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.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus, Metarhizium anisopliae var. acridum, Part 1: Comparison of cell wall characteristics and drying stability among three spore types

Metarhizium anisopliae var. acridum (IMI 330189) can produce at least three spore types in vitro; blastospores, submerged conidia, and aerial conidia, as defined by culturing conditions, sporogenesis, and spore morphology. This study compares morphological characteristics (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), and performance (germination rate and drying stability) among these three spore types. Submerged conidia and aerial conidia both possessed thick, double-layered cell walls, with hydrophobic regions on their surfaces. However, in contrast to aerial conidia, submerged conidia have: (1) a greater affinity for the lectin concanavalin-A; (2) more anionic net surface charge; and (3) a less distinct outer rodlet layer. Blastospores were longer and more variable in length than both submerged conidia and aerial conidia, and had thinner single-layered cell walls that lacked an outer rodlet layer. Also, blastospores had a greater affinity than either conidia type for the lectin, wheat germ agglutinin. Blastaspores lacked hydrophobic regions on their surface, and had a lower anionic net surface charge than submerged conidia. In culture, blastospores germinated the fastest followed by submerged conidia, and then aerial conidia. Survival of submerged conidia and aerial conidia were similar after drying on silica gel, and was greater than that for blastospores. We provide corroborating information for differentiating spore types previously based on method of production, sporogenesis, and appearance of spores. These physical characteristics may have practical application for predicting spore-performance characteristics relevant to production and efficacy of mycoinsecticides.     

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     

A comparison of the virulence,stability and cell-wall-surface characteristics of three spore types produced by the entomopathogenic fungus Beauveria bassiana

Summary Beauveria bassiana can produce three spore types; aerial conidia, submerged conidia and blastospores. We have examined the spore surface characteristics (hydrophobicity and cell-wall surface lectins), thermal inactivation and the virulence towards the migratory grasshopper, Melanoplus sanguinipes, of each of the three spore types. The hydrophobicities of the aerial and submerged conidia were quite similar. Blastospores were less hydrophobic than either of the two types of conidia. Hydrophobic interactions are thought to play a significant role in attachment of the spore to the host organism. However, the less hydrophobic blastospores were slightly more virulent (LT₅₀ of 6.50 days) when compared to the aerial and submerged conidia (7.12 and 7.24 days), respectively. The lectin-binding characteristics of the aerial and submerged conidia were very similar but differed from that of blastospores. Growth of blastospores on a variety of carbohydrates did not affect their lectin-binding characteristics. Spore viability measurements showed that aerial and submerged conidia retained their viability for a longer period than blastospores. The similarity in hydrophobicity, stability, virulence and lectin-binding of aerial and submerged conidia make the latter an ideal candidate for mycoinsecticide production since they can be recovered after growth on inexpensive substrates.Offprint requests to: G. G. Khachatourians     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.     

Keimungsprozente und Keimungskurven der Konidien und Submers Gebildeten Blastosporen eines Stammes vonBeauveria bassiana (Bals.) Vuill.

Zusammenfassung Für Infektionsversuche und biologische Bek?mpfung k?nnen Massenkulturen vonBeauveria bassiana entweder Konidien oder Blastosporen liefern. Die qualitative Bewertung solcher Sporen und Sporenpr?parate und ebenso deren Standardisierung setzen voraus, dass auch das Keimverhalten der Sporen bekannt ist. Deshalb wurden die zeitabh?ngigen Keimungsprozente und Keimungskurven der Blastosporen und Konidien eines Stammes vonB. bassiana auf Malzextrakt-Pepton-Agar bei 24 und 18°C ermittelt und damit auch die Unterschiede, die durch Temperatur und Sporenart bedingt sind. Für die Bewertung und Standardisierung von Sporen und Sporenpr?paraten k?nnen die Keimprozente nach 24 und 36 Stunden oder die Zeit für 50% Keimung Kriterien sein und mit den hier wiedergegebenen Standardwerten verglichen werden.

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Summary Mass cultures ofBeauveria bassiana provide either conidia or blastospores for laboratory experiments and biological control measures. For qualitative evaluation of spores and spore preparations it is of primary importance to know the germination behaviour of spores. Therefore, the percentage germination of blastospores and conidia in relation to time (0–36 hours) and temperature (24 and 18°C) have been determined on malt-extract peptone agar (Table 1) and demonstrated by graphs (Fig. 1). The periods of time necessary for 50% germination of blastospores and conidia show differences depending on temperature and type of spore (Table 2). Pronounced differences of percentage germination as caused by the two temperatures become obvious with blastospores after 6–10, with conidia after 15–20 hours; after 24 hours, such differences are relatively low (Table 3). For evaluation and standardization of spores and spore preparations ofB. bassiana the percentage germination at 24°C as obtained after 24 and 36 hours or the period of time for 50% germination can be used as criteria and compared with the corresponding standard values presented.

     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

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 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.     

Media and Fermentation Processes for the Rapid Production of High Concentrations of Stable Blastospores of the Bioinsecticidal Fungus Paecilomyces fumosoroseus

In shake flask and fermentor studies, various media components and culture inocula were tested to improve P. fumosoroseus spore production rates, yield and stability. To evaluate inoculum potential and inoculum scale-up for fermentor studies, conidia and liquid culture-produced spores of various strains of P. fumosoroseus were compared as inoculum. Inoculation of liquid cultures with blastospores at concentrations of at least 1×10⁶ spores mL^-1 resulted in the rapid production of high concentrations of blastospores (∼1×10⁹ spores mL^-1, 48 h fermentation time) for all strains tested. The rapid germination rate of blastospores (90% after 6 h incubation) compared to conidia (>90% after 16 h incubation) and the use of higher inoculum rates reduced the fermentation time from 96 to 48 h for maximal spore yields. A comparison of various complex nitrogen sources showed that liquid media supplemented with acid hydrolyzed casein or yeast extract supported the production of high concentrations of blastospores that were significantly more desiccation-tolerant (79-82% survival after drying) when compared to blastospores produced in media supplemented with other nitrogen sources (12-50% survival after drying). For rapid spore production, requirements for trace metals and vitamin supplementation were dependent on the type of hydrolyzed casein used in the medium. Fermentor studies with two strains of P. fumosoroseus showed that high concentrations (1.3-1.8×10⁹ spores mL^-1) of desiccation-tolerant blastospores could be produced in 48-h fermentations. These studies have demonstrated that the infective spores of various strains of the fungal bioinsecticide Paecilomyces fumosoroseus can be rapidly produced using deep-tank, liquid culture fermentation techniques.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

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.     

Adhesion of the Entomopathogenic Fungus Beauveria (Cordyceps) bassiana to Substrata

The entomopathogenic fungus Beauveria bassiana produces at least three distinct single-cell propagules, aerial conidia, vegetative cells termed blastospores, and submerged conidia, which can be isolated from agar plates, from rich broth liquid cultures, and under nutrient limitation conditions in submerged cultures, respectively. Fluorescently labeled fungal cells were used to quantify the kinetics of adhesion of these cell types to surfaces having various hydrophobic or hydrophilic properties. Aerial conidia adhered poorly to weakly polar surfaces and rapidly to both hydrophobic and hydrophilic surfaces but could be readily washed off the latter surfaces. In contrast, blastospores bound poorly to hydrophobic surfaces, forming small aggregates, bound rapidly to hydrophilic surfaces, and required a longer incubation time to bind to weakly polar surfaces than to hydrophilic surfaces. Submerged conidia displayed the broadest binding specificity, adhering to hydrophobic, weakly polar, and hydrophilic surfaces. The adhesion of the B. bassiana cell types also differed in sensitivity to glycosidase and protease treatments, pH, and addition of various carbohydrate competitors and detergents. The outer cell wall layer of aerial conidia contained sodium dodecyl sulfate-insoluble, trifluoroacetic acid-soluble proteins (presumably hydrophobins) that were not present on either blastospores or submerged conidia. The variations in the cell surface properties leading to the different adhesion qualities of B. bassiana aerial conidia, blastospores, and submerged conidia could lead to rational design decisions for improving the efficacy and possibly the specificity of entomopathogenic fungi for host targets.     

Liquid-culture production of blastospores of the bioinsecticidal fungus<Emphasis Type="Italic"> Paecilomyces fumosoroseus</Emphasis> using portable fermentation equipment

The production of fungal spores using on-site, non-sterile, portable fermentation equipment is technically constrained. Very little information is available on the production requirements, such as medium concentration, inoculum stabilization, required fermentation times, and maintenance of axenic growth. In this study, we developed a two-part, liquid concentrate of the production medium that remains stable and soluble at room temperature. We also examined inoculum stability and showed that freeze- or air-dried blastospore preparations were stable for 7 days after rehydration when stored at 4 °C. The use of a low-pH (pH 4), relatively rich complex medium provided a growth environment deleterious to bacterial growth yet conducive to rapid sporulation by Paecilomyces fumosoroseus. High concentrations of blastospores (7.9×10⁸/ml) of P. fumosoroseus were produced in a 40-h fermentation with very low levels of bacterial contamination when the fermentor was charged with a blastospore production medium with a starting pH of 4 and inoculated with blastospore concentrations greater than 1×10⁶ spores/ml. These studies demonstrate that the use of disinfected, portable fermentation equipment has potential for on-site production of high concentrations of blastospores of the bioinsecticidal fungus P. fumosoroseus.     

Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures

The effects of cultural parameters such as carbon and nitrogen source and environmental factors including temperature and pH were investigated on spore and mycelial yield of Trichoderma viride, which has potential as a biocontrol agent against species of Fusarium in batch culture and fed-batch culture where there was limiting nutrient. The results obtained indicated that growth and sporulation of T. viride were greatly influenced by various carbon and nitrogen sources, and by environmental factors such as pH and temperature. Mannitol, wheat bran and rice bran as sole carbon sources appear to stimulate high mycelial growth and spore yield in fed-batch culture. Growth and sporulation were also favoured by NaNO₃, peptone and NH₄SO₄ as the nitrogen sources in fed-batch and batch cultures_. Maximum growth and sporulation was between pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good for mycelium growth of T. viride while temperatures between 30 to 45 °C were good for sporulation. The amount of spore and mycelium produced and the time required for attainment of maximum spore yield increased with increasing carbon and nitrogen source in batch culture. The final spore yield obtained in fed-batch culture was two times higher than the apparent spore-carrying capacity of batch culture. These results show that T. viride is capable of growing and sporulating with varied nutritional and environmental conditions, and, therefore, this strain of T. viride may be useful as a biocontrol agent under diverse physiological and environmental conditions.     

Carbon Concentration and Carbon-to-Nitrogen Ratio Influence Submerged-Culture Conidiation by the Potential Bioherbicide Colletotrichum truncatum NRRL 13737

We assessed the influence of various carbon concentrations and carbon-to-nitrogen (C:N) ratios on Colletotrichum truncatum NRRL 13737 conidium formation in submerged cultures grown in a basal salts medium containing various amounts of glucose and Casamino Acids. Under the nutritional conditions tested, the highest conidium concentrations were produced in media with carbon concentrations of 4.0 to 15.3 g/liter. High carbon concentrations (20.4 to 40.8 g/liter) inhibited sporulation and enhanced the formation of microsclerotiumlike hyphal masses. At all the carbon concentrations tested, a culture grown in a medium with a C:N ratio of 15:1 produced more conidia than cultures grown in media with C:N ratios of 40:1 or 5:1. While glucose exhaustion was often coincident with conidium formation, cultures containing residual glucose sporulated and those with high carbon concentrations (>25 g/liter) exhausted glucose without sporulation. Nitrogen source studies showed that the levels of C. truncatum NRRL 13737 conidiation were similar for all protein hydrolysates tested. Reduced conidiation occurred when amino acid and inorganic nitrogen sources were used. Of the nine carbon sources evaluated, acetate as the sole carbon source resulted in the lowest level of sporulation.     

Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus

Several entomopathogenic fungi produce toxins that could be used as bioinsecticides in integrated pest management programs. Paecilomyces fumosoroseus is currently used for the biological control of the whiteflies Bemisia tabaci and B. argentifolii. Supernatants from submerged batch culture, where the fungus produced abundant dispersed mycelium, conidia and blastospores, were toxic to the whitefly nymphs. The most abundant metabolite was purified by HPLC and identified by mass spectrometry and NMR as dipicolinic acid. Both the dipicolinic acid produced by the fungus and the chemically synthesized compound had insecticidal activity against third-instar nymphs of the insect. Dipicolinic acid was toxic to the whitefly nymphs in bioassays involving topical applications. In submerged culture, the specific growth rate of P. fumosoroseus was 0.054 h⁻¹, the specific glucose consumption rate was 0.1195 g g⁻¹ h⁻¹ and the specific dipicolinic acid production rate was 0.00012 g g⁻¹ h⁻¹. Dipicolinic acid was detected after 24 h when the fungus started growing; and dipicolinic acid production was directly correlated with fungal growth. Nevertheless, the yield was low and the maximal concentration was only 0.041 g l⁻¹. The maximal concentrations of conidia and blastospores (per milliliter) were 1.4×10⁸ and 7×10⁷, respectively.     

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.     

Influence of Temperature,pH and Selected Growth Media on Germination,Growth and Sporulation of Aschersonia placenta and Hypocrella raciborskii

Tween 70 at 0.1% provided the best conditions for germination of Aschersonia placenta. Optimum germination and growth of the germ tube occurred over a temperature range of 25–30°C and a pH range of 5.0–6.0. A temperature of 30°C resulted in the longest germ tube at 45 μm. Apparently, temperature and pH did not affect the type of germination, with polar germination being consistently recorded for in excess of 60% of conidia. In general, growth and sporulation seemed to be much better in semi‐solid than in liquid media. Amongst several plant media tested, pumpkin consistently gave the most mycelial growth and sporulation. The ability of A. placenta to sporulate on the surface of liquid culture has increased the possibilty of its mass production for the purpose of formulating a microbial pesticide against the indigenous scale insects of tropical fruits such as durian and guava in Malaysia.