Microsclerotia production of Metarhizium spp. for dual role as plant biostimulant and control of Spodoptera frugiperda through corn seed coating

The fungal genus Metarhizium comprises entomopathogenic species capable of producing overwintering structures known as microsclerotia. These structures offer many advantages in pest control due to the formation of infective conidia in situ and their persistence in the environment under adverse conditions. In addition, the in vitro production of Metarhizium microsclerotia under controlled liquid fermentation is faster and with greater process control than the production of aerial conidia. However, the potential of Metarhizium microsclerotia to control pests from the orders Lepidoptera and Hemiptera is unexplored. In this study, we examined the ability of Metarhizium spp. microsclerotia to promote corn growth and to provide plant protection against Spodoptera frugiperda (Lepidoptera: Noctuidae) and Dalbulus maidis (Hemiptera: Cicadellidae), through seed coating using microsclerotial granules. A screening to find higher microsclerotia producers was conducted by culturing 48 native Brazilian isolates of Metarhizium spp. (Metarhizium anisopliae, Metarhizium robertsii, Metarhizium humberi and Metarhizium sp. indeterminate). The best microsclerotia producers, M. anisopliae ESALQ1814, M. robertsii ESALQ2450 and M. humberi ESALQ1638 improved the leaf area, plant height, root length, and dry weight of plants compared to un-inoculated plants. Significant reduction in S. frugiperda survival (mortality > 55% after 7 days) was observed when larvae were fed on corn plants treated with any of the three Metarhizium species. Conversely, survival of D. maidis adults were unaffected by feeding on fungus-inoculated plants. Our results suggest that microsclerotia of Metarhizium spp. may act as biostimulants and to provide protection against S. frugiperda in corn through seed coating, thus adding an innovative strategy into the integrated management of this major worldwide pest.     

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.     

Development of pilot-scale fermentation and stabilisation processes for the production of microsclerotia of the entomopathogenic fungus Metarhizium brunneum strain F52

Using 100 L stirred-tank bioreactors, we evaluated the effect of fermentation parameters and drying protocols on the production and stabilisation of microsclerotia (MS) of the entomopathogenic fungus Metarhizium brunneum (formerly M. anisopliae F52). Results showed that stirred-tank bioreactors can be used to mass produce stable MS of Metarhizium and that culturing and drying protocols significantly affected MS yield and stability. Length of fermentation (4–7 days) for Metarhizium cultures had no significant impact on biomass accumulation, MS formation or the storage stability of the air-dried MS granules. Although cultures of Metarhizium grown on media with a carbon-to-nitrogen (C:N) ratio of 30:1 produced significantly more biomass when compared to cultures grown in media with a C:N ratio of 50:1, MS formation and desiccation tolerance following drying were similar. After storage for 1 year at 4°C, conidia production by air-dried MS granules from 50:1 media was significantly higher compared to MS granules from 30:1 media. The addition of diatomaceous earth (DE) to cultures of Metarhizium prior to drying at rates of 0–60 g L^?1 had no significant effect on MS desiccation tolerance but did impact conidia production. Air-dried MS granules without DE produced significantly more conidia g^?1 during the first 4 months of storage, but after 1 year, conidia production was similar regardless of DE content of the MS granule. Microsclerotial granules with higher moisture levels (2.6–5.0% w/w) produced significantly more conidia immediately after drying and MS granules with low moisture (0–2.5% w/w) produced more conidia after 12 months storage.     

Exploring virulence of new and less studied species of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. from Brazil for two-spotted spider mite control

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 10⁷ conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT₅₀). The lowest LT₅₀ (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.     

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12–16 mg ml⁻¹) and blastospore yields (6–11 × 10⁸ blastospores ml⁻¹) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml⁻¹) but produced similar blastospore concentrations (7.3–11.0 × 10⁸) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus. Received 27 October 1998/ Accepted in revised form 06 May 1999     

Comparison between conidia and blastospores of Esteya vermicola, an endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively.     

Impact of carbon and nitrogen nutrition on the quality, yield and composition of blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus

The impact of growing cultures of Paecilomyces fumosoroseus in liquid media containing four combinations of glucose and casamino acids (8 g l^–1 or 80 g l^–1 glucose, 1.32 g l^–1 or 13.2 g l^–1 casamino acids) was evaluated, based on blastospore production, germination rate, viability after freeze-drying and short-term storage stability. When blastospores were produced using a high casamino acid concentration, blastospore yields and germination rates were significantly higher (13.2–18.5×10⁷ blastospores ml^–1, 50–60% germination after 4 h), compared to cultures grown in media containing lower casamino acid concentrations (0.4–2.3×10⁷ blastospores ml^–1, 10–20% germination after 4 h). Chemical analyses of blastospore composition showed that accelerated blastospore germination may be related to increased proteinaceous reserves rather than to glycogen or lipid accumulation. Tolerance to freeze-drying by blastospores suspended in spent medium was enhanced by a high initial casamino acid concentration in the culture medium (75% survival) and by the residual glucose concentrations in the spent medium. Under the conditions of this study, the storage stability of blastospores of P. fumosoroseus was unaffected by the nutritional condition in which they were produced.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

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.     

Dissolved oxygen levels affect dimorphic growth by the entomopathogenic fungus Isaria fumosorosea

The entomopathogenic fungus Isaria fumosorosea is capable of dimorphic growth (hyphal or yeast-like) in submerged culture. Using 250-mL baffled flasks, culture volumes of 50, 100, 150, and 200 mL were grown in a shaker incubator at 350 rpm and 28°C. Dissolved oxygen (DO) was continuously monitored using a non-invasive oxygen monitoring system. Culture volumes of 50 mL maintained DO concentrations above 10% throughout the 3-day growth period and accumulated biomass and produced blastospores more rapidly (1.2×10⁹ blastospores mL^?1 in 2 days) than the other culture volumes tested. Dissolved oxygen was depleted in culture volumes of 100, 150, and 200 mL after 20.5, 16.8, and 13.5 h, respectively. The DO in 150 and 200 mL cultures remained exhausted (<3%) throughout the growth period resulting in significantly lower blastospore yields and increased hyphal growth. These results were used to establish oxygen levels (>20% DO) for I. fumosorosea growth in 100-L bioreactors resulting in blastospore production (1.1×10⁹ blastospores mL^?1 in 2 days) comparable to highly aerated, low volume shake flask cultures. In addition, maintaining higher DO levels resulted in increased blastospore production by cultures of I. fumosorosea grown on low-cost nitrogen sources (cottonseed meal and soy flour) that previously elicited excessive hyphal growth. These studies showed that oxygen availability is essential for significant yeast-like growth by I. fumosorosea cultures and that continuous monitoring of oxygen concentrations in shake flask cultures can be used to establish aeration conditions for bioreactors.     

Susceptibility of larvae of Trichoplusia ni and Anticarsia gemmatalis to intrahemocoelic injections of conidia and blastospores of Nomuraea rileyi

The LD₅₀ for larvae of Trichoplusia ni injected with blastospores of Nomuraea rileyi was 4.30 ± 1.16 hyphal bodies/larva; the LD₅₀ for injected conidia was ca. 25,000 conidia/larva. The dose-mortality regression line for blastospores was Y = 4.6504 + 0.5487 X. Larval mortalities of Anticarsia gemmatalis and T. ni at 100 blastospores/larva were 0.4 ± 0.5% and 96.7 ± 1.9%, respectively. At a dosage of 25,000 conidia/larva, larval mortalities for A. gemmatalis and T. ni were 0.4 ± 0.5% and 43.1 ± 8.7%, respectively. Thus, larvae of A. gemmatalis were > 100 times and >200 times more resistant to injected conidia and blastospores, respectively, than were larvae of T. ni. Resistance of A. gemmatalis to N. rileyi may not be solely at the integumental barrier, as is often believed, but may also be a function of an internal physiological response.     

Comparison of aerial conidia and blastospores from two entomopathogenic fungi against Diaphorina citri (Hemiptera: Liviidae) under laboratory and greenhouse conditions

This study compared the insecticidal activity of liquid culture-produced blastospores and solid substrate-produced aerial conidia of Beauveria bassiana GHA and Isaria fumosorosea ARSEF3581 strains against Diaphorina citri adults. Insects exposed to 10⁷ propagules/ml in a spray residue contact leaf bioassay died within 6 days at 25°C, with no significant differences between fungal treatments. At higher concentrations (10⁸ propagules/ml), I. fumosorosea conidia killed psyllids faster compared to its blastospore formulation, i.e. 4 versus 5 days, respectively. In greenhouse tests, the same treatments applied to infested citrus plants (2?×?10⁶ spores/ml) all significantly reduced the number of nymphs compared with the untreated controls over 3 weeks; however, only I. fumosorosea blastospores significantly reduced the number of F1 adult psyllids when compared with controls. Similar results were observed in the follow-up greenhouse test, where I. fumosorosea blastospores were the most effective treatment overall, reducing D. citri populations by about 60% after 21 days; by contrast, imidacloprid killed almost 100% of psyllids within a week in both tests. Fewer psyllids exhibited mycosis in the greenhouse (i.e. ≈20 versus?≥?87% in the laboratory). This is the first report comparing both conidial and blastospore formulations of B. bassiana and I. fumosorosea for the control of a psyllid pest. Field testing is required to determine how successful different spore formulations might be under various environmental conditions.     

MripacC regulates blastosphere budding and influences virulence of the pathogenic fungus Metarhizium rileyi

Fungal dimorphism is the ability of certain fungi to switch between two different cellular forms, yeast and mycelial forms, in response to external environmental factors. The pacC/Pal signal transduction pathway responds to neutral and alkaline environments and is also involved in the fungal dimorphic transition. In this study, we investigated the function of the pacC homolog, MripacC, which regulates the dimorphic transition and modulates virulence of the insect pathogenic fungus Metarhizium rileyi. MripacC expression was upregulated under alkaline condition, with increased number of yeast-like cells compared to the number of hyphae cells. A MripacC deletion mutant (ΔMripacC) was obtained by homologous replacement and exhibited decreased blastospore budding, with direct development of conidia into hyphae without entering the yeast-like stage when cultured on alkaline medium. Observation of host hemolymph morphology and analysis of samples to detect the main immune factors revealed a decreased ability of ΔMripacC to evade the host immune system. The results of insect bioassays showed that ΔMripacC had decreased virulence with extended median lethality time. Together, the results suggested that MripacC not only regulated adaptation to acidic and alkaline environments, but also influenced virulence by budding blastospores. This elucidation of the function of MripacC adds to our understanding of blastospore budding and virulence of this fungal pathogen.     

Toxicity of Khaya senegalensis (Meliaceae) Seed Oil Against the Larvae of Boophilus decoloratus: A One-Host Tick in Cattle

The in vitro toxicity of seed oil of Khaya senegalensis (family: Meliaceae) was tested against the larvae of a one host tick, Boophilus decoloratus (family: Ixodidae or hard tick) known as ‘blue tick’ parasitic mainly to cattle commonly found in savannas of tropical equatorial Africa. The 20, 40, 60, 80, and 100 % concentrations of seed oil were found to kill all (100 % mortality) the larvae after 28, 28, 24, 20 and 20 h respectively.     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.     

Differential susceptibility of blastospores and aerial conidia of entomopathogenic fungi to heat and UV-B stresses

We investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores were produced in solid or liquid Adámek-modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m⁻² under artificial UV-B radiation. Our results showed that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in regions where heat and UV-B are limiting environmental factors.     

Biodegradation of entomopathogenic hyphomycetes: Influence of clay coating on Beauveria bassiana blastospore survival in soil

The survival of naked and clay-coated Beauveria blastospores in soil was investigated with an experimental biodegradation method using a trap technique. At various times of incubation, traps were collected to study changes in biomass, inoculum potential, and infection potential of degraded blastospores, and to investigate the colonization of blastospore biomass by antagonistic soil-borne microorganisms. Infection potential was tested on larvae of Plutella maculipennis and Leptinotarsa decemlineata. Naked blastospores were inactivated after 3 weeks incubation, while clay-coated blastospores were still active after 2 months incubation in soil at 20°C. All investigations demonstrated the protective role of clay coating against biodegradation of fungal propagules by soil bacteria and protozoa. Consequently, clay coating of blastospores is proposed for the formulation of entomopathogenic hyphomycetes propagules. Moreover, antagonists implicated in lysis of blastospores, must be considered as an important part of the environmental response to a massive introduction of a fungus used for insect control.     

Performance of two isolates of Isaria fumosorosea from hot climate zones in solid and submerged cultures and thermotolerance of their propagules

Isaria fumosorosea frequently causes mycosis of agricultural pests in the hot semiarid and dry tropical regions of Mexico. Because temperature tolerance restricts the use of fungal biopesticides, we investigated two isolates from these areas for possible development into mycoinsecticides for use in hot weather agricultural zones. We studied the effects of culture system (solid or submerged cultures) and temperature on the fungal growth, extracellular enzyme production, pathogenicity, and thermotolerance of the produced propagules. Between 20 and 28 °C, the specific growth rates of the isolate PCC were higher on solid media, but in the submerged culture, the isolate P43A grew faster even at temperatures of up to 34 °C. On solid media, P43A produced 1.5-fold more proteases than PCC, but in the submerged culture, both strains had similar activities. Under the same culture conditions, PCC produced a blastospore:conidia ratio of 1:2, and P43A produced a ratio of 1:5. PCC aerial conidia had the shortest Lethal Time 50 (LT₅₀, the time to reach 50 % mortality) against Galleria mellonella larvae, but LT₅₀ was equal for the aerial conidia and the submerged propagules of P43A and PCC. The submerged and aerial propagules of P43A were more thermotolerant than those of PCC. Each isolate performed differently in each culture system, and we concluded that the intended production method should be included as a criterion for screening of entomopathogenic fungus. We found that thermotolerance is a specific characteristic of an isolate from a given species. Because of its specific characteristics, P43A shows more promise for the development of a submerged conidia-based mycoinsecticide for foliar application in aqueous form in hot climate regions.     

Production ofPaecilomyces Fumosoroseus conidia in submerged culture

Mexican isolates ofPaecilomyces fumosoroseus (Wize) Brown & Smith virulent to nymphs and adults ofBemisia tabaci Gennadius (Homoptera: Aleyrodidae) were screened in terms of spore production in submerged culture. Effects of light, temperature stress and yeast extract on sporulation were studied. Cycles of 12 hours light/12 hours dark increased spore production as well as an incubation for 24 hours at 37°C prior to incubation at 30°C. In absence of organic nitrogen both fungal growth and sporulation were very low. Spore production in fermentors with a culture media of a C:N ratio of 25 was doubled as compared to a media with a C:N ratio of 11. Both conidia and blastospores were produced. Production of conidia directly from blastospores through microcyclic sporulation was observed. The proportion of conidia obtained under optimal conditions was 88.8%. Submerged culture ofP. fumosoroseus seemed advantageous compared to ricefilled plastic bags production method because of shorter fermentation times, higher spore yields and substantially higher volumetric spore productivity. Results indicated that careful manipulation of nutritional and environmental conditions allowed for production of conidia during submerged growth ofP. fumosoroseus, microcyclic sporulation being induced under a set of environmental conditions including temperature stress and nutrients limitation.     

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.