Virulence assessment of enterohepatic Helicobacter species carried by dogs using the wax moth larvae Galleria mellonella as infection model.

BACKGROUND: Enterohepatic species of the genus Helicobacter (EHH) are emerging pathogens that have been associated with gastrointestinal and hepatobiliary diseases in humans. However, studies on their pathogenicity are scarce. Galleria mellonella is a recently proposed model for the study of virulence in different pathogens, such as Campylobacter spp. and Helicobacter pylori. Despite this, its usefulness in EHH has not yet been evaluated. Therefore, we determined the pathogenic potential of different EHH species isolated from dogs in this infection model. MATERIALS AND METHODS: Four species of EHH (H. bilis, H. canicola, H. canis, and 'H. winghamensis') isolated from fecal samples from domestic dogs were evaluated. Three strains of each species were inoculated in cohorts of G. mellonella at a concentration of 1 × 107  CFU/mL. Survival curves were determined by the Kaplan-Meier method. In addition, the quantification of melanin, bacterial load in hemolymph, and histopathology were evaluated daily post-infection (pi). RESULTS: G. mellonella larvae are susceptible to EHH infection, exhibiting intra- and inter-species variability. Melanin production became evident from 4 h pi and increased throughout the assay. All species were recovered from the hemolymph after 20 min pi; however, only H. canis could be recovered up to 48 h pi. Histopathology revealed cellular and humoral immune response, evidencing accumulation of hemocytes, nodulation, and melanin deposition in different tissues. CONCLUSION: EHH species carried by dogs have considerable pathogenic potential, being H. canicola the species with the highest degree of virulence. Thus, G. mellonella is a useful model to assess virulence in these emerging pathogens.

Antifungal Activity of Beauveria bassiana Endophyte against Botrytis cinerea in Two Solanaceae Crops.

Botrytis cinerea causes substantial losses in tomato and chili pepper crops worldwide. Endophytes have shown the potential for the biological control of diseases. The colonization ability of native endophyte strains of Beauveria bassiana and their antifungal effect against B. cinerea were evaluated in Solanaceae crops. Root drenching with B. bassiana was applied, and endophytic colonization capacity in roots, stems, and leaves was determined. The antagonistic activity was evaluated using in vitro dual culture and also plants by drenching the endophyte on the root and by pathogen inoculation in the leaves. Ten native strains were endophytes of tomato, and eight were endophytes of chili pepper. All strains showed significant in vitro antagonism against B. cinerea (30-36%). A high antifungal effect was observed, and strains RGM547 and RGM644 showed the lowest percentage of the surface affected by the pathogen. Native strains of B. bassiana colonized tomato and chili pepper tissues and provided important levels of antagonism against B. cinerea.

Intraspecific differentiation of Chilean isolates of the entomopathogenic fungi Metarhizium anisopliae var. anisopliae as revealed by RAPD, SSR and ITS markers

The genus Metarhizium consists of a diverse group of asexual entomopathogenic fungi, which have a wide geographical distribution. The Chilean National Agricultural Research Institute (Instituto de Investigaciones Agropecuarias - INIA, Quilamapu Chile) has collected about 350 isolates of Metarhizium anisopliae var. anisopliae from central and southern Chile. These isolates have been partially characterized using morphological traits such as conidia size and shape, colony color, growth pattern and the efficiency of the isolates in controlling specific pests. However, further characterization with molecular markers could detect differences in DNA which could help to better understand the genetic diversity and structure of Chilean populations of this fungus. We analyzed approximately 10 percent of the INIA collection (39 isolates selected at random) collected from different geographical origins using the polymerase chain reaction (PCR)-random amplified polymorphic DNA (RAPD) method, simple sequences repeat (SSR or microsatellites) analysis and the PCR-restriction fragment length polymorphism (RFLP) assay of internal transcribed spacer (ITS)-rDNA sequences. The RAPD data revealed high genetic diversity in this fungus and an average of 41 percent of similarity while SSR analysis detected 45.2 percent similarity and the ITS markers 70.2 percent similarity. For the three molecular markers, this diversity was not associated with the geographical origin of these isolates.