Detection of dsRNA from Cleistothecia and Conidia of the Grape Powdery Mildew Pathogen, Uncinula Necator

Double-stranded RNA species ranging in molecular weight from 0.95 to 6.3 × 106 were detected in grapevines in New York. We recently showed that two of the species (Mr = 5.3 and 4.4 × 106) are associated with rupestris stem pitting disease. In this report, we show that the other eight detectable dsRNA species are associated with the powdery mildew fungus, Uncinula necator. These dsRNAs associated with the powdery mildew fungus were previously detected in leaves and epidermal stem tissue of grapevines infected with powdery mildew. The same dsRNA species were also detected from extracts of isolated cleistothecia and conidia of U. necator devoid of plant tissue. Isometric and rigid rodlike particles were observed in single cleistothecia preparations when examined under transmission electron microscopy.

Patterns of release of the secondary conidia of Claviceps africana, the sorghum ergot pathogen in Australia

Trials were conducted in southern Queensland, Australia between March and May 2003, 2004 and 2005 to study patterns of hourly and daily release of the secondary conidia of Claviceps africana and their relationships with weather parameters. Conidia were trapped for at least one hour on most (> 90%) days in 2003 and 2004, but only on 55% of days in 2005. Both the highest daily concentration of conidia, and the highest number of hours per day when conidia were trapped, were recorded 1-3 days after rainfall events. Although the pattern of conidial release was different every day, the highest hourly conidial concentrations occurred between 10.00 hours and 17.00 hours on 73% of all days in the three trials. Hours when conidia were trapped were characterized by higher median values of temperature, windspeed and vapour pressure deficit, lower relative humidity, and leaf wetness values of 0%, than hours when no conidia were recorded. The results indicate that fungicides need to be applied to the highly ergot-susceptible male sterile (A-) lines of sorghum in hybrid seed production blocks and breeders' nurseries as soon as possible after rainfall events to minimize ergot severity.

Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability

The insect pathogen Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosos can be effective biocontrol agents when relative humidity (RH) is close to 100%. At reduced water availability, germination of propagules, and therefore host infection, cannot occur. Cultures of B. bassiana, M. anisopliae and P. farinosus were grown under different conditions to obtain conidia with a modified polyol and trehalose content. Conidia with higher intracellular concentrations of glycerol and erythritol germinated both more quickly and at lower water activity (a(w)) than those from other treatments. In contrast, conidia containing up to 235.7 mg trehalose g-1 germinated significantly (P < 0 05) more slowly than those with an equivalent polyol content but less trehalose, regardless of water availability. Conidia from control treatments did not germinate below 0.951 - 0.935 a(w) (≡ 95.1 - 93.5% RH). In contrast, conidia containing up to 164.6 mg glycerol plus erythritol g-1 germinated down to 0.887 a(w) (≡ 88.7% RH). These conidia germinated below the water availability at which mycelial growth ceases (0.930 - 0.920 a(w)). Germ tube extension rates reflected the percentage germination of conidia, so the most rapid germ tube growth occurred after treatments which produced conidia containing the most glycerol and erythritol. This study shows for the first time that manipulating polyol content can extend the range of water availability over which fungal propagules can germinate. Physiological manipulation of conidia may improve biological control of insect pests in the field.

Effects of KCl concentration on accumulation of acyclic sugar alcohols and trehalose in conidia of three entomopathogenic fungi

Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were grown on Sabouraud Dextrose Agar (SDA) modified with KCl to give a range of water activity (a(w)) from 0.938 to 0.998. Growth of all three species was optimal at 0.983 a(w) and growth occurred over the a(w) range tested. Acyclic sugar alcohol (polyol) and trehalose content of conidia was determined by HPLC and found to vary with species and a(w). Conidia of B. bassiana and P. farinosus were found to contain totals of 1.5% and 2.3% polyols respectively at 0.998 a(w), and double these amounts at <0.950 a(w). Conidia of M. anisopliae contained from 5.7% to 6.8% polyols at each a(w) tested. In conidia of all three species the predominant polyol was mannitol. The lower molecular weight polyols, arabitol and erythritol, were found to accumulate at reduced a(w). Small amounts of glycerol were present in conidia of each species; <15% total polyols. Conidia of B. bassiana and M. anisopliae contained about 0.5% trehalose from 0.970 to 0.998 a(w), but only trace amounts below 0.950 a(w). Conidia of P. farinosus contained 2.1% trehalose at 0.998 a(w) and this decreased to <0.1% below 0.950 a(w). Potential to manipulate the endogenous reserves of conidia of these biological control agents to enhance viability and desiccation tolerance is discussed.

Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi

The entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were cultured on solid agar media containing different carbohydrate components (glycerol, glucose, trehalose or starch) at concentrations of ≤ 142.7 g added carbon 1-1 for 30 d at 25°C. The water activity (a(w)) of the media ranged from 0.925 to 0.998. Growth of M. anisopliae and P. farinosus was stimulated between 0.975 and 0.995 a(w) on glucose media and that of P. farinosus at 0. 975 a(w) on glycerol media. At < 0.970 a(w), growth of each fungal species was significantly reduced (P < 0.05). Polyhydroxy alcohols (polyols) and trehalose were extracted from conidia produced on different media and quantified using HPLC. Total polyol content of conidia produced on glucose media varied between 5.2 and 52.2 mg g-1 for B. bassiana, 77.3 and 90.3 mg g-1 for M. anisopliae, and 26.7 and 76.1 mg g-1 for P. farinosus. The amounts of specific polyols in conidia varied significantly from media of different glucose concentrations. Mannitol was the predominant polyol in conidia of all three species, with conidia of M. anisopliae, for example, containing as much as 75.2 mg mannitol g-1 when cultured on glucose media. The amount of the lower molecular mass polyols glycerol and erythritol was greater in conidia produced on glucose media with > 50.0 g added carbon 1-1 than that in conidia produced at lower glucose concentrations. Conidia contained between 10.8 and 20.8 mg glycerol plus erythritol g-1 on glucose media with 142.7 g added carbon 1-1, depending on species. Conversely, conidia of B. bassiana and P. farinosus contained maximum amounts of trehalose ( ≤ 23.5 mg g-1) when produced on glucose media with < 50.0 g added carbon l-1, and trehalose content was considerably less at higher glucose concentrations. There were accumulations of glycerol and erythritol in conidia of all three species when grown on glycerol media with > 25.0 g added carbon 1-1; conidia of B. bassiana contained up to 154.0 mg glycerol plus erythritol g-1. hen B. bassiana and P. farinosus were grown on trehalose media, conidia contained up to 222.1 mg trehalose g-1. By contrast, conidia of M. anisopliae contained < 17.0 mg trehalose g-1 under all conditions tested. The water availability of solutions of different polyols is discussed in relation to their potential to act in osmotic adjustment during germination. The ability to manipulate polyol and trehalose content of fungal propagules may be critical in enhancing the storage life and efficacy of biological control agents.

Saturated salt solutions for humidity control and the survival of dry powder and oil formulations of Beauveria bassiana conidia

Oil-based formulated conidia sprayed on steel plates and conidia powder (control) of Beauveria bassiana isolate IMI 386243 were stored at temperatures from 10 to 40 degrees C in desiccators over saturated salt solutions providing relative humidities from 32 to 88%, or in hermetic storage at 40 degrees C, and moisture contents in equilibrium with 33 or 77% relative humidity. The negative semi-logarithmic relation (P < 0.005) between conidia longevity (at 40 degrees C) and equilibrium relative humidity did not differ (P > 0.25) between formulated conidia and conidia powder. Despite this, certain saturated salts provided consistently greater longevity (NaCl) and others consistently shorter longevity (KCl) for formulated conidia compared to conidia powder. These results, analysis of previous data, and comparison with hermetic storage, indicate that storage of conidia over saturated salt solutions provides inconsistent responses to environment and so may be problematic for bio-pesticide research. In hermetic storage, oil formulation was not deleterious to longevity and in the more moist environment enhanced survival periods. (c) 2005 Elsevier Inc. All rights reserved.

Proteins and Peptidases from Conidia and Mycelia of Scedosporium apiospermum Strain HLPB

The conidia-mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62-48 and 22-18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis.

Improving photoprotection of Beauveria bassiana conidia for biological control of the cactus pest Dactylopius opuntiae in the semiarid region northeast of Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of culture media in viability test of conidia of entomopathogenic fungi

O presente trabalho objetivou investigar se meios de cultura utilizados em teste de viabilidade afetam a germinação de conídios de cinco isolados de Lecanicillium lecanii, cinco de Beauveria bassiana e quatro de Paecilomyces fumosoroseus. Os testes foram realizados em lâminas de microscopia contendo um dos seguintes meios de cultura: Ágar-água (AA), Meio Mínimo (MM), Batata, dextrose e ágar (BDA), Batata, dextrose, ágar e 1% de extrato de levedura (BDAL), Sabouraud, dextrose, ágar e extrato de levedura (SDAL) e Meio Completo (MC). Delimitaram-se três áreas por lâmina e em cada uma aplicou-se 0,05mL de uma suspensão com concentração de 5,5 x 105 conídios ml-1. Para cada isolado foi realizado um bioensaio, com seis tratamentos e cinco repetições. Avaliou-se a germinação 15 horas após incubação, a 26±0,5ºC. Os meios de cultura influíram na capacidade de germinação das três espécies estudadas, ocorrendo variações inter e intraespecíficas. Verificou-se que os meios Completo e BDA proporcionaram as maiores porcentagens de germinação dos isolados de L. lecanii, sendo que e as menores foram obtidas nos meios SDAL e AA. Os meios ricos em nutrientes (BDA, BDAL e Completo) favoreceram a germinação dos isolados de B. bassiana, o que não ocorreu com os meios pobres (AA e MM). Nos meios Completo e BDA foram obtidas as maiores porcentagens de germinação dos isolados de P. fumosoroseus. As menores percentagens, por sua vez, foram obtidas no meio SDAL. Entretanto, alguns isolados apresentaram alta germinação em meios pobres em nutrientes (AA e MM).

Persistence of conidia of Entomophthora muscae in relation to age, temperature, and humidity

In this paper, the effect of age, humidity, and temperature on the conidial survival of Entomophthora muscae was evaluated. E. muscae was obtained from Musca domestica in a dairy in Itatiba (São Paulo, Brazil) and maintained in the laboratory by continuous passage through flies. Furthermore, the ability of conidia to infect flies at three temperatures (17, 21, and 27 degrees C), four ages of conidia (12, 72, 96, and 154 hours) and two humidities (100 and 60% RH) was evaluated. The temperature of 21 degrees C was the most favorable for the infection of house flies. Humidity was a cause of variation at 27 degrees C when the conidia were up to 12 hours old, but had no effect at lower temperatures. Conidia held at 100% RH and aged 72 hours caused no infection at 17 degrees C, but were infective at 21 degrees C. In the present study, conidia retained viability much longer than previously observed. Finally, the effect of humidity, temperature, and conidial age is discussed.

Potential misidentification of in situ archaeological tool-residues: starch and conidia

Microscopic identification of organic residues in situ on the surface of archaeological artefacts is an established procedure. Where soil components morphologically similar to use-residue types exist within the soil, however, there remains the possibility that these components may be misidentified as authentic residues. The present study investigates common soil components known as conidia, fungal spores which may be mistaken for starch grains. Conidia may exhibit the rotating extinction cross under cross-polarised light commonly diagnostic of starch, and may be morphologically indistinguishable from small starch grains, particularly at the limits of microscope resolution. Conidia were observed on stone and ceramic archaeological artefacts from Honduras, Palau and New Caledonia, as well as experimental artefacts from Papua New Guinea. The findings act as a caution that in situ analysis of residues, and especially of those less than 5 mu m in size, may be subject to misidentification. (c) 2005 Elsevier Ltd. All rights reserved.