Characterization of Growth and Virulence of Five Nigerian Isolates of Entomopathogenic Fungi Using Galleria mellonella Larvae for Pathogenicity Testing

Entomopathogenic fungi (EF) are naturally occurring insect population regulators, with several species that are exploited as biocontrol agents against insect pests. Five EF consisting of two strains of Isaria farinosa, (IF-I and IF-II) and one strain each of Metarhizium anisopliae, Beauveria bassiana and Entomophaga sp. (Sensu lato) were isolated from soil using Tribolium castaneum (Coleoptera: Tenebrionidae) (Herbst) larvae as bait. The isolates were cultured on standard Potato Dextrose Agar (PDA) (Sigma-Aldrich, UK) and identified based on phenotypic appearance and micro-morphology. Growth rates (mm day-1), number of conidia per cm2 colony area after incubation for 14 days at ambient temperature (25±2°C), viability of conidia (% germination), based on 24-hours incubation period and virulence of the infective conidia against Galleria mellonella were evaluated. The data on growth was subjected to analysis of Variance (ANOVA) procedure and means were separated using Tukeys Honestly Significant Difference (P=0.05). The number of conidia produced by Entomophaga sp was (7.0×105 conidia cm 2 per colony area), while the isolate of M. anisopliae produced (5.2×104 conidia cm2 per colony area). The number of conidia produced by the two isolates of I. farinosa, IF-I and IF-II and B. bassiana were 9.4×104, 7.2×104 and 2.1×105 conidia cm2 per colony area respectively. Eighty percent of Entomophaga sp conidia germinated after incubating for 24 hours at 25°C while 100% germination occurred in the other fungal isolates. There were statistically significant variabilities in the rates of growth of the EF isolates F(4,2.064) =12.97, P=0.001. The B. bassiana isolate had the fastest growth rate, with statistically significant value of 3.3 mm day-1. The rates of growth of the two I. farinosa isolates: IF-I, IF-II and M. anisopliae were comparable, being 1.53, 1.4 and 1.28 mm day-1 respectively, without statistically significant difference. The growth rate of Entomophaga sp was 2.0 mm day-1, which was significantly higher than the growth rates of I. farinosa and M. anisopliae. The mean percentage mortality values of, G. mellonella larvae treated with 1×108 conidia ml-1 of the infective conidia of I. farinosa, IF-I, IF-II, M. anisopliae, after five days were 70, 60, 60% respectively while Entomophaga sp and B. bassiana caused 50% mortality. The results suggest that the five isolates examined can potentially be developed into experimental formulations and tested against important horticultural pests in future studies.

Effect of calcium on sporulation of Taiwanofungus camphoratus in submerged fermentation / 生物工程学报

Taiwanofungus camphoratus is a valuable and rare medicinal mushroom with various bioactivities, such as liver protection and anti-cancer. T. camphoratus can produce many arthroconidia at the end of submerged fermentation, but molecular mechanism underlying this submerged conidiation remains unknown. In this study, we found that Ca²⁺ concentration in culture medium significantly affected the arthroconidium production of T. camphoratus. Then, we identified two proteins (CaM and HSP90) involved in Ca²⁺/calmodulin signaling pathway and one protein (AbaA) involved in FluG-mediated conidiation pathway by two-dimensional electrophoresis analyses. Furthermore, we proposed a Ca²⁺/calmodulin- and FluG-mediated signaling pathway by bioinformatics analysis. By real-time quantitative PCR analyses of 23 key genes in the Ca²⁺/calmodulin- and FluG-mediated conidiation pathway, we found that expression levels of 7 genes (crz1, hsp90, flbB, brlA, abaA, wetA and fadA) showed significant responses to Ca²⁺ concentration in fermentation medium. Our research is beneficial for elucidating the underlying mechanism of submerged fermentation conidiation for T. camphoratus.

Microcyle Conidiation in Filamentous Fungi

The typical life cycle of filamentous fungi commonly involves asexual sporulation after vegetative growth in response to environmental factors. The production of asexual spores is critical in the life cycle of most filamentous fungi. Normally, conidia are produced from vegetative hyphae (termed mycelia). However, fungal species subjected to stress conditions exhibit an extremely simplified asexual life cycle, in which the conidia that germinate directly generate further conidia, without forming mycelia. This phenomenon has been termed as microcycle conidiation, and to date has been reported in more than 100 fungal species. In this review, first, we present the morphological properties of fungi during microcycle conidiation, and divide microcycle conidiation into four simple categories, even though fungal species exhibit a wide variety of morphological differences during microcycle conidiogenesis. Second, we describe the factors that influence microcycle conidiation in various fungal species, and present recent genetic studies that have identified the genes responsible for this process. Finally, we discuss the biological meaning and application of microcycle conidiation.

Regulation of Development in Aspergillus nidulans and Aspergillus fumigatus

Members of the genus Aspergillus are the most common fungi and all reproduce asexually by forming long chains of conidiospores (or conidia). The impact of various Aspergillus species on humans ranges from beneficial to harmful. For example, several species including Aspergillus oryzae and Aspergillus niger are used in industry for enzyme production and food processing. In contrast, Aspergillus flavus produce the most potent naturally present carcinogen aflatoxins, which contaminate various plant- and animal-based foods. Importantly, the opportunistic human pathogen Aspergillus fumigatus has become the most prevalent airborne fungal pathogen in developed countries, causing invasive aspergillosis in immunocompromised patients with a high mortality rate. A. fumigatus produces a massive number of small hydrophobic conidia as the primary means of dispersal, survival, genome-protection, and infecting hosts. Large-scale genome-wide expression studies can now be conducted due to completion of A. fumigatus genome sequencing. However, genomics becomes more powerful and informative when combined with genetics. We have been investigating the mechanisms underlying the regulation of asexual development (conidiation) and gliotoxin biosynthesis in A. fumigatus, primarily focusing on a characterization of key developmental regulators identified in the model fungus Aspergillus nidulans. In this review, I will summarize our current understanding of how conidiation in two aspergilli is regulated.

Morphological Characteristics of Conidiogenesis in Cordyceps militaris

Conidial development of Cordyceps militaris was observed from germinating ascospores and vegetative hyphae through light and scanning electron microscopy (SEM). Ascospores were discharged from fresh specimens of C. militaris in sterile water as well as Sabouraud Dextrose agar plus Yeast Extract (SDAY) plates. We observed ascospore germination and conidial formation periodically. Under submerged condition in sterile water, most part-spores germinated unidirectionally and conidia were developed directly from the tips of germinating hyphae of part-spores within 36 h after ascospore discharge, showing microcyclic conidiation. First-formed conidia were cylindrical or clavate followed by globose and ellipsoidal ones. Germination of ascospores and conidial development were observed on SDAY agar by SEM. Slimy heads of conidia on variously arranged phialides, from solitary to whorl, developed 5 days after ascospore discharge. Besides, two distinct types of conidia, elongated pyriform or cylindrical and globose, were observed in the same slimy heads by SEM. Conidia were shown to be uninucleate with 4,6-diamidino-2-phenylindole staining. Conidiogenous cells were more slender than vegetative hyphae, having attenuated tips. Microcyclic conidiation, undifferentiated conidiogenous hyphae (phialides), polymorphic conidia and solitary, opposite to whorled type of phialidic arrangement are reported here as the characteristic features of asexual stage of C. militaris, which can be distinguished from other Cordyceps species.