Spatiotemporal association between the mite Brevipalpus yothersi and Citrus leprosis virus C in orange orchards.

Citrus leprosis virus C (CiLV-C) is an economically important pathogen and the main causative agent of leprosis disease in citrus orchards. The main vector of this disease, the mite Brevipalpus yothersi, is widely distributed in Mexican orchards on a wide range of citrus species. Despite the importance of both the virus and the mite, field studies recording their occurrence and co-occurrence are practically non-existent. We systematically sampled orange orchards for both CiLV-C and B. yothersi throughout the year. The distribution of the CiLV-C and B. yothersi was evaluated on each sampling occasion and their spatiotemporal associations were determined. Specifically, 100-112 orange trees, distributed in 18 rows (five or six trees per row), were sampled monthly between March 2017 and February 2018 (11 sampling dates). Twenty leaves per tree were sampled on each occasion. The number of mites per tree and the percentage of leaves per tree with disease symptoms were recorded. On each sampling occasion, spatiotemporal associations between mites and disease were determined using the Spatial Analysis by Distance Indices (SADIE) method. CiLV-C and B. yothersi were identified using molecular methods. Throughout the study, the distribution of CiLV-C was aggregated and the distribution of B. yothersi was random. No association was found between the virus and the mite on any of the sampling dates. In total, 173 mites were collected, but only 43 mites were found to be carrying CiLV-C. The reason for this lack of association between the virus and the mite, as well as the impact of our findings on the epidemiology of the disease in orange orchards, are discussed.

Susceptibility of Diaphorina citri (Hemiptera: Liviidae) and Its Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) to Entomopathogenic Fungi under Laboratory Conditions.

Diaphorina citri (Kuwayama) is a global pest of citrus that transmits the bacteria associated with the disease, Huanglongbing. Entomopathogenic fungi and the parasitoid Tamarixia radiata (Waterston) are important biological control agents of this pest and likely to interact in D. citri populations. As a basis for interaction studies, we determined the susceptibility of nymphs and adults of D. citri and adults of the parasitoid T. radiata to six fungal isolates from the species Beauveria bassiana s.l. (Bals.-Criv.) Vuill. (isolates B1 and B3), Metarhizium anisopliae s.s. (Metsch.) (Ma129 and Ma65) and Isaria fumosorosea Wize (I2 and Pae). We conducted experiments evaluating infection levels in all three insect groups following inoculation with a series of conidial concentrations (1 × 104-1 × 108 conidia mL-1). Results showed that D. citri nymphs and T. radiata were more susceptible to fungal isolates than D. citri adults. Overall, B. bassiana and M. anisopliae isolates caused the greatest infection compared with I. fumosorosea isolates in all three groups of insects. Isolates B1 (B. bassiana) and Ma129 (M. anisopliae) infected a greater proportion of adults and nymphs of D. citri, respectively. Both isolates of B. bassiana caused greater infection in T. radiata compared with isolates of the other fungal species. We propose that isolates B1 and Ma129 are the strongest candidates for control of D. citri. Our results represent the first report of entomopathogenic fungi infecting T. radiata, and the basis for future studies to design a biological control programme that uses both agents more efficiently against D. citri populations.

Specific Diversity of Metarhizium Isolates Infecting Aeneolamia spp. (Hemiptera: Cercopidae) in Sugarcane Plantations.

Spittlebugs from the genus Aeneolamia are important pests of sugarcane. Although the use of the entomopathogenic fungus Metarhizum anisopliae s.l. for control of this pest is becoming more common in Mexico, fundamental information regarding M. anisopliae in sugarcane plantations is practically non-existent. Using phylogenetic analysis, we determined the specific diversity of Metarhizium spp. infecting adult spittlebugs in sugarcane plantations from four Mexican states. We obtained 29 isolates of M. anisopliae s.str. Haplotype network analysis revealed the existence of eight haplotypes. Eight selected isolates, representing the four Mexican states, were grown at different temperatures in vitro; isolates from Oaxaca achieved the greatest growth followed by isolates from Veracruz, San Luis Potosi and Tabasco. No relationship was found between in vitro growth and haplotype diversity. Our results represent a significant contribution to the better understanding of the ecology of Metarhizum spp. in the sugarcane agroecosystem.

Bacteria Present in Comadia redtenbacheri Larvae (Lepidoptera: Cossidae).

The external and internal culturable bacterial community present in the larvae of Comadia redtenbacheri Hammerschmidt, an edible insect, was studied. Characterization of the isolates determined the existence of 18 morphotypes and phylogenetic analysis of the 16S rRNA gene revealed the existence of Paenibacillus sp., Bacillus safensis, Pseudomonas sp., Bacillus pseudomycoides, Corynebacterium variabile, Enterococcus sp., Gordonia sp., Acinetobacter calcoaceticus, Arthrobacter sp., Micrococcus sp., and Bacillus cereus. Greater diversity of bacteria was found in those larvae obtained from vendors than in those directly taken from Agave plants in nature. Many of the larvae obtained from vendors presented signs of potential disease, and after the analysis, results showed a greater bacterial community compared with the larvae with a healthy appearance. This indicates that bacterial flora can vary in accordance with how the larvae are handled during extraction, collection, and transport.

Temporal Variation in Immune Components of the White Grub Phyllophaga polyphylla (Bates) (Coleoptera: Melolonthidae).

Ecological immunology assumes that the host immune efficiency is correlated with its survival after pathogen challenge. To test this hypothesis, we challenged Phyllophaga polyphylla (Bates) larvae with the naturally occurring fungus Metarhizium pingshaense on two consecutive years (2011 and 2012). In each year, we injected the blastospores of M. pingshaense and then used levels of prophenoloxidase (proPO), phenoloxidase (PO) and total haemolymph serum protein as indicators of immune efficiency. Larvae were injected with (1) phosphate buffered saline (PBS) + Tween and viable blastospores of M. pingshaense, (2) PBS + Tween and non-viable blastospores of M. pingshaense, (3) PBS + Tween, or (4) non-manipulated. Overall, levels of PO, proPO and total haemolymph serum protein in larvae after 12 h were similar amongst treatments within each year of collection. However, larvae collected in 2011 showed higher PO and proPO activity but lower total haemolymph serum protein compared with larvae collected in 2012. A survival study injecting viable blastospores showed that larvae collected in both years died within 48 h; however, when non-viable blastospores were injected, which were still toxic to larvae, mortality was greater in larvae collected in 2011 compared with larvae collected in 2012. Altogether, these results indicate that PO, proPO and total haemolymph serum protein do not predict immune strength of P. polyphylla against blastospores of M. pingshaense, but higher values of PO and proPO were correlated with higher survival rates against non-infective but toxic agents. The possible role of some abiotic factors over the differences observed for immune components of P. polyphylla in different years of collection is discussed.

Metarhizium pingshaense applied as a seed treatment induces fungal infection in larvae of the white grub Anomala cincta.

Metarhizium pingshaense has potential as a control agent of the white grub Anomala cincta. We compared its ability to cause infection when applied as a seed treatment or directly to the compost around the plant roots. Although the greatest infection (93%) occured in the direct inoculation treatment, 50% of larvae still became infected by M. pingshaense applied as a seed treatment. The fungus persisted in the compost and also colonised the roots of the developing maize plants endophytically. More research is required but seed treatments with M. pingshaense have potential as cost-effective control options for A. cincta.

Occurrence of entomopathogenic fungi in tejocote (Crataegus mexicana) orchard soils and their pathogenicity against Rhagoletis pomonella.

AIMS: To determine the abundance and diversity of entomopathogenic fungi in tejocote orchard soils and evaluate their ability to infect Rhagoletis pomonella Walsh., the main pest of tejocote. METHODS AND RESULTS: Surveys were made in two locations in Mexico state and two in Puebla state. Soil from selected locations was baited for entomopathogenic fungi with Galleria mellonella (L.). All isolates were identified morphologically to genus level and to species level using Bloc and elongation factor 1-α gene sequence information, respectively; Beauveria bassiana ((Bals.-Criv.) Vuill.), B. pseudobassiana (S.A. Rehner & Humber) and Metarhizium robertsii (J.F. Bisch., Rehner & Humber) were found, with B. bassiana being the most abundant and widely distributed. Pathogenicity of five selected B. bassiana isolates and three M. robertsii isolates was evaluated against larvae and pupae of R. pomonella. All isolates infected larvae resulting in an average mortality of 35%. Pupae were not susceptible; however, adults emerging from inoculated pupae did die due to infection. CONCLUSIONS: At least three species of entomopathogenic fungi are present in the soil from tejocote orchards, with B. bassiana being the most abundant and widely distributed. Rhagoletis pomonella larvae were more susceptible to infection than pupae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study has produced new information about the distribution of entomopathogenic fungi in cultivated soils from this region of North America, contributing to a better understanding of their natural occurrence and underpinning the development of biological control approaches.

Contrasting effects of geographical separation on the genetic population structure of sympatric species of mites in avocado orchards.

Oligonychus punicae and Oligonychus perseae (Acari: Tetranychidae) are the most important mite species affecting avocado orchards in Mexico. Here we used nucleotide sequence data from segments of the nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes to assess the phylogenetic relationships between both sympatric mite species and, using only ITS sequence data, examine genetic variation and population structure in both species, to test the hypothesis that, although both species co-occur, their genetic population structures are different in both Michoacan state (main producer) and Mexico state. Phylogenetic analysis showed a clear separation between both species using ITS and COI sequence information. Haplotype network analysis done on 24 samples of O. punicae revealed low genetic diversity with only three haplotypes found but a significant geographical population structure confirmed by analysis of molecular variance (AMOVA) and Kimura-2-parameter (K2P) analyses. In addition, a Mantel test revealed that geographical isolation was a factor responsible for the genetic differentiation. In contrast, analyses of 22 samples of O. perseae revealed high genetic diversity with 15 haplotypes found but no geographical structure confirmed by the AMOVA, K2P and Mantel test analyses. We have suggested that geographical separation is one of the most important factors driving genetic variation, but that it affected each species differently. The role of the ecology of these species on our results, and the importance of our findings in the development of monitoring and control strategies are discussed.

Order of inoculation affects the success of co-invading entomopathogenic fungi.

The effect of order of inoculation of Pandora blunckii and Zoophthora radicans co-infecting Plutella xylostella (L.) (Lepidoptera: Plutellidae) was investigated. After co-inoculation, the proportion of larvae infected by either species was greatly reduced compared to when they were inoculated singly. The order of inoculation influenced the final outcome; the isolate inoculated last always killed more larvae than the isolate inoculated first.

Susceptibility of the cactus weevil Metamasius spinolae to Beauveria bassiana and Metarhizium anisopliae under laboratory and field conditions.

AIMS: To evaluate different entomopathogenic fungal isolates against the cactus weevil Metamasius spinolae under laboratory and field conditions, and select an isolate to be used as a tool in the management of this insect pest. METHODS AND RESULTS: Four experiments were carried out. The effect of temperature on the in vitro growth of eight isolates of entomopathogenic fungi (two Metarhizium anisopliae and six Beauveria bassiana) was assessed. The susceptibility of adult M. spinolae to the same isolates was evaluated. Using three selected isolates, the interaction between susceptibility and sex of the insect was studied. Finally, a field experiment was carried out to evaluate infection of adult M. spinolae by the same three isolates under natural abiotic conditions. Overall, growth rate was greatest at 25°C for all the isolates. In vitro growth of M. anisopliae was greater than B. bassiana. Mortality of adult M. spinolae was greater when inoculated with B. bassiana compared with isolates of M. anisopliae. Susceptibility had no interaction with the sex of the insect. The proportion of insects succumbing to infection was smaller when incubated under field conditions than when incubated under laboratory conditions. CONCLUSIONS: The experiments described here showed a complex interaction between entomopathogenic fungi and M. spinolae, and these data allows us to select isolate Bb107 as a first step towards its use in the management of this pest insect. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results showed that entomopathogenic fungi can be used for the control of M. spinolae, which may help reduce the use of chemical insecticides and, therefore, the exposure of Opuntia ficus-indica producers to pesticides.