Combined application of entomopathogenic fungi and predatory mites for biological control of Tetranychus urticae on chrysanthemum.

BACKGROUND: Most studies on efficacy of fungal pathogens and predatory mites against Tetranychus urticae have been done on individual species in the laboratory. We evaluated fungi and predatory mites separately and together against glasshouse populations of T. urticae on chrysanthemum plants. First, effectiveness of the fungal pathogens Beauveria bassiana (Bb88) and Metarhizium anisopliae (Ma129) was compared; then, effectiveness of the predatory mites Phytoseiulus persimilis and Neoseiulus californicus. Based on the results, N. californicus and isolate Ma129 were selected and evaluated in combination. In all experiments, treatment effects were assessed for eggs and motile stages of T. urticae. RESULTS: The first experiment showed no significant effect of either fungal isolate on T. urticae populations, except on plants initially infested with 20 mites, where more eggs were found in the control compared to the fungal treatments. In the second experiment, both predatory mites were equally effective at reducing T. urticae populations compared with the control, regardless of initial T. urticae population density. The last experiment demonstrated that populations of T. urticae were reduced most when M. anisopliae (Ma129) and N. californicus were applied together, compared with the control and when each natural enemy was applied separately. CONCLUSIONS: Metarhizium anisopliae (Ma129) and B. bassiana (Bb88) isolates did not have a significant effect on reducing T. urticae populations. Both predatory mites reduced T. urticae populations, regardless of T. urticae density. Combined application of M. anisopliae (Ma129) and N. californicus were more effective against T. urticae than the control or when each agent was applied separately. © 2024 Society of Chemical Industry.

Thirty-minute test to detect insecticide resistance under field conditions: the case of the Asian citrus psyllid.

BACKGROUND: Diaphorina citri Kuwayama is one of the most destructive citrus pests worldwide. It is controlled mainly through applications of conventional insecticides. Methodologies used to estimate resistance to insecticides do not correlate with field efficacy, and do not provide timely and reliable information to make decisions at a site where spraying is needed. The use of diagnostic doses with 30-min exposure is proposed for estimating the resistance of D. citri to imidacloprid, spinosad, malathion and chlorpyrifos at the orchard level. RESULTS: Under laboratory conditions, we estimated the lowest doses that caused 100% mortality within 30 min of exposure (diagnostic dose) in a susceptible D. citri colony. The diagnostic doses for imidacloprid, spinosad, malathion and chlorpyrifos were 7.4, 4.2, 1.0 and 5.5 mg a.i. L-1 , respectively. Under field conditions, we applied the diagnostic doses to D. citri feeding on Citrus aurantifolia Swingle at five localities in Michoacan state, Mexico (Nueva Italia, Santo Domingo, El Varal, Gambara and El Ceñidor). Additionally, the field efficacy of these insecticides against these populations was evaluated. A significant correlation between field efficacy and mortality was observed with the diagnostic doses for imidacloprid, malathion and chlorpyrifos (R2 ≥ 0.93). The correlation for spinosad could not be estimated because the mortality caused by the diagnostic dose and its field efficacy at all study sites was consistently >98%. CONCLUSIONS: Field efficacy and resistance were estimated based on the field diagnostic doses with 30-min exposure for all tested insecticides. Consequently, growers and pest management technicians can estimate the performance of the evaluated insecticides at the orchard level and before insecticide application. © 2023 Society of Chemical Industry.

Berry species and crop management approaches affect species diversity and abundance of predatory mites (Acari: Phytoseiidae).

Berries comprise an economically important group of crops. Knowledge about their arthropod pests and biological control agents is important in the development of more efficient integrated pest management programs. Identification of potential biocontrol agents based solely on morphological attributes may be difficult and so molecular techniques should be incorporated. Here we studied the species diversity of predatory mites in the family Phytoseiidae, and how this diversity is affected by the berry species and crop management approaches, specifically pesticide application regimes. We sampled 15 orchards in the State of Michoacán, Mexico. Sites were selected based on berry species and pesticide regimes. Mite identification was achieved by combining morphological attributes and molecular techniques. Phytoseiidae diversity was compared amongst blackberry, raspberry and blueberry. Subsequently we studied the effect of berry species and pesticide regime on the abundance of the most prevalent phytoseiid species. We identified 11 species of phytoseiid mites. The greatest species diversity was found in raspberry, followed by blackberry and then blueberry. The most abundant species were Typhlodromalus peregrinus and Neoseiulus californicus. The abundance of T. peregrinus was significantly affected by pesticide application but not by berry species. In contrast, abundance of N. californicus was significantly affected by berry species but not by pesticide regime.

Incidence of Citrus leprosis virus C and Orchid fleck dichorhavirus Citrus Strain in Mites of the Genus Brevipalpus in Mexico.

The incidences of Citrus leprosis virus C (CiLV-C) and Orchid fleck dichorhavirus Citrus strain (OFV-citrus) were determined in field populations of Brevipalpus mites from 15 citrus-producing states in Mexico. Mites were collected from orange, grapefruit, mandarin, lime, and sweet lime orchards. Brevipalpus yothersi (Baker) (Trombidiformes: Tenuipalpidae) was the most abundant species followed by Brevipalpus californicus (Banks) (Trombidiformes: Tenuipalpidae), which confirmed previous reports. The viruses CiLV-C and OFV-citrus were found in both mite species. The incidence of CiLV-C, OFV-citrus and both viruses simultaneously (CiLV-C and OFV-citrus) was 17.2, 10.3, and 3.4% (n = 116) for B. yothersi, and 12.5, 20.8, and 4.1% (n = 24) for B. californicus, respectively. No significant difference was found when the incidence of these viruses was compared between both mite species. The importance of our results in relation to the epidemiology of leprosis is discussed.

Differential Susceptibility of Bagrada hilaris (Hemiptera: Pentatomidae) to Different Species of Fungal Pathogens.

Bagrada hilaris Burmeister (Hemiptera: Pentatomidae) is a pest of Palearctic origin. Its presence in the United States was first reported in 2008 and in Mexico in 2014; it affects brassica crops. There are practically no reports of natural enemies of B. hilaris in America. Entomopathogenic fungi are strong candidates for microbial control of this pest. Evaluating the susceptibility of this pest to fungi that are native to the region where they will be used is a sensible first step to finding candidate biological control agents. The aim of our research was to select potential microbial agents to control B. hilaris. Eleven isolates of Beauveria bassiana, Beauveria pseudobassiana, Metarhizium anisopliae, and Isaria fumosorosea were evaluated to determine the susceptibility of B. hilaris. Isolates of B. bassiana caused the highest mortality due to infection (100%) compared with the other isolates. The I. fumosorosea isolate caused the lowest percent mortality (56%). The two B. bassiana isolates Bb88 and AP3 were more virulent than M. anisopliae isolate Ma129. The sex of the insect had no effect on infection levels achieved by B. bassiana isolates Bb88 and AP3. The results of our study contribute valuable information for the development of fungal species with potential to manage B. hilaris populations. Field studies are the next step in order to develop these isolates as biological control agents of B. hilaris.

Transmission of Citrus leprosis virus C by the Mite, Brevipalpus yothersi (Acari: Tenuipalpidae), on Four Species of Citrus.

Transmission of the virus, Citrus leprosis virus C (CiLV-C) (Cilevirus) by Brevipalpus yothersi Baker, on different citrus species was evaluated under greenhouse conditions. First, the relationship between acquisition access periods (AAPs; 1, 12, 24, 36, and 48 h) and virus concentration in mites was determined. Second, the ability of B. yothersi to transmit CiLV-C to orange, mandarin, grapefruit, and lime trees was measured. We then assessed the establishment of mites on the different citrus species as measured by their population increase on each species. We found no relationship between AAPs and virus load in mites. The virus was found in all mites tested but there was no difference in virus quantities among the treatments. We selected an AAP of 24 h for the transmission experiment. Brevipalpus yothersi transmitted the virus to all citrus species evaluated, but susceptibility was different. The number of infected leaves was greater on orange and mandarin compared with grapefruit and lime. Furthermore, populations of B. yothersi successfully established on orange and mandarin, but not on grapefruit and lime trees. The implications of our results in the virus-mite-citrus plant relationship are discussed.

Interaction of Beauveria bassiana and Metarhizium anisopliae with chlorpyrifos ethyl and spinosad in Spodoptera frugiperda larvae.

BACKGROUND: The high natural resistance of the fall armyworm (FAW) to entomopathogenic fungi and the speed at which it develops resistance to chemical insecticides make it difficult to control in several crops where it is a key pest. The aim of the study was to improve mortality by combining a 50% lethal concentration of chlorpyrifos ethyl or spinosad with the entomopathogenic fungus Beauveria bassiana (Bb88) or Metarhizium anisopliae (ETL). RESULTS: Three main results were obtained. (i) Both insecticides improved the performance of the fungus when the insecticide and fungus were applied simultaneously and when spinosad preceded fungi. In control larvae treated with fungi, <1% of larvae showed fungal sporulation, but under simultaneous application of the fungus with chlorpyrifos or spinosad, 31 and 47% did so, with 68 and 93% of cadavers showing sporulation, respectively. (ii) Synergistic mortality was observed when Bb88 and spinosad were applied simultaneously, which resulted in 34% more dead larvae than the spinosad control (44%). Finally, (iii) antagonism occurred when Bb88 was applied before chlorpyrifos, when Bb88 and chlorpyrifos were applied simultaneously, and when ETL was applied before chlorpyrifos, which reduced larval mortality by 27, 31 and 19%, respectively. CONCLUSION: The synergistic mortality and improved fungal performance observed here support the hypothesis that combinations of low insecticide doses and entomopathogenic fungi can enhance integrated pest management programs and reduce the environmental impact of insecticides. Antagonism shows that some particular combinations and application sequences may produce disadvantages in pest control. © 2018 Society of Chemical Industry.

Species Diversity and Population Dynamics of Entomopathogenic Fungal Species in the Genus Metarhizium-a Spatiotemporal Study.

We studied the species diversity and population genetic structure of isolates of fungi from the entomopathogenic genus Metarhizium that had been isolated from sugarcane crops and surrounding grass. Soil and leaf samples were taken on four sampling occasions over 13 months (October 2014-October 2015). Isolations were made using the Galleria mellonella baiting method and selective media. Phylogenetic placement of isolates was done by sequencing a fragment of the 5' of the elongation factor 1-α gene (EF1-α). Population genetic structure was determined by analysing this sequence information using AMOVA and Haplotype network analyses. Genotypic diversity was studied using microsatellite genotyping. The most abundant species was M. anisopliae s.s. (80 isolates), then M. pingshaense (three isolates), and M. guizhouense (one isolate). More than 50% of the genetic variation was explained by the time the samples were collected regardless of plant host association. Some haplotypes were found on the first sampling date and then not found on subsequent sampling dates, while other haplotypes were found initially, disappeared, but then found again on the last sampling date. To the best of our knowledge, this is the first report of the population genetic structure of M. anisopliae species in time and space. The effect of abiotic factors is discussed.

Differential Host Plant-Associated Genetic Variation Between Sympatric Mite Species of the Genus Oligonychus (Acari: Tetranychidae).

Adaptation to different host plants can lead to host-associated differentiation (HAD). The mites Oligonychus perseae and Oligonychus punicae have a broad range of host plants, but, to date, records of them coexisting sympatrically had only been reported on avocado. However, our field observations showed both species coexisting on host plants other than avocado. The lack of previous records of these mites on the host plants studied here suggests only recent divergence to new host plant species. Previous studies showed that O. punicae had a limited migration capacity compared with O. perseae, suggesting that O. punicae is more likely to develop a close host plant relationship leading to HAD. Adults of both species were collected from trees hosting both mite species. Three genera of host plants considered were Persea, Salix, and Alnus; two species within one genus were Alnus jorullensis and Alnus acuminata; and three varieties within one species were Persea americana var. Fuerte, var. Hass, and var. Criollo, a noncommercial variety. Using sequence data from a segment of the mitochondrial cytochrome oxidase subunit I, the phylogenetic relationships and genetic population structure of both mite species in relation to the host plant were determined. Oligonychus perseae populations showed a significant population structure in relation to host plant at the species and genus level, but there was no effect of variety. In contrast, host plant explained none of the genetic variation among O. punicae populations. The potential role of coexistence mechanisms in the contrasting genetic population structure of both mite species is discussed.

Analysis of Genetic Variation in Brevipalpus yothersi (Acari: Tenuipalpidae) Populations from Four Species of Citrus Host Plants.

We studied species diversity and genetic variation among populations of Brevipalpus mites from four species of citrus host plants. We sampled mites on orange, lime, grapefruit and mandarin trees from orchards at six localities distributed in the five most important citrus producing states in Mexico. Genetic variation among citrus host plants and localities were assessed by analysis of nucleotide sequence data from fragments of the mitochondrial cytochrome oxidase subunit I (COI). Both Brevipalpus yothersi and B. californicus were found at these sites, and B. yothersi was the most abundant species found on all citrus species and in all localities sampled. B. californicus was found mainly on orange and mandarin and only in two of the states sampled. AMOVA and haplotype network analyses revealed no correlation between B. yothersi genetic population structure and geographical origin or citrus host plant species. Considering that a previous study reported greater genetic diversity in B. yothersi populations from Brazil than we observed in Mexico, we discuss the possibility that the Mexican populations may have originated in the southern region of America.

Specific diversity of the entomopathogenic fungi Beauveria and Metarhizium in Mexican agricultural soils.

Prior knowledge of the local population structure of entomopathogenic fungi is considered an important requisite when developing microbial control strategies against major pests of crops such as white grubs. An extensive survey in the estate of Guanajuato, one of the main agricultural regions of Mexico, was carried out to determine the abundance and diversity of entomopathogenic fungi in soil. Soil collected from 11 locations was baited for entomopathogenic fungi using Galleria mellonella. In addition, all isolates were morphologically identified and selected isolates of Beauveria and Metarhizium isolates identified using Bloc and ITS or Elongation Factor 1-α and ITS sequence information respectively. Genotypic diversity was then studied using microsatellite genotyping. The proportion of isolates belonging to each genus varied amongst all locations. The species Beauveria bassiana, B. pseudobassiana and Metarhizium robertsii were found, with B. bassiana being the most abundant and widely distributed. Microsatellite genotyping showed that the 36 B. bassiana isolates were grouped in 29 unique haplotypes, but with no separation according to geographical origin.

Competition between isolates of Zoophthora radicans co-infecting Plutella xylostella populations.

Interactions between Zoophthora radicans isolates were studied in vitro and in vivo during infection of Plutella xylostella larvae. We distinguished between isolates within infected hosts using PCR-RFLP. Isolates obtained from P. xylostella larvae (NW386 and NW250) were more virulent than isolates from other insect hosts. Isolate NW250 was most virulent at 27°C and isolate NW386 was most virulent at 22°C. In vitro growth of all isolates except NW386 was affected by the presence of other isolates. During in vivo interactions between NW250 and NW386, the isolate with the greatest conidial concentration at inoculation infected more larvae than its competitor. Dual infected larvae were only found in treatments where inoculation concentrations of conidia were high for both isolates. Where concentrations of conidia at inoculation were low for both isolates, only NW250 caused successful infection. The implications of these results for the ecology of Z. radicans are discussed.

Selection of a fungal isolate for the control of the pink hibiscus mealybug Maconellicoccus hirsutus.

BACKGROUND: Maconellicoccus hirsutus Green is a widely distributed pest of numerous crops. Although synthetic pesticides are used to control this pest, entomopathogenic fungi may provide an alternative control mechanism. Three experiments were carried out to select a fungal isolate with the potential to be used as a microbial control agent. The in vitro growth of five isolates of Beauveria bassiana sensu lato (Bals.) Vuill and Metarhizium anisopliae sensu lato (Metschn.) Sorokín, along with three isolates of Lecanicillium lecanii (Zimm.) Zare & W. Gams and Isaria fumosoroseus (Wize), was assessed at four temperatures. The in vivo sporulation of eight selected isolates was then evaluated, followed by the susceptibility of third-instar M. hirsutus to a single dose (1 × 10(8) conidia mL(-1) ) of each of these isolates. RESULTS: Growth was greatest by isolates of I. fumosoroseus and L. lecanii at 15 and 20 °C and by isolates of M. anisopliae at 25 and 30 °C. In vivo conidium production was greatest when infected with B. bassiana isolate GHA and M. anisopliae isolate Ma65. Mortality was greatest when inoculated with M. anisopliae isolates Ma65 and Ma129. CONCLUSION: Isolate Ma65 shows the best potential to be developed as a microbial control agent for M. hirsutus.

Diversity and genetic population structure of fungal pathogens infecting white grub larvae in agricultural soils.

White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and ß-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.

Use of quantitative PCR to understand within-host competition between two entomopathogenic fungi.

Interactions between the entomopathogenic fungi Zoophthora radicans and Pandora blunckii infecting larvae of Plutella xylostella were investigated. This is the first report to quantify within-host growth of one fungus in the presence of another competing for the same host resource using quantitative PCR (qPCR) at regular time points during the infection process. In larvae inoculated only with Z. radicans, there was a cumulative increase in the quantity of Z. radicans DNA throughout the time course of infection. However, in dual-inoculated larvae, there was an initial accelerated rate of growth of Z. radicans compared to when it was inoculated alone, but by the time of host death it had been effectively excluded by P. blunckii. The implications of these results for co-existence of these fungal pathogens in the field are discussed.

Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella.

The entomopathogenic fungi Zoophthora radicans and Pandora blunckii co-occur in field populations of Plutella xylostella and, therefore, are likely to interact during the infection process. We have investigated the possible outcomes of these interactions in the laboratory. Using four isolates, two of each fungal species, inter-specific interaction experiments were done in Petri dishes and on intact plants. In Petri dish experiments, larvae were inoculated directly using sporulating mats of mycelium, both species had the same opportunity to infect and only the relative concentration of conidia of each pathogen species applied was manipulated. In the intact plant experiments, larvae were placed onto fungus-contaminated plants, inoculation was passive and the probability of infection by either or both species of fungi depended on larval activity and proximity to inoculum. In the Petri dish experiment, the species with the largest concentration of conidia out-competed the other regardless of virulence, and results were similar in the intact plant experiment. The ecological implications for competition or co-existence of these two pathogens in the field are discussed.

Development of species-specific diagnostic primers for Zoophthora radicans and Pandora blunckii; two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella.

Species-specific primers for Zoophthora radicans and Pandora bluckii were developed. To achieve this, partial sequences of DNA that encode for rRNA, more specifically, the ITS region (rDNA-ITS) were obtained from different isolates and analysed. Seven Z. radicans isolates (four from P. xylostella, and three from other lepidopteran hosts) and one P. blunckii isolate (from P. xylostella) were used. These isolates were selected based on PCR-RFLP patterns obtained from 22 isolates of P. blunckii and 39 isolates of Z. radicans. All P. blunckii isolates were from the same host (P. xylostella); 20 isolates were from Mexico, one from the Philippines, and one from Germany. The Z. radicans isolates were more diverse in geographical origin (Mexico, Kenya, Japan, New Zealand, Australia, Taiwan, Philippines, Malaysia, Uruguay, France, USA, Poland, Indonesia, Switzerland, Israel, China, and Denmark) and host origin (Lepidoptera, Hemiptera, Hymentoptera, and Diptera). Using conventional PCR, each pair of species-specific primers successfully detected each species of fungus from DNA extracted from infected host larvae either single- or dual-inoculated with both fungal species. The PCR-RFLP analysis also showed that Z. radicans was genetically more diverse than P. blunckii, although only a limited number of P. blunckii isolates from one country were considered. There was no direct relationship between genetic diversity and host or geographical origin. The relationship between genetic variation within both fungal species and host specificity or ecological adaptation is discussed.