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.     

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.     

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.     

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%).     

Metarhizium anisopliae (Deuteromycetes: Moniliaceae) Pr1 activity: biochemical marker of fungal virulence in Rhipicephalus microplus (Acari: Ixodidae)

In the present study, the subtilisin-like protease Pr1 activity of five Metarhizium anisopliae s.l. isolates was evaluated together with their virulence against Rhipicephalus microplus females. Bioassays were performed with engorged females, and Pr1 activity was evaluated with the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe ρ-nitroanilide. All isolates were virulent to R. microplus, although the isolates CG 629, CG 148 and CG 32 caused higher mortality (P ≤ 0.05) than isolates CG 112 and CG 347 after eight days. No mortality was observed in the control group. Enzymatic assays revealed that the protease activity was greater in isolates CG 629, CG 148 or CG 32 than in isolates CG 112 or CG 347 after cultivation for 48 and 72 h. Based on these results, we suggest that the Pr1 activity may be used as a biochemical tool for screening the virulence among M. anisopliae s.l. isolates, and then selecting promising candidates for biocontrol of R. microplus ticks.     

Host-plant mediated interactions between two aphid species

Three isolates of Isaria fumosoroseus Wize (Hyphomycetes) were cultured on six media composed of differing amounts of chitin, carbon, and nitrogen. The effect of nutrition on growth and virulence was studied by measuring colony growth, spore yield, germination rate, spore-bound protease (Pr₁) and lipase activity, and virulence of inoculum produced by different media against second instars of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Chitin peptone nutrition media resulted in the highest colony growth but spore yields were lowest for this medium, whereas the osmotic stress medium resulted in the lowest colony growth with fewer spores. Highest lipase activity (4.14 µmol/ml/min) was observed for spores produced on high C/N medium for isolate IF28.2, whereas highest Pr1 activity (2.64 µmol/ml/min) was also observed on high C/N medium for isolate IF28.2. A higher rate of Pr1 and lipase activity for all three isolates was observed on low C/N, 2% peptone, and the osmotic stress media. Conidia from nutrient-poor media (2% peptone) proved to be the least virulent for all three isolates with median survival time values of 2.23, 2.05, and 1.79 days for IF32, IF28.2, and IF49, respectively. Median survival time values for the various nutrient media proved to be positively correlated with spore-bound Pr1 and lipase activity, having correlation coefficient values of 0.115 and 0.538, respectively.     

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.     

Effect of temperature and poultry litter in Beauveria bassiana (bals.) Vuill. and Metarhizium anisopliae (Metsch) virulence against the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate the influence of temperature and poultry litter on germination vegetative growth virulence and conidial production of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates on larvae and adults of the lesser mealworm (Alphitobius diaperinus) (Panzer). The vegetative growth and conidial production were evaluated on culture media. Virulence was studied submerging larvae and adults in a conidial suspension (1 x 10(8) conidia/ml). All the experiments were carried out in growth chamber (26 degrees C and 32 degrees C and 14h photophase). Fungus-killed insects were daily collected and used for microscopic conidial counts. The poultry litter effect was evaluated by submerging the insects in a fungal suspension (10(8) conidia/ml) and then transferring them to cups containing poultry litter (new and used). B. bassiana isolates were more sensitive than M. anisopliae to high temperature because conidia viability, vegetative growth and virulence were negatively affected (P < 0.05). The conidial production was higher to B. bassiana in 26 degrees C (7 to 11 x 10(8) conidia/larval cadaver and 8 x 10(8) conidia/adult cadaver) (P < 0,05). Larval stage was about 10 times more sensitive to M. anisopliae at 26 degrees C than adults stage. Regarding B. bassiana, differences on sensitivity between larval stages and adults were not observed at this temperature. However, at 32 degrees C, larval stage was more sensitive for CB116 and UEL50 isolates. Mortality was higher when larvae and adults (15.7 and 66.7% respectively) were treated by B. bassiana and maintained on new poultry litter at 26 degrees C) (P < 0.05).     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Propagule interactions and the evolution of virulence

The evolution of parasite virulence is thought to involve a trade‐off between parasite reproductive rate and the effect of increasing the number of propagules on host survivorship. Such a trade‐off should lead to selection for an intermediate level of within‐host reproduction (λ). Here I consider the effects of parasite propagule number on selection affecting λ when (i) the effect of each propagule is independent of propagule number, and (ii) when the effect of each propagule changes as a function of propagule number. Virulence evolves in these models as a correlated response to selection on λ. If each propagule has the same effect (s) as all previous propagules, the survivorship of infected hosts is reduced by more than 60% at equilibrium, independent of the value of s. If, instead, each propagule has a more negative effect on host survivorship than previous propagules, host survivorship at equilibrium is expected to increase as the effect becomes more pronounced. These results are directly parallel to results derived for population mean fitness at mutation‐selection balance; and they suggest that high virulence should be associated with parasites for which the effect of adding propagules either remains constant or diminishes with propagule number.     

Effect of culture media on virulence of Hirsutella thompsonii (Fischer) (Deuteromycetes) to control Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae)

The virulence of Hirsutella thompsonii (Fischer) to Brevipalpus phoenicis (Geijskes) was evaluated in laboratory, grown on complete and solid culture media (MC-S); complete and liquid culture media (MC-L); rice (APC) and powdered rice (APC-SM). Adults were confined to arenas prepared with citrus leaves in acrylic dishes containing water-agar. Conidial suspensions were prepared at different concentration (3.2 x 10(5) to 1 x 10(7) spores/ml) and applied on mites to establish the table curve-response on fourth day. For field evaluation, adults were maintained in arenas prepared with fruits which were placed in plants. In this test, four treatments were tried: H. thompsonii cultured on rice (APC) at two concentrations (20 kg/ha and 10 kg/ha), H. thompsonii produced by liquid fermentation (MC-L) (5 L/ha) and control (sterile water). Adult survival, number of eggs and nymphs per fruit were observed 10 and 20 days after the fungus application. The lowest LC25 value calculated was from pathogen produced in MC-S (1.9 x 10(5) conidia/ml).The LC25 values calculated to APC and APC-SM did not differ statistically. The LC25 values to MC-L and MC-S were 1.9 x 10(6) infective cells/ml and 2.2 x 10(5) conidia/ml. In the field, concentration and time to death differed between treatments and control. The applications resulted in reduction of adult survival and number of eggs.     

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

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

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

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

Development of a Spray-drying Technique for Submerged Spores of Entomopathogenic Fungi

The stability of blastospores or submerged conidia of entomopathogenic fungi is one of the key problems associated with the practical use of fungal biopreparations. A spray-drying technique was developed which allows the drying of blastospores and/or submerged conidia, here called submerged spores, without a significant loss of viability and with only a slight delay in eY cacy. The method was tested successfully using the fungal species Metarhizium anisopliae , M. flavoviride, Beauveria bassiana and Paecilomyces fumosoroseus . For all experiments, a laboratory spray dryer was used. The optimum inlet and outlet temperatures were found to be 64 2oC and 48 2oC respectively. The best protective agent for submerged spores was skimmed milk powder at concentrations of 10 or 20%. The addition of sugars, especially 2.5% sugar-beet syrup, slightly improved the viability after spray-drying. Submerged spores of eight isolates of M. anisopliae, M. flavoviride, B. bassiana and P. fumosoroseus were suspended in 20% skimmed milk powder and 2.5% sugar-beet syrup, and spray-dried. Germination rates of about 90% were achieved, which were comparable to those of freshly produced submerged spores. Finally, the virulences of spray-dried submerged spores of M. anisopliae (Ma 97) and M. flavoviride (Mfl 5) were tested in bioassays using third- and fourth-instar nymphs of the African locust, Locusta migratoria . In contrast to M. flavoviride , there was no significant diVerence in the median lethal time between spray-dried and fresh submerged spores of M. anisopliae . The investigations demonstrated that spray-drying is possible to preserve sensitive submerged spores of entomopathogenic fungi.     

两种杀虫真菌制剂与低用量吡虫啉对温室粉虱的协同防效评价

在昆明市郊蔬菜基地4间大棚(5m×100m·间^-1)栽培的生菜上,用球孢白僵菌(Beauveriabassiana)和玫烟色拟青霉(Paecilomyces fumasonoseus)的纯孢子乳悬剂(有效含孢量10^10·ml^-1)及其与1％和3％(w／v)的吡虫啉10％WP的混配剂对温室粉虱(Trialeurodes vaporariorum)进行了协同药效试验,吡虫啉添加量仅相当于推荐用量的6．4％～9．5％和19．1％～28．6％,试验含6个菌剂处理、2个低用量吡虫啉处理和1个清水对照,各有3个小区(7m×5m·小区^-1)重复,随机区组排列,稀释1000倍的菌液(含孢量10^7·ml^-1)和吡虫啉液于生菜移栽后第11d首次喷雾,15d后喷第二次,首次用菌前调查初始粉虱密度,用菌后每隔5d调查活虫数和死虫数,从8月20日起历时30d,结果表明,连续2次用菌有效地控制了粉虱对当季大棚生菜的危害,各菌剂处理的最终相对防效和虫口减退率均达到95％以上,极显著地高于低用量吡虫啉处理,其中,玫烟色拟青霉制剂对粉虱的控制效果一般优于球孢白僵菌制剂,且与吡虫啉的添加量呈正相关,但是,各菌剂处理间控虫效果的差异主要发生在首次用菌后第5d和10d,此后差异逐渐缩小,至第二次用菌后第10d各菌剂处理间无显著差异,本试验结果表明,两种真菌的孢子乳悬剂均可用于温室粉虱的防治,添加微量吡虫啉可增强菌剂的防效。     

Field studies using a recombinant mycoinsecticide (Metarhizium anisopliae) reveal that it is rhizosphere competent

In the summer of 2000, we released genetically altered insect-pathogenic fungi onto a plot of cabbages at a field site on the Upper Marlboro Research Station, Md. The transformed derivatives of Metarhizium anisopliae ARSEF 1080, designated GPMa and GMa, carried the Aequorea victoria green fluorescent protein (gfp) gene alone (GMa) or with additional protease genes (Pr1) (GPMa). The study (i) confirmed the utility of gfp for monitoring pathogen strains in field populations over time, (ii) demonstrated little dissemination of transgenic strains and produced no evidence of transmission by nontarget insects, (iii) found that recombinant fungi were genetically stable over 1 year under field conditions, and (iv) determined that deployment of the transgenic strains did not depress the culturable indigenous fungal microflora. The major point of the study was to monitor the fate (survivorship) of transformants under field conditions. In nonrhizosphere soil, the amount of GMa decreased from 10(5) propagules/g at depths of 0 to 2 cm to 10(3) propagules/g after several months. However, the densities of GMa remained at 10(5) propagules/g in the inner rhizosphere, demonstrating that rhizospheric soils are a potential reservoir for M. anisopliae. These results place a sharp focus on the biology of the soil/root interphase as a site where plants, insects, and pathogens interact to determine fungal biocontrol efficacy, cycling, and survival. However, the rhizospheric effect was less marked for GPMa, and overall it showed reduced persistence in soils than did GMa.     

Effect of Additives on the Production, Viability and Virulence of Blastospores of Metarhizium anisopliae

Studies on the blastospore production of Metarhizium anisopliae var. anisopliae were conducted in Adamek's medium used as a standard, enriched with lecithin, collagen, lactic acid or polyethylene glycol 200 (PEG 200) to increase spore yield and suppress mycelial pellet formation. The addition of 5% lecithin resulted in a significant 10-fold increase in spore yield up to 1.9 108 blastospores/ml compared with 1.9 107 spores/ml in the standard medium. Collagen (3%) increased the number of blastospores 3.7-fold, and lactic acid (1.5%) two-fold. A reduction of mycelial pellet formation in favour of spore production was noted with each additive. The viability of blastospores at 40IC from media with lecithin, collagen and lactic acid suspended in 25% Ringer's solution was comparable to that of spores produced in the standard medium. Striking differences were noticed in the viability of spores produced with 5% PEG 200 in standard medium. The half-life of blastospores produced in standard medium suspended in sunflower oil was 33.6 h and that of 5% PEG 200 spores only 25.2 h. In bioassays, the virulence of spores produced in standard medium to which 3% lecithin, 3% collagen, 1.5% lactic acid or 5% PEG 200 had been added was tested against third-instar nymphs of Locusta migratoria migratorioides (R. & F.). The median lethal time and the mortality of L. migratoria achieved with blastospores produced with 3% lecithin (5.7 days, 99%) was comparable to that of blastospores from standard medium (5.1 days, 98%). The virulence of blastospores from all other media with additives was significantly reduced.     

Successive subculturing alters spore-bound Pr1 activity,germination and virulence of the entomopathogenic fungus,Beauveria bassiana

One of the hurdles in the development of entomopathogenic fungi such as Beauveria bassiana is loss of virulence when successively maintained in vitro. This may result in products of inferior quality in mass production programs. Also, there are many contradicting data and unclear points in this case. Three isolates of B. bassiana were subcultured successively 15 times. Spore-bound Pr1 activity, germination rate, and virulence of conidia against mealworm (Tenebrio molitor L.) larvae were studied. Results showed that isolates normally retained their virulence during 10 subculturings. However, they clearly offered decreased virulence (elevated LT₅₀ values and lower percent mortality). The activity of Pr1 bound to conidia declined as subculturing continued; the lowest spore-bound activity and germination potential of conidia was recorded for the 15th subculture. Virulence data were in agreement with Pr1 activity and germination rate as there was a positive correlation between germination rate and spore-bound Pr1 activity with fungal virulence. This explains that at least a part of attenuation in fungal virulence can be explored in enzymatic activity, especially in the important cuticle-degrading protease, Pr1.     

Chlorine demand and inactivation of fungal propagules

Conidia of filamentous fungi, vegetative yeast cells, and coliform bacteria were tested to determine their chlorine demand and their sensitivity to chlorine inactivation. Levels of chlorine demand for the various conidia, yeast, and coliforms were, respectively, 3.6 x 10(-9) to 3.2 x 10(-8), 1.2 x 10(-9) to 8.0 x 10(-9), and 2.5 x 10(-11) to 6.3 x 10(-10) mg of chlorine per propagule. Preliminary evidence suggests that the chlorine demand per propagule increases as the number of propagules per milliliter decreases. In general, conidia showed greatest resistance to chlorine inactiviation, followed by the yeast and coliforms. Inactivation by chlorine was influenced by pH, with inactivation (chlorine activity) falling in the order pH 5 > 7 > 8.     

Cuticle-degrading proteases of entomopathogenic fungi: from biochemistry to biological performance

Abstract

Entomopathogenic fungi are among the most successful biocontrol agents for preventing economic loss from insects. The identification of virulent species or isolates, the development of formulation technology and the improvement of efficiency are avenues being pursuing by researchers in diverse scientific disciplines. A successful entomopathogenic fungus deploys a combination of mechanical and biochemical processes to overcome the first defensive barrier in insects, the integument. A precise understanding of the mechanisms underlying fungal pathogenicity, particularly the roles of enzymes such as proteases, is essential in order to highlight the potential of entomopathogenic fungi and increase their virulence via genetic modifications. Cuticle-degrading proteases are divided into subtilisin-like (Pr1) and trypsin-like (Pr2) proteases, which are secreted in the initial stages of penetration. The biochemical structure contains the catalytic triad Asp39, His69 and Ser224 in addition to Ca²⁺ binding sites. Studies have shown a molecular weight of almost 19–47?kDa, an optimal pH of 7–12 and an optimal temperature of 35–45?°C. Different species or isolates of entomopathogenic fungi exhibit differences in the secretion and activity of cuticle-degrading proteases, which may indicate their virulence capacity. Genetic engineering techniques have been developed to create isolates with protease overexpression. Such isolates have significantly higher virulence against the host because they not only ensure fungal penetration but also exhibit direct toxicity to insects.