Evaluation of Spore Acquisition, Spore Production, and Host Survival Time for Tea Shot-Hole Borer, Euwallacea perbrevis, Adults after Exposure to Four Commercial Products Containing Beauveria bassiana.

Euwallacea perbrevis, the tea shot-hole borer (TSHB), is an invasive ambrosia beetle that vectors several fungal pathogens that cause Fusarium branch dieback in avocado trees in southern Florida. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing adult TSHB beetles. Formulated products containing Bb strains to which adult beetles were exposed were BioCeres WP, BotaniGard WP, BotaniGard ES, and Velifer ES. Controls consisted of water only and BotaniGard ES and Velifer ES supernatant with spores removed. Acquisition of spores by adult beetles dipped in product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the Bb-based products in an in vivo avocado bark plug bioassay was determined. Production of Bb spores on beetles after being dipped in product suspensions and placed in a moistened bark-plug assay with water only was assessed. Significantly more spores were acquired by beetles exposed to Velifer ES and BotaniGard ES than beetles exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6-8 days) compared to beetles dipped in the other fungal products (10-11 days) and controls (12 days). Percentage of mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES (20 ± 6.4 × 105 spores/cadaver) was the highest among all treatments, whereas it was the lowest on cadavers of beetles dipped in BotaniGard ES (1 ± 0.2 × 105 spores/cadaver). All Bb-based products, especially Velifer ES, demonstrated potential to manage TSHB populations under laboratory conditions. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results.

In Vitro Effects of Leaf Extracts from Brassica rapa on the Growth of Two Entomopathogenic Fungi.

This study aimed to determine the inhibitive or stimulatory effects of leaf extracts from two Brassica rapa subspecies on the hyphal growth of two well-known entomopathogenic fungi, Cordyceps fumosorosea and Beauveria bassiana. Extract concentrations of 50, 25, and 10% w/v based on leaf fresh weight were prepared from turnip (B. rapa subspecies rapa) and bok choy (B. rapa subspecies chinensis) leaves. Each concentration was individually incorporated into potato dextrose agar plates for in vitro bioassays. The center of each plate was inoculated with 20 µL of a fungal suspension that was allowed 24 h to soak into the agar before sealing the plates and incubating them at 25 °C under a 14-h photophase. The fungal colony perimeter was marked 5 days after inoculation on two perpendicular lines drawn on the bottom of each plate. Radial colony growth was measured from 4 marks per plate 5, 10, and 15 days later. Radial growth rates for both fungi were 1.3-2.0 and 0.9-1.4 times faster with bok choy and turnip extracts, respectively, at the 25% and 50% concentrations compared to the no-extract control treatment. Therefore, bok choy and turnip leaf extracts can stimulate entomopathogenic fungus growth within 15 days. Biochemical compounds in the extracts include sesquiterpenes, α-copaene, ß-selinene, γ-gurjunene, calamenene, cubenene, and α-calacorene.

Field Efficacy of Cordyceps javanica, White Oil and Spinetoram for the Management of the Asian Citrus Psyllid, Diaphorina citri.

Citrus greening disease is devastating the citrus industry in Florida, and the conventional synthetic pesticide applications used to control the vector, the Asian citrus psyllid (AsCP), Diaphorina citri, are rapidly becoming unsustainable. Various laboratory experiments indicate that the entomopathogenic fungus Cordyceps javanica, alone and in combination with horticultural oils, may offer a more sustainable strategy for the management of AsCP. Field studies conducted in 2018 and 2019 in mature citrus indicated that C. javanica alone, C. javanica mixed with white oil, and the chemical standard spinetoram mixed with white oil significantly suppressed AsCP adult populations by 61-83% up to 14 days after treatment in 2018, although colony-forming units of C. javanica were still present on the leaves 21 days after treatment (DAT). Only spinetoram + oil significantly suppressed AsCP, by 100%, up to 7 DAT in 2019. Natural enemies of AsCP, including lady beetles, lacewing larvae and the parasitoid Tamarixia radiata, were observed in the fungal treatments and the untreated control. The AsCP suppression by C. javanica and its compatibility with beneficial organisms suggest the potential use of this entomopathogenic fungus in citrus-integrated pest management.

Mitochondrial sequence data clarify species concepts in the Cyclocephala mafaffa species complex (Coleoptera: Scarabaeidae: Dynastinae: Cyclocephalini).

The speciose genus Cyclocephala Dejean (Coleoptera: Scarabaeidae: Dynastinae: Cyclocephalini) has attracted research attention due to their diversity, agroeconomic importance, and floral visitation habits. Uniquely among Cyclocephala species, C. mafaffa Burmeister and C. deceptor (Casey), two nearly identical species, are diagnosed by a pronotal character: beaded or not beaded basal pronotal margin. We evaluated these morphological species hypotheses with a phylogenetic analysis of 12S and COI, neighbor-joining analysis, and several single-locus species delimitation procedures (automatic barcode gap analysis and three Poisson tree processes analyses). Together, these analyses supported the species concepts for C. deceptor and C. mafaffa. Delimitation procedures supported several distinct molecular operational taxonomic units among these taxa. We consider the separation of C. deceptor and C. mafaffa to be valid. We conservatively synonymize the West Indian subspecies C. mafaffa grandis Burmeister under C. mafaffa and offer a discussion on subspecific concepts in Cyclocephalini. We designate the lectotype of Stigmalia deficiens Casey. Implications of this study for other geographically widespread cyclocephalines or species with variable pronotal morphology are discussed.

Identification of the Achilles heels of the laurel wilt pathogen and its beetle vector.

Ambrosia beetles harbor fungal symbionts that serve as food sources for larvae and adults. These beetles lay their eggs along tunnels in xylem sapwood, which is the substrate for fungal growth. Symbiotic fungi of the genus Raffaelea found in invasive and indigenous ambrosia beetles include the highly virulent plant pathogen Raffaelea lauricola affecting members of the Lauraceae family. R. lauricola is responsible for the deaths of > 500 million trees since 2005. Infection by as few as 100 spores can kill a healthy tree within months. Our data show that R. lauricola is cold-adapted with optimal growth between 16 and 26 °C, with little to no growth at temperatures ≥ 30 °C. The fungus is halophilic and shows a dramatic decrease in growth at pH ≥ 6.8. Fungicide resistance profiling revealed sensitivity of R. lauricola to prochloraz, dichlorofluanid, most conazoles, dithiocarbamates, and zineb (zinc fungicide), whereas the related species Raffaelea arxii showed more limited fungicide sensitivity. Entomopathogenic fungi potentially useful for beetle control were generally highly resistant to most fungicides tested. Coupling pH decreased the concentration for 95% inhibition of fungal growth (IC95) of the most potent R. lauricola fungicides by 3-4-fold. Use of avocado bark plug insect bioassays revealed that commercially available Beauveria bassiana can be used as a biological control agent capable of effectively killing the beetle vectors. These data provide simple and practical recommendations to specifically target R. lauricola while having minimal effects on other symbiotic and entomopathogenic fungi, the latter of which can be used to manage the beetle vectors.

Synopsis of the cyclocephaline scarab beetles (Coleoptera, Scarabaeidae, Dynastinae).

The cyclocephaline scarabs (Scarabaeidae: Dynastinae: Cyclocephalini) are a speciose tribe of beetles that include species that are ecologically and economically important as pollinators and pests of agriculture and turf. We provide an overview and synopsis of the 14 genera of Cyclocephalini that includes information on: 1) the taxonomic and nomenclatural history of the group; 2) diagnosis and identification of immature life-stages; 3) economic importance in agroecosystems; 4) natural enemies of these beetles; 5) use as food by humans; 6) the importance of adults as pollination mutualists; 7) fossil cyclocephalines and the evolution of the group; 8) generic-level identification of adults. We provide an expanded identification key to genera of world Cyclocephalini and diagnoses for each genus. Character illustrations and generic-level distribution maps are provided along with discussions on the relationships of the tribe's genera.

Annotated catalog and bibliography of the cyclocephaline scarab beetles (Coleoptera, Scarabaeidae, Dynastinae, Cyclocephalini).

Cyclocephaline scarab beetles represent the second largest tribe of the subfamily Dynastinae, and the group includes the most speciose genus of dynastines, Cyclocephala. The period following publication of Sebo Endrodi's The Dynastinae of the World has seen a huge increase in research interest on cyclocephalines, and much of this research has not been synthesized. The objective of this catalog and bibliography is to compile an exhaustive list of taxa in Cyclocephalini. This paper provides an updated foundation for understanding the taxonomy and classification of 14 genera and over 500 species in the tribe. It discusses the history of cataloging dynastine species, clarifies issues surrounding the neotype designations in Endrodi's revision of Cyclocephalini, synthesizes all published distribution data for cyclocephaline species, and increases accessibility to the voluminous literature on the group by providing an easily searchable bibliography for each species. We propose the nomen novum Cyclocephala rogerpauli, new replacement name, for C. nigra Dechambre.

Spore Acquisition and Survival of Ambrosia Beetles Associated with the Laurel Wilt Pathogen in Avocados after Exposure to Entomopathogenic Fungi.

Laurel wilt is a disease threatening the avocado industry in Florida. The causative agent of the disease is a fungus vectored by ambrosia beetles that bore into the trees. Until recently, management strategies for the vectors of the laurel wilt fungus relied solely on chemical control and sanitation practices. Beneficial entomopathogenic fungi (EPF) are the most common and prevalent natural enemies of pathogen vectors. Laboratory experiments demonstrated that commercial strains of EPF can increase the mortality of the primary vector, Xyleborus glabratus, and potential alternative vectors, Xylosandrus crassiusculus, Xyleborus volvulus and Xyleborus bispinatus (Coleoptera: Curculionidae: Scolytinae). Our study provides baseline data for three formulated commercially-available entomopathogenic fungi used as potential biocontrol agents against X. crassiusculus, X. volvulus and X. bispinatus. The specific objectives were to determine: (1) the mean number of viable spores acquired per beetle species adult after being exposed to formulated fungal products containing different strains of EPF (Isaria fumosorosea, Metarhizium brunneum and Beauveria bassiana); and (2) the median and mean survival times using paper disk bioassays. Prior to being used in experiments, all fungal suspensions were adjusted to 2.4 × 106 viable spores/mL. The number of spores acquired by X. crassiusculus was significantly higher after exposure to B. bassiana, compared to the other fungal treatments. For X. volvulus, the numbers of spores acquired per beetle were significantly different amongst the different fungal treatments, and the sequence of spore acquisition rates on X. volvulus from highest to lowest was I. fumosorosea > M. brunneum > B. bassiana. After X. bispinatus beetles were exposed to the different suspensions, the rates of acquisition of spores per beetle amongst the different fungal treatments were similar. Survival estimates (data pooled across two tests) indicated an impact for each entomopathogenic fungus per beetle species after exposure to a filter paper disk treated at the same fungal suspension concentration. Kaplan⁻Meier analysis (censored at day 7) revealed that each beetle species survived significantly shorter in bioassays containing disks treated with EPF compared to water only. This study demonstrated that ambrosia beetles associated with the laurel wilt pathogen in avocados are susceptible to infection by EPF under laboratory conditions. However, the EPF needs to be tested under field conditions to confirm their efficacy against the beetles.

Compatibility and Efficacy of Isaria fumosorosea with Horticultural Oils for Mitigation of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae).

Horticultural oils are an important component of integrated management programs of several phytophagous arthropods and pathogens affecting fruit, ornamentals and vegetables in greenhouse and field production systems. Although effective against the target pest, their incompatibility with biological control agents can compromise efforts to develop eco-friendly management programs for important agricultural pests. In this study, we assessed the in vitro effect of selected refined petroleum oils used in citrus and other horticultural crops with a biopesticide containing the entomopathogenic fungi, Isaria fumosorosea (PFR-97) under laboratory conditions. Further, we used leaf disk bioassays to evaluate the combined efficacy of petroleum oils and I. fumosorosea against the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), a major pest of citrus in the United States. All five petroleum oil treatments (Orchex, Sun Pure, Conoco Blend -1, Conoco Blend -2, and JMS) were compatible with I. fumosorosea blastospores, as none of them were found to affect I. fumosorosea colony-forming units and radial fungal growth measured at 3, 6, 9, and 12 days post-inoculation. All mixed treatments performed better than I. fumosorosea alone against D. citri, where the highest mean survival time of D. citri was 12.5 ± 0.7 days. No significant differences in D. citri survival time and I. fumosorosea growth (fungal development index) on dead cadavers, which is important for determining their horizontal transmission, were observed when mixed with Orchex, Sun Pure, Conoco Blend -2, and JMS. Results indicated that horticultural oils in combination with I. fumosorosea could offer citrus growers an alternative treatment for integrating into their current management programs while battling against D. citri in citrus production systems. Due to their eco-friendly, broad-spectrum effect, it could provide control against various citrus pests, while also encouraging the retention of effective chemistries for a longer period in the marketplace. However promising, these combination treatments need to be tested further with I. fumosorosea under grove conditions to confirm their field efficacy.

Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae.

Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of D. abbreviatus exist in citriculture, and new methods of control are desperately sought. To protect citrus against D. abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin protein, was developed using Agrobacterium-mediated transformation of 'Carrizo' citrange [Citrus sinensis (L) Osbeck Poncirus trifoliate (L) Raf]. The transgenic citrus root stock expressed the cytolytic toxin Cyt2Ca1 constitutively under the control of a 35S promoter in the transgenic Carrizo citrange trifoliate hybrid including the roots that are the food source of larval D. abbreviatus. The engineered citrus was screened by Western blot and RT-qPCR analyses for cyt2Ca1 and positive citrus identified. Citrus trees expressing different levels of cyt2Ca1 transcripts were identified (Groups A-C). High expression of the toxin in the leaves (109 transcripts/ng RNA), however, retarded plant growth. The transgenic plants were grown in pots and the roots exposed to 3week old D. abbreviatus larvae using no-choice plant bioassays. Three cyt2Ca1 transgenic plants were identified that sustained less root damage belonging to Group B and C. One plant caused death to 43% of the larvae that fed on its roots expressed 8×106cyt2Ca1 transcripts/ng RNA. These results show, for the first time, that Cyt2Ca1 expressed in moderate amounts by the roots of citrus does not retard citrus growth and can protect it from larval D. abbreviatus.

An annotated checklist of the New World pentodontine scarab beetles (Coleoptera: Scarabaeidae: Dynastinae: Pentodontini).

An updated and annotated checklist of the Pentodontini (Coleoptera: Scarabaeidae: Dynastinae) of the New World is presented. The tribe is composed of 32 genera and 151 species, including the introduced species Heteronychus arator (Fabricius).

Suitability of Microtheca ochroloma (Coleoptera: Chrysomelidae) for the Development of the Predator Chrysoperla rufilabris (Neuroptera: Chrysopidae).

Microtheca ochroloma Stål, the yellowmargined leaf beetle, is one of the most destructive pests of crucifer vegetables on organic farms. Larvae of the green lacewing Chrysoperla rufilabris Burmeister have been observed preying on M. ochroloma, but no studies have evaluated the suitability of M. ochroloma as prey for C. rufilabris or the efficacy of this predator as a biological control agent of the pest. This study quantified the killing rate, developmental time, and survivorship of C. rufilabris when offered eggs and larvae of M. ochroloma at 10, 15, 20, or 25°C. Mean number of prey killed daily increased from 8.4 eggs and 4.0 larvae at 15°C to 18.6 eggs and 10.2 larvae at 25°C. However, predator larvae killed 78% fewer total eggs at 25°C than at 15°C; total number of first-instar prey killed did not vary significantly with temperature. Mean developmental time of predator larvae decreased from 75.5 d at 15°C to 26.6 d at 25°C when they were fed eggs, whereas it decreased from 54.0 d at 15°C to 21.4 d at 25°C when they were fed larvae. Predator survivorship was reduced by 80% at 15°C and no larvae survived at 10°C. We conclude that C. rufilabris can complete development on a diet of eggs of M. ochroloma, but its effectiveness to control M. ochroloma populations will be lessened during cool months, from November to April, when crucifers are produced in Florida and the beetle is actively developing, reproducing, and causing crop damage.

Evaluation of the predation capacity of Podisus maculiventris (Hemiptera: Pentatomidae) on Microtheca ochroloma (Coleoptera: Chrysomelidae) in field cages.

The predation capacity of the spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae), was evaluated at three release rates in field cages containing larvae of the yellowmargined leaf beetle, Microtheca ochroloma Stål (Coleoptera Chrysomelidae), a pest of organic crucifer (Brassicaceae) crops in the United States. The experiment was conducted twice, in February-March 2009 and in February-March 2010. On the same day as the introduction of 132 first instars of M. ochroloma into the cages, four (= low), 10 (= medium), or 16 (= high) first instars of P. maculiventris were released evenly among six turnip (Brassica rapa L.) plants in cages. Control treatment cages received no predators. In 2009, the mean number of M. ochroloma larvae declined progressively during the first four sampling dates. On the final (seventh) sampling date, the mean numbers of total M. ochroloma in the two higher release-rate treatments were significantly lower than in the low release-rate and control treatments. Overall survivorship of P. maculiventris was 39.1% in the high release-rate treatment. In 2010, the mean number of M ochroloma larvae declined progressively during the first four sampling dates. On the fourth sampling date and the final (ninth) sampling date, there were no significant differences among treatments involving predator releases. The lowest overall survivorship of P. maculiventris (3.1%) was in the high release-rate treatment. Two tentative recommendations for growers emerged from this study: 1) release 10 first instars of P. maculiventris per six plants if the plants are expected to have > or = 7 leaves per plant; or 2) release four first instars of P. maculiventris per six plants if the plants are expected to have < or = 6 leaves per plant.

Development time and predation rate of Podisus maculiventris (Hemiptera: Pentatomidae) feeding on Microtheca ochroloma (Coleoptera: Chrysomelidae).

Microtheca ochroloma Stål is a beetle native to South America and was introduced to the United States in 1945. Since then, M. ochroloma has become a serious pest in crucifer crops because of the lack of natural enemies. The objective of this study was to measure the predation rate and development time of the commercially available predator Podisus maculiventris (Say) feeding on M. ochroloma at four constant temperatures in the laboratory as a first step to evaluating the predator's capability as a biological control agent of the pest. Nymphal development of P. maculiventris increased from 23 d at 25°C to 99 d at 15°C. There was no development of first instar or egg hatch at 10°C. Number of fourth-instar M. ochroloma killed during nymphal development varied significantly from 65 at 15°C to 53 at 20°C because of length of the nymphal period. A mean total of 741 eggs of M. ochroloma were consumed during nymphal development at 25°C. Adult P. maculiventris preyed on nine and 12 times more fourth-instar M. ochroloma during 10 d at 20° and 25°C, respectively, than at 15°C. We conclude that P. maculiventris can develop successfully on a diet of eggs or fourth-instar M. ochroloma, but its predation and development rates will be significantly curtailed during the cool months from November to March when M. ochroloma is a key pest of organically grown crucifers in Florida.

Genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae).

The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 (HoCV-1) revealed a 9321-nt polyadenylated genome encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5'-proximal ORF (ORF1, nt 420-5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other insect-infecting picorna-like viruses. A structural model created using Mfold exposed a series of stem loop (SL) structures immediately preceding the second ORF which are analogous to an internal ribosome entry site (IRES), suggesting that ORF2 begins with a noncognate GCA triplet rather than the canonical AUG. This 3' ORF2 (5990-8740) showed significant similarity to the structural proteins of members of the family Dicistroviridae, particularly those belonging to the genus Cripavirus. Evidence demonstrating relatedness of these viruses regarding genome organization, amino acid sequence similarity, and putative replication strategy substantiate inclusion of HoCV-1 into this taxonomic position.