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.     

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.     

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.     

Comparison of air-drying methods for evaluating the desiccation tolerance of liquid culture-produced blastospores of Paecilomyces fumosoroseus

Various drying methods were tested to identify a standard procedure for evaluating the desiccation tolerance of liquid culture-produced blastospores of Paecilomyces fumosoroseus. Since our work is focused on optimizing fermentation conditions for the production of P. fumosoroseus, the criteria for selecting a drying method included ease of use, moderate spore survival after drying and limited variation in spore survival. Three air-drying methods were tested: P. fumosoroseus blastospores mixed with silica gel, with sand, or with diatomaceous earth. Humidity controlled drying was used in the diatomaceous earth drying method. Blastospore survivals after drying were 19% (C.V. range, 32 to 45%), 82% (C.V. range, 26 to 43%), and 2% (C.V. range 32 to 50%) for the silica gel, sand, and diatomaceous earth methods, respectively. Blastospores dried using the silica gel and sand methods had been rinsed in 0.7 m polyethylene glycol before drying and rehydrated in the same solution for determination of survival. The variation observed within each method was similar. The silica gel drying method was selected as most appropriate for our studies based on moderate blastospore survival (19%) and ease of use.     

Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens

AIMS: To test the suitability of the Agrobacterium tumefaciens-mediated transformation (AMT) method with Paecilomyces fumosoroseus, a fungal pathogen that causes diseases in a wide range of insects including whiteflies. METHODS AND RESULTS: Conidia of P. fumosoroseus were successfully transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selectable marker. Transformation frequencies were 58.3 +/- 18.5, 98.3 +/- 24.8 and 169.7 +/- 35.5 (+/-SEM) transformants per 10(5), 10(6) and 10(7) target conidia respectively. After confirmation by PCR, transformants were subjected to Southern analysis, and the results revealed that 45% (four of nine) of the transformants contained single-copy integration of the T-DNA. CONCLUSIONS: In our AMT system, we efficiently transformed conidia of P. fumosoroseus. The employment of this method circumvents time-consuming protoplast preparation and allows the isolation of transformants containing single-copy integration of the T-DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the efficiency of Ag. tumefaciens-mediated transformation, this method represents a useful tool for insertional mutagenesis to characterize genes that are important for the pathogenicity of P. fumosoroseus.     

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     

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.     

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。     

Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) as a potential mycoparasite on Sphaerotheca fuliginea (Ascomycotina: Erysiphales)

Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed.P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M.Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.     

Isolation and characterization of microsatellite loci from the entomopathogenic hyphomycete,Paecilomyces fumosoroseus

Nine microsatellite markers were isolated from the entomopathogenic haploid fungus Paecilomyces fumosoroseus. Genetic diversity was assessed in 26 P. fumosoroseus isolates originated from the whitefly Bemisia tabaci collected in various geoclimatic areas. Eight loci were polymorphic with an observed number of alleles ranging from two to six. The loci differentiated some isolates and group of isolates according to their geographical location, showing promise for the study of gene flow. All loci failed to give clear amplifications in P. fumosoroseus isolates from hosts other than B. tabaci. These microsatellite markers provide powerful tools for ecological, epidemiological, and population genetic studies.     

玫烟色拟青霉对小菜蛾致病力的时间-剂量-死亡率模型模拟

小菜蛾Plutella xylostella是我国南方十字花科蔬菜上的重要害虫,已对田间常用的化学杀虫剂产生了严重的抗性。为寻找有效的小菜蛾生物防治措施,本实验研究了一株分离自家白蚁的玫烟色拟青霉Paecilomyces fumosoroseus (SCAU-PFCF01)对小菜蛾2～4龄幼虫的致病力。实验采用浸液法,供试浓度为1×10³、1×10⁴、1×10⁵、1×10⁶和1×10⁷个孢子/mL。结果表明：随玫烟色拟青霉孢子浓度的升高,小菜蛾的感病死亡率增加,在浓度为1×10⁷ /mL时,小菜蛾2、3和4龄幼虫的累计死亡率分别为96%、85%和80%。玫烟色拟青霉对小菜蛾各龄幼虫的致病力与供试龄期有关,其感病的敏感顺序为2龄、3龄和4龄。用时间 剂量 死亡率模型（time-dose-mortality model,TDM）对各龄幼虫的致病力数据进行模拟,所建模型均顺利通过Hosmer-Lemeshow拟合异质性检验,表明模型拟合良好,并由模型估计出了该菌株对小菜蛾各龄幼虫的致死剂量与致死时间。2龄幼虫接种后第7天、3龄幼虫接种后第5天、4龄幼虫接种后第4天的LC₅₀估计值分别为1.17×10⁴、1.44×10⁴和5.21×10⁴ /mL,LC₉₀估计值分别为1.98×10⁶、3.82×10⁷和1.29×10⁸ /mL。玫烟色拟青霉对小菜蛾幼虫的致死时间与浓度相关,供试各龄幼虫的LT₅₀值随着孢子悬浮液浓度的增加而递减,在1×10⁵～1×10⁷ /mL的范围内,2龄幼虫的LT₅₀值从3.16天降低到1.72天,3龄幼虫的LT₅₀从3.21天降低到1.83天,4龄幼虫的LT₅₀从3.69天降低到2.04天。即2龄幼虫致死所需的时间最短,其次为3龄幼虫,4龄幼虫致死所需的时间最长。结果显示了该株玫烟色拟青霉在小菜蛾的生物防治中具较强的应用潜力。     

The entomopathogenic fungi Isaria farinosa (formerly Paecilomyces farinosus) and the Isaria fumosorosea species complex (formerly Paecilomyces fumosoroseus): biology,ecology and use in biological control

The entomopathogenic fungi, Isaria farinosa and Isaria fumosorosea were known as Paecilomyces farinosus and Paecilomyces fumosoroseus, for more than 30 years. Both fungi have a worldwide distribution and a relatively wide host range. While I. farinosa currently is of minor importance in research and as biocontrol agent, I. fumosorosea is regarded as a species complex, and various strains are successfully used for biocontrol of several pest insects, mainly whiteflies. During the past 40 years, numerous scientific papers on both fungal species covering various research areas were published. This is the first monographic review on the biology, ecology and use of I. farinosa and I. fumosorosea as biocontrol agents. In the following compilation, data on the identity, natural occurrence and geographical distribution, host range, production of metabolites, effect of abiotic and biotic factors (temperature, relative humidity, solar radiation, fungus, host plant) and on the use of these species in biocontrol experiments against pest insects, plant pathogens and nematodes in the laboratory and the field are summarised. Sections on their effects on non-target organisms and safety issues are also included. The aim of this review is to increase our knowledge on these entomopathogenic fungi.     

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.     

玫烟色拟青霉适温性及抗多菌灵特性的种内变异

在15 ℃～35 ℃的温度范围内比较测定了玫烟色拟青霉(Paecilomyces fumosoroseus) 6株野生菌的菌落生长、产孢量及孢子活力.结果显示,3个指标均以25 ℃左右最好,但同一温度下菌株间或同一菌株在不同温度下的差异显著, 菌株Pfr116和Pfr6206具有相对稳定优良的生长、产孢及活孢率性状.对不同浓度多菌灵对6株野生菌的菌落生长和单孢菌落形成的影响测定表明,菌株间存在较大差异.不同多菌灵浓度对单孢菌落形成的抑制率观察值可用逻辑斯蒂模型较好地拟合(r²≥0.90).用所获参数估计的最低禁菌浓度MIC值显示,Pfr4205和Pfr116对多菌灵表现为低抗性（MIC≤20.0 μg·ml^-1）;Pfr153、Pfr612和Pfr2175属于低水平中抗,MIC值仅略大于20 μg·ml^-1;而Pfr6206的MIC值高达93.5 μg·ml^-1,接近MIC>100 μg·ml^-1的高抗标准.因此,Pfr6206为适温性和抗多菌灵特性俱佳的菌株,可作为抗多菌灵和适应不同季节或地区的害虫生防菌剂的候选菌株.     

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.     

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.     

Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Effect of various liquid culture media on morphology,growth, propagule production,and pathogenic activity to Bemisia argentifolii of the entomopathogenic Hyphomycete,Paecilomyces fumosoroseus

The entomopathogenic Hyphomycete Paecilomyces fumosoroseus was grown in five different liquid media, which have been developed for mass production of Beauveria spp. or P. fumosoroseus. Production was followed for 96 h by measuring both biomass and concentration of propagules. Maximal biomass was obtained with two media, Jackson and Catroux media (40–60 mg ml-1 suspension produced after 42 h incubation), where the exponential phase of growth began earlier than in the other media. While high concentrations of propagules (1.4–5.5 × 108 propagules ml-1) were produced in three media (Jackson, Paris, and Catroux media) after 48–72 h incubation, production of propagules was lower in the two other media, containing maltose as carbon source (Goral and Kondryatiev media) with 0.4–3.7 × 107 propagules ml-1 after 96 h incubation. P. fumosoroseus produced oblong blastospores in the three most productive media and conidiospore-like (ovoid to subspherical) propagules in the two other media. Infection potential of produced propagules was tested on the silverleaf whitefly ( Bemisia argentifolii). Whiteflies were sprayed as 2nd instars with P. fumosoroseus propagules produced in the five liquid media (1.9 × 103 and 1.9 × 104 propagules cm-2). All the media produced propagules that were infectious for larvae. With the lower dosage, mortality rates were significantly lower with propagules produced in one of the two least productive media (57%) (in the Kondryatiev medium) compared to those obtained with the three most productive media (>90%). However, when whiteflies were treated with the higher dosage, mortality rates (91–99%) between media were not significantly different. This revised version was published online in June 2006 with corrections to the Cover Date.