The effect of insecticides on growth,germination and cuticle-degrading enzyme production by Isaria fumosorosea

The influence of six chemical insecticides on growth, sporulation, conidial germination and cuticle-degrading enzyme production by Isaria fumosorosea were investigated under laboratory conditions. Maximum reduction in vegetative growth, sporulation and conidial germination in relation to the control treatment was observed for Chloranthraniliprole whereas Indoxacarb proved to be the safest insecticide causing lowest reduction in these parameters. Chloranthraniliprole, Chlorpyrifos and Chlorfenapyr caused higher reduction in enzyme activities (chitinase, Pr1, Pr2 and lipase) at all three concentrations whereas very low reduction in enzyme activities was caused by Hubendamide+ Avermectin and Indoxacarb when used at 10 µg/ml. The data presented can be used for future recommendations of these insecticides in IPM programmes where I. fumosorosea is an important control agent.     

Hirsutella thompsonii, a fungal pathogen of mites. I. Biology of the fungus in vitro

Hirsutella thompsonii, a moniliaceous fungus pathogenic to mites, grew and sporulated on sterilised wheat bran. The effects of environmental factors were studied on the fungus grown on potato-dextrose-agar (PDA). The fungus was mesothermophilic. Growth, sporulation and conidial germination were best at 25^o-30 ^oC. Conidia kept at 37 ^oC for 5 days on PDA died, but those held at 5 ^oC germinated upon a subsequent removal to 25 ^oC. Almost all conidial germ tubes survived an 8 h exposure to 3–5% r.h. and to 60% r.h., but subsequently the former grew poorly at 100% r.h. H. thompsonii sporulated equally well in continuous darkness or light, and produced typical chlorinous to light olive-green mycelium and conidia under all conditions. A 2 h exposure of naked mycelium and conidia (which have melanised walls) to u.v. irradiation failed to kill the fungus.     

Conidial water affinity is an important characteristic for thermotolerance in entomopathogenic fungi

The interspecific thermotolerance of several species of entomopathogenic fungi was evaluated based on the conidial water affinity. The species were divided between hydrophilic and hydrophobic conidia. The species with hydrophobic conidia were Beauveria bassiana (ARSEF 252), Metarhizium brunneum (ARSEF 1187), Metarhizium robertsii (ARSEF 2575), Isaria fumosorosea (ARSEF 3889) and Metarhizium anisopliae s.l. (ARSEF 5749). The species with hydrophilic conidia were Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), Simplicillium lanosoniveum (ARSEF 6430), Lecanicillium aphanocladii (ARSEF 6433), S. lanosoniveum (ARSEF 6651), Aschersonia placenta (ARSEF 7637) and Aschersonia aleyrodis (ARSEF 10276). The conidial surface tension of each isolate was also studied. Conidial suspensions were exposed to 38, 41 or 45 °C. After exposure, the suspensions were inoculated on media and conidial germination was evaluated. Considerable differences in thermotolerance were found among the 12 entomopathogenic fungal species. Species with hydrophobic conidia were generally more thermotolerant than species with hydrophilic conidia. All isolates with hydrophobic conidia showed higher conidial surface tension than the isolates with hydrophilic conidia.     

Growth and sporulation of the fungus Claviceps purpurea in batch fermentation

Summary The sporulation of the fungus Claviceps purpurea is connected with the change in its respiration rate which is effected by deficiency of dissolved oxygen tension. It stops the vegetative growth of the fungus and induces the formation of conidiophores with conidia production until glucose is exhausted. With exhaustion of glucose the conidiophores continue to produce conidia by transforming vegetative cell material into conidia. Therefore final conidial concentration in batch fermentation depends on these two processes which can be regulated by oxygen input.     

The effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of Valdensinia heterodoxa

Valdensinia heterodoxa (Sclerotiniacae) is a potential fungal bioherbicide for control of salal (Gaultheria shallon). The effect of culture media, substrates and relative humidity (RH) on growth, sporulation and conidial discharge of V. heterodoxa was determined for two isolates PFC2761 and PFC3027 in vitro. Culture media significantly affected the growth, sporulation, and conidial discharge of V. heterodoxa. Of eight agar media used, colony radial growth was optimal on salal oatmeal agar and salal potato dextrose agar for isolates PFC2761 and PFC3027, respectively; whereas sporulation was at an optimum on salal oatmeal agar for both isolates. Of the eight liquid media tested, mycelial production was highest on wheat bran–salal–potato dextrose broth. Growth on solid substrates greatly stimulated sporulation and conidial discharge of V. heterodoxa. Of the 12 solid substrates used, the greatest numbers of discharged conidia were observed from wheat bran and wheat bran–salal within 14 d of sporulation. Sporulation on solid substrates continued for 42 d. RH significantly affected the sporulation and conidial discharge for both isolates across all solid substrates tested. No conidia were produced or discharged below 93 % RH on wheat bran–salal and millet. With an increase of the RH from 93 to 97 %, sporulation and the number of discharged conidia increased significantly for both isolates on wheat bran–salal, but not on millet.     

Host plants influence susceptibility of whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) to the entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae)

We quantified the tritrophic effect of host plant on the susceptibility of the sweetpotato whitefly Bemisia tabaci (Genn.) to a fungal pathogen in the laboratory. Second-instar whiteflies reared on cucumber, eggplant, tomato and bean plants for six generations were exposed to conidial suspensions of Isaria fumosorosea isolate IF-1106. Our results did not detect differences in response (proportional survival or median lethal time, LT₅₀ days) among insect populations derived from different plants that were treated with 10⁷ conidia/ml. However, at concentrations ≤ 5×10⁶ conidia/ml, whiteflies reared on bean and tomato died significantly more quickly (i.e. LT₅₀ of 4–5 days) compared with cucumber and eggplant reared populations (5–7 days). Bean and tomato-reared populations were also more susceptible to mycosis (LC₅₀ ≈ 6 × 10⁵ conidia/ml) compared with those reared on cucumber (1.9 × 10⁶ conidia/ml) and eggplant (1.5 × 10⁶ conidia/ml). A separate study confirmed that this differential response of whitefly populations to I. fumosorosea was not explained by differences in deposition rate of conidia on leaf surfaces (i.e. a dosage effect). Our findings show that host plants affect the pathogenicity and virulence of a herbivore pathogen, but depend on the rate of exposure (inoculum) applied.     

The relation between growth, conidiation and trehalase activity in Neurospora crassa

The extent to which trehalose is accumulated in the vegetative mycelium of strains of Neurospora crassa is significantly affected by conidiation. In heavily conidiating strains a rapid decrease in mycelial trehalose occurs following the initiation of conidiation. Meanwhile, trehalase activity in the vegetative mycelium of heavily conidiating strains increases rapidly following the initiation of conidiation, although apparently it is not directly caused by the sporulation process. High levels of both trehalase and trehalose appear concomitantly in the newly formed conidia.     

Nuclear distribution in Alternaria tenuis

Nuclear distribution and behaviour during vegetative growth and spore formation inAlternaria tenuis was studied utilising the HCl — Giemsa staining technique. The vegetative mycelium and conidia are predominantly monokaryotic. Anastomoses, followed by nuclear migrations, have been recorded. Intercellular nuclear migrations have only been observed in germinating conidia. Nuclear behaviour during conidial formation indicates that the conidia are homokaryotic. Cytological differences have been found in different monocoidal isolates of A. tenuis.     

Production ofBeauveria bassiana [Deuteromycotina: Hyphomycetes] in different liquid media and subsequent conidiation of dry mycelium

Mycelium ofBeauveria bassiana can be grown in liquid culture, filtered, and the mycelium dried. After rehydration the mycelium sporulates. Two carbohydrate sources (sucrose and maltose), and one nitrogen/vitamin source (yeast extract) were tested for mycelium growth and subsequent conidial production. Maximum mycelium growth (12.31 mg/ml), in liquid culture, was in the sucrose (3.5%)/yeast extract (3.5%) medium, but mycelium from a maltose (2%)/yeast extract (0.75%) medium produced the maximum of 4.62×10⁶ conidia/mg dry mycelium after incubation in moist Petri dishes. Using the data on mycelium yield (in liquid culture) and conidial production (by dry mycelium) it is calculated that the sucrose (3.5%)/yeast extract (3.5%) and the maltose (2%)/yeast extract (0.75%) media produce most conidia per media volume (an equivalent of 3.52–3.72×10⁷ conidia/ml).      

An Isaria fumosorosea SCAR marker for evaluation of soil,insect, and airborne samples

Molecular detection markers are needed for ecological studies of entomopathogenic fungi. In this study, a sequence-characterized amplified region (SCAR) marker of Isaria fumosorosea was used to detect the fungus in soil, insects, and airborne samples. These were artificially added with different fungal conidial concentrations. Specificity and sensitivity were tested with semi-nested PCR using oligonucleotides E-AA/M-CTA₁₂₄ F and E-AA/M-CTA₁₂₄ R for the first amplification and E-AA/M-CTA₁₂₄ F and E-AA/M-CTA₁₀₃ R for the second amplification. Specificity assays showed a specific band of 103 bp for DNA samples from 10 I. fumosorosea strains used. Negative results were observed for DNA samples from other species of Isaria, including I. amoene-rosea, I. farinosa, and Paecilomyces carneus as well as with other entomopathogens such as Metarhizium acridum, M. anisopliae, M. majus, M. flavoviride Type E, and Lecanicillium lecanii. Sensitivity assays showed that the specific SCAR marker detected 10⁴ conidia from I. fumosorosea EH-511/3 that were artificially mixed with soil, from 1 to 10⁴ conidia artificially mixed with Galleria mellonella (Lepidoptera: Pyralidae), and from 10 to 10⁵ conidia in Melinex tape for airborne samples. The marker was also able to detect conidia in airborne samples from cotton wicks in a mini wind tunnel. These SCAR markers for I. fumosorosea had excellent specificity and sensitivity and are relevant tools for ecological studies of this fungus.     

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).     

Effects of culture media, temperature, pH, and light on growth, sporulation, germination, and bioherbicidal efficacy of Phoma exigua, a potential biological control agent for salal (Gaultheria shallon)

In order to evaluate the potential use of Phoma exigua isolate PFC 2705 (PFC2705) as a biological control agent for salal (Gaultheria shallon), effect of cultural and environmental parameters on growth, conidia production, and pathogenicity of P. exigua were characterized in studies conducted under laboratory and greenhouse conditions. Within a range of 5-30°C, the optimum growth and germination temperature range was 20-25°C. The effect of pH on mycelial growth and conidial germination was not significant from pH 5 to 10. Fluorescent light significantly enhanced sporulation of the fungus on most agar media tested, yet was not necessary for growth. The type of culture media significantly affected mycelium growth, sporulation, and conidia germination. Age of mycelia used as inoculum affected the disease severity on salal. PFC2705 suppressed the growth of mature salal plant by inciting lesions on leaves, branch tips, and axillary buds and caused 56% death of the total biomass above ground. Characteristics such as easy inoculum production, wide range of growth environments, and high infectivity on salal increased the potential of P. exigua as a biocontrol agent for management of salal.     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Downstream processing of Beauveria bassiana and Metarhizium anisopliae-based fungal biopesticides against Riptortus pedestris: solid culture and delivery of conidia

We established a fungal production platform by focusing on substrates of solid culture for conidial productivity and thermotolerance, and focusing on surfactants to effectively deliver fungal conidia to the Riptortus pedestris cuticles. First, to produce thermotolerant fungal conidia, 2 of each Beauveria bassiana and Metarhizium anisopliae isolates were cultured on 13 cereal substrates for 10 days. Overall, five substrates (millet, non-glutinous Italian foxtail millet, barley, glutinous Italian foxtail millet, and brown rice) produced greatest number of conidia with thermotolerant conidia. When the selected substrates were mixed with minerals, zeolite, perlite or vermiculite to reduce the amount of cereal grains, vermiculite combination showed relatively high conidial production compared to the other mineral combinations. Next, to efficiently deliver the fungal conidia to the cuticles of R. pedestris, six surfactants, CO-2.5, CO-12, LE-7, PE-61, TED-3, and siloxane were each combined with the fungal conidia. The 0.01% combination showed significantly increased insect mortality, which varied depending on isolate. Virulence tests against R. pedestris were performed with conidial suspensions of isolates to assess their virulence. As a result, isolates showed the highest virulence when a virulence test was conducted at 25°C, rather than 20°C, 30°C and 35°C. This work suggests that the combination of cereal grain substrates and vermiculite could be considered for economic conidial production with high thermotolerance, and the CO-12 surfactant is the most suitable for effective delivery to target insects, followed by the information on optimal temperature for virulence against R. pedestris.     

A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata

Summary A diffusible self-inhibitor of germination of conidia of Glomerella cingulata appears to act as a regulator of protein synthesis. Both uptake of labeled amino acids and their incorporation into protein are reduced by the inhibitor or by crowding. Compared to conidia incubated without self-inhibitor, conidia incubated with self-inhibitor incorporated no labeled amino acids into protein in the first hour and 80% less in 6h. Thoroughly washed conidia were more permeable to amino acids and incorporated 6 times more precursor into proteins than unwashed conidia. At high density in nutrient medium, conidia of G. cingulata preferentially form secondary conidia instead of germ tubes and a mycelium. This inhibition of germination of conidia and regulation of development is mimicked by exposing them to an auto-inhibitor extracted from used culture medium and conidial washings. Germination of conidia of G. cingulata involves two steps, an initial step of 5 h duration which continues unaffected by crowing (1.7×10⁸/ml) and a subsequent 2 h step which conidia do not take unless they are sufficiently diluted. It is this step for which protein synthesis may be required.Non-Standard Abbreviations CHM cyloheximide - NM Neurospora minimal medium - psi pound per square inch - RPH reconstituted algal protein hydrolysate - TCA trichloroacetic acid     

MoSNF1 regulates sporulation and pathogenicity in the rice blast fungus Magnaporthe oryzae

The protein kinase Snf1 is a major component of the glucose derepression pathway in yeast and a regulator of gene expression for the cell wall degrading enzyme (CWDE) in some plant pathogenic fungi. To address the molecular function of Snf1 in Magnaporthe oryzae, which causes the rice blast disease, MoSNF1 was cloned and functionally characterized using gene knock-out strategies. MoSNF1 functionally complemented the growth defect of the yeast snf1 mutant on a non-fermenting carbon source. However, the growth rate of the Δmosnf1 mutant on various carbon sources was reduced independent of glucose, and the expression of the CWDE genes in the mutant was induced during derepressing condition like the wild type. The pre-penetration stage including conidial germination and appressorium formation of the Δmosnf1 was largely impaired, and the pathogenicity of the Δmosnf1 was significantly reduced. Most strikingly, the Δmosnf1 mutant produced only a few conidia and had a high frequency of abnormally shaped conidia compared to the wild type. Our results suggest that MoSNF1 is a functional homolog of yeast Snf1, but its contribution to sporulation, vegetative growth and pathogenicity is critical in M. oryzae.     

Entomopathogenic fungi-mediated biological control of the red palm weevil Rhynchophorus ferrugineus

The red palm weevil (RPW), Rhynchophorus ferrugineus, is an important pest of palms, and difficult to control by conventional methods. Therefore, microbial control is an alternative strategy for controlling RPW. Herein, a total of 15 entomopathogenic fungi (EPFs) were subjected to primary pathogenicity screening against last stage of RPW larvae. The preliminary data showed that four Beauveria bassiana isolates (JEF-484, 158, 462 and 507) and one Isaria fumosorosea isolate (JEF-014) resulted in 100 % mortality within 5–10 days post inoculation (d.p.i.), respectively. According to the time required for RPW mortality, JEF-484, 158, 462 and 014 were further subjected to bioassays using 10⁷ conidia/ml suspensions by spraying method. Based on the results, JEF-484 showed the highest mortality and shortest LT₅₀ on the last stage of RPW larvae, followed by JEF-158. The two isolates also showed good conidial production and high thermal stability compared to the other isolates. Therefore, JEF-484 and JEF-158 were selected for bioassays against RPW egg and the last larval stage with different concentrations of 10⁵, 10⁶ and 10⁷ conidia/ml conidial suspensions by spraying method. For the bioassay at the egg stage, JEF-158 showed a significantly higher ovicidal effect than JEF-484. In the larval bioassay, both EPF isolates showed a dosage-dependent effect on the RPW larvae. JEF-484 caused higher mortality in RPW larvae than JEF-158. In summary, the combination of the 2 promising EPF isolates might provide an opportunity for the practical microbial control of RPW at different life stages in palm tree fields.     

Effect of relative humidity,temperature and fungicide on germination of conidia of Cercospora canescens caused the Cercospora leaf spot disease in mungbean

The aim of the present investigation was to determine the impact of relative humidity (RH) and temperature on conidial germination, nuclear position and effect of important fungicides on growth and conidial germination of Cercospora canescens. Germination of conidia was observed at RH range 92–100% at 5–35°C. Significant interaction between temperature and RH indicated that higher humidity and high temperature promoted quick germination both in the presence and absence of free moisture. Although in absence of free moisture at 92–95% RH higher temperatures 25–35°C promoted quick evaporation of moisture and no conidial germination. Number of germtube was increased significantly at the optimum temperature 25–30°C and higher humidity (98–100%). But higher temperature 25–35°C with lower RH did not support the conidial germination. This finding is very important for disease forecasting using meteorological data. The spray of Carbendazim as contact fungicide may not be useful since it is not effective against the conidia of C. canescens. Triadimefon did not inhibit the conidia germination but completely inhibited mycelium development at 50 μg/ml. Propriconazole inhibited both conidia germination and mycelial development. Therefore, Propiconazole may be taken as protective as well as curative spray. In non-systemic fungicide, Copper oxychloride gave anticipated result by inhibiting both conidial germination and mycelium development. Therefore, copper oxychloride can be used as protectant fungicides for Cercospora leaf spot caused by C. canescens.     

Development of Media and Automated Liquid Fermentation Methods to Produce Desiccation-Tolerant Propagules ofTrichoderma harzianum

A suitable medium was developed from modified Richard's medium plus V8 juice (RM8) to produce high levels of desiccation-tolerant conidia ofTrichoderma harzianumstrain 1295-22. The addition of 9% (v/v) glycerol to RM8 improved both biomass production and desiccation tolerance of the conidia ofT. harzianum.This medium was then used in a laboratory scale fermenter (1.5 liter) to determine optimal operating conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the fermenter was 32°C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate was 1000 revolutions per minute. The initial water potential of the medium (with 9% glycerol) was −3.7 MPa, the pH was 6, and neither was controlled during fermentation. Changes in medium pH and dissolved oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to 6.0–6.2 at phialide formation. Intensive conidiation occurred at pH 6.3–6.5 and reached its maximal level at 6.9–7.1. Changes in pH values could be used as indicators to monitor the morphological development and conidiation ofT. harzianumduring fermentation. The use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the time required to complete the shift from vegetative growth to phialide formation. Intensive conidiation occurred immediately after the addition of culture inoculum and reached maximum levels within 68 h of fermentation. Dry weight of biomass increased with the duration of fermentation and was greatest at 96 h. However, no improvements in conidia/gram and CFU/gram were achieved after 72 h of fermentation. The desiccation tolerance of conidia harvested at 72 or 96 h was significantly (P = 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further scale-up of the fermentation process.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.