Transmission of Xylella fastidiosa to grapevines by Homalodisca coagulata (Hemiptera: Cicadellidae)

Pierce's disease (PD) of grapevines is caused by a xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) that is transmitted to plants by xylem sap-feeding insects. The introduction of the sharpshooter leafhopper Homalodisca coagulata (Say) into California has initiated new PD epidemics in southern California. In laboratory experiments, the major characteristics of H. coagulata's transmission of X. fastidiosa to grapevines were the same as reported for other vectors: short or absent latent period; nymphs transmitted but lost infectivity after molting and regained infectivity after feeding on infected plants; and infectivity persisted in adults. Adult H. coagulata acquired and inoculated X. fastidiosa in <1 h of access time on a plant. Inoculation rates increased with access time, but acquisition efficiency (20% per individual) did not increase significantly beyond 6-h access. Estimated inoculation efficiency per individual per day was 19.6, 17.9, and 10.3% for experiments where plant access was 1, 2, and 4 d, respectively. Freshly molted adults and nymphs acquired and transmitted X. fastidiosa more efficiently than did older, field-collected insects. H. coagulata transmitted X. fastidiosa to 2-yr-old woody tissues of grapevines as efficiently as to green shoots. H. coagulata transmitted X. fastidiosa 3.5 mo after acquisition, demonstrating persistence of infectivity in adults. About half (14/29) of the H. coagulata from which we failed to culture X. fostidiosa from homogenized heads (with a detection threshold of 265 CFU/head) transmitted the pathogen to grape, and 17 of 24 from which we cultured X. fastidiosa transmitted.     

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID₅₀/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.     

Host specificity of <Emphasis Type="Italic">Anagrus epos</Emphasis>: a potential biological control agent of <Emphasis Type="Italic">Homalodisca vitripennis</Emphasis>

Anagrus epos Girault (Hymenoptera: Mymaridae) is a candidate for a classical biological control program targeting the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), in California. Because mass production of GWSS is expensive and labor-intensive, a factitious host that is more economical to produce is desirable to mass produce A. epos for colonization and augmentation efforts. Here, we report the results of host specificity tests and potential rearing techniques for A. epos under laboratory conditions. Females discriminated and oviposited into eggs of seven cicadellid species: H. vitripennis, Circulifer tenellus (Baker), Erythroneura variabilis Beamer, Amblysellus grex (Oman), Graphocephala atropunctata (Signoret), Macrosteles severini Hamilton, and H. liturata Ball, and two cerambycid species: Phoracantha recurva Newman and P. semipunctata (F.). Anagrus epos successfully completed development in the eggs of H. vitripennis, C. tenellus, E. variabilis, A. grex, G. atropunctata, M. severini, and H. liturata. The use of a factitious host and potential nontarget effects of this generalist parasitoid are discussed.     

Classical biological control of the glassy-winged sharpshooter,Homalodisca vitripennis,by the egg parasitoid Gonatocerus ashmeadi in the Society,Marquesas and Austral archipelagos of French Polynesia

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (= H. coagulata [Say]) (Hemiptera: Cicadellidae), was a major exotic pest in French Polynesia until a classical biological control program against this pest was conducted using the host-specific egg parasitoid Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae). After risk assessment studies indicated an acceptably low potential threat to non-target species, parasitoids were released on Tahiti in May 2005. One year after release, populations of H. vitripennis had decreased by more than 90% in Tahiti and nearby Moorea. Here we present results of impact studies obtained during the second post-release year for G. ashmeadi in Tahiti and Moorea; we also report for the first time on results for eight other H. vitripennis infested islands located in three different archipelagos (Society, Marquesas, and Austral) of French Polynesia. On all infested islands across the three archipelagos, arrival of G. ashmeadi slashed H. vitripennis densities by more than 95%. In Tahiti and Moorea, H. vitripennis populations were maintained at very low densities during the second post-release year. Seasonal fluctuations of H. vitripennis abundance were observed in Tahiti with pest populations being more abundant during the cooler dry season than during the warmer wet season because of lower parasitism rates. Hence, similar seasonal fluctuations of H. vitripennis abundance are expected across all infested archipelagos in French Polynesia.     

Gut content examination of the citrus predator assemblage for the presence of Homalodisca vitripennis remains

A two-year study was conducted in a citrus orchard, Citrus sinensis L., to determine frequency of predation on glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar). A total of 1,578 arthropod predators, representing 18 taxa, were collected and assayed for the presence of GWSS egg protein by an enzyme-linked immunosorbent assay using a Homalodisca-species and egg-specific monoclonal antibody and then by polymerase chain reaction using a H. vitripennis-specific DNA marker. The gut content analyses revealed the presence of GWSS remains in the gut of 2.28 % of the total arthropod predator population, with 3.09 % of the spiders and 0.59 % of the insect predators testing positive. Moreover, a comparison of the two assays indicated that they were not equally effective at detecting GWSS remains in predator guts. Low frequencies of GWSS detection in the gut of predators indicated that GWSS are not a primary prey and that predators may contribute little to suppression of this pest in citrus.     

Invasion dynamics of the glassy-winged sharpshooter <Emphasis Type="Italic">Homalodisca vitripennis</Emphasis> (Germar) (Hemiptera: Cicadellidae) in French Polynesia

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) [formerly Homalodisca coagulata (Say)] (Hemiptera: Cicadellidae), has recently emerged as a serious invasive pest. From its natural range in the southeast USA and northeast Mexico, it invaded successively California (late 1980s), French Polynesia (1999), Hawaii (2004), and recently Easter Island (2005) inadvertently through the transportation of infested plants. In French Polynesia, H. vitripennis has reached impressive densities becoming an important pest threatening agriculture, native biodiversity, as well as being a major social nuisance. Since 1999, H. vitripennis spread rapidly from Tahiti to neighboring islands, colonizing most of the archipelagos of French Polynesia. In this paper, we present the results of surveys of H. vitripennis populations from 15 islands of French Polynesia and use these data to investigate the invasion dynamics and colonization processes of this pest in a tropical climate. We found H. vitripennis present in 10 islands with two new records confirmed. Our analyses suggest that: (1) H. vitripennis abundance is strongly associated with urbanization, with highest pest densities found in the most developed coastal areas of infested islands, (2) H. vitripennis may exhibit an Allee effect during the early phase of an invasion, and (3) the invasion dynamics of H. vitripennis conform to a stratified dispersal model marked by rapid long-distance human-mediated movement.     

Engineering an invasion: classical biological control of the glassy-winged sharpshooter, <Emphasis Type="Italic">Homalodisca vitripennis</Emphasis>, by the egg parasitoid <Emphasis Type="Italic">Gonatocerus ashmeadi</Emphasis> in Tahiti and Moorea,French Polynesia

The glassy-winged sharpshooter, Homalodisca vitripennis Germar (=H. coagulata Say) (Hemiptera: Cicadellidae), invaded Tahiti in 1999 and spread rapidly to the main island groups of French Polynesia becoming an important pest. It threatened agriculture, native biodiversity, and created serious social and recreational problems. Further, massive uncontrolled populations on Tahiti presented an elevated invasion threat to other South Pacific nations. In 2004, a classical biological control program against H. vitripennis was initiated in French Polynesia using the highly host-specific egg parasitoid Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae). After risk assessment studies indicated an acceptably low level of risk to non-target species, 13,786 parasitoids were released at 27 sites in Tahiti between May and October 2005. Here we present the results of G. ashmeadi and H. vitripennis population surveys during the first year of their interaction in French Polynesia (until mid-May 2006). The impact of G. ashmeadi on H. vitripennis was extremely rapid and high. Parasitism of H. vitripennis egg masses by G. ashmeadi has averaged 80–100% in Tahiti since the introduction of the parasitoid, and populations of H. vitripennis nymphs and adults have decreased by more than 90% since December 2005. Populations of H. vitripennis have been successfully maintained at this low level for more than 1 year. The same results were obtained in nearby Moorea where the parasitoid was probably spread by the unregulated transport of plants infested with parasitized H. vitripennis eggs. Population monitoring continues in order to determine if a stable equilibrium between the pest and the parasitoid has been reached.     

Transmission of Pantoea agglomerans—A paratransgenic control agent—Within a Homalodisca vitripennis population

Paratransgenic control of pests can be an alternative to chemical control, which is associated with environmental contamination, and higher input cost. We have recently developed paratransgenic control strategy to block transmission of the Pierce's disease pathogen—Xylella fastidiosa by its arthropod vector—Homalodisca vitripennis (the glassy-winged sharpshooter) using antimicrobial peptide-expressing strains of Pantoea agglomerans. In the present study, we report horizontal transmission of one of such P. agglomerans strains from H. vitripennis. This process indicates that P. agglomerans can self-sustain in a population of H. vitripennis and, thus, can provide paratransgenic control of X. fastidiosa in field. These results also provide basis for future field studies.     

Characterization of cell lines developed from the glassy-winged sharpshooter, Homalodisca coagulata (hemiptera: Cicadellidae)

Summary Four continuous cell lines were established from the embryos of the glassy-winged sharpshooter, Homalodisca coagulata (Say), an economically important insect vector of bacterial pathogens of grape, almond citrus, oleander, and other agricultural and ornamental plantings. The cell lines were designated GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH. The GWSS-Z10, GWSS-Z15, and GWSS-G3 lines were cultured in Ex-Cell 401 medium supplemented with 10% fetal bovine serum (FBS), whereas the GWSS-LH line was cultured in LH medium supplemented with 20% FBS. The cell lines were characterized in terms of their morphology, growth, protein composition, and polymerase chain reactionamplification patterns of their chromosomal deoxyribonucleic acid. The population doubling times of GWSS-Z10, GWSS-Z15, GWSS-G3, and GWSS-LH were 46.2, 90.9, 100.3 and 60.2 h, respectively. These lines should be useful for the study of insect-pathogenic viruses of leafhoppers, aphids, treehoppers, and other related insects as well as plant-pathogenic viruses that are transmitted by these insects.     

Nematode Reproduction in Environments of Fluctuating Aeration

Reproduction of Aphelenchus avenae, reared on Rhizoctonia solani growing on steamed wheat seeds and Caenorhabditis sp., reared on a mixed bacterial culture grown on oatmeal, was significantly reduced at 5% oxygen and inhibited at 4% oxygen and below. Aeration ranging from atmospheric air (21%) to 10% oxygen had no effect on reproduction. Close interval (5 days or less) fluctuations, between high and low oxygen concentrations, inhibited population buildup of Hemicycliophora arenaria on tomato in soil, and of A. avenae and Caenorhabditis sp. in vitro. In soil tests with H. arenaria exposed to 12 hr of nitrogen every three days (in air) inhibited the rate of buildup compared to controls maintained in continuous air. With the in vitro studies, as little as 4 hr nitrogen every 3 days (stored in air) significantly influenced the population numbers.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

Effects of a Commercial Formulation of Bacillus firmus I-1582 on Golf Course Bermudagrass Infested with Belonolaimus longicaudatus

One of the primary pests of bermudagrass (Cynodon spp.) on golf courses in the southeastern United States is Belonolaimus longicaudatus (sting nematode). In 2011, a commercial formulation of Bacillus firmus I-1582, Nortica 5WG, was launched in the United States for management of plant-parasitic nematodes on turfgrasses. To test the efficacy of late winter/early spring application of this biopesticide on B. longicaudatus, two field trials in 2009 compared B. firmus with fenamiphos and untreated control treatments. In 2011, two additional field trials compared treatment with B. firmus with untreated control only. These trials measured treatment effects on the population density of B. longicaudatus, turf root length, and turf percent green cover. In all four trials, treatment with B. firmus improved root length and decreased numbers of B. longicaudatus in contrast to the untreated. These results indicate that late winter/early spring application of B. firmus is an effective biopesticide treatment for management of B. longicaudatus on golf course bermudagrass.     

Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.     

Short-distance dispersal behavior and establishment of the parasitoid Gonatocerus ashmeadi (Hymenoptera: Mymaridae) in Tahiti: Implications for its use as a biological control agent against Homalodisca vitripennis (Hemiptera: Cicadellidae)

The egg parasitoid Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae), was introduced into French Polynesia as a biological control agent to control the invasive plant feeding pest Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). The short-distance dispersal of G. ashmeadi was monitored as part of the biological control program. G. ashmeadi showed exponential dispersal capacity with 47 m/day being a minimum estimate of its natural rate of spread at high host densities (>150 nymphs per minute of sweep net sampling) in urbanized areas at sea level, which were characterized by a high diversity of exotic ornamental plants. This rate of spread contrasted starkly with almost nonexistent establishment and dispersal where host densities were very low (<2 nymphs per minute of sweep net sampling) at high elevation (800 m) with relatively undisturbed native vegetation. Survey results across different altitudes revealed an effect of vegetative diversity and host density on the measurable mobility and establishment of G. ashmeadi. In contrast, no significant influence of wind direction was found on G. ashmeadi dispersal rate or direction. Survey results for G. ashmeadi from French Polynesia suggest that the best release establishment strategies for classical biological control of H. vitripennis are: (1) many small releases where host density is high, or (2) larger and fewer releases where host densities are low.     

Evaluating the potential of buckwheat and cahaba vetch as nectar producing cover crops for enhancing biological control of Homalodisca vitripennis in California vineyards

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) is a significant pest of grapevines in California due to vectoring Xylella fastidiosa Wells et al., Buckwheat (Fagopyrum esculentum Moench) and vetch (Vicia sativa L. cv. ‘cahaba white’) are potential nectar-producing cover crop plants in California vineyards that could enhance Gonatocerus ashmeadi Girault, a parasitoid of H. vitripennis. Three aspects of these 2 plant species were evaluated. (1) Effect on parasitoid fitness: In the laboratory, buckwheat and vetch enhanced survival of G. ashmeadi by 9 and 6 days, respectively, compared to water. G. ashmeadi offspring production was 81% and 142% greater when females were provided vetch or buckwheat, respectively, compared to water. Buckwheat decreased G. ashmeadi female offspring by 19% compared to water and vetch. (2) Nectar production phenology in southern California: From April through September, buckwheat required only 23–32 days from sowing to nectar-producing flowers. Vetch required an additional 14–33 days to produce extrafloral nectar during June–August. The range in length of the nectar producing period between August 2007 and November 2007 was 41–52 days and 163–164 days for buckwheat and vetch, respectively. (3) X. fastidiosa Wells et al. host testing and H. vitripennis transmission studies: Needle inoculation of cover crop plants showed that X. fastidiosa successfully infected and replicated in buckwheat, vetch, sweet alyssum [Lobularia maritima (L.) Desv.] and coriander (Coriandrum sativum L.), plants that were assessed here or elsewhere as nectar producing cover crops for use in vineyards. H. vitripennis successfully transmitted X. fastidiosa between buckwheat and grapevine in the greenhouse and field. H. vitripennis successfully transmitted X. fastidiosa from grapevine to vetch in the field, while transmission studies investigating movement from vetch to grapevine in the greenhouse were inconclusive.     

Compromised autophagy by MIR30B benefits the intracellular survival of Helicobacter pylori

Helicobacter pylori evade immune responses and achieve persistent colonization in the stomach. However, the mechanism by which H. pylori infections persist is not clear. In this study, we showed that MIR30B is upregulated during H. pylori infection of an AGS cell line and human gastric tissues. Upregulation of MIR30B benefited bacterial replication by compromising the process of autophagy during the H. pylori infection. As a potential mechanistic explanation for this observation, we demonstrate that MIR30B directly targets ATG12 and BECN1, which are important proteins involved in autophagy. These results suggest that compromise of autophagy by MIR30B allows intracellular H. pylori to evade autophagic clearance, thereby contributing to the persistence of H. pylori infections.     

Seasonal flight activity of two Homalodisca species (Homoptera: Cicadellidae) that spread Xylella fastidiosa in southern California.

Homalodisca coagulata (Say) and Homalodisca lacerta (Fowler) are vectors of a new bacterial disease of oleander in California known as oleander leaf scorch, induced by the bacterium Xylella fastidiosa. H. coagulata also has been implicated in the spread of the strain of X. fastidiosa that induces Pierce's disease of grapevines in California. We monitored the flight activity of H. coagulata and H. lacerta in oleander and citrus by using yellow sticky cards at three southern California locations where outbreaks of oleander leaf scorch have been documented, and where vector compliments are different. Areas sampled included a mesic coastal area (Irvine, CA) that supports predominantly H. coagulata and few H. lacerta, a dry inland location (Palm Desert, CA) that supports predominantly H. lacerta and few H. coagulata, and an intermediate area (Riverside, CA) supporting both Homalodisca species. From November 1996 to October 1999 peak catches of both Homalodisca species occurred during the midsummer at all locations. H. coagulata was trapped in greater numbers in citrus than in oleander at both the Riverside and the Irvine sites. Likewise, H. lacerta in Riverside was more associated with citrus than oleander, yet H. lacerta in Palm Desert was trapped in greater numbers in oleander than citrus.