Tenebrio molitor-mediated entomopathogenic fungal library construction for pest management

Entomopathogenic fungi are soil-dwelling microorganisms that infect host insects, and some have been used as biological control agents. Construction of an entomopathogenic fungal library provides a strong platform for the development of highly effective biopesticides, but the traditional method using antibiotic media has low isolation efficiency. Herein, to increase the efficiency of isolation, a Tenebrio molitor pathogenicity-based fungal collection method was used for the construction of a library. The isolation efficiency using the T. molitor larvae-based pathogenicity assay was 55.4%, which was significantly higher than the method of antibiotic medium with dodine (6.0%). We named it the Jeonbuk (Chonbuk) National University Entomopathogenic Fungi (JEF) library. It consisted of 279 isolates belonging to 12 genera and 29 species. Main features of the species-representative 15 isolates were characterized in terms of morphology, virulence, conidiogenesis, thermotolerance and production of biologically active materials such as virulence-related enzymes. Some of the species-representative isolates showed higher virulence against T. molitor, Riptortus pedestris and Plutella xylostella in laboratory conditions. The production of enzymes such as chitinase, Pr1 protease and lipase all related to pathogenesis in LB medium was higher than in the SDB and PDB media. In the thermotolerance assay, a Purpureocillium lilacinum isolate showed higher thermotolerance than other isolates. This work reports an efficient library method using T. molitor for building a library for development of fungal biopesticides for pest management.     

Chemical stimulation of the virulence of entomopathogenic fungi

The effect of magnesium and manganese ions on the virulence of Beauveria bassiana, Metarhizium anisopliae, Paecilomyces farinosus, and Paecilomyces fumosoroseus entomopathogenic fungi was tested. The virulence of entomopathogenic fungi to Sitona lineatus (L) weevil, pupae and larvae, was increased by tested metal ions. Mg ions increased the virulence of B. bassiana against S. linatus (L) weevils 100%. The fungi exhibited various sensitivity to these metal ions.     

Cold activity and tolerance of the entomopathogenic fungus Tolypocladium spp. to UV-B irradiation and heat

Studies on the stress resistance of insect-pathogenic fungi are very important to better understand the survival of these organisms in the environment. In this study, we examined the cold activity (8 ± 1 °C for 7 days), UV-B tolerance (Quaite-weighted UV-B irradiance at 847.90 mW m⁻² for 1, 2, 3, and 4 h), and wet-heat tolerance (45 °C for 1, 2, 3, and 4 h) of two isolates of Tolypocladiumcylindrosporum (ARSEF 3392 and 5558), one isolate of Tolypocladium geodes (ARSEF 3275), and two isolates of Tolypocladium inflatum (ARSEF 4772 and 4877) based on their germination, compared with Metarhizium robertsii (ARSEF 2575). After 3 h of UV-B exposure, T. cylindrosporum germinated at a greater rate than the other Tolypocladium species and had similar viability to that of the M. robertsii. Most Tolypocladium isolates, however, were less UV-B tolerant than M. robertsii. The T.cylindrosporum isolates were also the most thermotolerant, with similar tolerance to the M. robertsii. The isolates of T. inflatum and T. geodes, which had similar heat tolerance, were the least heat tolerant compared with the isolates of T. cylindrosporum and M. robertsii. After 4 h of heat exposure, the germination of T. inflatum and T. geodes isolates was not significantly different. For cold activity, both T.cylindrosporum isolates germinated to ca. 100% in only 3 days. Approximately 50% of the two T. inflatum isolates germinated, and less than 5% of T. geodes germinated after 3 days. All fungal isolates, however, completely germinated by the seventh day, except M.robertsii. The isolates of T. cylindrosporum, therefore, were the most heat and UV-B tolerant, and had the highest cold activity compared to the other species. The tolerance of M. robertsii to UV-B radiation and heat was similar to that of T.cylindrosporum.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.     

Culture degeneration in conidia of Beauveria bassiana and virulence determinants by proteomics

The quality of Beauveria bassiana conidia directly affects the virulence against insects. In this study, continuous subculturing of B. bassiana on both rice grains and potato dextrose agar (PDA) resulted in 55 and 49 % conidial yield reduction after 12 passages and 68 and 60 % virulence reduction after 20 and 12 passages at four d post-inoculation, respectively. The passage through Tenebrio molitor and Spodoptera exigua restored the virulence of rice and PDA subcultures, respectively. To explore the molecular mechanisms underlying the conidial quality and the decline of virulence after multiple subculturing, we investigated the conidial proteomic changes. Successive subculturing markedly increased the protein levels in oxidative stress response, autophagy, amino acid homeostasis, and apoptosis, but decreased the protein levels in DNA repair, ribosome biogenesis, energy metabolism, and virulence. The nitro blue tetrazolium assay verified that the late subculture's colony and conidia had a higher oxidative stress level than the early subculture. A 2A-type protein phosphatase and a Pleckstrin homology domain protein Slm1, effector proteins of the target of rapamycin (TOR) complex 1 and 2, respectively, were dramatically increased in the late subculture. These results suggest that TOR signalling might be associated with ageing in B. bassiana late subculture, in turn affecting its physiological characteristics and virulence.     

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.     

Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella

The entomopathogenic fungi Zoophthora radicans and Pandora blunckii co-occur in field populations of Plutella xylostella and, therefore, are likely to interact during the infection process. We have investigated the possible outcomes of these interactions in the laboratory. Using four isolates, two of each fungal species, inter-specific interaction experiments were done in Petri dishes and on intact plants. In Petri dish experiments, larvae were inoculated directly using sporulating mats of mycelium, both species had the same opportunity to infect and only the relative concentration of conidia of each pathogen species applied was manipulated. In the intact plant experiments, larvae were placed onto fungus-contaminated plants, inoculation was passive and the probability of infection by either or both species of fungi depended on larval activity and proximity to inoculum. In the Petri dish experiment, the species with the largest concentration of conidia out-competed the other regardless of virulence, and results were similar in the intact plant experiment. The ecological implications for competition or co-existence of these two pathogens in the field are discussed.     

利用28S rDNAI型内含子研究琅琊山球孢白僵菌菌株的遗传多样性

通过四对引物对89株采自琅琊山的球孢白僵菌菌株进行PCR扩增,得到17类不同的单倍型,其中以BBBA型最多,占39．33％,而BABB型等其他9种单倍型较少,各占1．12％。该地区单倍型种类占已发现类型的57．7％,这说明在固定时间内同一地区的菌株也有较为丰富菌株类型,种群遗传结构是多样化的。具有较广寄主谱的BBBA型菌株是该地区的优势菌株,可作为生防菌株的重点筛选对象。     

Taxonomic and phylogenetic analysis of the Oomycota mosquito larvae pathogen Crypticola clavulifera

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.     

利用28S rDNA Ⅰ型内含子研究琅琊山球孢白僵菌菌株的遗传多样性

通过四对引物对89株采自琅琊山的球孢白僵菌菌株进行PCR扩增,得到17类不同的单倍型,其中以BBBA型最多,占39.33%,而BABB型等其他9种单倍型较少,各占1.12%。该地区单倍型种类占已发现类型的57.7%,这说明在固定时间内同一地区的菌株也有较为丰富菌株类型,种群遗传结构是多样化的。具有较广寄主谱的BBBA型菌株是该地区的优势菌株,可作为生防菌株的重点筛选对象。