Vital roles of Pks11, a highly reducing polyketide synthase, in fungal conidiation, antioxidant activity, conidial cell wall integrity, and UV tolerance of Beauveria bassiana.

Fungal polyketide synthases play important and differential roles in synthesizing secondary metabolites and regulating several cell events, including asexual development, environmental adaptation, and pathogenicity. This study shows the important functions of a highly reducing polyketide synthase, Pks11, in Beauveria bassiana, a filamentous fungal insect pathogen used worldwide for pest biocontrol. The deletion of pks11 led to severe defects in conidial yields on different media and a decrease of 36.27% in the mean thickness of conidial cell wall under normal conditions. Compared with the wild-type, Δpks11 showed higher tolerance to oxidation and increased sensitivity to high temperature during colony growth. Moreover, the lack of pks11 caused a decrease in conidial germination after exposure to UV radiation but did not affect the virulence of B. bassiana against Galleria mellonella larvae via typical cuticle infection. These findings concurred with the alteration in the transcript levels of some phenotype-related genes. These data suggested that pks11 played vital roles in the asexual development, cell wall integrity, and fungal responses to oxidation, high temperature, and UV irradiation of B. bassiana.

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.

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.

A polyketide synthase, BbpksP, contributes to conidial cell wall structure and UV tolerance in Beauveria bassiana.

Conidial pigments of filamentous fungi play vital roles in fungal biotic/abiotic stress tolerance and are usually synthesized by polyketide synthases or other pigment synthesis proteins. Beauveria bassiana, an important insect pathogenic fungus used worldwide for pest biocontrol, produces white conidia on artificial media, while no conidial pigment has been observed or reported in it. However, real-time PCR and promoter-report analyses reveal a polyketide gene of B. bassiana (named BbpksP), homologous to melanin synthesis genes, is specifically expressed in aerial conidia. We show that deletion of BbpksP does not result in changes in conidial yield, germination rate or colony radial growth; however, the defect impairs conidial cell wall structure. A dense electron layer appears in the outer edge of the cell envelope in wild-type conidia, as observed by TEM, but this dense layer is absent in the ΔBbpksP mutant. The lack of BbpksP gene also reduces the UV-B tolerance of B. bassiana conidia. Bioassay reveals that deletion of BbpksP decreased virulence of B. bassiana against Galleria mellonella larvae via topical infection. These data indicate that the product(s) of BbpksP contributes to the integrity of the B. bassiana conidial cell wall and further affects the tolerance of UV-B stress and insecticidal activity.

Efficiency of natural substances to protect Beauveria bassiana conidia from UV radiation.

BACKGROUND: Solar radiation is assumed to be a major factor limiting the efficacy of entomopathogenic fungi used as biocontrol agents in open field applications. We evaluated 12 natural UV-protective co-formulants for their effect on the survival of UV-exposed Beauveria bassiana spores on agar plates, colza leaf discs and in the field. RESULTS: Colony-forming unit (CFU) counts of unformulated conidia on agar plates and leaf discs dropped to ≤ 50% after exposure to UV radiation. The highest UV protection was achieved with humic acid, which provided > 90% protection of UV-B-exposed conidia in laboratory experiments. In the field, 10% humic acid increased spore persistence up to 87% at 7 days after application. Sesame and colza oil also provided high UV protection in both assays (> 73% and > 70%, respectively). CONCLUSIONS: This study shows that it is possible to increase the persistence of B. bassiana spores under exposure to UV radiation by formulation with natural UV-protective additives. UV protectants might, therefore, increase the efficacy of entomopathogenic fungi as biocontrol agents in open field applications. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Vital role for the J-domain protein Mdj1 in asexual development, multiple stress tolerance, and virulence of Beauveria bassiana.

Mdj1 is a member of the Hsp40 family containing a DnaJ or J domain. Here, we have examined the functions of an Mdj1 orthologue (56.68 kDa) in Beauveria bassiana, a filamentous fungal insect pathogen widely applied in biological control of insect pests. Deletion of mdj1 in B. bassiana resulted in significant growth defects on a variety of complex and minimal media. The Δmdj1 mutant exhibited not only a drastic reduction (92 %) in aerial conidiation during optimal cultivation but also a remarkable decrease (77 %) in submerged blastospore production. Compared to wild-type, the deletion mutant was significantly more sensitive to the stresses of cell wall perturbation, high osmolarity, oxidation, carbendazim fungicide, several metal ions, and acidic/alkaline pH during conidial germination and/or colony growth. In the mutant, conidial thermotolerance and UV-B resistance decreased by 61 and 25 %, respectively, and virulence to Galleria mellonella larvae was largely attenuated. Transcript levels of many phenotype-related genes were drastically suppressed in the absence of mdj1, accompanied with impaired cell walls and reduced intracellular anti-stress molecules, including superoxide dismutases, catalases, glycerol, trehalose, and mannitol. These data indicate that Mdj1 plays a vital role in normal fungal development and contributes significantly to the biological control potential of B. bassiana against insect pests.

WetA and VosA are distinct regulators of conidiation capacity, conidial quality, and biological control potential of a fungal insect pathogen.

Many filamentous fungi produce only conidia for dispersal and survival in vitro or in vivo. Here, we show that the developmental regulator WetA and the velvet protein VosA are not only required for conidial maturation but indispensable for conidiation in Beauveria bassiana, a filamentous entomopathogen. Deletion of wetA or vosA resulted in more than 90 % transcriptional depression of brlA and abaA, two activator genes in the central developmental pathway, during the critical period of conidiophore development and conidiation. Consequently, ΔwetA and ΔvosA strains lost 98 % in and 88 % of their conidiation capacities under optimal culture conditions, respectively. The conidia of ΔwetA showed more defective features than those of ΔvosA, including smaller size, lesser density, lower hydrophobicity, and impaired cell walls although intracellular trehalose content decreased more in the aging culture of ΔvosA than of ΔwetA. As a result, conidial sensitivity to cell wall perturbation was elevated in ΔwetA but unaffected in ΔvosA, which produced conidia more sensitive to the oxidant menadione and the wet-heat stress at 45 °C. Both deletion mutants showed similar defects in conidial tolerance to high osmolarity or UV-B irradiation but no change in conidial sensitivity to the other oxidant H2O2 or the fungicide carbendazim. Moreover, ΔwetA lost more virulence to Galleria mellonella larvae than ΔvosA. All these phenotypical changes were restored by either wetA or vosA complementation. Taken together, WetA and VosA are indispensable for asexual development and contribute differentially to conidial quality and hence the biological control potential of B. bassiana against insect pests.

Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana.

Three non-ionic surfactants: Tween20, Tween80 and Breakthru (®) were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations . Breakthru (®) had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25-30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses.

Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana

Three non-ionic surfactants: Tween20, Tween80 and Breakthru® were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations. Breakthru® had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25–30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses.

Distinct contributions of one Fe- and two Cu/Zn-cofactored superoxide dismutases to antioxidation, UV tolerance and virulence of Beauveria bassiana.

Beauveria bassiana, a filamentous entomopathogen, has five distinct superoxide dismutases (SODs), including cytosolic and mitochondrial MnSODs (Sod2/3) which have proved contributing primarily to intracelluar SOD activity and additively to antioxidation and virulence. Here we characterized cytosolic Cu/ZnSOD (Sod1), mitochondrial FeSOD (Sod4) and cell wall-anchored Cu/ZnSOD (Sod5). The latter two are unexplored despite existence in many filamentous fungi, and their subcellular localization was well confirmed with specifically stained cells expressing Sod4::eGFP or Sod5::eGFP fusion. Total SOD activity decreased by ∼15% in Δsod1 but increased by 11-20% in three sod4 knockdown mutants (Δsod4 was lethal) when co-cultivated with menadone and H2O2. Surprisingly, total catalase activity decreased much more in the sod4 mutants (69-75%) than in Δsod1 (27-33%) under normal and oxidative conditions. However, Δsod5 showed little change in either SOD or catalase activity. Transcript levels of SOD partners and five catalases also changed more dramatically in the sod4 mutants than in Δsod1 and Δsod5. As a consequence of global effect, intracellular ROS levels induced by both oxidants were higher in Δsod1 than in the sod4 mutants and Δsod5. All the mutants were differentially more sensitive to the two oxidants and UV-A/UV-B irradiations and less virulent to Galleria mellonella larvae but not responsive to high osmolarity, cell wall stress and high temperature. Taken together with previously characterized Sod2 and Sod3, our results provide full insight into the SOD family, unveiling the interactions of each SOD with other partners and catalases in the antioxidant reaction associated with the fungal biocontrol potential.

Enhanced conidial thermotolerance and field efficacy of Beauveria bassiana by saccharides.

Insecticide efficacy of Beauveria bassiana conidia was improved by optimizing the concentrations of conidial heat-protective saccharides (glucose, sucrose, maltose, trehalose, α-lactose, and mannitol) using response surface methodology. Two field trials in tea gardens were carried out to control leafhopper (Empoasca vitis) by spraying B. bassiana conidia together with the optimized saccharides (0.26 g glucose, 0.28 g lactose, 0.24 g mannitol per ml). In the field studies, B. bassiana conidia were applied to control Empoasca vitis with and without saccharides and compared with bifenthrin, a pyrethroid insecticide. With the optimal concentrations of saccharides, the conidial germination rate reached 72% and the control efficacy of the saccharides group (65.7%) was equal to the bifenthrin group (69.4%), which improved by about 55%.

Phytochrome controls conidiation in response to red/far-red light and daylight length and regulates multistress tolerance in Beauveria bassiana.

Phytochromes (Phy) in filamentous fungi are Group VIII histidine kinases that share a unique N-terminal photosensory core, but their functions are largely unknown. Here we show that Beauveria bassiana Phy (Bbphy) is functionally vital for growth, conidiation and multistress tolerance of the fungal entomopathogen lacking sexual stage. Colony growth of ΔBbphy was significantly slower in a nutrition-rich medium but faster in several minimal media. Conidial yield of ΔBbphy in the rich medium increased at the fitted rate of 3.4 × 10(7) conidia h(-1) white light in the light/dark cycles of 0:24 to 16:8 h, decreased greatly in the short-, long- and full-day cycles of red/far-red light, but was unaffected under full-day blue light. Moreover, ΔBbphy showed higher osmosensitivity, increased antioxidant capability, and decreased conidial thermotolerance and UV-B resistance, accompanied with downregulation of Hog1 phosphorylation and of four Hog1-related genes under osmotic stress, and upregulation of five superoxide dismutases and four catalases under oxidative stress. All the changes were restored by the gene complementation. Taken together, Bbphy controls conidiation by responding to daylight length and red/far-red light and regulates multistress responses perhaps because of an involvement in Hog1 pathway. Our findings highlight diverse functions of Bbphy in B. bassiana.

Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses.

The catalase family of Beauveria bassiana (fungal entomopathogen) consists of catA (spore-specific), catB (secreted), catP (peroxisomal), catC (cytoplasmic) and catD (secreted peroxidase/catalase), which were distinguished in phylogeny and structure and functionally characterized by constructing single-gene disrupted and rescued mutants for enzymatic and multi-phenotypic analyses. Total catalase activity decreased 89% and 56% in ΔcatB and ΔcatP, corresponding to the losses of upper and lower active bands gel-profiled for all catalases respectively, but only 9-12% in other knockout mutants. Compared with wild type and complement mutants sharing similar enzymatic and phenotypic parameters, all knockout mutants showed significant (9-56%) decreases in the antioxidant capability of their conidia (active ingredients of mycoinsecticides), followed by remarkable phenotypic defects associated with the fungal biocontrol potential. These defects included mainly the losses of 40% thermotolerance (45°C) in ΔcatA, 46-48% UV-B resistance in ΔcatA and ΔcatD, and 33-47% virulence to Spodoptera litura larvae in ΔcatA, ΔcatP and ΔcatD respectively. Moreover, the drastic transcript upregulation of some other catalase genes observed in the normal culture of each knockout mutant revealed functionally complimentary effects among some of the catalase genes, particularly between catB and catC whose knockout mutants displayed little or minor phenotypic changes. However, the five catalase genes functioned redundantly in mediating the fungal tolerance to either hyperosmotic or fungicidal stress. The differentiated roles of five catalases in regulating the B. bassiana virulence and tolerances to oxidative stress, high temperature and UV-B irradiation provide new insights into fungal adaptation to stressful environment and host invasion.

Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana.

Knockout and complement mutants of mannitol-1-phosphate dehydrogenase (MPD) and mannitol dehydrogenase (MTD) were constructed to probe the roles of both enzymes in the mannitol metabolism and multi-stress tolerances of entomopathogenic fungus Beauveria bassiana. Compared with wild-type and complement mutants, ΔBbMPD lost 99.5% MPD activity for reducing fructose-6-phosphate to mannitol-1-phosphate while ΔBbMTD lost 78.9% MTD activity for oxidizing mannitol to fructose. Consequently, mannitol contents in mycelia and conidia decreased 68% and 83% for ΔBbMPD, and 16% and 38% for ΔBbMTD, accompanied by greatly enhanced trehalose accumulations due to 81-87% decrease in their neutral trehalase expression. Mannitol as mere carbon source in a nitrate-based minimal medium suppressed the colony growth of ΔBbMTD instead of ΔBbMPD, and delayed more conidial germination of ΔBbMTD than ΔBbMPD. Based on median lethal responses, conidial tolerances to H(2) O(2) oxidation, UV-B irradiation and heat stress at 45°C decreased 38%, 39% and 22% in ΔBbMPD, and 18%, 16% and 11% in ΔBbMTD respectively. Moreover, ΔBbMPD and ΔBbMTD lost 14% and 7% of their virulence against Spodoptera litura larvae respectively. Our findings highlight the primary roles of MPD and MTD in mannitol metabolism and their significant contributions to multi-stress tolerances and virulence influential on the biocontrol potential of B.bassiana.

Improving UV resistance and virulence of Beauveria bassiana by genetic engineering with an exogenous tyrosinase gene.

Insect pathogenic fungi like Beauveria bassiana have been developed as environmentally friendly biocontrol agents against arthropod pests. However, restrictive environmental factors, including solar ultraviolet (UV) radiation frequently lead to inconsistent field performance. To improve resistance to UV damage, we used Agrobacterium-mediated transformation to engineer B. bassiana with an exogenous tyrosinase gene. The results showed that the mitotically stable transformants produced larger amounts of yellowish pigments than the wild-type strain, and these imparted significantly increased UV-resistance. The virulence of the transgenic isolate was also significantly increased against the silkworm Bombyx mori and the mealworm Tenebrio molitor. This study demonstrated that genetic engineering of B. bassiana with a tyrosinase gene is an effective way to improve fungal tolerance against UV damage.

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen.

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn(2+), suppressed by Cu(2+) and Zn(2+) but inhibited by Fe(3+). Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 +/- 6.1 U mg(-1) protein) by 4-10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 +/- 74.7 U mg(-1) was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC(50) = 2.41 +/- 0.03 versus 1.25 +/- 0.01 mM) and conidial germination (EC(50) = 0.89 +/- 0.06 versus 0.55 +/- 0.07 mM), and 23% more tolerant to UV-B irradiation (LD(50) = 0.49 +/- 0.02 versus 0.39 +/- 0.01 J cm(-2)). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT(50) = 4.5 (4.2-4.8) versus 5.7 (5.2-6.4) days]. Our study highlights for the first time that the Mn(2+)-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.

Evaluation of soyscreen in an oil-based formulation for UV protection of Beauveria bassiana conidia.

Soyscreen oil was studied as a formulation ingredient to protect Beauveria bassiana (Balsamo) Vuillemin conidia from UV degradation. Feruloylated soy glycerides, referred to as Soyscreen oil, are biobased UV-absorbing molecules made by combining molecules of soybean oil with ferulic acid. Conidia stored in Soyscreen oil for 28 wk at 25, 30, and 35 degrees C retained viability as well as conidia stored in sunflower oil, demonstrating that Soyscreen did not adversely affect viability with prolonged storage. For samples applied to glass and exposed to simulated sunlight (xenon light), conidia in sunflower oil with or without sunscreens (Soyscreen or oxyl methoxycinnimate) had similar conidia viability after exposure. These oil formulations retained conidia viability better than conidia applied as an aqueous treatment. However, the 10% Soyscreen oil formulation applied to field grown cabbage (Brassica oleracea L.) and bean (Phaseolus vulgaris L.) plants, did not improve residual insecticidal activity compared with aqueous applications of unformulated conidia or two commercial formulations when assayed against Trichoplusia ni (Hübner) larvae. Our results suggest that the oil applications lose UV protection because the oil was absorbed by the leaf. This conclusion was supported in subsequent laboratory exposures of conidia in oil-based formulations with UV screens applied to cabbage leaves or balsa wood, which lost protection as measured by decreased viability of conidia when exposed to simulated sunlight. As a result, additional formulation techniques such as encapsulation to prevent separation of the protective oil from the conidia may be required to extend protection when oil formulations are applied in the field.

Comparative tolerances of various Beauveria bassiana isolates to UV-B irradiation with a description of a modeling method to assess lethal dose.

The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1-1.6 J cm(-2) (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0-2.61 mW cm(-2). Irradiated conidia were then incubated for 24 h at 25 degrees C under saturated humidity. The ratio of germination at each dose over that in the blank control was defined as survival index (I (s)). For all isolates, the I (s) - D observations fit well with the survival model I (s) = 1/[1 + exp(a + bD)] (0.94 < or = r (2) < or = 0.99) generated widely spanned lethal doses of 0.154-0.928, 0.240-1.139, and 0.383-1.493 J cm(-2) for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm(-2) measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses.

Assessment of the suitability of Tinopal as an enhancing adjuvant in formulations of the insect pathogenic fungus Beauveria bassiana (Bals.) Vuillemin.

BACKGROUND: Biopesticides based on Beauveria bassiana (Bals.) Vuillemin hold great promise for the management of a wide range of insect pests. The conidia in the biopesticide formulation require an adjuvant to protect them from photoinactivation by sunlight. The suitability of Tinopal, an optical brightener used as sunscreen for baculovirus formulations, for use with B. bassiana was assessed. The aim was to study the effect of Tinopal on the growth and photoprotection of B. bassiana, and its effect on the susceptibility of insects to B. bassiana. RESULTS: Tinopal was found to have no adverse effect on the growth of B. bassiana. It was found to confer total protection (approximately 95% conidial germination at 10 g Tinopal L(-1)) from sunlight up to 3 h of exposure, and a better survival rate than controls even up to 4 h. Helicoverpa armigera Hübner larvae fed on diet with 5 g kg(-1) Tinopal were found to have reduced growth. The duration of the larval stage increased by 3-4 days in 1 and 5 g kg(-1) Tinopal treatments. Among the moths that emerged from larvae fed on diet with 5 g kg(-1) Tinopal, a significantly high number were malformed compared with controls. The larvae that were fed diet with Tinopal showed quicker and higher mortality and required a lower effective lethal dose (LC(50)) than the controls. Tinopal was found to have a synergistic effect with B. bassiana in causing insect mortality. CONCLUSIONS: Tinopal was found to be a suitable adjuvant for B. bassiana-based biopesticide formulations. It conferred tolerance to sunlight and caused stress in the insect, leading to a synergistic effect with B. bassiana.

Isolation and characterization of nucleotide excision repair deficient mutants of the entomopathogenic fungus, Beauveria bassiana.

To better understand DNA repair in the entomopathogenic fungus Beauveria bassiana, three ultraviolet (UV) light sensitive mutants were isolated and characterized to be deficient in nucleotide excision repair (NER). The UV sensitive mutants were scored by comparison to survival of the parental isolate, GK2016, after 36 J/m(2) UV-C irradiation. At this dose, conidial survival of GK2016 was 98% and the mutants LC75, LC194, and LC85 had survival values of 63%, 45%, and 31%, respectively. An immunological method which measured the removal of pyrimidine-(6-4)-pyrimidone photoproducts during repair confirmed the decreased ability of LC75, LC194, and LC85 to remove these UV-induced dimers by NER. The mutants were also found to be deficient in NER at swollen/ germinating conidia and blastospore life cycle stages. The germination of the moderately UV sensitive mutant, LC75, was similar to that of the parental isolate, GK2016, after UV irradiation and incubation to enhance NER. The more sensitive mutants, LC194 and LC85 were 2.1- or 2.7-fold, respectively, less likely to germinate after UV irradiation based on their ability to carry out NER. These NER deficient mutants, the first to be derived from B. bassiana, reveal the importance of NER in spore survival post-UV irradiation.