problem only recently (McMullen et al., 2012). Infection of wheat heads by F. graminearum reduces yield and can contaminate kernels with deoxynivalenol (DON), a trichothecene mycotoxin that reduces the market value of grain (Pirgozliev et al., 2003; Snijders, 1990) and can harm animals that consume contaminated grain (Awad et al., 2006; Rocha et al., 2005). While disease forecasting (Kriss et al., 2010; Prandini et al., 2009), resistant varieties (Sato et al., 2008; Skinnes et al., 2010), cultural controls (Dill-Macky and Jones, 2000; Guo et al., 2010; Lori et al., 2009), fungicides (Beyer et al., 2006; Paul et al., 2007, 2010) and biocontrol agents (Jochum et al., 2006; Khan and Doohan, 2009; Khan et al., 2004; Schisler et al., 2002; Xue et al., 2009a,b) are useful in reducing FHB in the field, control of the disease remains an intractable problem. The basidiomycetous yeast genus Cryptococcus contains several strains of biological control agents that are commonly associated with aerial plant parts (Fonseca et al., 2011; Perelló et al., 2002; Pusey et al., 2009; Schisler et al., 2011a; Sláviková et al., 2009). Cryptococcus flavescens OH 182.9 (NRRL Y-30216) was originally isolated from the anthers of flowering wheat heads (Khan et al., 2001) and reduces FHB and DON in greenhouse and field trials (Khan et al., 2004; Schisler et al., 2002) when assayed alone or in combination with other biocontrol agents (Kolombet et al., 2005; Yuen et al., 2010). Challenges remain regarding the use of strain OH182.9 to effectively manage FHB. For example, although consistent reductions in disease severity have been documented via application of OH 182.9 to wheat at Feekes growth stage 10.5.1 (beginning of flowering, (Large, 1954)) little is known about the population dynamics of this biological control agent on wheat heads and anthers. Likewise, information regarding whether the growth stage of wheat at the time of application influences the survival and efficacy of OH 182.9 is lacking. Wheat heads are vulnerable to F. graminearum infection and DON development in kernels from the beginning of flowering (Feekes 10.5.1) until soft dough (Feekes 11.2) development (Yoshida and Nakajima, 2010) yet minimum preharvest intervals for fungicide use restricts applications after wheat flowering. Determining the colonization dynamics of OH 182.9 on wheat heads and anthers would be especially useful in understanding if the strain has the potential to be active on infection courts throughout the period of head susceptibility to FHB. The goal of this study was to explore the population dynamics of OH 182.9 on wheat anthers and heads when applied at different stages of wheat head development including split boot (Feekes 10.1), heading complete (Feekes 10.5) and the beginning of flowering (Feekes 10.5.1) using traditional techniques for quantifying microbial populations supported by direct observation using scanning electron microscopy. In experiments where field conditions were suitable for FHB disease development, antagonist populations associated with the level of FHB severity and DON in harvested kernels were determined.

2. Materials and methods 2.1. Selection of cycloheximide-tolerant variant of antagonist C. flavescens OH 182.9

of the strain was isolated. A pure culture of C. flavescens OH 182.9 (NRRL Y-30216) was initiated on 1/5 strength Tryptic soy broth agar (TSBA/5) (Difco Laboratories, Detroit, MI) by transferring cells from 10% glycerol stocks stored at 80 °C. After 24 h, cells were transferred from plates using sterile cotton swabs to 10 ml of 1/5 strength Tryptic soy broth (TSB/5) containing 50 ppm cycloheximide (Sigma–Aldrich, St. Louis, MO) in 50 ml Erylenmeyer flasks (optical density (OD) of 0.1 at 620 nm wavelength light (A620) or 3  106 CFU/ml). Flasks were incubated in an shaker incubator for 5 days at 25 °C (250 rpm, eccentricity = 2.5 cm; Inova 4230, New Brunswick Scientific, Edison, NJ), and colonized broth were used to inoculate fresh TSB/5 broth containing 50 ppm cycloheximide for two additional growth cycles. Colonized broth was then serially diluted onto TSBA/5 with 100 ppm cycloheximide and resultant colonies compared in size with those of the progenitor strain plated on the same media. Cycloheximide tolerant variant C100R1 was selected for experimental use based on superior growth on TSBA/5 + 100 ppm cycloheximide and similar growth on TSBA/5 compared to the wild type progenitor strain. 2.2. Confirmation of cycloheximide tolerant variant OH 182.9 C100R1 identification To ensure that a species other than C. flavescens was not isolated during the selection of the variant, a phyllogenetic analysis of C100R1 and the progenitor wild type strain was conducted and results compared. The nucleotide sequence of the divergent domain (d1/d2) at the distal end of the 26S ribosomal RNA (rRNA) gene was obtained. Each polymerase chain reaction (PCR) consisted of the following reagent concentrations: 0.5 unit of Amplitaq DNA Polymerase (Invitrogen Life Technologies, Carlsbad, CA), 2.5 mM MgCl2, 200 lM dNTPs, 0.5 lM each of oligonucleotide primers NL1 and NL-4 (O’Donnell, 1993), 1X reaction buffer, and 50–100 ng template DNA. All PCR reactions were performed for 35 cycles, each consisting of a 30 s. denaturation step at 94 °C, a 30 s. annealing step at 50 °C, and a 1 min. extension step at 72 °C. Amplification products were purified using Montage PCR Cleanup Filter Plates (Millipore, Billerica, MA). Sequencing reactions were conducted using the ABI BigDye version 3.0 sequencing kit (Applied Biosystems, Foster City, CA) following the manufacturer’s suggested protocol but at onetenth the recommended volume. Reaction products were purified using the BigDye XTerminatorÒ Purification Kit (Applied Biosystems, Foster City, CA) following the manufacturer’s suggested protocol and sequenced on an ABI3730 genetic analyzer (Applied Biosystems, Foster City, CA) using the aforementioned oligonucleotide primers. After the resulting DNA sequences were verified, the online NCBI BLAST computer program was used to identify the closest sequence matches in GenBank, which included C. flavescens and several closely related species. An alignment was constructed using the program ClustalX (Thompson et al., 1997) for the sequences corresponding to the type strains of each of these species along with the sequence obtained in this study for strain C100R1 and the wild type progenitor strain; the resulting alignment was subsequently checked for errors by visual inspection. Each isolate was identified to species level through phylogenetic analyses with the aforementioned type strains; the analyses were conducted using the maximum likelihood method as implemented in the computer program MEGA 5 (Tamura et al., 2011) with Tamura and Nei (1993) distances. Rate variation among sites was modeled using a gamma distribution with shape parameter a = 0.455 and five rate categories. The reliability of internal branches was assessed using 1500 bootstrap pseudoreplicates. 2.3. Preparation of C. flavescens OH 182.9 C100R1 inoculum

To track populations of C. flavescens OH 182.9 on wheat anthers (greenhouse and year 1 and 2 field studies) and heads (year 3 and 4 field studies), a naturally occurring cycloheximide tolerant variant

Biomass was produced in liquid culture in shaker flasks for greenhouse and field trials (years 1 and 2) in Wooster, Ohio. Cells

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of C. flavescens OH 182.9 C100R1, hereafter designated as ‘‘OH 182.9’’, were stored frozen at 80 °C as 10% glycerol stocks. When needed, cell cultures were initiated on plates of TSBA/5. After 24 h, cells were used to inoculate 50 ml of semi-defined complete liquid medium (SDCL; Slininger et al., 2010) in 250-ml Erlenmeyer flasks (OD = 0.10, A620). Flasks were incubated in a shaker incubator (250 rpm, 25 °C, 24 h) and the colonized broth used to inoculate 1 L Erlenmeyer flasks containing 500 ml SDCL (OD = 0.10, A620). Cultures were incubated at 250 rpm and 25 °C for 48 h, then chilled in shaved ice and used for greenhouse or field experiments within 24 h. Production of OH 182.9 inoculum for use in field experiments conducted at Peoria, IL (years 3 and 4) was similar except that liquid cultures used for experimental inoculum were grown in 1.5 L of SDCL medium in 2.8 L Fernbach flasks. 2.4. Preparation of plant material for greenhouse and year 1 and 2 field studies in Wooster, OH For greenhouse studies, the hard red spring wheat cultivar Norm was grown in 400-ml pots (one seedling per pot) containing air–steam pasteurized (60 °C for 30 min) potting mix (Terra-lite Redi-earth mix, W.R. Grace, Cambridge, MA) in the greenhouse. Pots were fertilized with triple 15 Osmocote (N 15%, P 15%, K 15%; 3 g/pot; Scotts, Inc., Marysville, OH) granules at planting and then with Peter’sÒ (N 20%, P 20%, K 20%; 4.79 g/ L water; 20 ml/pot) fertilizer solution (Scotts, Inc., Marysville, OH) once every week thereafter. Plants were treated with Abamectin (AvidÒ, Syngenta Crop Protection, Inc., Greensboro, NC; 0.0132 g ai/L water) twice to control aphids. Plants were utilized for colonization experiments in the greenhouse after approximately 7 weeks. For year 1 and 2 field studies, non-irrigated wheat plots were established at the Ohio Agriculture Research and Development Center in Wooster, Ohio with cultivation, fertilization, planting, and fungicide application at early boot (Feekes 10.1) to control powdery mildew and Stagonospora glume blotch as described previously (Khan et al., 2004). Moderately resistant soft red winter wheat cultivar Freedom was used in year 1 studies. Elkhart, a more FHB susceptible cultivar, was used to increase the likelihood of obtaining more severe disease in year 2 field studies. Prior to initiating experiments, corn kernel inoculum of F. graminearum was produced to supplement natural inoculum for field studies in Wooster, OH. Yellow dent corn was moistened, sterilized in milk bottles on each of 2 consecutive days, inoculated with F. graminearum, incubated for 2 weeks on a laboratory bench, and harvested as described previously (Campbell and Lipps, 1998). Colonized kernels were then scattered in the field (30 g/m2) 3 weeks prior to wheat flowering. The development of perithecia on kernels was verified microscopically after 2 weeks. 2.5. Preparation of plant material for year 3 and 4 field studies in Peoria, IL For experiments in Peoria, wheat was produced in pots as previously described (Schisler et al., 2011b) and subsequently moved to the field for colonization experiments. Briefly, two seedlings of cultivar Norm were grown per 2500 ml plastic pot. Each pot contained air–steam pasteurized potting mix as described above (section 2.4). Plants in two sets of 18 pots were initiated 1 week apart, grown in a growth chamber (20 °C night and 25 °C day, 14-h photoperiod, 600 lmol/[m2/s]), and both sets transferred to a greenhouse bench on the same day when plants were either 6 or 7 weeks old. Both sets were held on benches for 1 week (17– 20 °C night and 25–28 °C day) with natural sunlight supplemented by high-pressure sodium lights for 14 h/day. Pots were fertilized 1 week after seeding and weekly thereafter with 50 ml of a solution containing 1.25 g/L Peters 20-20-20 (Grace-Sierra

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Horticultural Products, Milpitas, CA) and 0.079 g/L iron chelate (Sprint 330, Becker Underwood, Inc., Ames, IA). 2.6. Colonization of wheat anthers by strain OH 182.9 on greenhouse wheat Three-hundred flowering (Feekes 10.5.1) wheat plants and 320 with heads fully emerged but not flowering (Feekes 10.5) were selected and marked with different colors of yarn to distinguish growth stage. Immediately prior to use as treatments, 48 h cultures of OH 182.9 were mixed (1:1, v/v) with phosphate buffer (pH 7.2, 0.004% [wt/v] KH2PO4 buffer with 0.019% [wt/v] MgCl2) and Tween-80 (final concentration of 0.036% (v/v); Fisher Scientific, Pittsburgh, PA) and applied (188 L/ha to visibly wet heads without run-off; 1  108 CFU/ml) to half of the plants in each group using a hand-held, CO2-powered boom sprayer equipped with 6503 T-jet nozzles and charged at 2.8 kg/cm2. The remaining plants in each group were treated with the buffer/tween solution only and served as controls. Two replicate pots of each treatment were arranged in a randomized complete block design. The population of OH 182.9 on treated and untreated anthers was enumerated immediately following treatment application and after every 48 h for 10 days. Samples consisting of 100 anthers (10 anthers from each of 10 heads) were suspended in 0.5 ml phosphate buffer in 1.7 ml Eppendorf tubes and placed on ice for 2 h. Extruded anthers were removed from flowering heads using sterilized jewelers forceps. For heads that were not flowering, glumes were removed, lemmas and paleas were separated, and anthers were removed. Samples were mixed for 30 s using a vortex mixer and serially diluted onto TSBA/5 + 100 ppm cycloheximide. The number of CFU’s of OH 182.9 was recorded following plate incubation at 29 °C for 2 days. Two, 100-anther samples were collected and plated per treatment per harvest time and the experiment was performed twice. All CFU data was log transformed, mean standard errors calculated for all combinations of treatment by plating time data, and population by time curves plotted for both experiments. 2.7. Colonization of wheat anthers for year 1 and 2 field studies in Wooster, OH Randomized complete block experimental designs were employed. For year 1 studies, blocks (n = 4) consisted of 1-m2 plots of the moderately FHB resistant, soft red winter wheat cultivar Freedom separated on all sides by 0.6-m alleys. For year 2 studies, blocks (n = 4) consisted of 3-m2 plots of the FHB-susceptible, soft red winter wheat cultivar Elkhart separated as above by 0.6-m alleys. Individual treatment plots were surrounded by non-treated buffer strips to minimize cross-plot contamination. Flowering and heading plants within each treatment were marked with yarn as described for the greenhouse studies prior to applying strain OH 182.9. Inoculum of OH 182.9 was prepared and applied as described for the greenhouse studies. Identical inoculum concentrations of 1.5A620 optical density that were applied in field trials varied in the CFU/ml represented. For the year 1 field trial, cells of the antagonist were applied at 5  107 CFU/ml at Feekes 10.5 and 10.5.1 while for the year 2 trial, cells were applied 1  108 CFU/ml at Feekes 10.5 and 2  108 CFU/ml at GS 10.5.1. A small delay at spraying GS 10.5.1 heads in the year 2 trial resulted in heads being treated at near 100% anther extrusion instead of 50%. Populations of OH 182.9 on anthers were enumerated via dilution plating on cycloheximide-amended 1/5 TSBA immediately following treatment and after every 48 h for 10 days. Anthers collected from field grown wheat were stored in 10% glycerol at 80 °C for up to 21 days prior to enumeration. All CFU data was log transformed, mean standard errors calculated for all combina-

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tions of treatment by plating time data, and population by time curves plotted for each treatment for both experiments. Wheat was scored for disease severity and incidence, 100 kernel weight, and DON content of grain. Disease severity was assessed when heads were between mid-milk and soft dough development (Feekes 11.1, 11.2, respectively) by evaluating 60 heads per plot using a visual rating scale for non-awned soft red winter wheat varieties (Engle et al., 2004). Disease incidence was determined by counting heads with symptoms and dividing by the total number heads scored (60 heads/treatment/replicate). In year 2 field studies, a 10–20 g sample was evaluated for DON content using Veratox enzyme-linked immunosorbent assay (ELISA) kits (Neogen, Lansing, MI) per the manufacturer’s instructions for grain with a DON concern level of 0.5–5 ppm. One-hundred kernel weight was determined in the year 2 study by weighing 5 cm3 of kernels, dividing by the number of kernels contained within the sample and multiplying by 100. Three samples were evaluated for each treatment and the means were used for analysis. Differences between treatments were determined using analysis of variance (ANOVA). Disease data were normalized when needed using the arcsine transformation before ANOVA. Means were separated from the controls using Fisher’s protected LSD test (P 6 0.05, FPLSD; Statistix 7.0, Tallahassee, FL). 2.8. Colonization of wheat heads by OH 182.9 in year 3 and 4 field studies in Peoria, IL To further understand colonization of wheat by strain OH 182.9, cells of the antagonist were applied at different stages of plant development and quantified on wheat head tissues, rather than anthers. Head tissue samples also were observed using SEM. Inoculum of OH 182.9 was prepared and applied as described for greenhouse studies. Immediately prior to use as treatments, 48 h cultures of OH 182.9 were mixed (1:1, v/v) with a solution of phosphate buffer and Tween-80 (section 2.6) described earlier. Wheat heads of seedlings that had been grown in pots for 7 or 8 weeks and were at split boot (Feekes 10.1) or flowering (Feekes 10.5.1), respectively, were then treated with OH 182.9 (4  108 CFU/ml) or PO4 buffer and tween-80 (control). Immediately after treatment, pots of seedlings were placed in a completely randomized design on gray landscape fabric that completely covered a 45 by 45 foot plot in a freshly tilled field with full sun exposure. The trials were not irrigated. Pots were separated by 0.6 m to minimize the possibility of microbial transfer between treatments and the entire plot was protected by rabbit fencing and bird netting. Samples for determining colonization of wheat head tissues as determined by dilution plating and observation using scanning electron microscopy (SEM) were taken the morning after the late afternoon application of treatments (1 day post inoculation) as well as 4, 7 and 10 days post inoculation. For dilution plating, separate counts from three heads from three plants were obtained by harvesting three spikelets per head, separating palea, lemma, and glume tissues using sterile forceps, and pooling all tissues from a head in 50 ml of sdH2O. Samples were agitated for 60 s at medium speed in a Stomacher 80 tissue processor (Seward Laboratory Systems, Port Saint Lucie, FL) and serially diluted. Cell counts of OH 182.9 and of total recoverable microbes per gram fresh tissue were obtained on TSBA/5 containing 100 ppm cycloheximide and 50 ppm streptomycin (Sigma–Aldrich, St. Louis, MO) and on unamended medium, respectively. Count data was normalized using log10 transformation before ANOVA. Means were separated from the controls (P 6 0.05, FPLSD). Spikelets from additional wheat heads were obtained at each sample time from plants treated or not with strain OH 182.9 at flowering (Feekes 10.5.1); palea, glume and lemma tissues separated from spikelets, and the tissues prepared for SEM using

one of two methods. Samples were fixed in 3% gluteraldehyde and 2% paraformaldehyde in a 0.1 M phosphate buffer, followed by dehydration in a series of ethanol and water (Schisler and Jackson, 1996). Tissue samples were then critical-point dried in an Auto Samdri-814 drier (Tousimis Research Corp., Rockville, MD), mounted on aluminum stubs with conductive sticky tabs (Ted Pella, Tustin, CA), and coated with gold palladium in a sputter coater (Hummer VII, Anatech, Hayward, CA). Alternatively, to facilitate viewing of large numbers of samples with minimal sample preparation time, fresh tissue was mounted directly on aluminum stubs with conductive sticky tabs and coated with gold (2 nm) to prevent charging and distortion artifacts from evaporating water when viewed using SEM. Specimens were examined with a scanning electron microscope (Hitachi Cold-Field Emission scanning electron microscope, Model S-4700, Hitachi High Technologies America Inc., Pleasanton, CA) operated with a 20.0 kV accelerating current and a 12 mm working distance. 3. Results 3.1. Selection and identification of cycloheximide tolerant variant of C. flavescens OH 182.9 Cycloheximide tolerant variant C100R1 of C. flavescens OH 182.9 was selected for use in all studies. Data from phylogenetic analysis of the variant show that strain C100R1 clusters with Cryptococcus strains that include the type strain of C. flavescens (Fig. 1). This grouping is supported by high bootstrap values (96% for the entire C. flavescens clade, and 86% for the grouping with the type strain and several previously identified reference strains) indicating that strain C100R1 can be classified as C. flavescens. 3.2. Colonization of wheat anthers by strain OH 182.9 on greenhouse wheat Populations of cycloheximide tolerant variant C. flavescens OH 182.9 C100R1 increased or were maintained on wheat anthers in both greenhouse (Fig. 2A and B) experiments. Populations of OH 182.9 remained stable on anthers for heads treated with OH 182.9 at Feekes GS 10.5 between the time of initial application (0 days post inoculuation = 0 DPI) and extrusion of anthers (3 DPI; Fig. 2A and B), followed by a population increase through 6 DPI. Populations then decreased by 2.5–3 log units by 10 DPI (Fig. 2A and B). Populations of OH 182.9 increased on anthers by approximately 1.5 log CFU/ml for 4 days after inoculation of spikes at flowering (Feekes GS 10.5.1) and then were unchanged for the remaining 6 days of the experiment (Fig. 2A and B). For both greenhouse studies, OH 182.9 was recovered from anthers on heads treated with buffer at full heading (Feekes 10.5) and flowering (Feekes 10.5.1), though populations were multiple log units less than the comparable post-application-day populations on anthers from OH 182.9-treated heads and dropped to unrecoverable levels after 9 days (Fig. 2 A and B). Temperatures were similar for the two experiments for the first 4 days after inoculation but average temperatures for trial A were as much as 20 °C cooler than for trial B from 6 to 10 days after inoculation. 3.3. Colonization of wheat anthers for year 1 and 2 field studies in Wooster, OH Populations of OH 182.9 increased or, less frequently, remained steady throughout the sampling period, regardless of the study year or application time (Fig. 3A and B). Populations of OH 182.9 peaked at 1–2 log units higher on anthers in the field studies (Fig. 3 A and B) compared to the greenhouse studies (Fig. 2A and

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Fig. 1. Phylogenetic placement of strain Cryptococcus flavescens OH 182.9 C100R1. Numbers along branches represent bootstrap percentage values. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Type strains are indicated with a ‘‘T’’ superscript at the end of each strain designation.

Fig. 2. Population dynamics of Cryptococcus flavescens OH 182.9 C100R1 liberated from anthers of hard red spring wheat cultivar Norm in two greenhouse trials (A and B). 4 Feekes 10.5, non-treated; h Feekes 10.5.1, non-treated; N Feekes 10.5, treated; j Feekes 10.5.1 treated; ; = anther extrusion for plants treated with strain OH 182.9 at Feekes GS 10.5. Bars represent standard error of means.

Fig. 3. Population dynamics of Cryptococcus flavescens OH 182.9 C100R1 liberated from anthers of soft red winter wheat in two successive years of field trials (A, B). (A) First year trial on cultivar Freedom. (B) Second year trial on cultivar Elkhart. 4 Feekes 10.5, non-treated; h Feekes 10.5.1, non-treated; N Feekes 10.5, treated; j Feekes 10.5.1 treated; ; = anther extrusion for plants treated with OH 182.9 at Feekes 10.5. Bars represent standard error of means.

B) and OH 182.9 populations on anthers peaked at similar levels in the field for both Feekes 10.5 and 10.5.1 treated heads. Populations at flowering and for several days after were 2–3 log units higher on anthers in year 2 compared to year 1 studies, regardless of Feekes stage when inoculated (Fig. 3). Cell populations of OH 182.9 recovered from anthers on buffer-treated spikes reached levels as high 3  102 CFU/ml by day 12 on Freedom and day 17 on Elkhart though these levels were always at least 3 log unit less than the comparable post-application-day populations on anthers from OH 182.9-treated heads (Fig. 3A and B). Disease severity and incidence was low in the year 1 study on wheat cultivar Freedom and OH 182.9 treated heads did not differ from the controls (P 6 0.05, FPLSD; Table 1). In the year 2 field experiment on cultivar Elkhart, heads treated at Feekes 10.5 and 10.5.1 with OH 182.9 had 34% and 37% less disease severity, respectively, than the associated control

(P 6 0.05, FPLSD; Table 1). No significant differences in disease incidence, 100-kernel weight or DON concentrations in harvested grain were detected in either field trial (P 6 0.05, FPLSD, Table 1). Environmental data for the two Wooster trials was similar in average temperature and rainfall (Table 2) while average humidity was higher in the second year trial (Table 2).

3.4. Colonization of wheat heads by OH 182.9 in year 3 and 4 field studies in Peoria, IL In the year 3 trial, spikelet populations of strain OH 182.9 on heads treated at split boot (Feekes 10.1) versus flowering (Feekes 10.5.1) did not differ at any of the DPI’s monitored (Table 3, P 6 0.05). In both cases, populations tended to decrease from 1

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Table 1 Efficacy of Cryptococcus flavescens OH 182.9 C100R1 on FHB development on soft red winter wheat in two field trials at Wooster, Ohioa,b,c. Treatment, timing

DS (%)

Control, Feekes 10.5 Control, Feekes 10.5.1 OH 182.9, Feekes 10.5 OH 182.9, Feekes 10.5.1

DI (%)

100-KW (g)

DON (ppm)

Year 1

Year 2

Year 1

Year 2

Year 2

Year 2

4A 5A 3A 3A

50A 46B 33C 29D

23A 23A 22A 21A

100A 100A 99A 99A

2.1A 2.2A 2.2A 2.3A

23.6A 23.2A 19.2A 19.6A

a

Soft red winter wheat was used in year 1 and year 2 field studies; Year 1 = cultivar Freedom; Year 2 = cultivar Elkhart. Timing = Feekes stage of wheat development when treated. Wheat heads at Feekes 10.5 (heading complete) and 10.5.1 (flowering) were spray inoculated with a suspension of cells of the antagonist (OH 182.9) or with water (control). DS = disease severity (=average % of individual head visually diseased), DI = disease incidence, 100KW = one hundred kernel weight, DON = deoxynivalenol. c Within a column, means followed not followed by the same letter are significantly different (FPLSD, P 6 0.05). b

Table 2 Environmental data during two successive years of field monitoring of inoculant Cryptococcus flavescens OH 182.9 C100R1 populations on anthers of soft red winter wheat in Wooster, Ohio. Days post inoculationb

Environmental parametera Year 1

1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Avg

Year 2

Precip (cm)

Temp (Avg, °C)

RH (Avg)

Precip (cm)

Temp (Avg, °C)

RH (Avg)

0.0 0.0 0.3 0.7 0.0 0.0 1.0 0.4 2.5 0.2 0.0 0.0 0.1 1.4 – – – – – 0.5

8 12 16 16 13 16 18 19 20 23 20 18 16 22 – – – – – 17

68 58 83 79 80 76 83 85 90 76 58 56 74 79 – – – – – 75

0.2 0.0 0.1 0.0 1.7 0.0 0.0 1.0 0.2 0.0 0.0 0.1 0.3 0.0 0.0 1.0 3.1 0.8 0.1 0.4

12 14 17 16 12 10 11 12 14 13 14 18 18 17 17 20 20 22 19 16

89 86 78 83 98 71 69 97 98 90 89 90 93 80 79 93 98 92 94 88

a Precip = precipitation (cm), Temp = temperature (°C), RH = relative humidity. Wheat plots also received supplemental irrigation applied aerially that totaled 2.5 cm of water/day during 6:00–10:00 AM and 8:00–10:00 PM. b Wheat heads were spray inoculated with a suspension of cells of a cycloheximide tolerant variant (C100R1) of yeast C. flavescens OH 182.9 on ‘‘day 0’’.

DPI through 7 DPI and then recover by 10 DPI to 1 DPI levels (Table 3, P 6 0.05). Strain OH 182.9 was not recovered from any treatment at 7 DPI and was not recovered from non-inoculated heads at any monitoring time. Strain OH 182.9 tended to comprise a higher percentage of the total recoverable microbial population when applied at Feekes 10.1 compared to Feekes 10.5.1 though the trend was not significant (Table 4, P 6 0.05). Strain OH 182.9 comprised as high as 59% of the total microbial population recovered from inoculated heads 1 and 4 DPI and 35% at 10 DPI in the case of the Feekes 10.1 treatment (Table 4). No precipitation occurred from 1 DPI until 7 DPI when 2.4 cm of rain was recorded after harvesting 7 DPI head samples (Table 7). Average daily temperatures were higher in the second half of the trial with a maximum of 22 C and average of 17 C overall. Symptoms of FHB infection were not detected during the 10 day study through head ripening. Spikelet populations of OH 182.9 on heads treated at Feekes 10.5.1 in the year 4 trial maintained statistically similar levels at each DPI sampling time and ranged from 4.36 to 5.77 CFU g1 spikelet tissue (Table 5). Fewer cells of OH 182.9 were recovered from heads treated at Feekes 10.1 than heads treated at Feekes 10.5.1 at 4, 7, and 10 DPI with the strain at undetectable levels at 7 and 10 DPI (P 6 0.05, Table 5). Strain OH 182.9 was not recovered from non-inoculated heads at any monitoring time (Table 5). For

Table 3 Population of Cryptococcus flavescens OH 182.9 C100R1 on wheat spikelets over time when applied at two.a,b,c. Treatment, timing

Control, Feekes 10.1 Control, Feekes 10.5.1 OH 182.9, Feekes 10.1 OH 182.9, Feekes 10.5.1

Days post inoculation 1

4

7

10

0.00B/a 0.00B/a 4.92A/a 3.92A/a

0.00B/a 0.00B/a 2.88A/b 3.55A/a

0.00A/a 0.00A/a 0.00A/c 0.00A/b

0.00B/a 0.00B/a 3.90A/ab 3.92A/a

a Table values are log10 counts of C. flavescens OH 182.9 C100R1 CFU g1 fresh weight of spikelet tissue. b Timing = Feekes stage of wheat development when treated. Wheat heads at Feekes 10.1 (split boot) and 10.5.1 (flowering) were spray inoculated with a suspension of cells of the antagonist (OH 182.9) or with water (control) on ‘‘day 0’’. c Values in columns followed by the same capital letter, and values in rows followed by the same lower case letter, are not significantly different (FPLSD, P 6 0.05).

each sampling time, cells of OH 182.9 comprised a higher percentage of the total recoverable microbial population for heads treated at Feekes 10.5.1 compared to Feekes 10.1 (P 6 0.05, Table 6). Percentages ranged from 81 to 36 for heads treated at Feekes 10.5.1 but were lower at each DPI sampling time for heads treated at

D.A. Schisler et al. / Biological Control 70 (2014) 17–27 Table 4 Cryptococcus flavescens OH 182.9 C100R1 population on wheat spikelets over time expressed as a percentage of the total recoverable microbial population when applied at two stages of head development in Peoria, IL (year 3)a,b,c. Treatment, timing

Days post inoculation

Control, Feekes 10.1 Control, Feekes 10.5.1 OH 182.9, Feekes 10.1 OH 182.9, Feekes 10.5.1

1

4

7

10

0B/a 0B/a 59A/a 26A/ab

0B/a 0B/a 52A/a 53A/a

0A/a 0A/a 0A/a 0A/c

0B/a 0B/a 35A/a 7A/b

a Table values are the percentage of the total recoverable microbial population that was comprised of cells of C. flavescens OH 182.9 C100R1. b Timing = Feekes stage of wheat development when treated. Wheat heads at Feekes 10.1 (split boot) and 10.5.1 (flowering) were spray inoculated with a suspension of cells of the antagonist (OH 182.9) or with water (control) on ‘‘day 0’’. c Values in columns followed by the same capital letter, and values in rows followed by the same lower case letter, are not significantly different (FPLSD, P 6 0.05).

Table 5 Population of Cryptococcus flavescens OH 182.9 C100R1 on wheat spikelets over time when applied at two stages of head development in Peoria, IL (year 4).a,b,c,d. Treatment, Timing

Control, Feekes 10.1 Control, Feekes 10.5.1 OH 182.9, Feekes 10.1 OH 182.9, Feekes 10.5.1

Days post inoculation 1

4

7

10

na 0.00 na 4.36

0.00C/a 0.00C/a 4.09B/a 5.77A/a

0.00B/a 0.00 B/a 0.00 B/b 5.13A/a

0.00B/a 0.00B/a 0.00B/b 5.01A/a

B/a

A/a

a Table values are log10 counts of C. flavescens OH 182.9 C100R1 CFU g1 fresh weight of spikelet tissue. b Timing = Feekes stage of wheat development when treated. Wheat heads at Feekes 10.1 (split boot) and 10.5.1 (flowering) were spray inoculated with a suspension of cells of the antagonist (OH 182.9) or with water (control) on ‘‘day 0’’. c Values in columns followed by the same capital letter, and values in rows followed by the same lower case letter, are not significantly different (FPLSD, P 6 0.05). d na = not attempted due to heads not fully emergent from flag leaf sheath on day 1.

Table 6 Cryptococcus flavescens OH 182.9 C100R1 population on wheat spikelets over time expressed as a percentage of the total recoverable microbial population when applied at two stages of head development in Peoria, IL (year 4).a,b,c,d Treatment, timing

Control, Feekes 10.1 Control, Feekes 10.5.1 OH 182.9, Feekes 10.1 OH 182.9, Feekes 10.5.1

Days post inoculation 1

4

7

10

na 0 B/a na 66A/a

0C/a 0C/a 25B/a 81A/a

0B/b 0 B/a 0B/b 36A/b

0B/a 0B/a 0B/b 77A/a

a Table values are the percentage of the total recoverable microbial population that was comprised of cells of C. flavescens OH 182.9 C100R1. b Timing = Feekes stage of wheat development when treated. Wheat heads at Feekes 10.1 (split boot) and 10.5.1 (flowering) were spray inoculated with a suspension of cells of the antagonist (OH 182.9) or with water (control) on ‘‘day 0’’. c Values in columns followed by the same capital letter, and values in rows followed by the same lower case letter, are not significantly different (FPLSD, P 6 0.05). d na = not attempted due to heads not fully emergent from flag leaf sheath on day 1.

Feekes 10.1 (P 6 0.05, Table 6). Precipitation occurred daily through 5 DPI but not through the remainder of the study (Table 7). Average daily temperatures were higher in the second half of the year 4 trial but lower than the year 3 trial with a maximum of 17 C and average of 13 C overall (Table 7). Symptoms of FHB infection were not detected.

23

For the year 3 and 4 trials, cells of OH 182.9 were detected using SEM on each of the spikelet tissues examined (Fig. 4A–F) and at each DPI sampling time. Detection of OH 182.9 was more sporadic on heads inoculated at Feekes 10.1 then Feekes 10.5.1. One and 4 DPI, cells of strain OH 182.9 were detected most often on the exterior (abaxial) surfaces of lemmas (Fig. 4A and B) and glumes (Fig. 4D) and near the apex of palea tissues (Fig. 4C), though cells were infrequently found on the adaxial surfaces of lemma and palea tissues. Cells of OH 182.9 tended to be found in larger microcolonies 1 and 4 DPI compared to more scattered small groups or individual cells predominating 7 (Fig. 4E and F) and 10 DPI (no micrograph shown). Cells on the abaxial surface of lemma tissues were most commonly detected near the juncture of the lemma surface and the overlapping glume tissue while cells on the abaxial surface of glumes were often near the edges and base. In only one instance were cells comparable to known C. flavescens morphology seen in SEM observation of tissues not inoculated with OH 182.9.

4. Discussion The selection of fungicide-tolerant variants of fungal biocontrol agents has been demonstrated previously (Shapiro-Ilan et al., 2002). When selecting for cell lines with enhanced ability to grow in the presence of a compound that restricts cell growth, contaminant cell lines can inadvertently be fostered instead of a variant of the progenitor strain. In the present study, we selected for cycloheximide-tolerant variants of the wild type strain C. flavescens OH 182.9 wt. Phylogenetic analysis showed that the cycloheximide-tolerant variant C100R and the progenitor strain OH 182.9 wt cluster within a clade of Cryptococcus strains that includes the C. flavescens type strain. Additionally, strains OH 182.9 C100R and wt possessed identical ITS1 sequences. These results, coupled with the observations that OH 182.9 wt and C100R possessed visually identical colony and cell morphology (data not shown) strongly suggest that C. flavescens OH 182.9 wt and the cycloheximide tolerant variant C100R are conspecific members of the C. flavescens species sensu stricto. In greenhouse trials, populations of OH 182.9 recovered from anthers on heads treated at Feekes 10.5 consistently dropped several log units by 5 days after flowering compared to heads treated at Feekes 10.5.1 (Fig. 2). In contrast, populations on anthers from heads treated at these same two Feekes growth stages in the field were similar in most cases (Fig. 3). One possible explanation for these population differences between greenhouse and field grown plant is how the plants were watered. In greenhouse trials at Wooster, OH, wheat was watered with a wand directly onto the surface of potting soil, while field plots were subjected to rain and wind events of varying intensity The comparative lack of free moisture on heads in greenhouse versus field grown wheat would contribute to limited growth and then decline of OH 182.9 in the greenhouse trials. A longer exposure to adverse osmotic conditions also may have contributed to the consistent multiple log unit reduction in OH 182.9 populations on anthers in greenhouse wheat inoculated at Feekes 10.5 compared to Feekes 10.5.1 (Fig. 2). Host cultivar can influence biocontrol efficacy (Schisler et al., 2000) and could contribute to differences in OH 182.9 populations observed in the greenhouse and field studies. In Wooster field studies, OH 182.9 populations on anthers increased in size at anther extrusion indicating that the strain not only survived but also reproduced under favorable environmental conditions (Fig. 3) and the likely increased availability of nutrients from moribund cells of dehiscent anthers. Interestingly, OH 182.9 cells were able to penetrate inside of florets to reach anthers not yet extruded when heads were inoculated at Feekes 10.5 in

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D.A. Schisler et al. / Biological Control 70 (2014) 17–27

Table 7 Environmental data during two successive years of field monitoring of inoculant Cryptococcus flavescens OH 182.9 C100R1 populations on heads of hard red spring wheat variety Norm in Peoria, IL. Days post inoculationb

Environmental Parametera Year 3

1 0 1 2 3 4 5 6 7 8 9 10 Avg a b

Year 4

Precip (cm)

Temp (Avg, °C)

RH (Avg)

Precip (cm)

Temp (Avg, °C)

RH (Avg)

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.4 0.0 0.1 0.0 0.2

19 15 19 15 9 11 17 20 22 16 16 22 17

67 73 72 65 60 62 50 47 62 82 62 48 62

0.1 0.1 0.1 0.1 0.1 0.6 0.1 0.0 0.0 0.0 0.0 0.0 0.1

17 12 7 7 10 13 16 17 14 16 14 17 13

85 59 78 85 85 86 81 67 48 51 57 46 69

Precip = precipitation (cm), Temp = temperature (°C), RH = relative humidity. Wheat heads were spray inoculated with a suspension of cells of a cycloheximide tolerant variant (C100R1) of yeast C. flavescens OH 182.9 on ‘‘day 0’’.

Fig. 4. A. Scabrous, denticulate margin of the abaxial (exterior) surface of lemma tissue exhibiting a colony of cells of yeast OH 182.9 1 day post inoculation (DPI); B. scattered, individual and several cell colonies of OH 182.9 and a fungal hyphae on the abaxial surface of lemma tissue1 DPI; C. loose aggregates of cells of OH 182.9 at the base of hairs on the adaxial surface near the apex of palea tissue 1 DPI; D. close up of budding cells of OH 182.9 on the abaxial surface of glume tissue 4 DPI; E. margin of the abaxial surface near the base of glume tissue with a scattered distribution of cells of OH 182.9 7 DPI; and F. small aggregates and individual cells of OH 182.9 between the apex and midpoint of lemma tissue (abaxial surface, 7 DPI). Scale bars represent 10 lm for panels A, B and F; 20 lm for panel C, 2 lm for panel D and 4 lm for panel E.

greenhouse and field experiments (Figs. 2 and 3). Competitive inhibition via aggressive colonization of potential infection courts of F. graminearum is a possible mechanism of biological control for OH 182.9, a strain originally isolated from extruded anthers of wheat (Khan et al., 2001). Strain OH 182.9 moved from inoculated heads to uninoculated extruded anthers on control plants (Fig. 3), an attribute that suggests the strain would readily spread on heads receiving only partial coverage during field inoculation. Anthers

of control plants were not colonized until after extrusion (Fig. 3) suggesting that large-scale penetration inside of untreated florets by OH 182.9 did not occur during spread of OH 182.9 from inoculated to uninoculated heads. Previously, in field studies that evaluated the efficacy of OH 182.9, the antagonist was applied at the beginning of flowering and regularly resulted in reduction of symptoms of FHB and DON (Khan et al., 2004; Schisler and Boehm, 2012; Schisler et al.,

D.A. Schisler et al. / Biological Control 70 (2014) 17–27

2002). In year 1 field studies, treatments did not influence FHB severity or incidence when disease pressure was light (P 6 0.05, FPLSD; Table 1). In year 2, wheat treated with OH 182.9 at Feekes 10.5 and 10.5.1 exhibited 34% and 37% less severity, respectively, compared to their respective controls (P 6 0.05, Table 1) but other disease parameters were not significantly reduced. Higher populations of OH 182.9 on extruded anthers (Fig. 3) may have improved biocontrol in the year 2 studies. Cultivars with a phenotype of partial anther extrusion are more prone to FHB infection (Graham and Browne, 2009; Kubo et al., 2013) and removal of anthers can reduce FHB severity (Engle et al., 2003; Strange and Smith, 1971). High OH 182.9 populations on anthers would likely limit nutrients available for supporting growth and infection by F. graminearum. In year 3 trials where colonization of wheat heads, rather than anthers, was determined, OH 182.9 populations progressed similarly whether treated at Feekes 10.1 or 10.5.1 and whether expressed in terms of counts per fresh tissue weight (Table 3) or the percentage of the total recoverable microbial count (Results, 3.4). A lack of recovery of OH 182.9 at 7 DPI is likely at least partially due to the paucity of rain experienced during the first 7 days of the trial (Table 7). Total recoverable microbial counts on head tissues were also lower at 7 DPI than at any other time (data not shown), but counts of OH 182.9 increased significantly by 10 DPI (Table 3) after two rain events occurred between these sampling times (Table 7). Osmotic stress can substantially reduce the recoverable population of a microbial strain even while its non-culturable population is largely unchanged (Cabrefiga et al., 2011). Producing OH 182.9 under conditions that promote drying stress tolerance has potential for enhancing cell survival and concomitantly, biological control (Dunlap et al., 2007; Palazzini et al., 2009). Interestingly, unlike the year 3 study, colonization of heads by OH 182.9 in the year 4 study was reduced on heads inoculated at Feekes 10.1 compared to Feekes 10.5.1 inoculated heads (Tables 5 and 6). At Feekes 10.1, heads by definition are still partially in boot and thus can range from being minimally to largely enclosed by the leaf sheath. The degree of spray coverage of the underlying head tissues will therefore vary considerably. While colonization of Feekes 10.1 inoculated heads can be at levels equivalent to Feekes 10.5.1 inoculated heads (Tables 3 and 4), timing of inoculation to ensure heads are nearly fully emerged from boot appears critical to obtaining maximum colonization of heads. Unlike heads inoculated with OH 182.9 at 10.5.1 in year 3, OH 182.9 consistently was recovered at high populations and comprised a high percentage of the total recoverable microbial population at every DPI sampling in year 4 (Tables 3–6). In addition to consistent rainfall events over the course of the first 6 days of the year 4 study (Table 7), relative humidity values measured over this time period were higher in the year 4 versus year 3 study and temperatures were more moderate (Table 7). Both high relative humidity and moderate temperatures have been associated with enhancing microbial colonization of plant surfaces (Pusey et al., 2009; Xu and Butt, 1998). Evidence of FHB symptoms was lacking in both of the study years in Peoria, IL. The lack of a nearby inoculum source combined with unfavorable environmental conditions for infection early in the study (Table 7) (Gautam and Dill-Macky, 2012; Kriss et al., 2010) likely were causal of this result. There was no evidence of OH 182.9 cells on untreated control plants in Peoria year 3 and 4 field trials and in only one instance were cells comparable to known C. flavescens morphology seen in SEM observation of tissues not inoculated with OH 182.9. Yet data from plating serial dilutions of anthers from control treatments in year 1 and 2 trials in Wooster, OH indicated spread of OH 182.9 from inoculated plants (Fig. 3). In contrast, plant density was much lower in the Peoria, study. Higher average precipitation (Tables 2 and 7), associated rain-aided dispersal (McManus and Jones, 1994; Monaghan and Hutchison, 2012; Mundt et al., 1999) and lar-

25

ger sinks of OH 182.9 inoculum (Upper et al., 2003) for the trials in Wooster likely would enhance movement of OH 182.9 to control plants. One and 4 DPI, cells of strain OH 182.9 were detected most often on the exterior (abaxial) surfaces of lemmas (Fig. 4A and B) and glumes (Fig. 4D) and near the apex of palea tissues (Fig. 4C), though cells were infrequently found on the adaxial surfaces of lemma and palea tissues. Hairs that cover the apex of lemma and palea tissues may trap spores of F. graminearum, restricting the pathogen’s access to susceptible tissues in the interior of florets (Bushnell et al., 2003). Cells of OH 182.9 were commonly found at the apex of lemma and palea tissues (Fig. 4C) though a portion of cells of OH 182.9 did penetrate to the interior of florets as evidenced by our SEM observations and the colonization of anthers prior to extrusion (Figs. 2 and 3). The process of flowering is generally completed in less than 60 min. Flowering temporarily provides microbial propagules easier access to the interior of florets when anthers commonly are extruded through an opening that forms at the apex of a floret when lemma and palea tissues separate (De Vries, 1971). A narrower aperture during the extrusion of anthers is associated with less FHB disease (Gilsinger et al., 2005). The timing of flowering and the size of the aperture at the floret apex is influenced by tightness of spikelet packing on heads, drought, high temperatures, and wheat variety (Gilsinger et al., 2005). Theoretically, these factors also would impact how many cells of yeast OH 182.9 would reach the interior of florets, the colonization success of those cells, and the subsequent level of biological control achieved. In this study, cells of OH 182.9 were not commonly observed on the adaxial surfaces of lemma and palea tissues using SEM. Further studies are needed to determine if the ingress of OH 182.9 to floret interiors can be improved through the use of surfactants tailored to the physiochemical characteristics of floret tissues (Dunlap and Schisler, 2009) and the effect on biocontrol. While F. graminearum rarely directly infects through the thick walled cells of the adaxial surface of glume and lemma tissues, crevices between the palea and lemma expand as the caryopisis enlarges providing a possible avenue for infective hyphae of F. graminearum to more easily access the caryopsis and interior surfaces of the palea and lemma (Boenisch and Schäfer, 2011; Kang and Buchenauer, 2002). SEM frequently revealed cells of OH 182.9 on the abaxial surface of lemma and glume tissues (Fig. 4) often near the juncture of the lemma surface with the overlapping glume tissue and at the base of the abaxial surface of glumes. Studies to specifically quantify populations of OH 182.9 on each tissue type over time would help determine if the degree of colonization of this potential avenue of pathogen ingress correlates with the level of biocontrol achieved. The application of thiophanate-methyl fungicide 20 days after anthesis (late milk or Feekes 11.1) reduced DON though not FHB severity (Yoshida et al., 2012). However, preharvest restrictions on fungicide use against FHB render this option untenable for reducing late kernel infection by F. graminearum. Treatment of heads at flowering could position OH 182.9 to reduce late infections and DON. Our studies indicated that yeast OH 182.9 can survive on anthers for at least 18 days and on wheat heads for at least 12 days and under some conditions even longer (Rong, 2013). In the current study, lack of rainfall was associated with reduced survival of the inoculated yeast antagonist. Identifying genes in OH 182.9 involved in dehydration tolerance (Rodríguez-Porrata et al., 2012) would be a critical first step in developing production and formulation protocols that enhance expression of such genes and concomitantly, enhance survival of biomass during stabilization, formulation, and application in field environments. The recent sequencing of the genome of C. flavescens OH 182.9 3C (McSpadden Gardener, personal communication) will enable this type of study to proceed.

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D.A. Schisler et al. / Biological Control 70 (2014) 17–27

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