Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l^?1 with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS–DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or ?20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1–7.3 × 10⁶ l^?1 after 3 days growth with maximum MS yields (0.7–1.1 × 10⁷ l^?1) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS–DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g^?1). All MS granules showed similar stability after storage at either 26 or ?20 °C for 3.5 months.     

The impact of temperature on the production and fitness of microsclerotia of the fungal bioherbicide Mycoleptodiscus terrestris

The impact of growth temperature was evaluated for the fungal plant pathogen Mycoleptodiscus terrestris over a range of temperatures (20–36°C). The effect of temperature on biomass accumulation, colony forming units (cfu), and microsclerotia production was determined. Culture temperatures of 24–30°C produced significantly higher biomass accumulations and 20–24°C resulted in a significantly higher cfu. The growth of M. terrestris was greatly reduced at temperatures above 30°C and was absent at 36°C. The highest microsclerotia concentrations were produced over a wide range of temperatures (20–30°C). These data suggest that a growth temperature of 24°C would optimize the parameters evaluated in this study. In addition to growth parameters, we also evaluated the desiccation tolerance and storage stability of air-dried microsclerotial preparations from these cultures during storage at 4°C. During 5 months storage, there was no significant difference in viability for air-dried microsclerotial preparations from cultures grown at 20–30°C (>72% hyphal germination) or in conidia production (sporogenic germination) for air-dried preparations from cultures grown at 20–32°C. When the effect of temperature on germination by air-dried microsclerotial preparations was evaluated, data showed that temperatures of 22–30°C were optimal for hyphal and sporogenic germination. Air-dried microsclerotial preparations did not germinate hyphally at 36°C or sporogenically at 20, 32, 34, or 36°C. These data show that temperature does impact the growth and germination of M. terrestris and suggest that water temperature may be a critical environmental consideration for the application of air-dried M. terrestris preparations for use in controlling hydrilla.     

Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects

Microsclerotia (MS), overwintering structures produced by many plant pathogenic fungi, have not been described for Metarhizium anisopliae. Three strains of M. anisopliae – F52, TM109, and MA1200 – formed MS in shake flask cultures using media with varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under the conditions of this study, all strains produced MS, compact hyphal aggregates that become pigmented with culture age, in addition to more typical blastospores and mycelia. While all strains formed desiccation tolerant MS, highest concentrations (2.7–2.9 × 10⁸ L⁻¹ liquid medium) were produced in rich media with C:N ratios of 30:1 and 50:1 by strain F52. All three strains of M. anisopliae produced similar biomass concentrations when media and growth time were compared. Strain MA1200 produced higher concentrations of blastospores than the other two strains of M. anisopliae with highest blastospore concentrations (1.6 and 4.2 × 10⁸ blastospores ml⁻¹ on days 4 and 8, respectively) in media with the highest carbon and nitrogen concentrations. Microsclerotial preparations of M. anisopliae containing diatomaceous earth survived air-drying (to <5 % moisture) with no significant loss in viability. Rehydration and incubation of air-dried MS granules on water agar plates resulted in hyphal germination and sporogenic germination to produce high concentrations of conidia. Bioassays using soil-incorporated, air-dried MS preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis. This is the first report of the production of sclerotial bodies by M. anisopliae and provides a novel approach for the control of soil-dwelling insects with this entomopathogenic fungus.     

Efficacy of Metarhizium anisopliae microsclerotial granules

The entomopathogenic fungus Metarhizium anisopliae has very recently been shown to produce microsclerotia (MS) – compact, heavily melanised, hyphal aggregates – in liquid media. Soil incorporation bioassays of dried MS preparations of three isolates of M. anisopliae were conducted using third instar Tetanops myopaeformis (sugarbeet root maggot) in clay and/or clay loam field soils as a model system to demonstrate efficacy. At rates as low as 23 mg MS granules/100 g dry soil, the biocontrol efficacy of MS granules of M. anisopliae Strain F52 produced in liquid media with a high carbon concentration (36 g/L) and high C:N ratios (30:1, 50:1) were superior to MS preparations produced in low carbon (8 g carbon/L) media and a high carbon medium with a 10:1 C:N ratio. Bioassays using MS formulations of M. anisopliae strains MA1200 and TM109 produced in high carbon and high C:N ratio media were superior in efficacy to the other MS production media tested. MS preparations of M. anisopliae F52 showed superior efficacy against the sugarbeet root maggot in comparison with more conventional, conidia-covered nutritive (corn grit) granules in a clay and clay soil. The MS granules were also highly efficacious against the sugarbeet root maggot at soil moisture levels as low as 0.983 A _w (?2.33 MPa). Granular preparations incorporating Metarhizium MS can serve as a viable formulation for the use of this fungus against soil insects.     

Evaluating different carriers of Metarhizium brunneum F52 microsclerotia for control of adult Asian longhorned beetles (Coleoptera: Cerambycidae)

Microsclerotia (MS) of Metarhizium brunneum strain F52 (Hypocreales: Clavicipitaceae) were processed as granules using three carriers: kaolin clay, microcrystalline cellulose (MCC), or diatomaceous earth (DE). In a series of experiments aimed at comparing viable conidial production, each MS carrier type was hydrated using a decreasing range of water activities on glycerol or polyethylene glycol (PEG200)-amended media. Conidial density and percent germination of conidia declined significantly as water activity was lowered. All three carrier types produced >2–6?×?10⁹ viable conidia/gram at higher water activities (а_w >0.987) but were still capable of producing 2.9?×?10⁷?±?1.6?×?10⁶ conidia/g (30% glycerol а_w?=?0.883) and 1.9?×?10⁷?±?6.5?×?10⁶ conidia/g (30% PEG200 а_w?=?0.924) at the lowest tested water activities. MS carrier types were sprayed onto 0.4?m length logs with or without hydromulch formulation. The median survival times of Asian longhorned beetles, Anoplophora glabripennis (Coleoptera: Cerambycidae), exposed by climbing double the length of the logs of hydromulch-treated bark, were 16.5–20.5?d while beetles exposed to logs without hydromulch formulation had median survival times of 22–25.5?d. Overall, experiments showed that there were few biologically significant differences between the MS carrier types.     

Modified Adamek's medium renders high yields of Metarhizium robertsii blastospores that are desiccation tolerant and infective to cattle-tick larvae

Blastospores are yeast-like cells produced by entomopathogenic fungi that are infective to arthropods. The economical feasible production of blastospores of the insect killing fungus Metarhizium spp. must be optimized to increase yields. Moreover, stabilization process is imperative for blastospore formulation as a final product. In this sense, our goal was to increase blastospore production of two Metarhizium isolates (ESALQ1426 and ESALQ4676) in submerged liquid cultures. A modified Adamek's medium was supplemented with increased glucose concentrations and the fermentation time was accelerated by using a blastospore pre-culture as inoculum. Virulence of air-dried stable blastospores was compared with conidia toward larvae of the cattle tick, Rhipicephalus microplus. Our results revealed that blastospore production of Metarhizium is isolate- and species-dependent. Glucose-enriched cultures (140 g glucose/L) inoculated with pre-cultures improved yields with optimal growth conditions attained for Metarhizium robertsii ESALQ1426 that rendered as high as 5.9 × 10⁸ blastospores/mL within 2 d. Resultant air-dried blastospores of ESALQ1426 were firstly proved to infect and quickly kill cattle tick larvae with comparable efficiency to conidia. Altogether, we argue that both osmotic pressure, induced by high glucose titers, and isolate selection are critical to produce high yields of blastospores that hold promise to control cattle-tick larvae.     

Production Factors Involved in the Formulation of Erynia neoaphidis as Alginate Granules

Granular formulations of the aphid-pathogenic fungus Erynia neoaphidis were produced by entrapping mycelia in alginate polysaccharide polymers. Four Swiss isolates were compared for the numbers of conidia discharged from the surface of alginate granules in standardized laboratory assays and two were considered to be suitable for further development. Conidiation was achieved from granules produced using nozzle diameters of 2.0. 1.0 and 0.5 mm from glass burettes or a novel vibrating tip apparatus. The mean diameters of dried granules varied from 0.5 to 1.8 mm. The addition of sucrose, potato starch or chitin in alginate solutions significantly improved the numbers of discharged conidia. W ith freshly produced granules, there was a 14.2- fold increase in sporulation from 6.3 to 89.7 conidia mm - 2 using 2% (w/v) sucrose. Increases of 1.6-to 2.3-fold, from 11.0 to 17.7 and 25.2 conidia mm - 2, were observed using 5% (w/v) starch or chitin respectively. The overnight drying of granules in a laminar flow hood and storage for 4 days at 4 C made differences in sporulation more obvious. There was a 15.5-fold difference in conidial numbers of 12.4 and 0.8 conidia mm - 2 from granules with and without sucrose respectively. For starch and chitin, there were 76.0-and 46.5-fold increases from 0.4 to 30.4 and 18.6 conidia mm - 2respectively. Fresh or dried alginate granules containing 2% sucrose and 5% starch gave 8.6-26.6% infection in laboratory bioassays with nymphs of pea aphid, Acyrthosiphon pisum , which were not significantly different when compared with infections of 6.7-22.9% using agar cultures or unsupplemented granules. Further studies on desiccation and storage regimes are required in order to improve the short-term shelf-life of E. neoaphidis alginate granules.     

Effects of Nutrition on Desiccation Tolerance and Virulence of Colletotrichum truncatum and Alternaria alternata Conidia

The promising mycoherbicides Colletotrichum truncatum and Alternaria alternata were grown respectively in liquid and solid semi-defined media. C. truncatum conidia produced in a medium with a C:N ratio of 5:1 showed higher desiccation tolerance (survival during storage) at 15% relative humidity and 25°C, greater germination on the host leaf and greater disease expression on Sesbania exaltata than those produced in media with C:N ratios of 15:1 or 40:1. Similar results were obtained with conidia of A. alternata produced on a medium with a C:N ratio of 15:1. Conidia washed with 0.9% (w/v) NaCl produced higher tolerance to desiccation, and greater disease incitement, than unwashed conidia of C. truncatum or conidia washed with water. In contrast, washing had no positive effect on desiccation tolerance in A. alternata .     

Sporulation,storage and infectivity of obligate aphid pathogen Pandora nouryi grown on novel granules of broomcorn millet and polymer gel

Aims: Producing granular cultures of obligate aphid pathogen Pandora nouryi for improved sporulation and storage. Methods and Results: Small millet–gel granules were made of the mixtures of 80–95% millet powder with 5–20% polymer gel (polyacrylamide, polyacrylate or acrylate‐acrylamide copolymer) and inoculated with mycelia at 30 mg biomass g^?1 dry granules plus 87·5% water, followed by static incubation at 20°C for 4–12 days. The fungus grew well on 12 preparations but best on that including 10% copolymer. An 8‐day culture of this preparation discharged maximally 58·5 × 10⁴ conidia mg^?1 granule at 100% RH and was capable of ejecting conidia at the nonsaturated regimes of 86–97% RH. During storage at 6°C, granular cultures with >85% water content had twofold longevity (120 days) and half‐decline period (34–36 days) of those stored at room temperature. The steadily high water content preserved the cultures better than that decreasing at 6°C. However, conidia from 70‐day‐stored granules were less infective to Myzus persicae nymphs than those from fresh ones based on their LC₅₀s. Conclusions: The millet–gel granules had higher sporulation capacity than reported Pandora cultures and a capability of spore discharge at nonsaturated humidity. Significance and Impact of the Study: The granular cultures are more useful for aphid control.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Influence of two formulation types and moisture levels on the storage stability and insecticidal activity of Beauveria bassiana

The formulation of mycopesticides may require a physical separation of conidia from the substrate and subsequent drying. In the present study, Beauveria bassiana conidia produced by solid-state fermentation were harvested either through a dry or washing protocol. Washed conidia were used to design a water-dispersible granule (WG) formulation, whereas sieved conidia were mixed with an emulsifiable oil to achieve an oil-based formulation (OD). Potential harmful effects caused by the formulation type on the storage stability and insecticidal activity against Hypothenemus hampei were assessed. As expected, the time for initial conidial germination to drop 50% (GT₅₀) in all treatments was deeply influenced by storage temperatures, which varied from over 180 days at 4 °C to less than 90 days at 35°C. In all four tested temperatures, GT₅₀s for unformulated dry conidia were significantly higher than for those formulated as WG, and the latter was similar to conidia formulated as OD in the two highest temperatures. Residual water content in the OD formulation (1,600 vs. 340?ppm) had a negative influence on conidial survival under storage, whereas WG granules immediately dried after the washing protocol showed conidial germination similar to granules exposed to a slower dehydration regime. Mortality of H. hampei adults exposed to different concentrations of B. bassiana formulated as WG was slightly lower (10–15%) than either the OD or the unformulated conidia. In brief, we have demonstrated that formulation type and their moisture level can affect the storage stability and insecticidal activity of B. bassiana conidia toward the coffee berry borer. Of particular importance, we have shown that drying oils prior to formulation could improve the storage of mycopesticides, an approach that may find industrial applications.     

Effect of liquid culture media on morphology,growth, propagule production,and pathogenic activity of the Hyphomycete,Metarhizium flavoviride

Two isolates of Metarhizium spp. were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M. flavoviride (or M. anisopliae var. acridum) strain IMI 330189, and Mf324 = M. flavoviride strain ARSEF324). Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects. Shake-flask experiments were carried out at 28 °C in the dark. Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity. Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules. Submerged propagule yields were higher withMf189 than with Mf324 in all seven media. While high concentrations of propagules (1.4 to 2.4 × 10⁸ propagules ml^-1 for MF189 and1.4 to 8.3 × 10⁷ propagules ml^-1 for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins–Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media). Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules. In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides. In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia. Infection potential of submerged propagules was assayed on Schistocerca gregaria. Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 10⁷ propagules ml^-1. All seven media produced submerged propagules that were highly infectious for S. gregaria larvae. Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins–Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control. This revised version was published online in June 2006 with corrections to the Cover Date.     

Drying and formulation of blastospores of Paecilomyces fumosoroseus (Hyphomycetes) produced in two different liquid media

Formulation matrices can play an important role in improving the storage survival and biocontrol efficacy of microorganisms used for the control of pest insects. In this study, liquid culture-produced blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with different inert and organic materials prior to air-drying. Paecilomyces fumosoroseus blastospores were produced in two different liquid media, a basal salts medium supplemented with Casamino acids and glucose (LM1) and a medium containing peptone of collagen and glucose (LM2). Blastospores produced in the two test media were formulated with various supports. The formulation supports were cornstarch, rice flour, talc powders, Mexican lime, calcined kaolin clay, and diatomaceous earth. Several of the supports were tested at different concentrations. The initial and long-term (after storage at 4 and 28 °C) survival of the formulated, air-dried blastospores were evaluated. Initial blastospore viabilities were affected by the formulation material and by the blastospore production medium. Medium composition, drying support and storage temperature had an impact on the long-term survival of the blastospores. Under the conditions of the study, LM1 produced higher concentrations of blastospores that not only survived drying better than blastospores produced in LM2 but also maintained viability longer during storage in the formulation supports tested. The nature of the drying supports was shown to have a significant impact on the storage stability of all blastospores, particularly those produced in LM1. Under the production, drying and storage conditions used in the study, calcined kaolin clay formulations stored at 4 °C had the best storage stability. In all formulations tested, spore survival over time was reduced for blastospore formulations stored at 28 °C rather than 4 °C.     

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT₅₀ of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60–75 g/kg rice), faster sedimentation time (ST₅₀) (2.3–2.65 h in 0.1% (w/v) Tween 80) and lower LC₅₀ (1.4–5.7×10³ conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).     

Optimising inoculum yield and shelf life of Plectosphaerella cucumerina,a potential bioherbicide for Cirsium arvense

Plectosphaerella cucumerina was identified as a potential bioherbicide for controlling Cirsium arvense in Canada and New Zealand. The current study evaluated production conditions using two isolates (one from each country) to determine whether the yield and shelf life of inoculum are suitable for mass production. Mycelial growth and sporulation in culture both increased from 15°C to 25°C and declined at higher temperatures with no mycelial growth at 37°C. The Canadian isolate produced fewer conidia than a New Zealand isolate. Potato dextrose-based liquid media with moderate to high concentrations of carbohydrates (25%, 50%, and 100%) maximised conidia production and these base media produced conidia with the highest germination rate (>80%) both at harvest and after 4 weeks stored at 4°C in 2.5% glycerol, 40% milk glycerol or after air drying. However, after 10-week storage, the conidia failed to germinate. Sporulation occurred during growth on all solid substrates tested (rice, rolled barley, and triticale), but conidial germination was highest on rice and barley, both before and after air drying. By contrast to conidia, 90% of mycelia-infested barley grains were viable after 3 years of storage at room temperature, although viability was lost by this time on the other substrates. This study has shown that the nutritional base is an important determinant of sporulation and shelf life for P. cucumerina. Although the yield of conidia in liquid medium was adequate to justify further development of P. cucumerina as a bioherbicide, improvement in its shelf life, or alternate formulation types that extend the shelf life, must be made for commercial efficiency.     

Oxidative stress in Nostoc flagelliforme subjected to desiccation and effects of exogenous oxidants on its photosynthetic recovery

The metabolism of reactive oxygen species in Nostoc flagelliforme and effects of exogenous oxidants on its photosynthetic recovery were investigated to obtain insight into oxidative stress in desiccation and its possible damaging impact on photosynthetic apparatus. No ascorbate was detected with ascorbate oxidase in N. flagelliforme. Superoxide dismutase (SOD) remained active even after three years drying storage and its activity was 78% of that in fully recovered samples. The SOD activity decreased during desiccation or in drying storage. Intracellular active oxygen production was studied by incubating samples in BG₁₁ medium for 2 h and measuring the oxidation of 2,7 -dichlorohydrofluorescein diacetate. The production rate was 38.11 nmol DCF_g (d.wt)^-1 h^-1 in dried field samples and was significantly higher than in fully recovered or air-dried samples. The balance between intracellular active oxygen production and the defense systems mightbreak down in air-dried and dried field samples. Treatment with exogenous oxidants slowed the photosynthetic recovery especially with singlet oxygen. Oxidative stress might play an important role in desiccationinduced damages to the photosynthetic apparatus.     

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

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

Comparison of the relative efficacy of an insect baiting method and selective media for diversity studies of Metarhizium species in the soil

Metarhizium are a commonly occurring group of entomopathogenic fungi normally found in soil. The most common methods to assess the diversity of Metarhizium species in soil are (i) the use of selective media and (ii) insect baiting using Galleria mellonella larvae. We compared the recovery efficiency from soil of four common species of Metarhizium (Metarhizium anisopliae, Metarhizium pingshaense, Metarhizium brunneum and Metarhizium robertsii) using these two methods. Firstly, we compared the number of colony forming units (CFU) produced in vitro when grown on two selective media, one containing chloramphenicol, thiabendazole and cycloheximidethe (CTC) and one based on the fungicide dodine (n-dodecylguanidine acetate) (DOD). Secondly, we artificially inoculated natural/non-sterile soil with the four fungal species at a rate of 2×10² and 2×10³ conidia g^?1of soil, baited with G. mellonella, and processed for evaluation using the selective media. The in vitro results showed that the greatest number of CFUs were recorded for M. brunneum. In contrast, when inoculated into soil, more G. mellonella larvae became infected by M. anisopliae. Finally, when using selective media, most CFUs recovered were for M. robertsii. The importance of our results in selecting a method to study the natural occurrence of Metarhizium in soil are discussed.     

Fluidized-bed drying and storage stability of Cryptococcus flavescens OH 182.9, a biocontrol agent of Fusarium head blight

A method to produce dried granules of Cryptococcus flavescens (formerly Cryptococcus nodaensis) OH 182.9 was developed and the granules evaluated for storage stability. Small spherical granules were produced and dried using a fluidized-bed dryer. A drying and survival curve was produced for the process of fluidized-bed drying at 30°C. The granules were dried to different moisture contents (4, 7, 9 and 12%) and evaluated for storage stability at 4°C for up to a year. These different moisture contents granules had the following respective water activities (0.22, 0.38, 0.47 and 0.57 a _w). The results show the storage stability varied significantly across this moisture content range. The 9% moisture content sample had the best short-term stability (up to 4 months), while 4% moisture content had the best long-term survival (1 year). A desorption isotherm of C. flavescens was determined and modeled. The results of the storage stability and drying studies are interpreted in context of the desorption isotherm.     

Media and culturing protocol using a full 25 factorial design for the production of submerged aggregates by the potential bio-herbicide Plectosporium alismatis against weed species of Alismataceae

The potential bio-herbicide Plectosporium alismatis produces drying and UV tolerant micro-sclerotia-like structures named aggregates, effective against the weed Alisma plantago-aquatica. In this study, we evaluated (i) optimal liquid culture conditions that supported the high dry weight, conidia and aggregate yields and (ii) stress-tolerance and effectiveness of aggregates. Using a full factorial 2⁵ design, we studied the impact of agitation (A), glucose concentration (B), Tween 80 concentration (C), malt extract concentration (D), and inoculum density (E), on P. alismatis growth yields. The inoculum density (E), the agitation rate (A), and 3-factor interactions involving E and/or A had significant effects on aggregate yields (average 213 aggregates mL^?1; main effect of E: +112.87; main effect of A: ?74.81), but no significant effect on conidial yields and dry weight. The agitation was maintained at 100?rpm and increasing numbers of conidia were inoculated into the culture media. Maximal aggregate yields (3.6?×?10³ aggregates mL^?1) were obtained with an initial conidial density of 4?×?10⁶ conidia mL^?1. While freshly-harvested, dried and/or UV exposed aggregates germinated at high rates (respectively: 100%; 99%; 76%; 85%), sporogenesis significantly decreased after stress exposure. In bioassays using leaf discs of A. plantago-aquatica, chlorosis and/or necrosis was observed after 8 days incubation, regardless of whether aggregates had been exposed to desiccation and/or UV-radiations prior to application on leaf discs. These data provide a cultural protocol for the production of high numbers of UV and drying resistant aggregates effective against weed species of Alismatacae.