Abstract: Multistrain mixtures of biocontrol agents which can reduce plant disease to a greater extent than the individual strains of the mixture, commonly, are prepared by blending separately produced fermentation products. Co-cultivation of strains to equivalent biomass yields would provide mixture advantages without incurring the cost disadvantages of multiple fermentation and processing protocols. Fusarium Head Blight (FHB) antagonists Cryptococcus flavescens OH 182.9 (NRRL Y-30216), C. aureus OH 71.4 (NRRL Y-30213) and C. aureus OH 181.1 (NRRL Y-30215), were grown in two- and three-strain co-cultures to assess the quality and efficacy of the fermentation end products produced. Final cell counts of component strains of all co-cultures produced were equivalent when plated on a medium that contained the trisaccharide melezitose as a sole carbon source and produced colonies of strain-distinguishable sizes. Co-cultures of C. flavescens OH 182.9 and C. aureus OH 71.4 significantly reduced FHB disease severity (32%, p = 0.05, Dunnett’s t-test) when averaged across four greenhouse studies. In wheat field trials, biomass from co-cultures of these two strains reduced FHB incidence in some cases but rarely other FHB disease parameters (p = 0.05, Bonferoni mean separation). Relative Performance Index (RPI) analysis of the overall effect of treatments at both field sites revealed that treatment with the OH 71.4 and OH 182.9 co-culture significantly reduced FHB, as evidence by a higher RPI value than for the control, while the individual strains did not. The potential for obtaining superior efficacy and cost benefits with multi-strain cultures of biocontrol agents justifies additional research effort.
INTRODUCTION
Losses due to Fusarium head blight (FHB), a devastating disease of wheat and barley, are considered to be among the greatest known for a plant disease (McMullen et al., 1997; Goswami and Kistler, 2004). In North America, FHB is caused predominantly by Fusarium graminearum Schwabe (teleomorph: Gibberella zeae (Schwein Petch) (Aoki and O'Donnell, 1999), a phylogenetically diverse species (Akinsanmi et al., 2006). The pathogen not only reduces grain yield (Paul et al., 2010) but produces mycotoxins including deoxynivalenol (DON), a trichothecene mycotoxin that can reduce the market value of grain (Snijders, 1990; Pirgozliev et al., 2003) and be harmful to animals that consume the contaminated grain (Rocha et al., 2005; Awad et al., 2006).
A variety of approaches to limiting disease such as disease forecasting, resistant varieties, fungicides, cultural controls and biocontrol agents can contribute to reducing FHB (Dill-Macky and Jones, 2000; Schisler et al., 2002a; Khan et al., 2004; Beyer et al., 2006; Paul et al., 2008; Pereyra and Dill-Macky, 2008; Khan and Doohan, 2009; Xue et al., 2009; Skinnes et al., 2010) but significant reduction of FHB and DON contamination of wheat and barley on a consistent basis has not been achieved.
Utilizing an integrated pest management approach against the disease, such as combining resistant wheat varieties with fungicides, has been beneficial (Willyerd et al., 2010). Using biological control as part of the integrated management of FHB remains understudied yet has considerable potential for aiding in the reduction of FHB and DON (Schisler et al., 2002a).
Discovery, fermentation and formulation are crucial steps in the process of developing biological control products, with each step contributing to final product performance (Schisler and Slininger, 1997). Though fermentation as it pertains to the production of pharmaceuticals has been extensively studied (Walsh et al., 2001), fermentation research has been limited in regards to factors that impact the quantity and quality of biocontrol biomass produced. Fermentation protocols can be optimized to enhance strain efficacy, as demonstrated in selecting fungicide tolerant variants of yeast Cryptococcus flavescens OH 182.9, that exhibit enhanced efficacy in reducing FHB of wheat (Schisler and Boehm, 2011). Decreasing the fermentation temperature by 10°C as cells of strain OH 182.9 transition from logarithmic to stationary phase growth resulted in the production of cells with enhanced tolerance to hyperosmotic shock and increased membrane wall spring constants, survival during formulation and biocontrol effect compared to non-adapted cells (Zhang et al., 2006; Dunlap et al., 2007). Including broth supernatants in formulations of biocontrol agents can be beneficial or harmful to agent survival and should be considered when optimizing fermentation schemes. The polysaccharide marginal, produced in liquid culture by cells of biocontrol agent Pseudomonas fluorescens S11:P:12, improved cell tolerance to rapid drying when used in cell formulations (Slininger et al., 2010a).
Work in our laboratory and others has documented that combinations of separately produced biocontrol agents can improve the level and consistency of plant disease control obtained compared to treatment with a single strain (Janisiewicz, 1996; Schisler et al., 1997; Kolombet et al., 2005; Domenech et al., 2006; Karthikeyan et al., 2008; Karthikeyan and Gnanamanickam, 2008; Johnson et al., 2009). Formulations consisting of mixes of biocontrol agents also have been tailored to increase efficacy based on combining strains with different pathogen targets (Kim et al., 2008). However, a draw-back to this approach is that, compared to a single large volume process, running several smaller volume cultivations of pure strains and then blending the cells produced represents significantly higher manufacturing costs due to loss of the economy of scale (Searcy and Flynn, 2009) and, potentially, increased registration costs to prospective commercial manufacturers. An approach to avoiding these disadvantages would be to produce multiple strains of antagonists in a single, co-culture. The study of Slininger and co-workers (Slininger et al., 2010b), is the only example where mixtures of microbial antagonists were cultivated to near equivalent populations and evaluated for efficacy in reducing plant disease. Slininger and co-workers produced a tripartite culture of Psuedomonas spp., that reduced disease on stored potatoes to a greater extent than mixtures of the same strains produced individually.
We have demonstrated the efficacy of three different yeast strains of the genus Cryptococcus in reducing FHB (Schisler et al., 2002a; Khan et al., 2004) but have not determined whether these strains can be co-cultured to near equivalent cell counts and, if so, whether the resultant co-cultured strain mixture would reduce FHB on wheat. The objectives of this study were (1) to develop methodologies for quantifying cell growth of two- and three-strain co-cultures of Cryptococcus spp.; (2) to produce near equivalent cells numbers of each component strain of a co-cultivated mixture in shake flasks; (3) to evaluate the efficacy of individual strains and co-cultivated strain mixtures against FHB on wheat in greenhouse tests and (4) to evaluate in field tests those co-cultured strain mixtures that were effective in reducing FHB in greenhouse tests.
MATERIALS AND METHODS
Growth and quantification of cells in co-culture: When needed, pure cultures of C. flavescens OH 182.9 (NRRL Y-30216), C. aureus OH 71.4 (NRRL Y-30213) and C. aureus OH 181.1 (NRRL Y-30215) were initiated on 1/5 strength Tryptic soy broth agar (TSBA, Difco Laboratories, Detroit, MI) from 10% glycerol stocks of the strains stored at -80°C. After growth at 25°C for 24 h, cells from plates were used to inoculate precultures of each of the strains. Precultures were produced in non-baffled, 125 mL Erlenmeyer flasks containing 25 mL of liquid medium (SDCL) (Slininger et al., 2010a) that was incubated at 250 rpm, 2.5 cm orbit and 25°C for 24 h. Prior to evaluating the amenability of the strains to being co-cultured, methods for quantifying the number of cells of each strain in cell mixtures had to be developed due to cells of the 3 strains producing colonies of similar size, color and morphology on standard laboratory media. To identify a carbon source that the three strains utilized to differing degrees, cells of the three strains were grown on plates of BUY™ Agar medium (Biolog Inc., Hayward, CA) overnight. Cells were harvested from agar plates, washed twice in sterile distilled water and applied to YT Microplate™ plates that enabled a wide variety of carbon sources to be assayed for utilization as sole carbon sources by the yeast strains. Color intensity and turbidity of wells were read after 48 h incubation at 26°C using a Biolog Model 21907 turbidimeter. The trisaccharide melezitose was identified as differentially supporting growth of the 3 strains. A minimal medium containing the carbohydrate as a sole carbon source was then prepared and included: melezitose (14 g L-1), KH2PO4 and K2HPO4 (2 g L-1 each), MgSO4 (0.3 g L-1), CaCl2 (0.4 g L-1), FeSO4 (0.10 g L-1), (NH4)2SO4 (0.7 g L-1), vitamins (0.001 g L-1 each of thiamine, riboflavin, calcium pantothenate, niacin, pyridoxamine and thioctic acid) and agar (18 g L-1). All ingredients were autoclaved, except vitamins which were filter sterilized.
Co-cultures using the three yeast antagonists were then initiated using the SDCL liquid medium. Precultures of each strain first were established as described above. For all two-strain and the three-strain co-cultures, growth rate inequalities between strains were overcome by adjusting strain precultures in late log growth to produce equivalent (OD) seed inocula of all strains and using weighted ratios (Table 1) of each of the inocula to seed test cultures to an initial OD of approximately 0.100 at 620 nm wavelength light (A620). Populations of all cultures were determined every 12 h for 72 h by plating duplicate samples on the melezitose minimal medium. Co-culture component populations in shake flasks were compared at regular intervals using ANOVA and means separated using Tukey’s HSD (p = 0.05). Shake flask experiments were conducted three times and results of all trials were pooled prior to statistical analysis.
Assay of efficacy of co-cultures in reducing fusarium head blight on wheat in the greenhouse: To produce wheat for experimental use, two plants of FHB susceptible hard red spring wheat cultivar Norm were grown per 19-cm-diameter plastic pot. Each pot contained air-steam pasteurized (60°C for 30 min) potting mix (Terra-lite Redi-earth mix, W.R. Grace, Cambridge, MA) and plants were grown in a growth chamber (25°C, 14 h photoperiod, 600 ρ mol/[m2/s]) for 7-8 weeks prior to transfer to greenhouse benches. Pots were fertilized after 1 week and weekly thereafter with 50 mL of a solution containing 1.25 g L-1 Peters 20-20-20 (Grace-Sierra Horticultural Products, Milpitas, CA) and 0.079 g L-1 iron chelate (Sprint 330, Becker Underwood, Inc., Ames, IA). Each experiment to determine the efficacy of co-cultures and individual strains of yeasts was conducted in a climate-controlled greenhouse where temperatures ranged from 17 to 20°C at night and 25 to 28°C during the day. Natural sunlight was supplemented by high-pressure sodium lights for 14 h day-1.
Biomass of strains OH 182.9, OH 181.1 and OH 71.4 was produced in single or mixed strain co-cultures as described (above and Table 1), harvested at 48 h and diluted to ¼ strength with weak PO4 buffer before use. Macroconidia of G. zeae isolate Z-3639 were produced on clarified V8 juice agar (CV8 agar) under a regime of 12 h day-1 fluorescent light for seven days at 24°C (Schisler et al., 2002b). Colonized plates were flooded with weak PO4 buffer to obtain conidial suspensions of the pathogen (Schisler et al., 2002b).
Approximately one week after transferring plants to the greenhouse, wheat heads were inoculated at anthesis by spraying 25% freshly harvested liquid cultures of individual antagonist strains and co-cultures (5x107 cfu mL-1) in PO4 buffer and a final concentration of 0.036% Tween 80 (Sigma Chemical Co., St. Louis, MO). Antagonist cells were applied in the greenhouse at a field equivalent rate of approximating 375 L h-1a in replicated experiments. For each treatment application, 50 mL of inoculum was used to treat four plants representing a total of 12 to 16 heads. Heads were then challenged immediately by spraying 12 mL of a conidial suspension of G. zeae (1.5x104 conidia mL-1) in PO4 buffer with 0.036% Tween 80. Wheat heads treated only with a conidial suspension of G. zeae served as a disease control and untreated pots were used to insure pathogen inoculum was not spreading between treatments. Treated plants were misted lightly with distilled water and incubated in a plastic humidity chamber at 17 to 20°C at night and 25 to 28°C during the day for 3 days before being transferred to greenhouse benches. Treatments were arranged in a completely randomized design with four replicate plants for each treatment. FHB severity was visually estimated using a 0 to 100% scale (Stack and McMullen, 1995) at 10 to 14 days after inoculation. All greenhouse experiments were conducted 4 times. Disease severity data from repeated experiments were combined and analyzed using one-way analysis of variance (ANOVA) after preliminary analysis revealed that experiment by treatment interactions were not significant (p = 0.05). Means were separated using Dunnett’s t-test (p = 0.05).
Biocontrol assay on fusarium head blight in field grown wheat: Soft red winter wheat cultivars Freedom (moderately FHB resistant) and Pioneer Brand 2545 (FHB susceptible) were utilized for field trials conducted at Peoria, IL and Wooster, OH. Methods for conducting field trials in both locations have been described in detail elsewhere (Schisler et al., 2006). In summary, for trials conducted in Peoria, IL, alternating rows of both wheat cultivars were planted in the fall with a length of 1.8 and 0.3 m between rows. The following spring, 2 to 3 weeks before the anticipated date of wheat flowering, 25 to 35 kernels m-2 of yellow dent corn colonized by G. zeae Fg 111-B (Schisler et al., 2006) were applied uniformly by hand to the site. Perithecia appeared on the kernels after about 12 days and were releasing ascospores at the time of wheat anthesis.
| Table 1: | Description of single strain liquid cultures and multiple strain co-cultures of antagonists of Fusarium head blight of wheat and culture influence on symptoms of Fusarium head blight of wheat in greenhouse tests |
| 1 OH 181.1 (A), OH 182.9 (B) and OH 71.4 (C) are abbreviations for strains Cryptococcus aureus OH 181.1 (NRRL Y-30215), C. flavescens OH 182.9 (NRRL Y-30216) and C. aureus OH 71.4 (NRRL Y-30213), respectively. 2 Varying ratios of seed inoculum for co-cultures were utilized to insure near equivalent final cell counts of each strain utilized in a co-culture. NA= Not applicable. 3Within the column values followed by * differ significantly from the Gibberella zeae control (Dunnett’s t-test, p = 0.05) | |
Biomass of C. flavescens OH 182.9, C. aureus OH 71.4 and a co-culture of the two strains was produced in B Braun Biostat B fermentors (B. Braun Biotech Inc., Allentown, PA) charged with 1.5 L of SDCL medium. Antifoam 204 (Sigma, St Louis, MO) was added prior to medium sterilization and cultures were not pH controlled after inoculation at pH 7.0. Log-growth cells of strains OH 182.9 and OH 71.4 served as 5% total seed inoculum when produced individually and the co-culture was inoculated at a ratio of 1:4, respectively (Table 1). Fermentors were operated at 25°C, 1.5 L min-1 aeration and 200 rpm agitation. After dissolved oxygen had recovered to saturation at 48 h, cells were harvested for use in field trials. The three biological treatments were applied at early wheat flowering (Feekes 10.5.1 (Large, 1954) with two adjacent rows (one each of Freedom and Pioneer 2545) receiving treatment and the next row serving as a buffer row throughout the plot. Adjacent rows of the two varieties were also randomly assigned as untreated controls. Biomass was applied at a final spray concentration of ~1.5x108 cfu mL-1 and all treatments were applied at 375 L ha-1. Treatment suspensions were applied using a CO2-pressurized backpack sprayer charged at 2.8 kg cm-2 and attached to a boom equipped with 2, #6 Cone-jet® nozzles (R and D Sprayers, Opelousas, LA) spaced 30 cm apart and mounted pointing downward and toward the center of the boom at 45°. Treatment suspensions were charged with CO2 just prior to application. Treatments were applied in the late afternoon and there were 5 replicates per treatment arranged in a randomized block design. From the morning after treatment application until midmilk kernel development (Feekes 11.1), wheat heads were misted during the night with city water at a rate of approximately 1 cm water/day to supplement rainfall events. Field assessments of FHB severity and incidence were made by evaluating 75 heads per replicate (375 heads/treatment) using a 0 to 100% scale when heads were between mid-milk and soft dough development (Feekes 11.1 to 11.2). Wheat heads were harvested by hand and threshed using an Almaco single plant and head thresher (Almaco, Nevada, IA) when grain reached full maturity. Grain samples obtained from each replicate row were evaluated for 100 kernel weight and for deoxynivalenol content using GC/MS and published methods (Mirocha et al., 1998; Fuentes et al., 2005). Disease severity, incidence, 100 kernel weights and DON data were subject to analysis of variance (ANOVA) and the Bonferroni mean comparison test (p = 0.05) was used to compare treatment means (Statistix 7.0, Tallahassee, FL).
For field work conducted in Wooster, OH, Pioneer Brand 2545 and Freedom wheat were planted at a rate of approximately 79 seeds m-1 of row in Ravenna silt loam using a Hege 1000 Series plot planter (Wintersteiger, Inc., Salt Lake City, UT) at the Ohio Agricultural Research and Development Center near Wooster, OH. Prior to planting, the field was mold-board plowed and 84.2 kg ha-1 of ammonium nitrate was broadcast over the field and incorporated with a disc. The experimental treatments were arranged in a randomized block design with 6 replicate plots. Each experimental unit consisted of a 7-row plot that measured 1.7x4.6 m. Additional nitrogen was applied the following spring as 109 kg ha-1 of ammonium nitrate. Plots were inoculated by broadcasting corn kernels infested with a mixture of 10 G. zeae strains approximately 3 weeks prior to wheat flowering. Plots were mist irrigated each day from 1 week prior to flowering to 2 weeks after flowering at a volume of approximately 2.5 cm of water/day. Experimental treatments were made at early wheat flowering (Feekes 10.5.1) as described for the Peoria, IL trial with the following exceptions. Identical amounts of total active ingredients or colony forming units were applied at 188 L ha-1 with a CO2-pressurized back pack sprayer that used 2 Twinjet XR8001VS nozzles were mounted 38 cm apart and at 60° inwards to allow simultaneous coverage of each side of heads in individual rows of wheat. Field assessments of FHB severity and incidence were made by evaluating 75 heads per replicate (450 heads/treatment) when plants were between mid-milk and soft dough development. When grain reached full maturity, wheat heads were harvested using a Hege plot combine (Hege Maschinen, Waldenburg, Germany) set on the lowest blower setting to retain lighter weight kernels infected by G. zeae. Harvested wheat was evaluated for yield, test weight and DON. Data were subject to ANOVA and the Bonferroni mean comparison test (p = 0.05) was used to compare treatment means (Statistix 7.0, Tallahassee, FL).
The overall efficacy of treatments at the Peoria, IL and Wooster, OH field sites was determined by calculating a Relative Performance Index (RPI) (Slininger et al., 2010b) for the treatment efficacy observed for each combination of site (Peoria, IL and Wooster, OH), wheat variety (Pioneer 2545 and Freedom) and disease parameter (disease severity, disease incidence and DON) measured. The 12 RPI’s obtained for each treatment (2 sitesx2 varietiesx3 disease parameters) were then subject to ANOVA and the Bonferroni mean comparison test (p = 0.05) was used to compare overall RPIefficacy treatment means. RPI’s allow treatment data obtained on dissimilar population types (such as identical treatments applied at different locations or to wheat varieties that differ in susceptibility to G. zeae) using different types of quantitative measures of disease intensity (such as disease severity, diseases incidence and DON data) to be compared directly using a standardized scale. RPI’s are dimensionless and theoretically range in value from 0 to 100 for normally distributed data. In the present study, RPIefficacy was calculated by: | (F-2) | *25 where F = (X-Xavg)/s, X is a single disease measurement and Xavg and s are the average and standard deviation, respectively, of the data obtained for each unique combination of field location, wheat variety and disease intensity variable. Treatments with higher RPIefficacy values have enhanced efficacy in reducing FHB disease parameters.
RESULTS AND DISCUSSION
Growth and quantification of cells in co-culture: Cells of the three biocontrol strains (C. flavescens OH 182.9 (NRRL Y-30216), C. aureus OH 71.4 (NRRL Y-30213) and C. aureus OH 181.1 (NRRL Y-30215)) utilized in producing co-cultures in liquid medium did not produce colonies that were distinguishable from each other when grown on common nutrient media. Results from studies using Biolog YT Microplate™ plates revealed that the trisaccharide melezitose supported 3 distinct levels of growth for the three yeast strains (Fig. 1). Growing individual strains and known ratios of mixtures of the three strains on a solid medium containing melezitose as a sole carbon source resulted in three distinguishable colony sizes and counts which agreed with expected counts from mixing known cell numbers of the three strains. After 4 days’ incubation at 28°C, strains OH 182.9, OH 71.4 and OH 181.1 produced the largest, intermediate and smallest colonies, respectively, on the melezitose medium. Slininger and co-workers relied on differential growth rates, colony morphology and colony color produced on two types of media to separate counts of three strains of Pseudomonas biocontrol agents produced in mixed strain fermentations (Slininger et al., 2010a).
| Fig. 1: | Distinguishable colony sizes of strains Cryptococcus flavescens OH 182.9 and C. aureus OH 71.4 on a medium containing melezitose as a sole carbon source. Under identical growth conditions, C. aureus OH 181.1 produces smaller colonies than either of the strains pictured |
| Fig. 2: | Growth of component strains of a co-culture of Cryptococcus aureus strains OH 181.1 and OH 71.4 in SDCL medium in shake flasks when inoculated at an initial ratio of 14:1, respectively. An “*” above an incubation time indicates that the populations of individual strains of the co-culture differed significantly at the time point (Tukey HSD, p = 0.05). Means represent averages from three separate shake flask experiments |
All possible combinations of three yeast antagonists were co-cultivated in shake flasks to produce near equivalent levels of each component strain after 48, 60 and 72 h (Fig. 2-4; data not shown for the co-culture of OH 181.1 and OH 182.9) demonstrating for the first time, the feasibility of producing multiple strains of yeast biocontrol agents in a single batch fermentation, thereby reducing the manufacturing and possibly registration costs associated with conducting separate fermentations of each component strain.
| Fig. 3: | Growth of component strains of a co-culture of Cryptococcus flavescens strain OH 182.9 and C. aureus OH 71.4 in SDCL medium in shake flasks when inoculated at an initial ratio of 1:4, respectively. An “*” above an incubation time indicates that the populations of individual strains of the co-culture differed significantly at the time point (Tukey HSD, p = 0.05). Means represent averages from three separate shake flask experiments |
| Fig. 4: | Growth of component strains of a co-culture of Cryptococcus aureus OH 181.1, C. flavescens OH 182.9 and C. aureus OH 71.4 in SDCL medium in shake flasks when inoculated at an initial ratio of 45:1:4, respectively. An “*” above an incubation time would indicate that the populations of individual strains of the co-culture differed significantly at the time point (Tukey HSD, p = 0.05). Means represent averages from three separate shake flask experiments |
While populations of component strains in co-cultures were frequently statistically different during the early stages of culture growth, in nearly every instance, populations of each component of the two- and three-strain co-cultures were not statistically separable after 36 h of incubation. Significant strain population differences in the early incubation stages of some co-cultures were associated with disparities in the ratio of seed inocula used to initiate the cultures. The inoculum of strain OH 182.9 was reduced compared to the other strains used to initiate co-cultures because strain OH 182.9 has the fastest doubling time of the three strains studied.
Efficacy of co-cultures in reducing Fusarium head blight on wheat in the greenhouse: The co-culture of C. flavescens OH 182.9 and C. aureus OH 71.4 was the only treatment that reduced FHB disease severity significantly compared to the control (32%, p = 0.05, Dunnett’s t-test) when averaged across four greenhouse studies, though all of the antagonist treatments reduced disease severity arithmetically. The co-culture of strains OH 182.9 and OH 71.4 reduced FHB severity by 10% to 25% more than the single antagonist cultures and other co-cultures. Disease severity averaged 65% for the control and 44 to 59% for wheat treated with the various strain cultures.
Our work and that by others has reported that mixtures of strains can enhance and/or improve the consistency of biological control (Janisiewicz, 1996; Schisler et al., 1997; Johnson, 2010). Published reports on this subject have dealt with applications of strains that have been mixed after cultivation. In a recent study Slininger et al., (2010b), co-cultures of strains proved to be more efficacious and, significantly, consistent in inciting biological control of various maladies of potatoes than were mixtures of the same strains produced individually. Additional work on co-culturing microorganisms to develop biocontrol products is limited to one study and involves solid substrate, rather than liquid culture, fermentation (Olivain et al., 2004). The limited literature on multi-strain liquid cultures concerns producing biochemical products or the degradation of toxic compounds (Hanly and Henson, 2011; Nachiappan et al., 2011; O-Thong et al., 2011; Zhong et al., 2011). Interestingly, Johnson (2010), regarding the topic of pathogen refuge, postulates that some potentially infectious pathogen propagules are not susceptible to the influence of an antagonistic microbial agent but that the size of the pathogen refuge can be reduced via a number of measures including antagonist mixtures. Combinations of antagonist strains, such as produced via co-culturing in this study and that of Slininger et al. (2010a, b) could owe their enhanced efficacy to the co-cultured strains possessing compatible abilities for reducing pathogen refuge size.
In the present study, assays were conducted to determine how to produce equivalent numbers of antagonist cells in co-cultures and this was accomplished by varying the ratio of cells of the strains used in a particular co-culture.
| Table 2: | 2008 field trial results at Peoria, IL: Influence of Cryptococcus flavescens OH 182.9, C. aureus OH 71.4 and a co-culture of strains OH 182.9 and OH 71.4 on winter wheat cultivar Freedom |
| 1 OH 182.9 and OH 71.4 are abbreviations for strains C. flavescens OH 182.9 (NRRL Y-30216) and C. aureus OH 71.4 (NRRL Y-30213), respectively. OH 71.4 + OH 182.9 co-culture was produced by inoculating culture broth with both strains to produce a culture at harvest with near equivalent cell numbers of each strain (see Methods). 2 DS = Disease severity (= Average % of individual heads visually diseased), DI = Disease incidence, DON = Deoxynivalenol. 3 Within a column, means not followed by the same letter are significantly different (p = 0.05, Bonferoni mean separation) | |
| Table 3: | 2008 field trial results at Wooster, OH: Influence of C. flavescens OH 182.9, C. aureus OH 71.4 and a co-culture of strains OH 182.9 and OH 71.4 on winter wheat cultivar Freedom |
| 1 OH 182.9 and OH 71.4 are abbreviations for strains Cryptococcus flavescens OH 182.9 (NRRL Y-30216) and C. aureus OH 71.4 (NRRL Y-30213), respectively. OH 71.4 + OH 182.9 co-culture” was produced by inoculating culture broth with both strains to produce a culture at harvest with near equivalent cell numbers of each strain (see Methods). 2DS = Disease severity (= Average % of Individual heads visually diseased), DI = Disease incidence, DON = Deoxynivalenol. 3 Within a column, means not followed by the same letter are significantly different (P=0.05, Bonferoni mean separation) | |
In previous studies with FHB antagonist OH 182.9, we have demonstrated the importance of optimizing carbon loading, C:N ratio (Zhang et al., 2005) and reducing incubation temperature in the late log growth stage of the strain (Zhang et al., 2006; Dunlap et al., 2007) in order to enhance the efficacy and stress tolerance of the antagonist. We have not attempted to alter growth conditions to optimize cell biocontrol efficacy and/or stress tolerance in yeast co-cultures but previous results indicate such studies have the potential to increase the efficacy of the product produced.
Influence of strains OH 182.9, OH 71.4 and a co-culture of both on FHB in field grown wheat: For studies on Pioneer Brand 2545 and Freedom wheat in Peoria, IL, high variability in field data precluded statistically separating disease data obtained for individual disease parameters in most cases. On both wheat varieties, single antagonist and the co-culture treatments did not differ significantly from the control regarding disease severity though antagonist OH 71.4 alone or in co-culture with strain OH 182.9 arithmetically reduced FHB severity to the greatest extent (Table 2). On wheat variety Pioneer 2545, the same two treatments significantly reduced disease incidence (p = 0.05, Bonferoni mean separation) while only the co-culture of OH 71.4 and OH 182.9 reduced incidence on wheat variety Freedom. None of the treatments significantly altered DON (Table 2) or 100 kernel weights (data not shown). Data variability similarly limited statistical separation of treatment means in the trial conducted in Wooster, Ohio. On Pioneer Brand 2545, the co-culture of strains OH 71.4 and OH 182.9 reduced disease severity and incidence arithmetically but not significantly, compared to the other treatments and the control (Table 3). On wheat cultivar Freedom, treatments had no significant effect on disease severity, incidence, DON (Table 3) or test weight (data not shown).
RPI analysis of the overall effect of treatments across both field sites, wheat varieties and disease parameters used revealed that treatment with the co-culture of strains OH 71.4 and OH 182.9 significantly reduced disease parameters overall, as evidence by a higher RPI value for this treatment, compared to the untreated control (p = 0.05, Bonferoni mean separation) (Table 4). The RPIs calculated for treatment with strain OH 182.9 alone and OH 71.4 alone indicated these treatments reduced FHB disease parameters arithmetically, but not significantly, compared to the control. Interestingly, in field work conducted in four States of the U.S. over two years, treatment with the fungicide Prosaro® (Bayer CropScience, Research Triangle PK, NC) at flowering and a co-culture of strains OH 182.9 and OH 71.4 five days later significantly reduced the mycotoxin deoxynivalenol (DON) by an average of 36% (Yuen et al., 2010).
| Table 4: | Overall relative performance index values for treatment efficacy (RPIefficacy) in reducing FHB disease parameters in 2008 Peoria, IL and Wooster, OH field trials conducted to determine the influence of C. flavescens OH 182.9, C. aureus OH 71.4 and a co-culture of strains OH 182.9 and OH 71.4 on Fusarium head blight |
| 1 OH 182.9 and OH 71.4 are abbreviations for strains Cryptococcus flavescens OH 182.9 (NRRL Y-30216) and C. aureus OH 71.4 (NRRL Y-30213), respectively. OH 71.4 + OH 182.9 co-culture” was produced by inoculating culture broth with both strains to produce a culture at harvest with near equivalent cell numbers of each strain. 2 Overall RPIefficacy value is an average of 12 individual RPI values obtained for each combination of site (Peoria, IL and Wooster, OH), wheat variety (Pioneer 2545 and Freedom) and disease parameter (disease severity, disease incidence and DON) measured in 2008 field trials. 3 RPIefficacy means not followed by the same letter are significantly different (p = 05, Bonferoni mean separation) | |
Treatment with the co-culture alone at flowering significantly reduced disease severity across all locations compared to the control both years of the study. Because co-cultured inoculum for the studies of Yuen et al. (2010) was produced in 100 L, rather than 1.5 L fermentors and was separated from spent broth before use, it is possible that improvements in inoculum efficacy could have been achieved in the present study if a similar scale and handling of the co-cultured inoculum was utilized.
The potential for obtaining superior efficacy with co-cultures, such as one produced using strains C. flavescens OH 182.9 and C. aureus OH 71.4, compared to the individual component strains and cost benefits in producing, processing and registering a co-cultured biocontrol product justifies additional research effort on this concept. Genomic and proteomic research tools should foster further understanding of the complexity of relationships between host, biocontrol agents and pathogen. Concomitantly, research of this nature should provide additional guidance on how to improve the quality of biomass produced in liquid culture and its efficacy when deployed in field applications alone or in combination with other control measures.
ACKNOWLEDGMENTS
The expertise of Jennifer Sloan, Kelly Hall, Todd Hicks, Joe Rimelspach, Matthew Wallhead, William Bardall and Karlten Austin in conducting experiments is greatly appreciated as are the efforts of Dr. Yanhong Dong, University of Minnesota, in conducting the DON analysis. Studies were made possible, in part, by a grant from the U.S. Wheat and Barley Scab Initiative. Salaries and additional research support for P.A. Paul and M.J. Boehm were provided by state and federal funds to the Ohio Agricultural Research and Development Center, The Ohio State University.