Analysis of the virulence, infection process, and extracellular enzyme activities of Aspergillus nomius against the Asian corn borer, Ostrinia furnacalis guenée (Lepidoptera: Crambidae).

The control of Ostrinia furnacalis, a major pest of maize in Xinjiang, is challenging owing to the occurrence of resistant individuals. Entomopathogenic fungi (EPF) are natural insect regulators used as substitutes for synthetic chemical insecticides. The fungus Aspergillus nomius is highly pathogenic to O. furnacalis; however, its virulence characteristics have not been identified. This study aimed to analyse the lethal efficacy, mode of infection on the cuticle, and extracellular enzyme activity of A. nomius against O. furnacalis. We found that the mortality and mycosis of O. furnacalis were dose-dependent when exposed to A. nomius and varied at different life stages. The egg-hatching and adult emergence rates decreased with an increase in conidial suspension. The highest mortality (83.33%, 7 d post-infection [DPI]) and mycosis (74.33%, 7 DPI) and the lowest mortality response (8.52 × 103 conidia mL-1) and median lethal time (4.91 d) occurred in the 3rd instar larvae of O. furnacalis. Scanning electron microscopy indicated that numerous conidia germination and infection structure formation may have contributed to the high pathogenicity of A. nomius against O. furnacalis. There were significant correlations between O. furnacalis mortality and the activities of extracellular protease, lipase, and chitinase of A. nomius. This study revealed the infection process of the highly pathogenic A. nomius against O. furnacalis, providing a theoretical basis and reference for strain improvement and field application of EPF.

Identification and Observation of Infection Processes of Colletotrichum Species Associated with Persimmon Anthracnose in Guangxi, China.

Persimmon originated from China where it has a long cultivation history. Anthracnose fruit rot and leaf blight caused by Colletotrichum species are major diseases of persimmon in China and cause severe economic losses. To determine the species causing anthracnose of persimmon in Guilin, Guangxi Province, diseased samples were collected from the four local counties: Gongcheng, Yangshuo, Pingle, and Lipu. Seventy-five isolates were obtained from persimmon samples with anthracnose symptoms and had similar morphological characteristics. Isolates were identified using a BLAST search and phylogenetic analysis of the internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, partial actin, ß-tubulin, chitin synthase genomic regions, Apn2-Mat1-2 intergenic spacer, and the partial mating type gene and calmodulin genes. Five species (C. fructicola, C. horii, C. karstii, C. cliviicola, and C. siamense) accounted for 54.7, 25.3, 12.0, 5.3, and 2.7%, respectively, of the total isolates. All five Colletotrichum species were pathogenic on attached leaves and detached fruits of persimmon (cultivar Gongcheng Yueshi) in pathogenicity assays. The infection processes of the five Colletotrichum species were observed on persimmon leaves using light microscopy. Conidia of C. fructicola germinated at 12 h post inoculation (hpi) and quickly formed acervuli at 6 days post inoculation (dpi) and were the most aggressive. By contrast, conidia of C. cliviicola germinated at 3 hpi but produced the acervuli at 8 dpi and were the least aggressive. This is the first description of C. fructicola, C. cliviicola, and C. siamense as causal agents of persimmon anthracnose in Guangxi Province, China.

Systemic Colonization of Potato Plants by Verticillium dahliae Leads to Infection of Tubers and Sprouting Buds.

A green fluorescent protein (GFP)-tagged isolate of Verticillium dahliae was used to study its colonization in potato plants and tubers. Three-week-old potato plants of the highly susceptible cultivar 'Shepody' were inoculated with a conidial suspension of a GFP-tagged isolate of V. dahliae using a wound inoculation method. Colonization was studied using confocal microscopy combined with tissue sections. Conidia germinated and hyphae grew along the root hairs, elongation zones, and root caps between 24 and 96 h postinoculation (HPI). At 7 days postinoculation (DPI), the pathogen advanced to cortical tissues and grew into the root vascular bundles. At 8 weeks postinoculation (WPI), the stem epidermal cells, cortical tissues, vascular elements, and petioles were fully colonized by the mycelium of V. dahliae. At 11 WPI, the pathogen was detected in the stolon and progeny tubers, as confirmed by both GFP signals in tissues and reisolation of the pathogen on the semiselective NP-10 medium. Progeny potato tubers were harvested from the inoculated potato plants, and the GFP-signal was observed in the epidermal cells and vascular elements of sprouting buds that emerged from the harvested tubers. The infection rate of progeny tubers detected on semiselective NP-10 medium ranged from 34.55 to 55.56%, with an average of 45.31%. In conclusion, we report, for the first time, the entire progression of colonization by V. dahliae in potato plant tissues, progeny tubers, as well as of the sprouting buds that emerged from progeny tubers.

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae.

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.