Principal component analysis of the biological characteristics of entomopathogenic fungi in nutrient-limited and cuticle-based media.

Media formulated with insect cuticle (0.5% and 1%; Sphequit Sph®), with a reduction in nutrients (» Sabouraud dextrose agar + yeast [SDAY]) and commercial media (potato dextrose agar, Sabouraud dextrose agar) were evaluated for the cultivation of Beauveria bassiana, Cordyceps javanica (Isaria javanica [Bally] Samson & Hywel-Jones), and Metarhizium robertsii. By using principal component analysis, it was determined that the » SDAY and Sph formulations have greater advantages than commercial media for the development of fungi. The » SDAY and Sph (0.5% and 1%) improved hydrophobicity, radial growth rate, germination, conidia yield, and virulence in B. bassiana; in M. robertsii, they favored conidia yield, germination, and virulence, and in C. javanica, the » SDAY and Sph 0.5% media enhanced conidia yield, germination, radial growth rate, and virulence. We suggest that these formulations are an alternative to commercial culture media as they are cheaper and appropriate to improve the growth characteristics and virulence of the three strains evaluated. Some applications of culture media are suggested, and the importance of multivariate analysis as an exploratory tool to carry out the choice of culture media in a suitable way for the development of mycoinsecticides is also discussed.

Selection of Beauveria bassiana (Hypocreales: Cordycipitaceae) strains to control Xyleborus affinis (Curculionidae: Scolytinae) females.

BACKGROUND: Xyleborus affinis Eichhoff (Coleoptera: Curculionidae) is an ambrosia beetle reported to affect avocado trees (Persea americana Mill.). The use of the entomopathogenic fungus (EPF) Beauveria bassiana (Bals.-Criv.) Vuill. for ambrosia beetle control represents an alternative to insecticides. METHODS: This study was designed in two stages to select B. bassiana strains with potential to control X. affinis females. In the first stage, 19 B. bassiana Mexican strains from EPF collection, isolated from Coleoptera (CHE-CNRCB, http://www.gob.mx/senasica/documentos/coleccion-de-hongos-entomopatogenos), were tested. Analyses included radial growth rate, conidial yield, spore germination, and germ tube length. Results were analysed by Principal Component Analysis (PCA) to identify clusters within favourable growth phenotypes. For the second stage, 10 selected strains were re-analysed for virulence-related metabolic characteristic, including cell wall-bound cuticle-degrading enzymes-Pr1-like proteases and ß-N-acetyl glucosaminidases (NAGase) chitinases, conidial hydrophobicity and monopolar germination parameters. A second PCA analysis was run for those virulence parameters analysed, and upon results strains CHE-CNRCB 44, 171, 431 and 485 were selected and tested against X. affinis females. Females were treated with a 1 × 108 conidia mL-1 suspension (recommended rate), using a Potter Tower. RESULTS: All strains showed insecticidal activity, inducing up to 58% mortality; about 30% dead beetles developed aerial mycelia (CHE-CNRCB 485) and the fastest mortality rate was t0 = 1.95 (CHE-CNRCB 44). CONCLUSION: Since all selected strains showed virulence against X. affinis females, results indicated the possibility of selecting B. bassiana strains based on multiple metabolic attributes, as a preliminary test to perform bioassays against order-related target insects.

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.

Molecular characterization of Chrysoperla carnea (Neuroptera: Chrysopidae) from commercial insectaries in Mexico.

The larvae of the Chrysoperla carnea-group (Neuroptera: Chrysopidae) are recognized among the most effective larval predators of various phytophagous arthropods. Therefore, green lacewings are commonly grown by commercial insectaries and released as biological control agents. Previous work has shown that commercial laboratories frequently supply indeterminate species of the large C. carnea cryptic species complex. In Mexico, at least 20 biological control companies have commercialized the species C. carnea, but none of the products reared by those companies have been analyzed scientifically. Thus, the goal of this work was to molecularly characterize nine C. carnea populations from Mexican insectaries using the most efficient molecular markers available: the mitochondrial genes COI, COII, ND2, and ND5. Phylogenetic analysis demonstrated a unique mitochondrial haplotype in seven commercial insectaries showing 100% similarity to the reference specimen C. plorabunda E100. In contrast, we observed two and four different mitochondrial haplotypes of the carnea-group in two commercial insectaries. More precisely, three specimens possessed the mitochondrial haplotype of the species C. zastrowi, suggesting possible natural occurrence of this haplotype in Mexico. Consequently, this study demonstrated the need for an extensive survey of the different laboratories and insectaries producing C. carnea in Mexico, including unambiguous species identification by song recordings to confirm the species identity of the observed mitochondrial haplotypes.

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.

Viability, purity, and genetic stability of entomopathogenic fungi species using different preservation methods.

Preservation methods for entomopathogenic fungi (EPF) require effective protocols to ensure uniform processes and to avoid alterations during storage. The aim of this study was to preserve Beauveria bassiana, Metarhizium acridum, M. anisopliae, M. rileyi, Isaria javanica, Hirsutella thompsonii, H. citriformis and Lecanicillium lecanii in mineral oil (MO), sterile water (SW), silica gel (SG), lyophilisation (L), ultracold-freezing at -70 °C, and cryopreservation at -196 °C. The viability and purity of the fungi were then verified: phenotypic characteristics were evaluated qualitatively at 6, 12 and 24 m. Genetic stability was tested by amplified fragment length polymorphisms (AFLP) analysis at 24 m. Of the eight species of EPF, three remained viable in SW, five in MO and L, six at -70 °C, seven in SG, and eight at -196 °C. No significant changes were observed in AFLP patterns at 24 m of storage. The most effective preservation methods for EPF were SG, L, -70 and -196 °C. Beauveria bassiana, M. acridum, M. anisopliae, M. rileyi and I. javanica remained stable with all methods, while the remaining species were less compatible. The optimisation of preservation methods for EPF facilitates the development of reliable protocols to ensure their inherent characteristics in culture collections.

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.

Biological pest control in Mexico.

Mexico is a megadiverse country that forms part of the Mesoamerican biological corridor that connects North and South America. Mexico's biogeographical situation places it at risk from invasive exotic insect pests that enter from the United States, Central America, or the Caribbean. In this review we analyze the factors that contributed to some highly successful past programs involving classical biological control and/or the sterile insect technique (SIT). The present situation is then examined with reference to biological control, including SIT programs, targeted at seven major pests, with varying degrees of success. Finally, we analyze the current threats facing Mexico's agriculture industry from invasive pests that have recently entered the country or are about to do so. We conclude that despite a number of shortcomings, Mexico is better set to develop biological control-based pest control programs, particularly on an area-wide basis, than many other Latin American countries are. Classical and augmentative biological control and SIT-based programs are likely to provide effective and sustainable options for control of native and exotic pests, particularly when integrated into technology packages that meet farmers' needs across the great diversity of production systems in Mexico.