The major antigenic membrane protein of Candidatus Phytoplasma asteris selectively interacts with ATP synthase and actin of leafhopper vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of Candidatus Phytoplasma asteris, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.     

Chromosomal polymorphism in an Australian leafhopper (Homoptera: Cicadellidae)

Variation in chromosome number from 2n=9 to 2n=7 was found in a species of leafhopper, Alodeltocephalus draba. It is argued that this variation is due to chromosome fusion and that A. draba is presently evolving to a lower number. The process of reduction is more advanced in some populations than in others.Seconded from: Department of Biology, University of Oregon, Eugene, Oregon, U.S.A.     

Comparative genome analysis of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A) and "Ca. Phytoplasma asteris" Strains OY-M and AY-WB

The chromosome sequence of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of "Ca. Phytoplasma australiense" with "Ca. Phytoplasma asteris" strains OY-M and AY-WB showed that the gene order was more conserved between the closely related "Ca. Phytoplasma asteris" strains than to "Ca. Phytoplasma australiense." Differences observed between "Ca. Phytoplasma australiense" and "Ca. Phytoplasma asteris" strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function.     

Two high-copy plasmids found in plants associated with strains of "Candidatus Phytoplasma asteris"

Complete sequences for two plasmids associated with two strains of “Candidatus Phytoplasma asteris” have been obtained. The plasmid named pPARG1 was found in Rehmannia glutinosa L. associated with phytoplasma classified in the 16Sr I-C subgroup. Plasmid pPABN1 was from phytoplasma associated with infected winter oilseed rape and classified in the 16Sr I-B subgroup. The plasmids pPARG1 (4371 nt) and pPABN1 (3529 nt) have high A + T content of about 75%, similar to that of phytoplasma genomes. Six and five open reading frames longer than 100 amino acids and organized on the same strand were recognized on pPARG1 and pPABN1, respectively. A segment about 1.6 kbp long sharing high sequence identity with the Onion yellows phytoplasma genome was found in pPABN1.     

Fixed precision sampling plans for white apple leafhopper (Homoptera: Cicadellidae) on apple

Constant precision sampling plans for the white apple leafhopper, Typhlocyba pomaria McAtee, were developed so that it could be used as an indicator species for system stability as new integrated pest management programs without broad-spectrum pesticides are developed. Taylor's power law was used to model the relationship between the mean and the variance, and Green's constant precision sequential sample equation was used to develop sampling plans. Bootstrap simulations of the sampling plans showed greater precision (D = 0.25) than the desired precision (Do = 0.3), particularly at low mean population densities. We found that by adjusting the Do value in Green's equation to 0.4, we were able to reduce the average sample number by 25% and provided an average D = 0.31. The sampling plan described allows T. pomaria to be used as reasonable indicator species of agroecosystem stability in Washington apple orchards.     

Transmission of Spiroplasma citri by the aster leafhopper Macrosteles fascifrons (Homoptera: Cicadellidae)

The aster leafhopper (Macrosteles fascifrons), injected with an isolate of Spiroplasma citri obtained from brittle root-diseased horseradish (Armoracia rusticana), transmitted the spiroplasma to horseradish and China aster (Callistephus chinensis.) After feeding on plants infected with S. citri, M. fascifrons transmitted the spiroplasma from aster to aster and horseradish, from yellow rocket (Barbarea vulgaris) to aster, and from turnip (Brassica rapa) to turnip. Symptoms in infected horseradish were chlorosis and stunting of newly formed leaves, discoloration of root phloem, and reduced plant growth typical of brittle root disease. Chlorosis, stunting, and asymmetry of young leaves occurred in affected aster and turnip. Flowers of infected aster were small and pale in colour and occasionally showed other symptoms including asymmetry, petal distortion, or light green petals. Spiroplasmas were isolated from all plants showing symptoms. Transmission rates by M. fascifrons which acquired S. citri by feeding on infected plants were very low, but injected leafhoppers transmitted more frequently. This is the first report of the transmission of S. citri from diseased to healthy plants by M. fascifrons.     

Glandular-haired alfalfa resistance to potato leafhopper (Homoptera: Cicadellidae) and hopperburn: development of resistance indices

Eight proprietary genotypes of glandular-haired alfalfa, Medicago sativa L., supplied by two different companies, were compared for the degree and types of resistance to the potato leafhopper, Ernpoasca fabae (Harris), and hopperburn. A tube cage no-choice bioassay was developed to test leafhopper mortality, feeding, settling preferences, severity of hopperburn symptoms (in this case, defined as both yellowing and stem growth reduction), and trichome density and type on feeding sites. Leafhopper mortality was both strongly and significantly associated with feeding and leaf trichome density; decreased hopperburn symptom severity was weakly, although significantly, associated with increased mortality. To quantify hopperburn in terms of both yellowing and stem growth reduction, we developed a ranking system that reduces overall hopperburn expression to a single number that considers the varying responses to both types of symptoms. Great variability in leafhopper settling, leafhopper mortality, and stem glandular trichome density was detected among alfalfa genotypes, suggesting that genotypic differences may be based on the concentration and/or chemical constituency of the trichome exudates. We postulate that, among variably resistant genotypes of glandular-haired alfalfa, differences among leafhopper responses and hopperburn severity are linked to forced movement from the stems to the leaves as refuge feeding sites. Principal component analysis was performed to reduce the 10 variables down to five biologically significant factors. Scores for these factors were then used to develop resistance indices for potato leafhopper resistance, hopperburn resistance, and an overall glandular-haired alfalfa resistance index.     

Potato leafhopper (Homoptera: Cicadellidae) injury disrupts basal transport of 14C-labelled photoassimilates in alfalfa

The potato leafhopper, Empoasca fabae (Harris), is a key pest of alfalfa, Medicago sativa L., in part because of the leafhopper's ability to disrupt upward translocation within phloem tissues. To determine if leafhopper injury also disrupts basal translocation necessary for regrowth and perenniality of alfalfa, we used radiolabeled 14CO2 to measure the basal transport of photoassimilates in injured and healthy plants. In one experiment, less 14C was transported to lower stem tissue of leafhopper-injured plants in comparison to the same tissue of healthy plants in early vegetative and early reproductive stages of alfalfa development. In a second experiment, less 14C was transported to lower stem, crown, and root tissues of injured plants in comparison to the same tissues of healthy, early reproductive plants. The disruption of basal transport caused by potato leafhopper may impact carbon storage and mobilization subsequent to defoliation, winter survival, and nitrogen fixation.     

A new megophthalmine leafhopper from Oregon,with notes on its biology and behaviour (Homoptera: Cicadellidae)*

A new leafhopper species of the megophthalmine genus Tiaja Oman, found in the coastal dune community of north-western North America, is described. An account is given of the biology and behaviour of the species.     

Within-field manipulation of potato leafhopper (Homoptera: Cicadellidae) and insect predator populations using an uncut alfalfa strip

The effects of a 3-m wide uncut alfalfa strip on the within field distribution of Empoasca fabae (Harris) (Homoptera: Cicadellidae) and several species of insect predators were examined for 3 yr (1998-2000). The objectives were to determine whether this uncut strip would serve as a trap crop for E. fabae and a refuge for insect predators at first harvest. Empoasca fabae and insect predators in the families Coccinellidae, Nabidae, Anthocoridae, Chrysopidae, and Hemerobiidae were collected weekly using sweep samples and sticky traps from the uncut strips and up to 40 m into the alfalfa regrowth. For 2 yr, both small- (0.34 ha) and large-scale (approximately 11.3 ha) field trials showed higher numbers of E. fabae in 73% of the uncut strips for 2-3 wk after harvest. Similarly, the number of insect predators found within <50% of the uncut strips was also higher during the first or second week after harvest. In 1999, however, we did not observe higher numbers of E. fabae in the uncut strips. Differences may be because of higher E. fabae population numbers in 1999 compared with 1998 and 2000. This research provides alfalfa growers a potential cultural management technique for E. fabae while conserving predatory insects.     

Management of corn leafhopper (Homoptera: Cicadellidae) and corn stunt disease in sweet corn using reflective mulch

Plastic reflective mulches significantly reduced populations of corn leafhopper, Dalbulus maidis (DeLong & Wolcott), adults and the incidence of corn stunt disease caused by Spiroplasma kunkelii (CSS) in late planted sweet corn (Zea mays L.). The reflective mulches were more effective than were either foliar or soil applied insecticides in managing both the leafhopper and the pathogen it transmits. Yields of marketable ears were 1.5 to 2 times greater in reflective mulch plots than from fallow plots. This was due to larger ears (individual ear weight and length) rather than an increase in the number of ears. The use of reflective mulches provides an alternative strategy to insecticides in the management of both D. maidis and corn stunt disease. Such a strategy may prove useful to growers in Latin America and to limited resource growers and organic growers in the United States who wish to grow corn without the use of insecticides.     

Conversion of spheroplast symbiotes in a leafhopper, Helochara communis Fitch (Cicadellidae: Homoptera).

In mycetomes of leafhoppers, Helochara communis, ultrastructural and histochemical studies revealed that spheroplast symbiotes (t-symbiotes) were converted to 'a-symbiotes' (so-called), with apparent loss of DNA--a phenomenon reminiscent of rhizobium-bacteroid conversion in certain legume nodules. Additional t-symbiotes were incorporated into the substance of these 'a-symbiotes'.     

Host effects of glandular-haired alfalfa on alfalfa weevil (Coleoptera: Curculionidae) and potato leafhopper (Homoptera: Cicadellidae) populations in Virginia

Cultivars of glandular-haired alfalfa, Medicago sativa L., such as '54H69', are currently available and marketed as being resistant to potato leafhopper, Empoasca fabae (Harris). Between 2000 and 2002, studies were conducted to compare the effects of '54H69' and a standard, nonglandular-haired alfalfa cultivar, 'Choice', on alfalfa weevil, Hypera postica (Gyllenhal), and potato leafhopper populations at Campbell and Montgomery counties, Virginia. '54H69' had no effect on alfalfa weevil populations. At each location, densities of alfalfa weevil in '54H69' and 'Choice' were similar, but pest pressure was higher at Campbell Co. than at Montgomery Co. and always exceeded the economic threshold before insecticide was applied. Densities of potato leafhopper also did not differ between '54H69' and 'Choice' in any year at the two locations. Insecticide treatment effectively reduced potato leafhopper densities in the two cultivars, although populations were below the economic threshold at both locations when the insecticides were applied. Overall, postinsecticide treatment comparisons showed that the densities of alfalfa weevil and potato leafhoppers were similar or higher in untreated '54H69' compared with insecticide-treated 'Choice'. In addition, there were no differences in seasonal dry yields between '54H69' and 'Choice' in any year at either location. Our results indicate that the glandular-haired alfalfa '54H69' does not provide acceptable resistance to potato leafhopper and also does not offer a yield advantage to growers in Virginia.     

Role of the glandular trichomes in resistance of perennial alfalfa to the potato leafhopper (Homoptera: Cicadellidae)

Experiments were performed to elucidate resistance of glandular-haired alfalfa, Medicago sativa L., to the potato leafhopper, Empoasca fabae (Harris). The primary objective was to examine the potential role of the glandular trichomes and stem lignification for imparting resistance to this pest. During free-choice foliar discoloration experiments, the resistant alfalfa clone FGplh13 expressed lower levels of injury than the susceptible P5373 after 13 d. No-choice tests performed using nymphal potato leafhoppers showed higher levels of mortality associated with the resistant glandular-haired clone FGplh13, and no nymphs survived after 48 h. Additional no-choice experiments using FGplh13 and P5373 alfalfa with the glandular and nonglandular trichomes intact or removed show that mortality of nymphs and adults decreased after removal of the glandular trichomes from FGplh13. Nymphal and adult mortality and nymphal development time did not differ on FGplh13 with the trichomes removed and P5373 with the trichomes intact or removed. Firstinstar, and possibly second-instar, potato leafhoppers were entrapped by an exudate produced by the glandular trichomes on the FGplh13 alfalfa. Stem lignification and the number of vascular bundles did not differ between FGplh13 and P5373 alfalfas. The glandular trichomes on FGplh13 alfalfa appeared to provide the major host resistance factor, with resistance to adults being chemically based and resistance to nymphs being chemically and mechanically based. To maintain levels of potato leafhopper resistance, breeders would appear to benefit by continuing to select for the expression of the glandular trichome phenotype.     

Typhlocybinae of New Zealand (Homoptera: Cicadellidae)

Two tribes of the subfamily Typhlocybinae are represented in New Zealand, as follows. Erythroneurini: the cosmopolitan genus Zygina is represented by 4 endemic species—toetoe, ramsayi n.sp., dumbletoni, and agni n.sp.—plus zealandica, which occurs in Australia also. Typhlocybini: Typhlocyba froggatti, T. lethierryi, Ribautiana tenerrima, Eupteryx melissae, Kybos smaragdula, and K. betulicola are introduced Northern Hemisphere elements; Matatua montivaga n.gen.&sp. is endemic; M. maorica (formerly Dikraneura maorica), also endemic, is of uncertain identity in the absence of male specimens. Keys are given for the separation of all taxa, the genera and species are described, and all species except Matatua maorica are figured.     

Deltocephalinae of New Zealand (Homoptera: Cicadellidae)

The subfamily Deltocephalinae is represented in New Zealand by 3 tribes, 9 genera, and 20 species, of which 3 genera and 13 species are new to science. All taxa are described, and keys are given for their separation. Three of the genera are endemic, 3 are Australian, 2 are Holarctic, and 1 is cosmopolitan. Nesoclutha and Macrosteles are recorded in New Zealand for the first time, and the presence of Deltocephalus is authenticated. Fourteen species are endemic, 4 are Australian, 1 is widespread in the Pacific area, and 1 is Holarctic. Nesoclutha pallida, Macrosteles fieberi, and Deltocephalus hospes are recorded in New Zealand for the first time.     

Amino acids as determinants of host preference for the xylem feeding leafhopper,Homalodisca coagulata (Homoptera: Cicadellidae)

Summary Homalodisca coagulata is a highly polyphagous xylem feeder with distinct seasonal patterns in it's selection of host plants. These patterns were examined in relation to the amino acid content of the xylem for four common host species; Lagerstroemia indica, Baccharis halimifolia, Prunus persica, and Prunus salicina. Xylem fluid was collected from each host species at times when numbers of feeding leafhoppers were both low and high. In each case, concentrations of amino acids were greatest when numbers were high. Similarly, comparisons between host species at given times showed that concentrations of amino acids were positively correlated with host selection. In a second study, amino acids of xylem were manipulated by budding scions of a non-preferred host (P. persica) on rootstocks of preferred (P. salinica) and non-preferred (P. persica) hosts. Morphology and phenology of the budded trees were similar to that of the scion species yet the xylem composition of amino acids was primarily dependent on the rootstock. Concentrations of amino acids and the preference of leafhoppers were roughly two-fold greater for scions of the preferred than the non-preferred rootstock. In both studies, amides (glutamine plus asparagine) were the amino acids most highly correlated with host selection. These compounds are the predominant amino acids in xylem fluid, have high nitrogen to carbon ratios, and account for a high percentage of the caloric value in xylem fluid. Many of the less abundant amino acids were positively correlated with host preference, but the correlations were less consistent and correlation coefficients were generally lower.Florida Agricultural Experiment Station Journal Series No. 9672     

The distribution of a typhlocybine leafhopper, Ribautiana ulmi (Homoptera: Cicadellidae) on a specimen wych elm tree

ABSTRACT.

• 1 Higher numbers of feeding and resting Ribautiana ulmi (L.) were found on the more highly illuminated areas of the canopy of a wych elm tree, Ulmus glabra Huds. cv. Camperdownii.
• 2 Higher numbers were also found on the basal leaves of each branch when compared with the distal leaves, and on exposed areas of individual leaves when compared with the shaded areas which they overlapped.
• 3 Leaf thickness, tannin concentrations and leaf toughness were greater, and water content lower, in leaves from exposed areas of the canopy than from shaded ones.
• 4 R.ulmi feeds selectively on the contents of the palisade mesophyll cells, and therefore was assumed to ingest the highest available levels of tannins.

     

Effect of environmental conditions and leafhopper gender on Maize chlorotic dwarf virus transmission by Graminella nigrifrons (Homoptera: Cicadellidae)

To determine the most economical and efficient means to maintain cultures of Maize chlorotic dwarf virus (MCDV) and to screen for host plant resistance to MCDV, we evaluated the effects of temperature, light intensity, daylength, atmospheric pressure, and leafhopper gender on the frequency of transmission of MCDV by Grarminella nigrifrons Forbes (Homoptera: Cicadellidae). Female leafhoppers transmitted at higher frequencies than males under most conditions. In temperature studies, transmission rates for both male and female leafhoppers progressively increased as temperatures rose from 20 to 30 degrees C. At high light intensities, both males and females transmitted at greater frequencies than they did at low. Similarly, longer day lengths were correlated with higher transmission rates for both sexes. No significant differences in transmission rates were observed in response to differences in atmospheric pressure. The results also showed that transmission rates under most conditions are high enough to overcome potential ambiguities caused by inoculated susceptible plants that do not become infected (disease escapes) when screening for resistance.