Development of SCAR Markers for Genetic Authentication of Metarhizium acridum.

In this study, molecular typing using Randomly Amplified Polymorphic DNA (RAPD-PCR) was conducted on 16 original isolates of Metarhizium acridum obtained from locusts (Schistocerca piceifrons ssp. piceifrons.) in Mexico (MX). The analysis included reference strains of the genus Metarhizium sourced from various geographical regions. The isolates were identified by phenotypic (macro and micromorphology) and genotypic methods (RAPD-PCR and Amplified Fragment Length Polymorphisms (AFLP), through a multidimensional analysis of principal coordinates (PCoA) and a minimum spanning network (MST). Subsequently, Sequences-Characterized Amplified Region (SCAR) markers were developed for the molecular detection of M. acridum, these markers were chosen from polymorphic patterns obtained with 14 primers via RAPD-PCR. Phenotypic and genotypic characterization identified the MX isolates as M. acridum. Of all the polymorphic patterns obtained, only OPA04 and OPA05 were chosen, which presented species-specific bands for M. acridum, and further utilized to create SCAR markers through cloning and sequencing of the specific bands. The specificity of these two markers was confirmed via Southern hybridization. The SCAR markers (Ma-160OPA-05 and Ma-151OPA-04) exhibit remarkable sensitivity, detecting down to less than 0.1 ng, as well as high specificity, as evidenced by their inability to cross-amplify or generate amplification with DNAs from other strains of Metarhizium (as Metarhizium anisopliae) or different genera of entomopathogenic fungi (Cordyceps fumosorosea and Akanthomyces lecanii). These SCAR markers yield readily detectable results, showcasing high reproducibility. They serve as a valuable tool, especially in field applications.

A review of described cases of mycotic keratitis and sclerokeratitis related to entomopathogenic fungi from 1984 to 2021.

Infectious keratitis and sclerokeratitis caused by filamentous fungi prevail in agricultural regions with tropical and subtropical climates and are related mostly to mild abrasive corneal trauma especially after vegetable matter related injury. Biotechnological advances have introduced biological control agents in agriculture such as fungal-based biocontrol agents that use Beauveria and Metarhizium species as bioinsecticides. Keratitis and sclerokeratitis are the most frequent pathologies associated to Beauveria and Metarhizium infection that are the main entomopathogenic fungi used in biological control, although other clinical cases such as sinus, skin lesions, and disseminated infections have been reported. Search of publications was carried out using the databases: Scopus, Pubmed, ScienceDirect, MedLine Scielo. A total of 30 articles were retrieved from 1984 - 2021. From these, 17 keratitis and one sclerokeratitis clinical cases were related to Beauveria infection, while Metarhizium was linked to 13 keratitis cases and two sclerokeratitis clinical cases. Female sex predominated in both Metarhizium and Beauveria clinical cases, there was no significant difference in sclerokeratitis / keratitis by sex. Contact lenses use was a factor reported in 66.6% cases of infection with Metarhizium and 22.2% with Beauveria. The review of clinical cases of keratitis and sclerokeratitis related to Beauveria and Metarhizium suggests the need to consider entomopathogenic fungi in ocular pathologies and the risk that imply the misuse of contact lenses and agricultural/gardening activities.

Genetic diversity of Histoplasma and Sporothrix complexes based on sequences of their ITS1-5.8S-ITS2 regions from the BOLD System.

High sensitivity and specificity of molecular biology techniques have proven usefulness for the detection, identification and typing of different pathogens. The ITS (Internal Transcribed Spacer) regions of the ribosomal DNA are highly conserved non-coding regions, and have been widely used in different studies including the determination of the genetic diversity of human fungal pathogens. This article wants to contribute to the understanding of the intra- and interspecific genetic diversity of isolates of the Histoplasma capsulatum and Sporothrix schenckii species complexes by an analysis of the available sequences of the ITS regions from different sequence databases. ITS1-5.8S-ITS2 sequences of each fungus, either deposited in GenBank, or from our research groups (registered in the Fungi Barcode of Life Database), were analyzed using the maximum likelihood (ML) method. ML analysis of the ITS sequences discriminated isolates from distant geographic origins and particular wild hosts, depending on the fungal species analyzed. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Acute oral intragastric pathogenicity and toxicity in mice of Paecilomyces fumosoroseus isolated from whiteflies.

Paecilomyces fumosoroseus, monospore culture EH-506/3, isolated in Mexico from Bemisia tabaci whitefly was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated by gavage with only one dose (10(8) conidia/animal) of viable (72 mice), heat-killed (24 mice) fungus and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. No mice were clinically ill or died. At the end of the study, their mean weight corresponded to healthy adults. Positive fungal cultures of feces were obtained only 24 h after inoculation. Positive cultures were found in 15 out of 360 organs (liver, spleen, kidney, brain, lung) in 12 of 72 mice inoculated with viable conidia. Gross pathology exhibited splenomegaly and liver paleness in mice inoculated with viable and heat-killed fungus. Non-germinated conidia were observed in studied organs, without any pathological tissue reaction, suggesting no mycological or histopathological evidence of fungal multiplication. The fungus was able to persist, but did not cause permanent damage to the host. This study supports the non-pathogenic/toxic status of P. fumosoroseus EH-506/3 when administered intragastrically in mice.

Phenotyping and genotyping of Sporothrix schenckii isolates according to geographic origin and clinical form of Sporotrichosis.

Sporothrix schenckii isolates of fixed and lymphocutaneous clinical forms from Mexico (MX), Guatemala (GT), and Colombia (CO) as well as environmental isolates from MX were studied by analyzing their phenotypic characteristics (conidial length, thermotolerance by percent growth inhibition [GI] at 35 and 37 degrees C, median lethal dose [LD(50)]) and genotypic characteristics (by random amplified polymorphic DNA [RAPD] analysis-PCR). A significant difference (P < 0.01) in the mean conidial length of S. schenckii clinical isolates from CO ( = 4.03 +/- 1.04 microm) compared with those of clinical isolates from MX ( = 2.06 +/- 0.53 microm) and GT ( = 2.68 +/- 0.83 microm) was observed. The lowest thermotolerance, as determined by measurement of percent GI, was exhibited by isolates from CO at 35 degrees C ( = 50.1% +/- 15.9%) and 37 degrees C ( = 72.7% +/- 10.9%). In general, the highest virulence, as determined by measurement of the LD(50) for mice, was observed for the MX environmental isolates. RAPD analysis-PCR with 10-mer primers OPBG-01, OPBG-14, and OPBG-19 generated 52 reproducible bands. The 44 Sporothrix isolates fell into four major groups by hierarchical cluster analysis. The first group (group I), formed by 25 (of 27) isolates from MX, had two subgroups: subgroup Ia with 10 environmental isolates and subgroup Ib with 14 clinical isolates. The second group (group II) had two subgroups: subgroup IIa, formed by isolates from CO, and subgroup IIb, formed by isolates from GT. Groups III and IV each had only one clinical isolate from MX. A principal-component analysis of the same data yielded three distinct groups, depending on the geographical origins of the isolates, including the isolates in groups III and IV from MX, which were grouped with the isolates from MX by principal-component analysis. This study revealed that isolates from CO had low thermotolerances at 35 and 37 degrees C and could be associated with superficial skin lesions in patients with fixed clinical forms of sporotrichosis, the most frequent form of the disease in CO. Distinct patterns dependent on geographical origins were also revealed by RAPD analysis-PCR, but these had no relation to the clinical form of the disease.