Low- or high-white light irradiance induces similar conidial stress tolerance in Metarhizium robertsii.

White light during mycelial growth influences high conidial stress tolerance of the insect-pathogenic fungus Metarhizium robertsii, but little is known if low- or high-white light irradiances induce different stress tolerances. The fungus was grown either in the dark using two culture media: on minimal medium (Czapek medium without sucrose = MM) or on potato dextrose agar (PDA) or PDA medium under five different continuous white light irradiances. The stress tolerances of conidia produced on all treatments were evaluated by conidial germination on PDA supplemented with KCl for osmotic stress or on PDA supplemented with menadione for oxidative stress. Conidia produced on MM in the dark were more tolerant to osmotic and oxidative stress than conidia produced on PDA in the dark or under the light. For osmotic stress, growth under the lower to higher irradiances produced conidia with similar tolerances but more tolerant than conidia produced in the dark. For oxidative stress, conidia produced under the white light irradiances were generally more tolerant to menadione than conidia produced in the dark. Moreover, conidia produced in the dark germinated at the same speed when incubated in the dark or under lower irradiance treatment. However, at higher irradiance, conidial germination was delayed compared to germination in the dark, which germinated faster. Therefore, growth under light from low to high irradiances induces similar conidial higher stress tolerances; however, higher white light irradiances cause a delay in germination speed.

Timing and duration of light exposure during conidia development determine tolerance to ultraviolet radiation.

Metarhizium is an important genus of soil-inhabiting fungi that are used for the biological control of insects. The efficiency of biocontrol is dependent on the maintenance of inoculum viability under adverse field conditions such as solar ultraviolet (UV) radiation. Therefore, increasing the tolerance of Metarhizium to UV radiation is necessary. It was previously established that, in mycelium, exposure to visible light increases tolerance to UV radiation. Similarly, growth under visible light for 14 days induces the production of tolerant conidia. However, a study evaluating if and how visible light affects conidia and their relationship with UV radiation was never performed. Here, we report that a relatively short and timed exposure to light around the time of conidiation is sufficient to induce the production of conidia with increased photoreactivating capacity and UV tolerance in Metarhizium acridum. Conidia produced by this method retain their characteristic higher tolerance even after many days of being transferred to the dark. Furthermore, we show that mature conidia of M. acridum and Metarhizium brunneum can still answer to light and regulate UV tolerance, suggesting that gene expression is possible even in dormant spores. Being able to respond to light in the dormant stages of development is certainly an advantage conferring improved environmental persistence to Metarhizium.

UV-B tolerances of conidia, blastospores, and microsclerotia of Metarhizium spp. entomopathogenic fungi.

The aim of the present study was to analyze ten native Metarhizium spp. isolates as to their UV-B tolerances. Comparisons included: different fungal propagules (conidia, blastospores, or microsclerotia [MS]); conidia in aqueous suspensions or in 10% mineral oil-in-water emulsions; and conidia mixed with different types of soil. The UV-B effect was expressed as the germination of conidia or culturability of blastospores and MS relative to nongerminated propagules. Metarhizium anisopliae LCM S05 exhibited high tolerance as blastospores and/or MS, but not as conidia; LCM S10 and LCM S08 had positive results with MS or conidia but not blastospores. The formulations with 10% mineral oil did not always protect Metarhizium conidia against UV-B. Conidia of LCM S07, LCM S08, and LCM S10 exhibited the best results when in aqueous suspensions, 24 h after UV-B exposure. In general, conidia mixed with soil and exposed to UV-B yielded similar number of colony forming units as conidia from unexposed soil, regardless the soil type. It was not possible to predict which type of propagule would be the most UV-B tolerant for each fungal isolate; in conclusion, many formulations and propagule types should be investigated early in the development of new fungal biocontrol products.

Two white collar proteins protect fungal cells from solar UV damage by their interactions with two photolyases in Metarhizium robertsii.

The photolyases PHR1 and PHR2 enable photorepair of fungal DNA lesions in the forms of UV-induced cyclobutane pyrimidine dimer (CPD) and (6-4)-pyrimidine-pyrimidone (6-4PP) photoproducts, but their regulation remains mechanistically elusive. Here, we report that the white collar proteins WC1 and WC2 mutually interacting to form a light-responsive transcription factor regulate photolyase expression required for fungal UV resistance in the insect-pathogenic fungus Metharhizum robertsii. Conidial UVB resistance decreased by 54% in Δwc1 and 67% in Δwc2. Five-hour exposure of UVB-inactivated conidia to visible light resulted in photoreactivation rates of 30% and 9% for the Δwc1 and Δwc2 mutants, contrasting to 79%-82% for wild-type and complemented strains. Importantly, abolished transcription of phr1 in Δwc-2 and of phr2 in Δwc1 resulted in incapable photorepair of CDP and 6-4PP DNA lesions in UVB-impaired Δwc2 and Δwc1 cells respectively. Yeast two-hybrid assays revealed interactions of either WC protein with both PHR1 and PHR2. Therefore, the essential roles for WC1 and WC2 in both photorepair of UVB-induced DNA lesions and photoreactivation of UVB-inactivated conidia rely upon their interactions with, and hence transcriptional activation of, PHR1 and PHR2. These findings uncover a novel WC-cored pathway that mediates filamentous fungal response and adaptation to solar UV irradiation.

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.

Outcome of blue, green, red, and white light on Metarhizium robertsii during mycelial growth on conidial stress tolerance and gene expression.

Fungi sense light and utilize it as a source of environmental information to prepare against many stressful conditions in nature. In this study, Metarhizium robertsii was grown on: 1) potato dextrose agar medium (PDA) in the dark (control); 2) under nutritive stress in the dark; and 3) PDA under continuous (A) white light; (B) blue light lower irradiance = LI; (C) blue light higher irradiance = HI; (D) green light; and (E) red light. Conidia produced under these treatments were tested against osmotic stress and UV radiation. In addition, a suite of genes usually involved in different stress responses were selected to study their expression patterns. Conidia produced under nutritive stress in the dark were the most tolerant to both osmotic stress and UV radiation, and the majority of their stress- and virulence-related genes were up-regulated. For osmotic stress tolerance, conidia produced under white, blue LI, and blue HI lights were the second most tolerant, followed by conidia produced under green light. Conidia produced under red light were the least tolerant to osmotic stress and less tolerant than conidia produced on PDA medium in the dark. For UV tolerance, conidia produced under blue light LI were the second most tolerant to UV radiation, followed by the UV tolerances of conidia produced under white light. Conidia produced under blue HI, green, and red lights were the least UV tolerant and less tolerant than conidia produced in the dark. The superoxide dismutases (sod1 and sod2), photolyases (6-4phr and CPDphr), trehalose-phosphate synthase (tps), and protease (pr1) genes were highly up-regulated under white light condition, suggesting a potential role of these proteins in stress protection as well as virulence after fungal exposure to visible spectrum components.

UV sensitivity of Beauveria bassiana and Metarhizium anisopliae isolates under investigation as potential biological control agents in South African citrus orchards.

Seven indigenous entomopathogenic fungal isolates were identified as promising biocontrol agents of key citrus pests including false codling moth, Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), citrus thrips, Scirtothrips aurantii Faure (Thysanoptera: Thripidae) and citrus mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae) under laboratory conditions. Even though field trials using the two most virulent isolates (Beauveria bassiana G Ar 17 B3 and Metarhizium anisopliae FCM Ar 23 B3) against soil-dwelling life stages of T. leucotreta were positive, foliar application against citrus mealybugs and thrips, has been disappointing. Thus, the UV sensitivity of the seven initial promising isolates (four B. bassiana and three M. anisopliae) in comparison with two commercial isolates (M. anisopliae ICIPE 69 and B. bassiana PPRI 5339) and their formulated products were investigated in this study. All isolates investigated were highly sensitive to UV radiation, and a 2 h exposure to simulated full-spectrum solar radiation at 0.3 W/m2 killed conidia of all tested isolates. Nonetheless, variability in susceptibility was found amongst isolates after exposure for 1 h. The most virulent M. anisopliae isolate, FCM Ar 23 B3, was the most susceptible to UV radiation with <3 % relative germination, 48-51 h post-exposure. Whilst isolates of the two mycoinsecticides showed similar susceptibility to UV radiation, their formulated products (vegetable oil and emulsifiable concentrate) were tolerant, when tested for 1 h. These findings indicate that a suitable UV protectant formulation of these fungi or a different application strategy will be required for success against P. citri and S. aurantii.

The phosphatase gene MaCdc14 negatively regulates UV-B tolerance by mediating the transcription of melanin synthesis-related genes and contributes to conidiation in Metarhizium acridum.

Reversible phosphorylation of proteins regulated by protein kinases and phosphatases mediate multiple biological events in eukaryotes. In this study, a dual-specificity cell division cycle 14 phosphatase, MaCdc14, was functionally characterized in Metarhizium acridum. Deletion of MaCdc14 decreased branch numbers, affected septum formation and resulted in multiple nuclei in each hyphal compartment, indicating nuclear division and cytokinesis defects. The spore production capacity was severely impaired with decreased conidial yield and delayed conidiation in MaCdc14-deletion mutant (ΔMaCdc14). The transcription levels of conidiation-related genes were significantly changed after MaCdc14 inactivation. The morphology of conidia was uneven in size and the germination rate of conidia was increased in ΔMaCdc14. In addition, ΔMaCdc14 displayed significantly enhanced conidial tolerance to ultraviolet (UV) irradiation but had no significant effect on the thermotolerance, the sensitivities to cell wall damage reagents, osmotic and oxidative stresses, and virulence compared to the wild-type strain and complementary transformant. Furthermore, the pigmentation of ΔMaCdc14 was increased by the upregulated expression of melanin synthesis-related genes, which may result in the enhanced UV-B tolerance of ΔMaCdc14. In summary, MaCdc14 negatively regulated UV-B tolerance by mediating the transcription of melanin synthesis-related genes, contributed to conidiation by regulating the expression levels of conidiation-related genes and also played important roles in cytokinesis and morphogenesis in Metarhizium acridum.

Possible source of the high UV-B and heat tolerance of Metarhizium acridum (isolate ARSEF 324).

The isolate ARSEF 324 of Metarhizium acridum is very tolerant to UV-B radiation and heat, but the intrinsic traits behind the extreme tolerance of this isolate to both stress conditions have not been elucidated. Because trehalose and mannitol are documented stress reducers in fungi, we investigated the accumulation of these compounds in conidia of ARSEF 324 compared with the accumulation of these two compounds in conidia of M. robertsii (ARSEF 23 and ARSEF 2575), which are considerably more susceptible to UV-B radiation and heat than ARSEF 324. Conidia of ARSEF 324 produced on potato dextrose agar plus yeast extract accumulated two-fold more trehalose and mannitol than conidia of ARSEF 23 and ARSEF 2575 produced on the same medium. The high accumulation of trehalose and mannitol in conidia of ARSEF 324 suggests one mechanism that it uses to attain its high tolerance to UV-B radiation and heat.

Effect of UV-B Irradiation on Water-Suspended Metarhizium anisopliae s.l. (Hypocreales: Clavicipitaceae) Conidia and Their Larvicidal Activity in Aedes aegypti (Diptera: Culicidae).

Ultraviolet (UV) radiation is a key limiting factor for biological pest control with entomopathogenic fungi. While little is known about the impact of UV on Metarhizium anisopliae Metchnikoff (Sorokin) (Hypocreales: Clavicipitaceae) conidia in aquatic mosquito-breeding sites, this study determined the effect of UV-B on the viability and virulence of M. anisopliae sensu lato (s.l.) strain IP 46 in the laboratory against Aedes aegypti (L.) (Diptera: Culicidae) larvae. Conidia were treated in cups under defined water depths (0, 1, 2, and 3 cm) to six different UV-B doses (0, 0.657, 1.971, 3.942, 7.884, 11.826, or 15.768 kJ m-2) at 27 ± 2°C. The ability of treated conidia to germinate up to 24 h postexposure on PDAY + benomyl + chloramphenicol medium at 25 ± 1°C was adversely affected by higher doses of UV-B radiation regardless of the water depth. Germination, however, did not fall below 70% regardless of the test conditions. In fact, conidial virulence against second-instar larvae was not affected by either the water depth (F3,84 = 0.3, P = 0.85) or any tested levels of UV-B radiation (F6,21 ≤ 1.2, P ≥ 0.39) including those distinctly higher than might be expected for tropical sites. These findings strengthen previous observations that IP 46 has significant potential for use against A. aegypti larvae, even when exposed to elevated UV-B irradiance levels in the small breeding sites that are common for this important vector.

The Rab GTPase activating protein Gyp2 contributes to UV stress tolerance in Metarhizium acridum.

GTPase activation protein (GAP) for Rab GTPases can accelerate GTP hydrolysis to alter the activity of Rab GTPases. To explore the function of GAP in entomopathogenic fungi, we constructed a deletion mutant of Gyp2 gene, a member of the Gyp (GAP for Ypt/Rab proteins) family in the locust-specific fungal pathogen, Metarhizium acridum. Results showed that the ∆MaGyp2 mutant had dramatically decreased tolerance to ultraviolet irradiation compared to wild type strain. Quantitative real-time PCR revealed that UV irradiation repair related genes Uve1 and WC1 were downregulated in ∆MaGyp2 mutant. Seven of other ten Gyp family members had significantly increased transcription in ∆MaGyp2 mutant compared with wild type, which may partly rescue the deficiency of MaGyp2.

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence.

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m-2); 3) PDA medium under blue light (4.8 W m-2); 4) PDA medium under red light (2.8 W m-2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.

Responses of entomopathogenic fungi to the mutagen 4-nitroquinoline 1-oxide.

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC50 and IC90, respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC50 > 4.7 µM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC50 < 0.2 µM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC50 between 0.4 and 0.9 µM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC50 between 1.0 and 1.2 µM). In cluster 4 were species with moderate to high tolerance (IC50 between 1.3 and 1.6 µM). Cluster 5 contained the species with high tolerance (IC50 between 1.9 and 4.0 µM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi.

Exposing Metarhizium acridum mycelium to visible light up-regulates a photolyase gene and increases photoreactivating ability.

Metarhizium acridum is an entomopathogen currently used against acridids. We have previously reported that exposing mycelium to visible light increases M. acridum tolerance to ultraviolet-B (UV-B) radiation. Here we evaluated if light could also increase tolerance to ultraviolet-C (UV-C) radiation. We observed that, as opposed to UV-B radiation, light did not increase tolerance to UV-C radiation under dark repair conditions. However, light did increase tolerance to UV-C radiation if photoreactivating light was present after UV-C exposure. Quantitative PCR experiments revealed that light up-regulates a photolyase gene. This is the first report showing that light regulates photoreactivating ability in M. acridum.

Riboflavin induces Metarhizium spp. to produce conidia with elevated tolerance to UV-B, and upregulates photolyases, laccases and polyketide synthases genes.

AIMS: The effect of nutritional supplementation of two Metarhizium species with riboflavin (Rb) during production of conidia was evaluated on (i) conidial tolerance (based on germination) to UV-B radiation and on (ii) conidial expression following UV-B irradiation, of enzymes known to be active in photoreactivation, viz., photolyase (Phr), laccase (Lac) and polyketide synthase (Pks). METHODS AND RESULTS: Metarhizium acridum (ARSEF 324) and Metarhizium robertsii (ARSEF 2575) were grown either on (i) potato dextrose agar medium (PDA), (ii) PDA supplemented with 1% yeast extract (PDAY), (iii) PDA supplemented with Rb (PDA+Rb), or (iv) PDAY supplemented with Rb (PDAY+Rb). Resulting conidia were exposed to 866·7 mW m-2 of UV-B Quaite-weighted irradiance to total doses of 3·9 or 6·24 kJ m-2 . Some conidia also were exposed to 16 klux of white light (WL) after being irradiated, or not, with UV-B to investigate the role of possible photoreactivation. Relative germination of conidia produced on PDA+Rb (regardless Rb concentration) or on PDAY and exposed to UV-B was higher compared to conidia cultivated on PDA without Rb supplement, or to conidia suspended in Rb solution immediately prior to UV-B exposure. The expression of MaLac3 and MaPks2 for M. acridum, as well as MrPhr2, MrLac1, MrLac2 and MrLac3 for M. robertsii was higher when the isolates were cultivated on PDA+Rb and exposed to UV-B followed by exposure to WL, or exposed to WL only. CONCLUSIONS: Rb in culture medium increases the UV-B tolerance of M. robertsii and M. acridum conidia, and which may be related to increased expression of Phr, Lac and Pks genes in these conidia. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhanced UV-B tolerance of Metarhizium spp. conidia produced on Rb-enriched media may improve the effectiveness of these fungi in biological control programs.

UV-B radiation-related effects on conidial inactivation and virulence against Ceratitis capitata (Wiedemann) (Diptera; Tephritidae) of phylloplane and soil Metarhizium sp. strains.

Recent studies have demonstrated the presence of Metarhizium species on the epigeal areas of weeds and woody plants in various Mediterranean ecosystems, and the question arises whether isolates from the phylloplane, which experiences greater exposure to environmental UV-B radiation than soil isolates do, could have better UV-B radiation tolerance. The in vitro response of 18 Metarhizium strains isolated from phylloplane and soil of several Mediterranean ecosystems to UV-B radiation and the in vitro and in vivo effects of UV-B radiation on the viability and virulence of a selected M. brunneum strain against C. capitata were determined. The conidial germination, culturability and colony growth of these strains exposed to 1200mWm-2 for 2, 4 or 6h were evaluated. Germination rates below 30% and poor conidia recovery rates were observed for all strains. However, no relationship between the Metarhizium species or isolation habitat and the effect of UV-B radiation was found. Strain EAMa 01/58-Su, which showed a high tolerance to UV-B inactivation in terms of relative germination, was subsequently selected to investigate the UV-B related effects on virulence toward C. capitata adults. In a series of bioassays, the virulence and viability was determined using pure dry conidia, which were irradiated with 1200mWm-2 for 6h prior or after adult flies were inoculated, which resulted in a significant 84.7-86.4% decrease in conidial viability but only a slightly significant reduction of virulence, with 100.0% and 91.4% adult mortality rates and 4.6 and 5.9days average survival time for the no UV-B and UV-B treatments, respectively. A second series of experiments was performed to determine whether the UV-B effects on strain EAMa 01/58-Su were dose- or exposure time-dependent. Adult flies were inoculated with five doses (1.0×104-1.0×108conidiaml-1) and then irradiated at 1200mWm-2 for 6h, and similar LC50 values, 3.8×107 and 4.3×107conidiaml-1, were determined for the UV-B and no UV-B treatments, respectively. However, the LT50 values for flies inoculated with 1.0×108conidiaml-1 and with1.0×107conidiaml-1 were 15.1% and 30.8% longer for UV-B treatments than no UV-B treatments, respectively. Next, adult flies were treated with 1.0×108conidiaml-1 and then exposed to 1200mWm-2 for 0, 6, 12, 24, 36 and 48h, and the relationships among exposure time and conidia viability and fly mortality losses were determined. The exposure time for adult flies at 1200mWm-2 to achieve a 50% reduction in fly mortality was 47.2h, which was longer than that of 5.6h required for a 50% reduction in conidia viability. Our results show that the UV-B radiation significantly affected the virulence of EAMa 01/58-Su strain against C. capitata adults, with this effect being dependent on the exposure time but not related to fungal dosage.

UV-B radiation reduces in vitro germination of Metarhizium anisopliae s.l. but does not affect virulence in fungus-treated Aedes aegypti adults and development on dead mosquitoes.

AIMS: Control of diurnal Aedes aegypti with mycoinsecticides should consider the exposure of fungus-treated adults to sunlight, and especially to UV-B radiation that might affect activity of conidia applied on the mosquito's surface. METHODS AND RESULTS: Germination of Metarhizium anisopliae s.l. IP 46 conidia on SDAY medium was not affected at the lowest level of radiation with UV-B, 0·69 kJ m-2 , but was retarded and reduced at higher 2·075 and 4·15 kJ m-2 , and completely inhibited at ≥8·3 kJ m-2 . In contrast, germination of conidia applied onto fibreglass nettings and exposed from 0 to 16·6 kJ m-2 did not differ significantly among levels of irradiance exposure and the controls. There was also no significant impact of UV-B up to 16·6 kJ m-2 on the adulticidal activity of IP 46 and on the subsequent conidiogenesis on cadavers. The Quaite-weighted UV-B irradiance in the laboratory (1152 mW m-2 ) was higher than the natural sunlight irradiance observed in the city of Goiânia in Central Brazil on midday (706 mW m-2 in August to 911 mW m-2 in October 2015). CONCLUSIONS: UV-B does not impair the activity of IP 46 conidia applied previously to radiation on A. aegypti adults. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings contribute to a better understanding of the effectiveness of M. anisopliae against day-active A. aegypti and its potential for biological mosquito control.

Screening of Metarhizium anisopliae UV-induced mutants for faster growth yields a hyper-virulent isolate with greater UV and thermal tolerances.

The insect pathogenic fungus Metarhizium anisopliae is an important insect biological control agent commercialized for use worldwide. Fungal infection is percutaneous, and rapid germination and growth has been linked to virulence. Using a simple in vitro growth screen to isolate mutants with increased virulence, M. anisopliae SM04 conidia were exposed to UV radiation for 20, 40, and 60 min, and mutants were subsequently screened for more rapid growth on standard potato dextrose agar. From a screen of >6,000 colonies, mutants were selected based on larger colony diameters as compared to the wild-type parent. Insect bioassays using the diamondback moth, Plutella xylostella, revealed one mutant, designated as MaUV-40.1 as displaying both more rapid growth and increased virulence. The mean lethal time to kill (LT50 using 106 conidia/ml) was 57.6 and 115.4 h for the MaUV-40.1 mutant and wild-type strains, respectively. Total conidial production, UV and thermal tolerances of the MaUV-40.1 strain were increased, but a reduced secretome was seen for the mutant compared to wild type. Analyses of culture supernatants indicated significant shifts in secondary metabolite production in the mutant. The insecticidal activity of EthOAc extracts derived from MaUV-40.1 mutant cell-free culture supernatants were ~20 times more potent that wild-type extracts. These data indicate that mutagenesis coupled to a growth screen can be a simple approach to isolate strains with greater stress resistance and virulence and that cell-free extracts may hold promise as an alternative to the living organism for insect control.

Exposure of Metarhizium acridum mycelium to light induces tolerance to UV-B radiation.

Metarhizium acridum is an entomopathogenic fungus commonly used as a bioinsecticide. The conidium is the fungal stage normally employed as field inoculum in biological control programs and must survive under field conditions such as high ultraviolet-B (UV-B) exposure. Light, which is an important stimulus for many fungi, has been shown to induce the production of M. robertsii conidia with increased stress tolerance. Here we show that a two-hour exposure to white or blue/UV-A light of fast-growing mycelium induces tolerance to subsequent UV-B irradiation. Red light, however, does not have the same effect. In addition, we established that this induction can take place with as little as 1 min of white-light exposure. This brief illumination scheme could be relevant in future studies of M. acridum photobiology and for the production of UV-B resistant mycelium used in mycelium-based formulations for biological control.

An ENA ATPase, MaENA1, of Metarhizium acridum influences the Na(+)-, thermo- and UV-tolerances of conidia and is involved in multiple mechanisms of stress tolerance.

In fungi, ENA ATPases play key roles in osmotic and alkaline pH tolerance, although their functions in thermo- and UV-tolerances have not been explored. Entomopathogenic fungi are naturally widespread and have considerable potential in pest control. An ENA ATPase gene, MaENA1, from the entomopathogenic fungus Metarhizium acridum was functionally analyzed by deletion. MaENA1-disruption strain (ΔMaENA1) was less tolerant to NaCl, heat, and UV radiation than a wild-type strain (WT). Digital Gene Expression profiling of conidial RNAs resulted in 281 differentially expressed genes (DEGs) between the WT and ΔMaENA1 strains. Eighty-five DEGs, 56 of which were down-regulated in the ΔMaENA1 strain, were shown to be associated with heat/UV tolerance, including six cytochrome P450 superfamily genes, 35 oxidoreductase genes, 24 ion-binding genes, seven DNA repair genes, and five other genes. In addition, eight genes were components of stress responsive pathways, including the Ras-cAMP PKA pathway, the RIM101 pathway, the Ca(2+)/calmodulin pathway, the TOR pathway, and the HOG/Spc1/Sty1/JNK pathway. These results demonstrated that MaENA1 influences fungal tolerances to Na(+), heat, and UV radiation in M. acridum, and is involved in multiple mechanisms of stress tolerance. Therefore, MaENA1 is required for the adaptation and survival of entomopathogenic fungi in stressful conditions in the environment and in their hosts.