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.     

Effect of Host Plant on the Potential ofPaecilomyces fumosoroseus(Deuteromycotina: Hyphomycetes) for Controlling the Silverleaf Whitefly,Bemisia argentifolii(Homoptera: Aleyrodidae) in Greenhouses

We determined host plant effect on susceptibility of the silverleaf whitefly,Bemisia argentifolii, to the entomopathogenic fungusPaecilomyces fumosoroseus. Whiteflies were reared on three vegetable species (cucumber, cabbage, and tomato) and three cultivars of tomato (Heatwave, Better Boy, and Rutgers). Second instars were sprayed with 5 × 10⁴conidia/cm²ofPfr97, aP. fumosoroseusstrain, used as a microbial control agent of whiteflies. Trials were conducted in an experimental greenhouse, where temperature and relative humidity were adjusted to favor infection (22–33°C, and 68–100% RH). Larval susceptibility to fungal infection was high and not significantly affected by the host plant. Mortality was > 70% 1 week after treatment and increased further during the second week. Percentages of cadavers with subsequent production of conidia observed in the greenhouse did not vary significantly either with the host vegetable species (85–93% 7 days after treatment and 99–100% 14 days after treatment), or with the cultivar of tomato (96–97% 7 days after treatment and 99–100% 14 days after treatment). After incubation under optimal laboratory conditions, the percentages based on the total number of sporulating cadavers (includingin situsporulating individuals and cadavers sporulating afterin vitroincubation) were not significantly influenced either by host vegetable or cultivar of tomato. According to the conditions prevailing in the series of experiments with the three vegetable species or in the series of experiments with the three cultivars of tomatoes, the production of newly formed conidia varied from approximately 10,000 to 18,000 conidia/cadaver. However, in both series, there was no significant influence of the host vegetable species or cultivar. The survival of the newly formed conidia harvested 7 days following treatment reached more than 50% but was not affected by host plant. These results indicate thatP. fumosoroseusshows potential as a microbial control agent for controllingB. argentifoliion greenhouse crops.     

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.     

A Paecilomyces fumosoroseus mutant over-producing chitinase displays enhanced virulence against Bemisia tabaci

A Paecilomyces fumosoroseus strain was mutagenized by u.v. Among 200 colonies, one mutant (M84), showed a large and stable chitin hydrolysis-halo. Glucose consumption and biomass production were similar for M84 and the parental strain. Chitinase was inducible by chitin and repressed by glucose in both strains but, when they were grown on minimal medium plus colloidal chitin as sole carbon source, the parental and M84 strains yielded 198 and 690 mol N-acetylglucosamine, respectively. This results indicate that the mutant strain synthesized a chitinase with a higher activity. Bioassays against Bemisia tabaci nymph, showed that M84 incited a 2-fold higher incidence of disease compared to the parental strain.     

Influence of formulation additives on the desiccation tolerance and storage stability of blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with 10% lactose/1% bovine serum albumin (BSA) or various compositions of Fantesk™, a starch-oil composite prepared by jet-cooking an aqueous dispersion of starch and oil. Storage stability studies with wet blastospore formulations showed that maximum blastospore survival was achieved during low-temperature storage at -20°C with lactose/BSA formulations or starch-oil formulations supplemented with sucrose, zein protein, and whole milk. Under conditions of wet storage at -20°C, the addition of whole milk to starch-oil formulations significantly improved blastospore stability while the addition of sucrose or zein protein had no effect. In freeze-drying studies, no significant differences were seen in blastospore desiccation tolerance or in stability during storage at either 4 or -20°C when blastospores of P. fumosoroseus were formulated with lactose/BSA or starch-oil formulations with sucrose, zein protein, and whole milk. Freeze-dried blastospore formulations stored at 4°C showed no loss in blastospore viability after 3 months storage and blastospore formulations stored at -20°C showed no loss in viability during the entire 12-month study. For freeze-dried, starch-oil formulations, sucrose was shown to improve blastospore survival during the freeze-drying process. The addition of whole milk to starch-oil formulations significantly improved the stability of freeze-dried blastospores stored at 4°C. Compared to unformulated blastospore suspensions that showed blastospore settling after 30 min, suspensions of blastospores formulated with lactose/BSA or starch-oil composites remained stable for up to 2 h after mixing.     

菜青虫感染玫烟色拟青霉后血淋巴蛋白质含量及几种保护酶活力的变化

对玫烟色拟青霉Paecilomyces fumosoroseus侵染3、4龄菜青虫Pieris rapae后,其血淋巴中蛋白质含量、体内保护酶及谷胱甘肽S-转移酶活力进行了研究。结果表明,感病的3、4龄菜青虫血淋巴中蛋白质含量明显低于同期未感染的幼虫;感病虫体内超氧化物歧化酶、过氧化氢酶和过氧化物酶的活力发生了不同程度的改变,其中对3龄菜青虫体内酶活力的影响比4龄幼虫大。此外被侵染的3、4龄菜青虫体内谷胱甘肽S-转移酶活力在感病前期显著高于同期未感染的菜青虫,而在感病后期明显低于同期未感染的菜青虫。     

The effect of selected allelochemicals on germination of conidia and blastospores and mycelial growth of the entomopathogenic fungus,Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Selected allelochemicals that protect plants from invasion by plant pathogenic fungi were investigated for their activity against the entomopathogenic fungus, Paecilomyces fumosoroseus. The alkaloids tomatine, solanine, and camptothecin; the furanocoumarin, xanthotoxin; and the phenolic, tannic acid were tested for their effects on germination of conidia and blastospores and growth of mycelia. The LC50 values (corresponding to 50% inhibition of germination) for tomatine, solanine, camptothecin, xanthotoxin and tannic acid were 51.6, 95.9, 55.9, 83.0 and 72.8 mg/l respectively. When blastospores were placed on media containing a concentration of the individual allelochemicals that inhibit germination in approximately 50% of conidia, all but blastospores on tomatine had significantly less germination than did aerial conidia. Growth rates of mycelia were slowest in the camptothecin medium, followed by those of tomatine and xanthotoxin and were not significantly different from controls in the media containing solanine and tannic acid. A multitude of biotic and abiotic factors are responsible for specificity and degree of pathogenicity of entomopathogens. The effect of crop plant chemistry on the efficacy of entomopathogens should be quantified further in order to maximize their potential when used concomitantly with resistant plant varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes), a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)

Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpis Lutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalence of disease. A possible alternative to current strategies is the biological control of the vector using entomopathogenic fungi. This study evaluates the effects of the fungus, Beauveria bassiana (Bals.) Vuilleman, in different developmental stages of L. longipalpis. Five concentrations of the fungus were utilized ranging from 10⁴ to 10⁸ conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adults exposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified by polymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number of eggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower than that of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed to the positive chemical (pyrethroid, cypermetherin) control was less than 1 day. The effects of fungal infection on the hatching of eggs laid by infected females were also significant and dose-dependent (P < 0.05). With respect to fungal post-infection growth parameters, only germination and sporulation were significantly higher than the fungi before infection (P < 0.001). The identity of the reisolated fungus was confirmed by automated DNA sequencing post-passage in all insect stages. These data show that B. bassiana has good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of this fungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting in benefits to humans and the environment.     

Effects of temperature and solar radiation interactions on the survival of quiescent conidia of the entomopathogenic hyphomycetePaecilomyces fumosoroseus (Wize) Brown and Smith

The detrimental effect of solar radiation on the survival of conidia of the entomopathogenic fungusPaecilomyces fumoroseus was studied by monitoring germinability and ability to form colonies (CFU) of conidia irradiated at two temperatures, 25 and 35 °C, harmless to shaded conidia. There was no apparent effect when spores were exposed to a high level of artificial radiation (0.66 W m^–2 UVB). However, at a lower level of irradiance (0.33 W m^–2), effects of radiation occurred more quickly at 35 °C than at 25 °C. Under natural solar radiation, the rate of decrease in germinability or viability was doubled at 35 °C as compared to 25 °C, indicating an interaction between temperature and radiation effects under natural conditions. This interaction was not detected in indoor experiments, indicating that the spectral distribution of UV radiation has to be taken in account as well as its irradiance when studying its effects.Abbreviations CFU Colony Forming Units - UTC Universal Time Coordinates - UVB Ultra Violet B radiation (280–320 nm)     

Ovicidal and Larvicidal Activity of Conidia and Blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) Against Bemisia argentifolii (Homoptera: Aleyrodidae) with a Description of a Bioassay System Allowing Prolonged Survival of Control Insects

Fungi are the only effective entomopathogens of members of the Aleyrodidae and other homopterans because of the piercing and sucking feeding strategy of these insects. The laboratory assessment of the entomopathogenic activity of fungi often requires a prolonged period of observation. Leaf quality can be the limiting factor in bioassays of fungi against whiteflies that require longer observation periods or those that require conditions that are as close to natural ones as possible. A bioassay system is described that utilizes rooted cabbage leaves infested with the highly polyphagous whitefly, Bemisia argentifolii . Using this method, discriminating dosages of aerial conidia and blastospores of two isolates of Paecilomyces fumosoroseus (Pfr) were bioassayed against eggs and nymphs of B. argentifolii . Low, but significant, mortality (10-20%) of eggs was observed 14 days after exposure to 3.8 104 blastospores/cm2 for the Pfr 97 and European Biological Control Laboratory (EBCL) Pfr 42 isolates. Additionally, the majority of crawlers that had hatched from treated eggs died on the surface of the leaves. Exposure of early second-instar nymphs of B. argentifolii to 3.8 103 conidia or blastospores/cm2 of Pfr 97 resulted in mortalities of 27 and 77% respectively. Identical dosages of conidia and blastospores of the Pfr 42 isolate resulted in mortalities of 59 and 68% respectively. The bioassay method described enables the comparative evaluation of entomopathogenic fungi against whiteflies under diverse biotic and abiotic conditions.     

Phyllactinia chubutiana: a new species of Erysiphales from Patagonia (Argentina)

Phyllactinia chubutiana, a parasite on leaves of Lycium chilense (Solanaceae) collected in the arid Patagonian steppe, is proposed as a new species. The new combination Ovulariopsis insolita is introduced for its anamorph previously described as Oidium insolitum.     

Acute oral intragastric pathogenicity and toxicity in mice of <Emphasis Type="Italic">Paecilomyces fumosoroseus</Emphasis> isolated from whiteflies

Paecilomyces fumosoroseus, monospore culture EH-506/3, isolated in Mexico from Bemisia tabaci whitefly was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated by gavage with only one dose (10⁸ conidia/animal) of viable (72 mice), heat-killed (24 mice) fungus and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. No mice were clinically ill or died. At the end of the study, their mean weight corresponded to healthy adults. Positive fungal cultures of feces were obtained only 24 h after inoculation. Positive cultures were found in 15 out of 360 organs (liver, spleen, kidney, brain, lung) in 12 of 72 mice inoculated with viable conidia. Gross pathology exhibited splenomegaly and liver paleness in mice inoculated with viable and heat-killed fungus. Non-germinated conidia were observed in studied organs, without any pathological tissue reaction, suggesting no mycological or histopathological evidence of fungal multiplication. The fungus was able to persist, but did not cause permanent damage to the host. This study supports the non-pathogenic/toxic status of P. fumosoroseus EH-506/3 when administered intragastrically in mice.     

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.     

Notes on Erysiphales (Ascomycetes) from Patagonia, Argentina

Fifteen Erysiphaceous taxa found on 20 host plant species in Patagonia are documented. A new species Oidium maculatae (type host: Viola maculata) is described. Berberis linearifolia, Buddleja globosa, Prosopis alpataco and Viola maculata, are new host plants for Erysiphales. Three new combinations on fungi and host plant species were founded: Erysiphe howeana – Fuchsia magellanica; E. patagoniaca – Nothofagus pumilio and N. antarctica. The genus Sawadaea and the species S. bicornis on Acer negundo and A. pseudoplatanus, are new records for South America. New host plants recorded for Argentina: Consolida ajacis, Galega officinalis and Plantago lanceolata. New host plants recorded for Patagonia: Galium aparine, Melilotus albus, Petunia × hybrida, Potentilla anserina, and Spiraea × bumalda. Oidium longipes is a new record for Argentina and Golovinomyces riedlianus is a new record for Patagonia.     

Sublethal effects of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) on the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) under laboratory conditions

Sublethal effects were evaluated in subsequent generations of whiteflies Bemisia tabaci (Gennadius) arising from parental generations exposed in the four nymphal stages to the fungus Beauveria bassiana (Balsamo) Vuillemin. Examples of such effects include impaired fertility, production of malformations or external variations, and reduced survival of later generations. Malformations of the head, legs, wings or abdomen were not observed in adults derived from treated nymphs, nor were negative effects observed in the fecundity and fertility of the descendants of the whiteflies treated with the fungus. However, moulting problems were observed in insects descended from B. bassiana-treated whiteflies. This is the first time that such effects have been reported, with almost 30% of imagos resulting from treated nymphs unable to detach completely from the exuvia. A gradual reduction in mortality rates between subsequent generations was observed. The importance of these results is discussed in the light of findings from other studies into the effects of entomopathogenic fungi on pest insects.     

Evaluation of the Entomopathogenic Fungi Metarhizium anisopliae and Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) for Control of the Green Leafhopper Empoasca decipiens (Homoptera: Cicadellidae) and Potential Side Effects on the Egg Parasitoid Anagrus atomus (Hymenoptera: Mymaridae)

The efficacy of Metarhizium anisopliae strain Ma43 and Paecilomyces fumosoroseus strain Pfr12 (both Deuteromycotina: Hyphomycetes) against adults of Empoasca decipiens (Homoptera: Cicadellidae) and potential side effects on the egg parasitoid Anagrus atomus (Hymenoptera: Mymaridae), were investigated in greenhouse cage and laboratory experiments. Treating leafhopper-infested faba bean plants at a dose rate of 1×10⁷ conidia mL^-1 resulted in up to 97% mortality 7 days after application and a 100% infection rate. Experiments on the residual effects revealed a significant decrease in adult E. decipiens mortality with increasing time from application to insect release. The decrease in mortality over time corresponded well with data from conidia germination tests. The germination of conidia on agar medium after washing them from the surafce of sprayed plants declined significantly from 95 and 96% immediately after application for M. anisopliae Ma43 and P. fumosoroseus Pfr12, respectively, to 29 and 27% 5 days later. Experiments on potential side effects of the entomopathogenic fungi on A. atomus showed that the tested isolates had no influence on adult emergence and longevity; however, the rates of parasitism were significantly reduced by the fungal treatments. The latter might be due to either density effects and/or could indicate that A. atomus avoids fungal-treated plants. However, the parasitoid is substantially less susceptible to the fungal strains tested than the host itself.     

Evaluation of the desiccation tolerance of blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) using a lab-scale, air-drying chamber with controlled relative humidity

The stabilization of living microbial agents for use as biological control agents is often accomplished through desiccation. Our air-drying studies with the entomopathogenic fungus Paecilomyces fumosoroseus have shown that the relative humidity (RH) of the drying air significantly affects the desiccation tolerance and the storage stability of blastospores. Drying air with a RH of more than 40% supported significantly higher rates of initial blastospore survival (68-82%) after drying compared to drying with lower relative humidity air. Drying air with a RH above 50% improved the shelf-life of the air-dried blastospore preparations. Adjustment of the pH or replacement of the spent medium with deionized water (d-H₂O) in the blastospore suspension had no significant impact on blastospore desiccation tolerance or storage stability. We have developed and describe a lab-scale, air-drying chamber that delivers air flow over the sample and that can be operated at controlled relative humidity.     

Evaluation of an entomopathogenic fungus, Paecilomyces fumosoroseus (Wize) Brown and Smith (Deuteromycota: Hyphomycetes) obtained from Formosan subterranean termites (Isop., Rhinotermitidae)

Abstract:  An isolate of Paecilomyces fumosoroseus was obtained from Coptotermes formosanus collected in Hong Kong, and a commercially available isolate of Metarhizium anisopliae , were both tested against C. formosanus shipped live from China. Survivorship of termites treated with a suspension of 5 × 10⁵ M. anisopliae conidia/ml and kept alone declined more rapidly than for those treated at the same concentration of P. fumosoroseus conidia. At a 5 × 10⁶ conidia/ml concentration, no significant differences in terms of termite survivorship were observed between the two fungal species. However, among termites kept in groups of 10 after treatment, those sprayed with P. fumosoroseus conidia at either 5 × 10⁵ or 5 × 10⁶ conidia/ml had significantly lower survivorship than those sprayed with M. anisopliae conidia. All the cadavers of termites treated with P. fumosoroseus and kept alone sporulated and among grouped termites 29% of the cadavers sporulated. By comparison, 53% of the cadavers of termites treated with M. anisopliae and kept alone sporulated, and only 4% of the cadavers of treated termites kept in groups sporulated.     

New bioassay method to assess the pathogenicity of Colombian strains of Metarhizium anisopliae (Metsch.) Sorokin and Paecilomyces sp. (Deuteromycotina: Hyphomycetes) against the subterranean burrower bug Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae)

Using a new sand bioassay method, the pathogenicity of three Colombian strains of Metarhizium anisopliae (i.e., CIAT 224, 245, and 230) and one strain of Paecilomyces sp. (CIAT 308) were tested against fourth instars of the subterranean burrower bug Cyrtomenus bergi Froeschner (Hem.: Cydnidae) in the laboratory. The proposed bioassay simulates the habitat of C. bergi and at the same time enables the evaluation of a large number of insects in a short period of time and under controlled conditions. All tested fungal species/strains were pathogenic against C. bergi. However, only low levels of mortality were obtained, and never exceeded 55%. In the screening experiment, M. anisopliae strains CIAT 224 and CIAT 245 caused the highest levels of mortality. The use of the bioassay procedure described herein is discussed.     

Predation potential of three flatworm species (Platyhelminthes: Turbellaria) on mosquitoes (Diptera: Culicidae)

We conducted a field survey for flatworms to select species as potential biological control agents against Aedes aegypti and Culex pipiens (Diptera, Culicidae) breeding in artificial containers. Laboratory experiments were performed to determine the daily predation rate, differential predation on each mosquito larval instar, selective predation on either A. aegypti or C. pipiens, and predator tolerance to water from artificial containers. Girardia anceps (Tricladida, Paludicola, Dugesiidae), Mesostoma ehrenbergii and Bothromesostoma cf. evelinae (Rhabdocoela, Typhloplanoida, Typhloplanidae) were found in temporary puddles and permanent pools. In the laboratory, they killed between 52% and 100% of immature mosquitoes coexisting in the same habitat. No preference of flatworms for mosquito preys was detected. Predation rate was related to predator size and instar of preys. Girardia anceps and B. evelinae survived after a dry period and when re-flooding occurred, they laid eggs. Tolerance to water from artificial containers was highest in G. anceps and this species could be a suitable predator to reduce mosquito populations from artificial containers using an inoculative approach.