Genetic diversity of the Metarhizium anisopliae complex in Colima, Mexico, using microsatellites.

Metarhizium anisopliae is a complex of cryptic species with wide geographical distribution and versatile lifestyles. In this study, 45 isolates of the Metarhizium genus harbored in the "Colección de Hongos Entomopatógenos" of the "Centro Nacional de Referencia de Control Biológico" from different substrates, insect-host, and localities from Colima, Mexico, were phylogenetically identified using the 5'end of translation elongation factor 1-α (5'TEF) and intergenic nuclear region MzFG543igs. Seven species were recognized, M. acridum (n = 26), M. pemphigi (n = 1), and within the PARB and MGT clades: M. anisopliae (N = 7; sensu stricto: n = 2; sensu lato: n = 5), M. brunneum (n = 2), M. guizhouense (n = 2), M. pingshaense (n = 2), and M. robertsii (n = 5). Twenty-nine SSR markers were developed for M. acridum; according to the analysis of 12 polymorphic SSR loci, M. acridum showed low genetic diversity, revealing five genotypes with a dominant one (n = 21). Based on the analysis of 13 specific SSR loci, 14 genotypes were identified within the PARB and MGT clades. This study contributes to generating valuable information about the community structure and genotypic diversity of Metharhizum species in the state of Colima, Mexico.

High genetic diversity of the entomopathogenic fungus Beauveria bassiana in Colima, Mexico.

The entomopathogenic fungus Beauveria bassiana is used widely as a biological control agent against a wide range of insect pests globally. In this study, 44 Beauveria isolates from the state of Colima, Mexico harbored in the "Colección de Hongos Entomopatógenos" of the "Centro Nacional de Referencia de Control Biológico" and from different substrates, insect-hosts, and localities were characterized with molecular markers. All isolates were identified using a Bayesian phylogenetic analysis of translation elongation factor 1-α (TEF) and nuclear intergenic Bloc region. Forty-three isolates were identified as B. bassiana and grouped into two sub-clades, i.e., AFNEO_1 (n = 22; previously defined as a clade with African and Neotropical origin) and Bb clade (n = 21; closely associated with ex-type strain ARSEF 1564), and one isolate was identified as B. pseudobassiana. The fixation index (FST = 0.493) established the genetic differentiation between AFNEO_1 and Bb clades. High genotype richness and genetic diversity of AFNEO_1 and Bb clades were revealed in sequence analysis of Bloc region and SSR genotyping. Moreover, the AFNEO_1 and Bb clades were confirmed as two independent clonally structured assemblages. Finally, the AMOVA detected no significant association between any combination of substrate, insect-host or geographical origin. High genetic variation of B. bassiana in Colima, Mexico could suggest a functional diversity among isolates that may include those effective against a specific insect pest.

Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi.

Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/ß-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications.

Species clarification of Isaria isolates used as biocontrol agents against Diaphorina citri (Hemiptera: Liviidae) in Mexico.

Entomopathogenic fungi belonging to the genus Isaria (Hypocreales: Cordycipitaceae) are promising candidates for microbial control of insect pests. Currently, the Mexican government is developing a biological control program based on extensive application of Isaria isolates against Diaphorina citri (Hemiptera: Liviidae), a vector of citrus huanglongbing disease. Previous research identified three promising Isaria isolates (CHE-CNRCB 303, 305, and 307; tentatively identified as Isaria fumosorosea) from Mexico. The goal of this work was to obtain a complete morphological and molecular characterization of these isolates. Comparative analysis of morphology established that the isolates showed similar characteristics to Isaria javanica. Multi-gene analysis confirmed the morphological identification by including the three isolates within the I. javanica clade. Additionally, this work demonstrated the misidentifications of three other Isaria isolates (CHE-CNRCB 310 and 324: I. javanica, formerly I. fumosorosea; CHE-CNRCB 393: I. fumosorosea, formerly Isaria farinosa), underlying the need for a full and correct characterization of an isolate before developing a biological control program. Finally, the inter-simple sequence repeat (ISSR) genotyping method revealed that the CHE-CNRCB 303, 305, and 307 isolates belong to three different genotypes. This result indicates that ISSR markers could be used as a tool to monitor their presence in field conditions.