Identification of the pigment and its role in UV resistance in Paecilomyces variotii, a Chernobyl isolate, using genetic manipulation strategies.

Fungi produce secondary metabolites that are not directly involved in their growth, but often contribute to their adaptation to extreme environmental stimuli and enable their survival. Conidial pigment or melanin is one of the secondary metabolites produced naturally by a polyketide synthesis (PKS) gene cluster in several filamentous fungi and is known to protect these fungi from extreme radiation conditions. Several pigmented or melanized fungi have been shown to grow under extreme radiation conditions at the Chernobyl nuclear accident site. Some of these fungi, including Paecilomyces variotii, were observed to grow towards the source of radiation. Therefore, in this study, we wanted to identify if the pigment produced by P. variotii, contributes to providing protection against radiation condition. We first identified the PKS gene responsible for synthesis of pigment in P. variotii and confirmed its role in providing protection against UV irradiation through CRISPR-Cas9 mediated gene deletion. This is the first report that describes the use of CRISPR methodology to create gene deletions in P. variotii. Further, we showed that the pigment produced by this fungus, was not inhibited by DHN-melanin pathway inhibitors, indicating that the fungus does not produce melanin. We then identified the pigment synthesized by the PKS gene of P. variotii, as a naptho-pyrone Ywa1, by heterologously expressing the gene in Aspergillus nidulans. The results obtained will further aid in understanding the mechanistic basis of radiation resistance.

Modeling the inactivation of Aspergillus fischeri and Paecilomyces niveus ascospores in apple juice by different ultraviolet light irradiances.

The present work aimed to evaluate and to model the influence of UV-C light treatments with different irradiances (6.5, 13, 21, and 36 W/m2) on Aspergillus fischeri and Paecilomyces niveus ascospores inactivation in clarified apple juice. Approximately 5.0 and 6.0 log CFU/mL spores of P. niveus and A. fischeri, respectively, were suspended in 30 mL of clarified apple juice (pH 3.8, 12 ± 0.1°Brix) and exposed to UV-C light at different irradiances (as above) and exposure times (0 to 30 min). The first-order biphasic model was able to describe the experimental data with good statistical indices (RMSE = 0.296 and 0.308, R2 = 0.96 and 0.98, for P. niveus and A. fischeri respectively). At the highest irradiance level tested (36 W/m2), the UV-C light allowed the reduction of 5.7 and 4.2 log-cycles of A. fischeri and P. niveus ascospores, respectively, in approximately 10 min. P. niveus was the most UV-C resistant mould. The results showed that, to a defined UV-C fluence, a change in the level of either time or UV-C irradiance did not affect the effectiveness of UV-C light for A. fischeri and P. niveus inactivation. Thus, the modeling of the inactivation as a function of the UV-C fluence allowed the estimation of the primary model parameters with all experimental data and, consequently, no secondary models were needed. The model parameters were validated with experiments of variable UV-C fluences. Accordingly, experimental results allowed to conclude that UV-C treatment at the irradiances tested is a promising application for preventing A. fischeri and P. niveus spoilage of juices.

[The effect of low radiation doses on the growth of Aspergillus versicolor and Paecilomyces lilacinus].

Peculiarities of growth of the strains of two species of microscopic fungi Aspergillus versicolor and Paecilomyces lilacinus were analyzed under exposure to chronic irradiation. It was shown that the rate of radial growth increased nonuniformly in the range of doses from 0 to 250 mGy but with maximums at a certain dose of radiation. It was shown that the absorbed dose of radiation to 2 Gy did not influence the survival of investigated strains in comparison with the control ones without irradiation. It was shown that the doses of irradiation to 2 Gy (at capacity exposure dose 0.955 microC/kg) are small for these species of microscopic fungi.

Long-term preservation, regeneration, and cultivation of Paecilomyces tenuipes (Peck) Samson (Ascomycetes), an entomopathogenic fungus inoculated into the silkworm larva of Bombyx mori.

Paecilomyces tenuipes reportedly have anticancer and immune activities, along with various other medicinal uses. Cultured products with P. tenuipes are certified for use in food in South Korea, and processed goods containing this fungus have been developed in many countries, particularly South Korea, Japan, and China. Research on mass production technology-procured raw materials for the manufacture of P. tenuipes is very important; however, cultures of the fungus have been unstable. This study identified stable cultivation conditions, focusing on growth inhibition and revitalization. Moisture regulation and preservation of pupae inoculated with P. tenuipes were used to control growth inhibition and revitalization. When inoculated silkworm pupae were dehydrated to 4% moisture and preserved freeze-dried or at -70 degrees C, -20 degrees C, or 4 degrees C, the mycelia in their bodies were able to survive for 14 d. Inoculated silkworm pupae were rehydrated for 3 h and the mycelia within their bodies were recovered at 94.3-96.3%. Silkworm pupae at 4% moisture were able to survive for 135 d at temperatures < 4 degrees C and for 1 y after freeze-drying. Optimal conditions for synnemata induction were 25 degrees C and 100-300 1x.

[Influence of ionizing radiation on activity of enzymes of antioxidant defense of Paecilomyces lilaclvus (Thom) Samson].

The level of activity of antioxidant protection enzymes (superoxide dismutase, catalase and peroxidase) under exposure to ionizing radiation and without it in strain Paecilomyces lilacinus, showing radioadaptive properties, and in control one has been investigated. It has been established that the researched strains are characterized by the high level activity of superoxide dismutase (200-800 AU/mg protein), extracellular and intracellular catalase (0.02-40 mmol min(-1) mg(-1) protein) and peroxidase (0.2-4 mmol min(-1) mg(-1) protein). Ionizing radiation was the inducer of significant changes in antioxidant enzyme activity of the control strain (from the lack of influence to the change of activity by an order) and showed considerably less influence on their activity in the strain, showing radioadaptive properties (the activity changes by 40-50%). The complex response of antioxidant enzymes in investigated strains under the exposure to ionizing radiation has been revealed.

[Influence of light of different spectral composition on growth characteristics of microscopic fungi].

The features of radial growth of three microscopic fungi species were investigated at illumination by yellow, blue, green and red light. The studied species of fungi differed by isolation site (some of them were isolated from the places of high radioactive pollution, others--from the places with background level of radioactivity) and pigmentation degree (melanin-containing and light-colored). The parameters of radial growth, which are the integral indexes of the fungal organism physiology state were investigated: radial growth rate, branching degree and summarizing index--the intensity of substrate consumption. It was shown that the melanin-containing fungi Cladosporium cladosporioides growth was twice more rapid in response to blue and yellow light. Whereas such light depressed growth of non-pigmented strain of the same species as compared to control. A light response of other pigment-containing species Hormoconis resinae was far less expressed, and the light-colored species Paecilomyces lilacinus had no response to any offered conditions of illumination.

[A comparative investigation of adaptation to oxidative stress factors a strain of mycelial fungus Paecilomyces lilacinus from Chernobyl Atomic Energy Station and strains of the same species from territories with basic level of radioactive pollution].

An oxidative stress resistance in Paecilomyces lilacinus strain from Chernobyl Atomic Energy Station (ChAES) as well as in P. lilacinus strains from zones with control level of radioactive pollution has been studied. It was shown that radial growth rate of the colony of the ChAES strain in a range of glucose concentrations 0.002%; 0.2%; 0.5%; 1.0% showed maximum on the medium with 0.2% glucose. It was 1.5 times higher than growth rate of the control strains. Adaptation slowdown reaction has been found as a result of the first 10-30 min H2O2 treatment most profound in the ChAES (No 1941) strain. Under 10(-3) mol/l H2O2 growth of No 1941 resumed with 20% loss in growth rate. Cessation of growth of the control strain (No 10) was observed under these conditions. It has been shown that in the strain from ChAES zone adaptation to low glucose content in the medium (0.2%) was coupled with an increased resistance to oxidative stress.

Effect of different photoperiods on the growth, infectivity and colonization of Trinidadian strains of Paecilomyces fumosoroseus on the greenhouse whitefly, Trialeurodes vaporariorum, using a glass slide bioassay

Growth, infectivity and colonization rates for blastospores and conidia of Trinidadian strains T, T10, and T11 of Paecilomyces fumosoroseus (Wize) Brown and Smith were assessed for activity against late fourth-instar nymphs of Trialeurodes vaporariorum (Westwood) (Homoptera:Aleyrodidae) under two different photoperiods (24 and 16 hour photophase). A glass-slide bioassay and a fungal development index, modified for both blastospores and conidia, were used to compare the development rates of the fungal strains on the insect hosts. Fewer adult whiteflies emerged from nymphs treated with blastospores and reared under a 16:8 hour light:dark photoperiod than a 24:0 hour photoperiod. Eclosion times of whitefly adults that emerged from nymphs treated with the different strains of conidia were similar over the 8 day experimental period at both light regimes. The percent eclosion of adult whiteflies seems to be directly correlated with the speed of infection of the blastospore or conidial treatment and the photoperiod regime. The longer photophase had a significant positive effect on development index for blastospores; however, a lesser effect was observed for the conidia at either light regime. Blastospore strain T11 offered the most potential of the three Trinidadian strains against T. vaporariorum fourth-instar nymphs, especially under constant light. The glass-slide bioassay was successfully used to compare both blastospores and conidia of P. fumosoroseus. It can be used to determine the pathogenicity and the efficacy of various fungal preparations against aleyrodid pests.

Light-regulated asexual reproduction in Paecilomyces fumosoroseus.

The entomopathogenic fungus Paecilomyces fumosoroseus has been successfully used in the control of several insect pests. Asexually produced spores (conidia) are the means for dispersal and transmission of the entomopathogen; upon contact with the insect cuticle they germinate and penetrate the host. In model fungal systems it has been found that phototropism, resetting of the circadian rhythm, the induction of carotenogenesis and the development of reproductive structures are controlled by blue light. The effect of light quality on conidial yield of P. fumosoroseus was investigated. Incubation in total darkness resulted in continued vegetative growth and lack of reproductive structures. In contrast, growth of the fungus in continuous illumination or under a night-day regime resulted in prolific formation of conidiophores bearing abundant mature conidia. Conidiation was photoinduced in competent mycelia by a single pulse of blue light and colonies were competent only after they had grown at least 72 h under total darkness. The fluence-response curves generated with blue light indicated that the minimal fluence required for the photomorphogenetic response was 180 micro mol m(-2) and the half-maximal response was at 400 micro mol m(-2). A fluence of 540 micro mol m(-2) was enough to saturate the system, inducing the maximum production of 2.12x10(8) conidia per colony. Higher light intensities markedly decreased conidiation, suggesting the occurrence of a process of adaptation. The authors propose the existence of a dual light-perception system with at least two photoreceptors in P. fumosoroseus, one promoting and one inhibiting conidiation.

Effect of different photoperiods on the growth, infectivity and colonization of Trinidadian strains of Paecilomyces fumosoroseus on the greenhouse whitefly, Trialeurodes vaporariorum, using a glass slide bioassay.

Growth, infectivity and colonization rates for blastospores and conidia of Trinidadian strains T, T10, and T11 of Paecilomyces fumosoroseus (Wize) Brown and Smith were assessed for activity against late fourth-instar nymphs of Trialeurodes vaporariorum (Westwood) (Homoptera:Aleyrodidae) under two different photoperiods (24 and 16 hour photophase). A glass-slide bioassay and a fungal development index, modified for both blastospores and conidia, were used to compare the development rates of the fungal strains on the insect hosts. Fewer adult whiteflies emerged from nymphs treated with blastospores and reared under a 16:8 hour light:dark photoperiod than a 24:0 hour photoperiod. Eclosion times of whitefly adults that emerged from nymphs treated with the different strains of conidia were similar over the 8 day experimental period at both light regimes. The percent eclosion of adult whiteflies seems to be directly correlated with the speed of infection of the blastospore or conidial treatment and the photoperiod regime. The longer photophase had a significant positive effect on development index for blastospores; however, a lesser effect was observed for the conidia at either light regime. Blastospore strain T11 offered the most potential of the three Trinidadian strains against T. vaporariorum fourth-instar nymphs, especially under constant light. The glass-slide bioassay was successfully used to compare both blastospores and conidia of P. fumosoroseus. It can be used to determine the pathogenicity and the efficacy of various fungal preparations against aleyrodid pests.

Inactivation of conidia of Paecilomyces fumosoroseus by near-ultraviolet (UVB and UVA) and visible radiation.

The detrimental effects of solar radiation, especially the ultraviolet waveband, on quiescent conidia of Paecilomyces fumosoroseus were investigated. Conidia were irradiated by a high-intensity source, which emitted a continuous spectrum from 270 to 1100 nm and which was equipped with long-pass filters to block short wavelengths below 280, 295, 320, or 400 nm. After irradiation, conidia were tested for germinability, survival, and infectivity toward Spodoptera frugiperda larvae. It was demonstrated that the detrimental effects of light depended on irradiance in the shortest wavelengths. The UVB (280-320 and 295-320 nm) appeared to be the most detrimental part of natural radiation, although UVA (320-400 nm) was also harmful. Visible and near infrared radiations were less harmful than UV. Our results demonstrate that the irradiance of the UVB waveband should be considered as the pertinent factor for the detrimental effects of sunlight on the persistence of conidia of entomopathogenic fungi in insolated environments.