Genetic diversity of the entomopathogenic fungus Metarhizium rileyi based on de novo microsatellite markers.

Epizootics of the entomopathogenic fungus Metarhizium rileyi regulate lepidopteran populations in soybean, cotton, and peanut agroecosystems to the point that insecticide applications could be unnecessary. However, the contribution and how different strains operate during the epizootic are unknown. Several unanswered questions remain: 1. How many genotypes of M. rileyi are present during an epizootic? 2. Which genotype is the most common among them? 3. Are the genotypes involved in annual epizootics at the same location the same? Therefore, the development of molecular markers to accurately identify these genotypes is very important to answer these questions. SSR primers were designed by prospecting in silico to discriminate genotypes and infer the genetic diversity of M. rileyi isolates from the collection kept at Embrapa Soybean. We tested 13 SSR markers on 136 isolates to identify 43 clones and 12 different genetic clusters, with genetic diversity ranging from Hs = 0.15 (cluster I) to Hs = 0.41 (cluster IV) and an average diversity of 0.24. No clusters were categorically distinguished based on hosts or geographical origin using Bayesian clustering analysis. Nonetheless, some clusters comprised most of the isolates with a common geographic origin; for example, cluster VIII was mainly composed of isolates from Central-western Brazil, cluster II from Southern Brazil, and cluster XII from Quincy, Northern Florida, in the United States. Underrepresented regions (few isolates) from Pacific Island nations of Japan, the Philippines, and Indonesia (specifically from Java) were placed into clusters IX and X. Although the analyzed isolates displayed evidence of clonal structure, the genetic diversity indices suggest a potential for the species to adapt to different environmental conditions.

Tolerance to UV-B radiation of the entomopathogenic fungus Metarhizium rileyi.

Soybean, corn, and cotton crops are afflicted by several noctuid pests and the development of bioinsecticides could help control these pests. The fungus Metarhizium rileyi has the greatest potential because its epizootics decimate caterpillar populations in the absence of insecticide applications. However, insect-pathogenic fungi when used for insect control in agriculture have low survival mainly due to the deleterious effects of ultraviolet radiation and heat from solar radiation. In this study, fourteen isolates of M. rileyi were studied and compared with isolates ARSEF 324 and ARSEF 2575 of Metarhizium acridum and Metarhizium robertsii, respectively, whose sensitivity to UV-B radiation had previously been studied. Conidia were exposed at room temperature (ca. 26 °C) to 847.90 mWm-2 of Quaite-weighted UV-B using two fluorescent lamps. The plates containing the conidial suspensions were irradiated for 1, 2, and 3 h, providing doses of 3.05, 6.10, and 9.16 kJ m2, respectively. A wide variability in conidial UV-B tolerance was found among the fourteen isolates of M. rileyi. Isolate CNPSo-Mr 150 was the most tolerant isolate (germination above 80% after 2 h exposure), which was comparable to ARSEF 324 (germination above 90% after 2 h exposure), the most tolerant Metarhizium species. The least tolerant isolates were CNPSo-Mr 141, CNPSo-Mr 142, CNPSo-Mr 156, and CNPSo-Mr 597. Nine M. rileyi isolates exhibited similar tolerance to UV-B radiation as ARSEF 2575 (germination above 50% after 2 h exposure). In conclusion, the majority of M. rileyi isolates studied can endure 1 or 2 h of UV-B radiation exposure. However, after 3 h of exposure, the germination of all studied isolates reduced below 40%, except for CNPSo-Mr 150 and ARSEF 324.

Characterization and genomic analyses of a novel alphabaculovirus isolated from the black armyworm, Spodoptera cosmioides (Lepidoptera: Noctuidae).

The black armyworm Spodoptera cosmioides is a pest of increasing importance in Cry1Ac-Bt toxin crops and non-Bt crops of soybean and cotton in Brazil. Here we characterized a baculovirus isolated from extracts of S. cosmioides that died with symptoms of nuclear polyhedrosis. The putative novel virus exhibited polyhedral occlusion bodies (OBs) with virions containing multiple rod-shaped nucleocapsids, characteristic of alphabaculoviruses. The virus isolate was named Spodoptera cosmioides nucleopolyhedrovirus isolate CNPSo-72 (SpcoNPV-CNPSo-72). SpcoNPV-CNPSo-72 was lethal to third-instar S. cosmioides caterpillars but not to S. frugiperda under the tested viral concentrations. Moreover, SpcoNPV-CNPSo-72 contained a circular 147,763 bp long genome and a G + C content of 44.8% with 151 annotated ORFs (10 unique for baculovirus) and five homologous regions (hrs). The 38 currently defined baculovirus core genes were found in the SpcoNPV-CNPSo-72 genome. After phylogenetic analysis, the novel virus was found to be closely related to other members of Alphabaculovirus, especially to the Spodoptera-infecting viruses, which included Spodoptera eridania nucleopolyhedrovirus isolate 251, Spodoptera litura nucleopolyhedrovirus isolate II, Spodoptera exigua multiple nucleopolyhedrovirus isolate US-1, Spodoptera eridania nucleopolyhedrovirus isolate CNPSo-165, and Spodoptera frugiperda multiple nucleopolyhedrovirus isolate 19. Surprisingly, the new baculoviral genome was found to code for a putative arginine-associated tRNA gene with a predicted intronic sequence of 105 nt. The gene was found inside the bjdp CDS. Overall, baculoviruses are pathogens that lethally infect insect larvae and their study allows a better understanding of large DNA virus evolution, which provides important insights for the development and improvement of biological control agents.

Helicoverpa armigera and Helicoverpa zea hybridization: constraints, heterosis, and implications for pest management.

BACKGROUND: The invasion of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) into the New World has made it possible for this pest to hybridize with a native American species, H. zea (Boddie), under natural conditions. We investigated the viability and development of hybrids of these two Helicoverpa species. We reared the parental species and evaluated crosses between H. armigera males and H. zea females and vice versa, two intercrosses between hybrids, and eight backcrosses between hybrids and parental species. We estimated the length of immature stages, fecundity, survival, sex ratio, and heterosis. RESULTS: Although hybridization occcurred, with heterosis during the development of immatures, reproductive incompatibilities also were observed between the parental species and between hybrids from subsequent crosses. The interspecific crosses between hybrids and backcrosses confirmed the possibility of introgression events and their perpetuation in field populations. The results indicate that hybridization events are favored at high population levels, while at low population levels the 'species identities' will be maintained. CONCLUSIONS: The possibility of interspecific gene flow and its perpetuation through successive crosses and backcrosses suggests several recommenations for management. Populations of both species should be maintained at an equilibrium level to reduce the chance of interspecific crosses, which are presumably more likely to occur during pest outbreaks. The existence of hybridization and resistance to different active pesticide ingredients should be monitored. All practices related to managing the resistance of these pests to chemical and biological insecticides should be systematized to reduce the chance of selecting for resistant individuals.

Resistance status of lepidopteran soybean pests following large-scale use of MON 87701 × MON 89788 soybean in Brazil.

Widespread adoption of MON 87701 × MON 89788 soybean, expressing Cry1Ac Bt protein and glyphosate tolerance, has been observed in Brazil. A proactive program was implemented to phenotypically and genotypically monitor Cry1Ac resistance in Chrysodeixis includens (Walker). Recent cases of unexpected injury in MON 87701 × MON 89788 soybean were investigated and a large-scale sampling of larvae on commercial soybean fields was performed to assess the efficacy of this technology and the distribution of lepidopteran pests in Brazil. No significant shift in C. includens susceptibility to Cry1Ac was observed eight years after commercial introduction of this technology in Brazil. F2 screen results confirmed that the frequency of Cry1Ac resistance alleles remains low and stable in C. includens. Unexpected injury caused by Rachiplusia nu (Guenée) and Crocidosema aporema (Walsingham) in MON 87701 × MON 89788 soybean was detected during the 2020/21 season, and studies confirmed a genetically based alteration in their susceptibility to Cry1Ac. MON 87701 × MON 89788 soybean remains effective against Anticarsia gemmatalis (Hübner), C. includens, Chloridea virescents (Fabricius) and Helicoverpa armigera (Hübner) in Brazil. However, there is evidence of field-evolved resistance to MON 87701 × MON 89788 soybean by the secondary soybean pests R. nu and C. aporema.

Characterization of a novel alphabaculovirus isolated from the Southern armyworm, Spodoptera eridania (Cramer, 1782) (Lepidoptera: Noctuidae) and the evolution of odv-e66, a bacterium-acquired baculoviral chondroitinase gene.

The Southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae) is native to the American tropics and a polyphagous pest of several crops. Here we characterized a novel alphabaculovirus isolated from S. eridania, isolate Spodoptera eridania nucleopolyhedrivurus CNPSo-165 (SperNPV-CNPSo-165). SperNPV-CNPSo-165 occlusion bodies were found to be polyhedral and to contain virions with multiple nucleocapsids. The virus was lethal to S. eridania and S. albula but not to S. frugiperda. The SperNPV-CNPSo-165 genome was 137.373 bp in size with a G + C content of 42.8%. We annotated 151 ORFs with 16 ORFs unique among baculoviruses. Phylogenetic inference indicated that this virus was closely related to the most recent common ancestor of other Spodoptera-isolated viruses.

Genome Sequence of Metarhizium rileyi, a Microbial Control Agent for Lepidoptera.

Metarhizium rileyi (formerly known as Nomuraea rileyi) is a potential agent for microbial control of many insect pests from the order Lepidoptera, the damages of which can cause considerable loss of productivity in agriculture. We report the genome sequence and annotation of M. rileyi strain Cep018-CH2/ARSEF 7053.

The complete genome of Rachiplusia nu nucleopolyhedrovirus (RanuNPV) and the identification of a baculoviral CPD-photolyase homolog.

We described a novel baculovirus isolated from the polyphagous insect pest Rachiplusia nu. The virus presented pyramidal-shaped occlusion bodies (OBs) with singly-embed nucleocapsids and a dose mortality response of 6.9 × 103 OBs/ml to third-instar larvae of R. nu. The virus genome is 128,587 bp long with a G + C content of 37.9% and 134 predicted ORFs. The virus is an alphabaculovirus closely related to Trichoplusia ni single nucleopolyhedrovirus, Chrysodeixis chalcites nucleopolyhedrovirus, and Chrysodeixis includens single nucleopolyhedrovirus and may constitute a new species. Surprisingly, we found co-evolution among the related viruses and their hosts at species level. Besides, auxiliary genes with homologs in other baculoviruses were found, e.g. a CPD-photolyase. The gene seemed to be result of a single event of horizontal transfer from lepidopterans to alphabaculovirus, followed by a transference from alpha to betabaculovirus. The predicted protein appears to be an active enzyme that ensures likely DNA protection from sunlight.

A Novel Betabaculovirus Isolated from the Monocot Pest Mocis latipes (Lepidoptera: Noctuidae) and the Evolution of Multiple-Copy Genes.

In this report, we described the genome of a novel baculovirus isolated from the monocot insect pest Mocis latipes, the striped grass looper. The genome has 134,272 bp in length with a G + C content of 38.3%. Based on the concatenated sequence of the 38 baculovirus core genes, we found that the virus is a betabaculovirus closely related to the noctuid-infecting betabaculoviruses including Pseudaletia unipuncta granulovirus (PsunGV), Trichoplusia ni granulovirus (TnGV), Helicoverpa armigera granulovirus (HearGV), and Xestia c-nigrum granulovirus (XecnGV). The virus may constitute a new Betabaculovirus species tentatively named Mocis latipes granulovirus (MolaGV). After gene content analysis, five open reading frames (ORFs) were found to be unique to MolaGV and several auxiliary genes were found including iap-3, iap-5, bro-a, bro-b, and three enhancins. The virus genome lacked both chitinase and cathepsin. We then looked at the evolutionary history of the enhancin gene and found that betabaculovirus acquired this gene from an alphabaculovirus followed by several duplication events. Gene duplication also happened to an endonuclease-like gene. Genomic and gene content analyses revealed both a strict collinearity and gene expansion into the genome of the MolaGV-related species. We also characterized the granulin gene using a recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and found that occlusion bodies were produced into the nucleus of infected cells and presented a polyhedral shape and no occluded virions within. Overall, betabaculovirus genome sequencing is of importance to the field as few genomes are publicly accessible. Mocislatipes is a secondary pest of maize, rice, and wheat crops in Brazil. Certainly, both the discovery and description of novel baculoviruses may lead to development of greener and safer pesticides in order to counteract and effectively control crop damage-causing insect populations.

The complete genome sequence of the first hesperiid-infecting alphabaculovirus isolated from the leguminous pest Urbanus proteus (Lepidoptera: Hesperiidae).

Baculoviruses are insect viruses largely used as expression vectors and biopesticides. These viruses can efficiently infect the larval stage of several agricultural pests worldwide causing a lethal disease. In this work, we found a novel baculovirus isolated from the larval stage of Urbanus proteus (L.), the bean leafroller and characterized its complete genome. This is an important pest of several leguminous plants in Brazil and belongs to the butterfly family Hesperiidae, from where no baculovirus genome sequence has been described. This new virus was shown to have the smallest genome among all alphabaculoviruses sequenced to date, with 105,555 bp and 119 putative ORFs. We found ten unique genes, seven bro, and the 38 baculovirus core genes. UrprNPV was found to be related to the Adoxophyes-infecting baculoviruses AdorNPV and AdhoNPV with high genetic distance and a long branch length. Interestingly, few individual core gene-based phylogenies were found to support the relationship of UrprNPV to both AdorNPV and AdhoNPV. Importantly, the increase in number of completely sequenced baculovirus points to a very exciting way to understand baculovirus and its evolution and could potentially help the use of baculovirus as both biopesticides and expression vectors.

Species From the Heliothinae Complex (Lepidoptera: Noctuidae) in Tucumán, Argentina, an Update of Geographical Distribution of Helicoverpa armigera.

The Heliothinae complex in Argentina encompasses Helicoverpa gelotopoeon (Dyar), Helicoverpa zea (Boddie), Helicoverpa armigera (Hübner), and Chloridea virescens (Fabricius). In Tucumán, the native species H. gelotopoeon is one of the most voracious soybean pests and also affects cotton and chickpea, even more in soybean-chickpea succession cropping systems. Differentiation of the Heliothinae complex in the egg, larva, and pupa stages is difficult. Therefore, the observation of the adult wing pattern design and male genitalia is useful to differentiate species. The objective of this study was to identify the species of the Heliothinae complex, determine population fluctuations of the Heliothinae complex in soybean and chickpea crops using male moths collected in pheromone traps in Tucuman province, and update the geographical distribution of H. armigera in Argentina. The species found were H. gelotopoeon, H. armigera, H. zea, and C. virescens. Regardless of province, county, crop, and year, the predominant species was H. gelotopoeon Considering the population dynamics of H. gelotopoeon and H. armigera in chickpea and soybean crops, H. gelotopoeon was the most abundant species in both crops, in all years sampled, and the differences registered were significant. On the other hand, according to the Sistema Nacional Argentino de Vigilancia y Monitoreo de Plagas (SINAVIMO) database and our collections, H. armigera was recorded in eight provinces and 20 counties of Argentina, and its larvae were found on soybean, chickpea, sunflower crops and spiny plumeless thistle (Carduus acanthoides). This is the first report of H. armigera in sunflower and spiny plumeless thistle in Argentina.

Genome sequence of Perigonia lusca single nucleopolyhedrovirus: insights into the evolution of a nucleotide metabolism enzyme in the family Baculoviridae.

The genome of a novel group II alphabaculovirus, Perigonia lusca single nucleopolyhedrovirus (PeluSNPV), was sequenced and shown to contain 132,831 bp with 145 putative ORFs (open reading frames) of at least 50 amino acids. An interesting feature of this novel genome was the presence of a putative nucleotide metabolism enzyme-encoding gene (pelu112). The pelu112 gene was predicted to encode a fusion of thymidylate kinase (tmk) and dUTP diphosphatase (dut). Phylogenetic analysis indicated that baculoviruses have independently acquired tmk and dut several times during their evolution. Two homologs of the tmk-dut fusion gene were separately introduced into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, which lacks tmk and dut. The recombinant baculoviruses produced viral DNA, virus progeny, and some viral proteins earlier during in vitro infection and the yields of viral occlusion bodies were increased 2.5-fold when compared to the parental virus. Interestingly, both enzymes appear to retain their active sites, based on separate modeling using previously solved crystal structures. We suggest that the retention of these tmk-dut fusion genes by certain baculoviruses could be related to accelerating virus replication and to protecting the virus genome from deleterious mutation.

A betabaculovirus encoding a gp64 homolog.

BACKGROUND: A betabaculovirus (DisaGV) was isolated from Diatraea saccharalis (Lepidoptera: Crambidae), one of the most important insect pests of the sugarcane and other monocot cultures in Brazil. RESULTS: The complete genome sequence of DisaGV was determined using the 454-pyrosequencing method. The genome was 98,392 bp long, which makes it the smallest lepidopteran-infecting baculovirus sequenced to date. It had a G + C content of 29.7% encoding 125 putative open reading frames (ORF). All the 37 baculovirus core genes and a set of 19 betabaculovirus-specific genes were found. A group of 13 putative genes was not found in any other baculovirus genome sequenced so far. A phylogenetic analysis indicated that DisaGV is a member of Betabaculovirus genus and that it is a sister group to a cluster formed by ChocGV, ErelGV, PiraGV isolates, ClanGV, CaLGV, CpGV, CrleGV, AdorGV, PhopGV and EpapGV. Surprisingly, we found in the DisaGV genome a G protein-coupled receptor related to lepidopteran and other insect virus genes and a gp64 homolog, which is likely a product of horizontal gene transfer from Group 1 alphabaculoviruses. CONCLUSION: DisaGV represents a distinct lineage of the genus Betabaculovirus. It is closely related to the CpGV-related group and presents the smallest genome in size so far. Remarkably, we found a homolog of gp64, which was reported solely in group 1 alphabaculovirus genomes so far.

High susceptibility and low resistance allele frequency of Chrysodeixis includens (Lepidoptera: Noctuidae) field populations to Cry1Ac in Brazil.

BACKGROUND: The soybean looper (SBL), Chrysodeixis includens (Walker), is one of the most important soybean pests in Brazil. MON 87701 × MON 89788 soybean expressing Cry1Ac has been recently deployed in Brazil, providing high levels of control against the primary lepidopteran pests. To support insect resistance management (IRM) programmes, the baseline susceptibility of SBL to Cry1Ac was assessed, and the resistance allele frequency was estimated on the basis of an F2 screen. RESULTS: The toxicity (LC50 ) of Cry1Ac ranged from 0.39 to 2.01 µg mL(-1) diet among all SBL field populations collected from crop seasons 2008/09 to 2012/13, which indicated approximately fivefold variation. Cry1Ac diagnostic concentrations of 5.6 and 18 µg mL(-1) diet were established for monitoring purposes, and no shift in mortality was observed. A total of 626 F2 family lines derived from SBL collected from locations across Brazil during crop season 2014/15 were screened for the presence of Cry1Ac resistance alleles. None of the 626 families survived on MON 87701 × MON 89788 soybean leaf tissue (joint frequency 0.0004). CONCLUSIONS: SBL showed high susceptibility and low resistance allele frequency to Cry1Ac across the main soybean-producing regions in Brazil. These findings meet important criteria for effective IRM strategy. © 2015 Society of Chemical Industry.

Characterization and phylogeny of Isaria spp. strains (Ascomycota: Hypocreales) using ITS1-5.8S-ITS2 and elongation factor 1-alpha sequences.

The elongation factor 1-alpha (EF1-α) and the internal transcribed spacer (ITS) regions ITS1 and ITS2 (ITS1-5.8S-ITS2) sequences were used to characterize and to identify Isaria isolates from Argentina, Mexico, and Brazil, as well as to study the phylogenetic relationships among these isolates and other related fungi from the order Hypocreales. The molecular characterization, which was performed by PCR-RFLP of EF1-α and ITS1-5.8-ITS2 genes, was useful for resolving representative isolates of Isaria fumosorosea, Isaria farinosa, and Isaria tenuipes and to confirm the taxonomic identity of fungi from Argentina, Mexico, and Brazil. The phylogenetic analyses showed three clades corresponding to three families of Hypocreales. The genus Isaria was confirmed as polyphyletic and in family Cordycipitaceae, Isaria species were related to anamorphic species of Beauveria, Lecanicillium, and Simplicillium and to teleomorphic Cordyceps and Torrubiella. Therefore, EF1-α and ITS1-5.8S-ITS2 genes were found to be powerful tools for improving the characterization, identification, and phylogenetic relationship of the Isaria species and other entomopathogenic fungi.

Biotic potential, fertility and life table of Spodoptera albula (Walker) (Lepidoptera: Noctuidae), under controlled conditions.

This study aimed to evaluate the biotic potential, life table parameters and fertility of Spodoptera albula (Walker, 1857) under controlled conditions (25 ± 1°C, 70 ± 10% RH and 14 hour photo phase). The longevity, pre, post and oviposition periods, fecundity and fertility of 13 couples were evaluated. The longevity of females (13.500 days) was significantly higher than those of males (11.154 days). The mean durations of the pre, post and oviposition periods were 2.615, 1.769 and 9.385 days, respectively. The mean fecundity was 1.417.69 eggs and mean fertility was 1.340.401 larvae, per female. On average, females copulated 1, 231 times. A strong positive correlation was observed between the number of copulations and fecundity (r = 0.847, p <0.001), as well as a strong negative correlation between the number of copulations and the duration of the pre-oviposition period (r = -0.762, p = 0.002), and longevity (r = -0.788, p = 0.001). The biotic potential of S. albula was estimated at 8.768 x 1022 individuals / female / year. The net reproductive rate (Ro) was 353,904 times per generation and the mean generation time (T) was 37.187 days. The intrinsic rate of increase (rm) was 1,105, with a finite rate of increase (λ) of 3,019.

Immature stages of Spodoptera albula (Walker) (Lepidoptera: Noctuidae): developmental parameters and host plants.

This study aimed to detail the temporal and morphological parameters of the immature stages of Spodoptera albula (Walker 1857) under controlled conditions (25 ± 1°C, 70 ± 10% RH and 14 hour photophase) and to gather information about their larval host plants. For this purpose, a new rearing method and artificial diet was employed and validated. The viability of the egg, larval, pupal and pre-pupal stages was 94.54, 97.33, 93.84 and 92.34%, respectively. The average duration of the egg, larval, pupal and pre-pupal stages was 4.14, 16.37, 1.69, and 9.34 days, respectively. During the larval stage, 80.85% of females and 93.99% of males passed through six and remaining through seven instars, with significant larval protandry. The larvae that developed through six and seven instars exhibited a mean growth rate of 1.58 and 1.48, respectively. Fifty five host plant species belonging to 29 families are listed. The female pupae were significantly larger, exhibiting protogyny. Both the rearing methods as well as the larval diet proved adequate, providing more detailled observations of the biological cycle, especially the larval stage, and resulting in an overall survival of almost 80%.

An overview of arthropod-associated fungi from Argentina and Brazil.

Arthropod pests in forest and agricultural systems are affected by many pathogenic organisms. Among them, entomopathogenic fungi are the one most common control agents that regulate their populations. This review compiles the information available from Argentina and Brazil about the entomopathogenic fungi occurring in agricultural and natural environments. The scientific names of the fungi are listed according to the latest phylogenetically based classification of fungi. We present an updated list of arthropod-pathogenic fungi occurring in 15 of the 23 provinces of Argentina and 20 of the 27 states of Brazil based on published literature and our personal observations. The list includes a total of 114 fungal species from 53 genera: of Blastocladiomycetes (2 genera), Entomophthorales (8 genera), Harpellales (13 genera), and a diverse assortment of ascomycetes (primarily from Hypocreales) in 22 anamorphic and 5 teleomorphic genera. In the both countries, molecular studies on arthropod-pathogenic fungi are still in their early stages and have focused primarily on intraspecific variability and adequate generic assignment. This listing seeks to encourage more active collection and characterization of these fungi by both traditional and molecular approaches from the obviously rich but underexplored flora of fungi affecting arthropods throughout this large region of South America.

Insecticide susceptibility of Euschistus heros (Heteroptera: Pentatomidae) in Brazil.

Euschistus heros (F.) (Heteroptera: Pentatomidae) is the most prevalent stink bug pest in Brazil, and populations can be difficult to manage using organophosphates or endosulfan. Because E. heros is difficult to rear in the laboratory, no baseline insecticide susceptibility data have been published. Therefore, we conducted dose-mortality studies using field-collected insects, and, using the results from the dose-mortality studies, we surveyed susceptibility to acephate, methamidophos, and endosulfan in populations from southern and central Brazil. In addition, esterase activity was evaluated among populations as these enzymes have been related to organophosphate resistance. Finally, newer chemistries were evaluated for E. heros population management in small-plot field studies. In the dose-mortality bioassays, variation in susceptibility to methamidophos and endosulfan among populations was significant, but the variation in susceptibility to acephate was not. The population from Londrina (at the Embrapa Soybean Research Station, northern Parana state) was among the most susceptible, whereas the population from Pedrinhas Paulista (southwestern Sao Paulo state) was among the least susceptible. Significant variation among populations was observed in esterase activity with the population from Pedrinhas Paulista having the highest esterase levels and the population from Londrina having among the lowest levels. To survey populations, we used the dose-mortality data for the Londrina population (because it was, consistently, one of the most susceptible populations) to estimate the insecticide concentration that killed 99% of the population (LC99). The corresponding doses were 5.9 microg (AI) of acephate adult(-1), 0.7 microg (AI) methamidophos adult(-1), and 1,600 microg (AI) endosulfan adult(-1). The survey data confirmed the data from the dose-mortality bioassays in that the population from Pedrinhas Paulista was identified as one of the least susceptible and the population from Londrina was one of the most susceptible. The field tests confirmed that older chemistries provided minimal control (< 50% control), whereas the products containing mixtures of pyrethroids and neonicotinoids provided better control (> 60%).

Variability of the mitochondrial SSU rDNA of Nomuraea species and other entomopathogenic fungi from hypocreales.

Hypocrealean arthropod pathogenic fungi have profound impact on the regulation of agricultural and medical pests. However, until now the genetic and phylogenetic relationships among species have not been clarified, such studies could clarify host specificity relationships and define species boundaries. Our purpose was to compare the sequences of the mitochondrial SSU rDNA fragments from several mitosporic entomopathogenic Hypocreales to infer relationships among them and to evaluate the possibility to use these sequences as species diagnostic tool in addition to the more commonly studied sequences of nuclear SSU rDNA. The SSU mt-rDNA proved to be useful to help in differentiation of species inside several genera. Clusters obtained with Parsimony, Bayesian, and Maximum Likelihood analyses were congruent with a new classification of the Clavicipitaceae (Sung et al. Stud Mycol. 2007;57:5-59) in which the anamorphic genera Nomuraea and Metarhizium species remain in the Clavicipitaceae and Isaria species sequenced here are assigned to the family Cordycipitaceae. Mitochondrial genomic information indicates the same general pattern of relationships demonstrated by nuclear gene sequences.