Survival in Dalbulus leafhopper vectors improves after exposure to maize stunting pathogens

Using D. maidis and pathogen isolates collected at three field sites along an altitudinal gradient in Mexico, we compared survival in leafhoppers exposed to healthy maize to those exposed to maize infected with one of four isolates of maize stunting pathogens: two isolates of the corn stunt spiroplasma (CSS, Spiroplasma kunkelii) and two of the maize busby stunt phytoplasma (MBSP). Survival improved after exposure to either plant pathogen under both the cooler and warmer environmental conditions D. maidis is likely to encounter during the dry season. Survival varied among leafhoppers from the different field sites, suggesting that gene flow between these populations is limited. The leafhoppers responded differently to the four isolates (i.e., we noted significant population by exposure interactions), but we found no difference between MBSP and CSS exposure. Finally, we found evidence of local adaptation in one leafhopper population to sympatric, as compared to allopatric, plant pathogens. We have shown with this and our earlier study that aspects of the interaction phenotype in the association between D. maidis and the plant pathogens are mutualistic and that this association has considerable potential as a model for studies of local adaptation.     

Effect of maize stunting mollicutes on survival and fecundity of Dalbulus leafhopper vectors

The effect of corn stunt spiroplasma (CSS) on survival and fecundity of three Dalbulus leafhopper species was determined. CSS significantly reduced the survival, as measured by the time to 50% (t₅₀) and 25% (t₂₅) survival, and by the scale parameter (b) of the Weibull model, for D. elimatus and D. gelbus. Fecundity of these two species, as measured by the net and gross reproductive rates, was also reduced by CSS. CSS did not significantly affect the corn leafhopper, D. maidis. In a separate experiment, maize bushy stunt mycoplasma (MBSM) reduced the survival and fecundity of D. maidis at temperatures from 20 to 29 °C. The effect of MBSM on D. maidis survival was less severe than CSS on D. elimatus and D. gelbus; t₂₅, but not t₅₀, was reduced by MBSM. Survival times and the cohort generation time generally declined with increasing temperature. Fecundity, however, generally increased with increasing temperature.     

Oviposition behavior of Coccidoxenoides peregrinus, a parasitoid of Planococcus ficus

We tested hypotheses concerning the specificity of interactions between insect vectors and mollicute plant pathogens in a 22-month study of leafhoppers collected at three agricultural field sites in Mexico. The common species collected, Dalbulus maidis, D. elimatus, D. gelbus, and D. guevari were equally likely to test positive for corn stunt spiroplasma (CSS) in ELISA, and to transmit maize bushy stunt phytoplasma (MBSP) to test maize seedlings. We documented intraspecific variation in the ability of D. maidis to transmit confirmed CSS infections. Dalbulus guevari and D. gelbus were less successful in transmitting CSS than D. maidis from the same population. Our results suggest this vector-plant pathogen interaction is not specific to a single Dalbulus-mollicute combination, and that both the range of potential vectors in agricultural fields, and intraspecific variation across populations of these vectors, should be the focus of future work.     

First host record for Anteon pilicorne (Ogloblin) (Hymenoptera: Dryinidae), a parasitoid of Cicadellidae, including the corn leafhopper (Hemiptera: Cicadellidae)

For the first time the dryinid wasp Anteon pilicorne (Ogloblin) is recorded as a parasitoid of two Macrostelini leafhoppers: Balclutha rosea (Scott) and the corn leafhopper Dalbulus maidis (DeLong & Wolcott). New distributional records are presented.     

Increased survival of Dalbulus maidis, a specialist on maize, on non-host plants infected with mollicute plant pathogens

The leafhopper Dalbulus maidis DeLong & Wolcott survived significantly longer on aster, Callistephus chinensis Nees, infected with any one of 3 strains of aster yellows (AY) mycoplasma-like organism (MLO) than on healthy asters. After 7 or more days on AY-diseased aster, females were conditioned to survive longer on healthy asters than were leafhoppers of the same age previously exposed only to maize. Females were also conditioned to survive longer on healthy aster by prior exposure to AY-MLO-infected celery (Apium graveolens L.). Males were not so conditioned. Leafhoppers injected with infectious extracts of AY-MLO dit not live longer on aster nor transmit the AY-MLO to aster. Conditioning on AY-diseased aster did not cause D. maidis to transmit AY-MLO and did not interfere with the transmission to maize of the mollicute (Spiroplasma kunkelii Whitcomb et al.) that causes corn stunt disease. Spiroplasma citri Saglio et al. infection of aster but not of turnip (Brassica rapa L.), Plantago major L. or periwinkle (Catharanthus roseus (L.)), improved the longevity of D. maidis on these plants and conditioned leafhoppers for enhanced subsequent survival on healthy asters.

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Résumé La cicadelle du maïs, D. maidis à survécu significativement plus longtemps sur aster (Callistephus chinensis Nees) contaminé par l'une des trois souches de l'agent mycoplasmique (MLO) de la jaunisse de l'aster (AY), que sur des asters sains. Après 7 jours ou plus sur des asters AY-MLO, les femelles (et non les mâles) étaient conditionnées de telle sorte qu'elles survivaient plus longtemps sur asters sains que des femelles du même âge précédemment sur maïs. Sur des céleris (Apium graveolens) atteints de AY, D. maidis a survécu plus longtemps que sur céleri sain, mais moins que sur aster AY. Des extraits, contaminés par AY injectés à D. maidis n'ont pas augmenté sa longévité sur aster, ni provoqué la transmission de AY-MLO. Le conditionnement sur aster AY n'a pas entrainé la transmission de AY-MLO par D. maidis, et n'a pas interferé avec son aptitude à transmettre l'agent du nanisme du maïs, Spiroplasma kunkelii. S. citri a aussi conditionné D. maidis sur aster, mais a été sans effet sur navet (Brassica rapa), sur plantain (Plantago major) et sur pervenche (Vinca).

     

Effect of temperature on the population dynamics of three Dalbulus leafhopper species

Adult survival and fecundity of three Dalbulus leafhopper species were determined at constant temperatures of 20, 23, 26 and 29°C. Survival was measured by quartiles (i.e. time to 75%, 50% and 25% survival) and estimated parameters of the Weibull model fitted to the survival distributions. D. gelbus lived as long or significantly (P= 0.05) longer than the other species at all temperatures. D. maidis (the corn leafhopper) had survival times equal to or significantly shorter than D. elimatus (the Mexican corn leafhopper) at all temperatures except 29°C where D. maidis lived the longest. The shape of the survival curves did not vary among species or change with temperature. The fecundity of D. gelbus, as measured by the average number of eggs laid per female per generation, was equal to or significantly lower than the other species at all temperatures. D. maidis and D. elimatus had similar fecundity at all temperatures except 29°C, where D. maidis produced significantly more eggs. The mean development time from egg to adult declined with temperature between 17 and 29°C. At all temperatures, D. maidis developed the fastest, D. gelbus the slowest, and D. elimatus was intermediate. The results can be explained on the basis of the geographic distribution, plant host species, and life-history strategies of the leafhoppers. Models for describing the population dynamics of leafhoppers are evaluated and discussed.     

Infection of strawberry flowers by Botrytis cinerea and its relevance to grey mould development

Corn stunt spiroplasma (CSS) multiplied in all injected Dulbulus maidis, reaching titres of over 1 × 10⁶ colony forming units (cfu)/insect and 1 × 10⁴ cfu/salivary gland of each insect. Spiroplasmas could be isolated from the haemolymph and from the salivary glands 1 h after injection and at any time subsequently. Insect extract at a concentration greater than the equivalent of 0.1 insects/ml was inhibitory to the growth of CSS in cultures. Helices could be seen in the haemolymph at any time after injection. However, distorted or partially deformed cells and small aggregates were not present until 2–3 wk after injection. The salivary gland cells of injected insects contained membrane-bound ‘pockets’ or ‘colonies’ packed with pleomorphic organisms, which included some filamentous forms. Intracellular colonies were always on the periphery of cells and were easily detectable by fluorescent microscopy. Both pleomorphic and filamentous forms were also seen intercellularly in the salivary glands. Following injection, transmission of CSS to maize and to sterile feeding solution were compared using 1 day feeding periods. A proportion of injected leafhoppers began to transmit to maize by the third day following injection (5%) and reached a maximum of 72% by day 14. By day 9 , 82% of the population had transmitted at least once to plants and by day 12 , 100% had transmitted. Similar insects transmitted through membranes to sterile feeding solution on day 4 (3%) reaching a maximum of 62% by day 14.     

Dose-dependent influence of Microplitis rufiventris factors on developmental rate and growth of last-instar Spodoptera littoralis larvae

The solitary endoparasitoid, Microplitis rufiventris, attacks and can develop in earlier instars of Spodoptera littoralis larvae with preference to third‐instar larvae. We used the last stadium (sixth instar), a stage which is not naturally parasitized. The newly moulted larvae (0–3 h old) of this stadium were more acceptable for parasitization by the wasp females than the older ones (24 h old). Parasitization by M. rufiventris wasp of last instar S. littoralis larvae leads to dose (no. of eggs + parasitoid factors)‐dependent effects which were more pronounced at 20°C than at 27°C. A single oviposition into a sixth instar host larva resulted in normal development of the host. However, superparasitization increased the proportions of developmentally arrested hosts and number of live wasp larvae. Development of supernumerary individuals of the parasitoid in the host larva leads to dose‐related adverse effects on host growth and development. The present study may provide interesting opportunities for studying the physiological bases of host–parasitoid interactions and parasitoid intra‐specific competition in the biological system considered.     

Do assortative mating and immigrant inviability help maintain population genetic structuring of an herbivore on a crop and a wild relative?

Population genetic structuring is common among herbivorous insects and frequently is associated with divergent host plants, such as crops and their wild relatives. Previous studies showed population genetic structuring in corn leafhopper Dulbulus maidis in Mexico, such that the species consists of two sympatric, host plant-associated populations: an abundant and widespread "pestiferous” population on maize (Zea mays mays), and a small and localized "wild" population on perennial teosinte (Zea diploperennis). a maize wild relative with a limited distribution. This study addressed whether assortative mating and immigrant inviability mediate genetic structuring of corn leafliopper by comparing the mating and reproductive successes of pestiferous and wild females that colonize their nonassociated host plants against the successes of females colonizing their associated host plants. Assortative mating was assessed by comparing mating frequencies and premating and mating times among females of each population on each host plant: immigrant inviability was assessed by comparing, across two generations, the fecundity, survival, development time, sex ratio, and population growth rate among leafhopper populations and host plants. Our results showed that on maize, and compared to resident, pestiferous females, wild females were more likely to mate, and greater proportions of their offspring survived to adult stage and were daughters;consequently, the per-generation population growth rate on maize was greater for immigrant, wild leafhoppers compared to resident, pestiferous leafhoppers. Our results suggested that wild leafhoppers emigrating to maize have a fitness advantage over resident, pestiferous leafhoppers, while immigrant pestiferous and resident wild leafhoppers on teosinte have similar fitnesses.     

Population genetic structure of a specialist leafhopper on Zea: likely anthropogenic and ecological determinants of gene flow

Corn leafhopper, Dalbulus maidis DeLong & Wolcott (Hemiptera: Cicadellidae), is a specialist herbivore on the genus Zea (Poaceae). The genera Dalbulus and Zea evolved in central Mexico. We sought to determine whether population genetic structuring is prevalent in corn leafhoppers inhabiting three of its host plants: (1) the highland species perennial teosinte (Zea diploperennis Iltis, Doebley & Guzman), (2) the mid‐ to lowland‐species Balsas teosinte (Zea mays ssp. parviglumis Iltis & Doebley), and (3) the ubiquitous domesticated maize (Zea mays ssp. mays L.). We used amplified fragment length polymorphisms to detect population structuring and genetic differentiation among corn leafhoppers on the three host plants in western‐central and ‐northern Mexico. Our results showed that corn leafhopper in Mexico is composed of at least two genetically discrete populations: an ‘Itinerant’ population associated with the annual hosts maize and Balsas teosinte, which appears to be widely distributed in Mexico, and a ‘Las Joyas’ population restricted to perennial teosinte and confined to a small mountain range (Sierra de Manantlán) in western‐central Mexico. Our results further suggested that population structuring is not due to isolation by distance or landscape features: Las Joyas and Itinerant corn leafhopper populations are genetically distinct despite their geographic proximity (ca. 4 km), whereas Itinerant corn leafhoppers separated by hundreds of kilometers (>800 km), mountain ranges, and a maritime corridor (Sea of Cortez) are not genetically distinct. Based on our results and on published ethnohistorical and archaeological data, we propose pre‐Columbian and modern scenarios, including likely ecological and anthropogenic influences, in which the observed genetic population structuring of corn leafhopper could have originated and could be maintained. Also, we hypothesize that after evolving on the lowland Balsas teosinte, corn leafhopper expanded its host range to include maize and then the highland perennial teosinte, following the domestication and spread of maize within the last 9 000 years.     

Host plant dispersion, leaf hopper movement and disease transmission

Abstract.

• 1 The plant-to-plant movement of the corn leafhopper, Dalbulus maidis Delong & Wolcott, and the spread of the leafhopper-borne maize rayado fino virus were investigated in four patterns of maize (Zea mays) dispersion.
• 2 D. maidis was less abundant and the spread of the virus was slower in dense stands of maize than in sparse stands.
• 3 When plant density was held constant, leafhoppers were more abundant in maize stands with relatively equidistant plant spacing (uniform dispersion) than in stands with densely-sown rows (linear dispersion) or double-sown hills (clumped dispersion), but there was no difference in virus incidence among these plant dispersion patterns.
• 4 Leafhoppers were less likely to move to adjacent plants in uniform plant dispersion patterns than in either linear or clumped dispersion patterns. This result may explain the lack of higher virus incidence in uniform stands, despite higher leafhopper abundance.
• 5 Leafhopper movement was consistent with a simple rule: the shorter the distance to the next adjacent plant, the more likely a leafhopper is to move between plants.
• 6 These results demonstrate that host plant dispersion can affect the abundance and behaviour of highly mobile herbivorous insects even when plant density is constant.

     

Egg Parasitoid Complex of the Corn Leafhopper, <Emphasis Type="Italic">Dalbulus maidis</Emphasis> (DeLong) (Hemiptera: Cicadellidae), in Argentina

The corn leafhopper, Dalbulus maidis (DeLong), is the most important leafhopper pest of maize, Zea mays, in the Americas. A survey of the diversity of its egg parasitoids was carried out in northwestern Argentina. During summer from 2004 to 2007, the samples were collected, using sentinel eggs of D. maidis on corn leaves, exposed in 48 cornfields. Sixteen species belonging to four families of Chalcidoidea (Hymenoptera) were identified. Among the parasitoid groups, Trichogrammatidae was the most represented family with eight species, followed by Mymaridae with six species. The mymarid Anagrus incarnatus Haliday and the trichogrammatid Pseudoligosita longifrangiata (Viggiani) were the most abundant and frequent parasitoids. The mean percentage of parasitism of D. maidis eggs was 16.4% and varied greatly among the sites, ranging from 0 to 56.7%; generally, it was higher in Yungas and lower in Monte province sites. The species richness was higher in the localities within the Yungas, with 13 parasitoid species, of which two species were dominant, comprising 83.6% of the collected individuals. Monte was the province that showed the highest diversity index (H´ = 1.62). In addition, we present information on the distribution, known host associations of each parasitoid species and an identification key to all species of egg parasitoids of D. maidis in Argentina.     

Field overwintering biology of Spiroplasma kunkelii (Mycoplasmatales: Spiroplasmataceae) and its vector Dalbulus maidis (Hemiptera: Cicadellidae)

We studied the corn stunt spiroplasma (CSS), Spiroplasma kunkelii (Mycoplasmatales: Spiroplasmataceae) and its vector the corn leafhopper Dalbulus maidis (Hemiptera: Cicadellidae) under field conditions in Mexico. We surveyed for the presence of CSS in D. maidis by using PCR on samples of adults collected during the 2000–01 and 2003–04 winter (dry) seasons from irrigated low‐elevation sites and un‐irrigated high‐elevation sites. Also, we determined the body size and number of mature eggs of D. maidis females collected during the dry season in 2004 and in females collected on maize seedlings in the first months (June and July) of the wet (summer) season in 2005. Our PCR results showed that CSS was present in leafhopper adults collected during the 2000–01 and 2003–04 dry seasons in irrigated low‐elevation sites. However, in un‐irrigated high‐elevation sites, CSS was present in corn leafhopper adults caught before, but not during, the dry seasons. In these un‐irrigated high‐elevation sites, adult leafhoppers without CSS were recovered during the first 2 months (November and December) of the dry season from the foliage of wild perennial grasses. Females collected on wild perennial grasses in December 2004 showed similar head width and wing length to females caught on maize seedlings in June 2005. However, females collected on maize seedlings in July 2005 had the widest heads, longest wings and highest number of mature eggs. The largest body size of these females that arrived in July 2005 indicates that they are immigrants and support the Roff’s hypothesis that insect migrants should be larger than nonmigrants.     

Inoculation and acquisition of maize bushy stunt mycoplasma by its leafhopper vector Dalbulus maidis

Maize bushy stunt mycoplasma (MBSM), a mycoplasma-like organism, is transmitted in a persistent manner by the corn leafhopper, Dalbulus maidis, to maize (Zea mays). The influence of the duration of acquisition access and inoculation access periods on the transmission of MBSM by D. maidis was investigated. The proportion of plants infected by D. maidis increased significantly from 0 to 0.51 as the inoculation access time to a plant increased from 10 min to 72 h (X²= 101.5, P < 0.001). Likewise, the proportion of insects acquiring MBSM from infected plants increased from 0 to 0.19 as the acquisition access time to the source plant increased from 10 min to 72 h (X²= 53.2, P < 0.001). The data were fitted to a loglinear regression model. No significant association was found between the sex of the insects and vector ability.     

Exitianus obscurinervis (Hemiptera: Cicadellidae), a new experimental vector of Spiroplasma kunkelii

"Corn stunt" caused by the mollicute Spiroplasma kunkelii (Whitcomb) is potentially one of the most severe diseases affecting the corn (Zea mays L.) crop in the Americas, and the leafhopper Dalbulus maidis (DeLong & Wolcott) is considered its most important vector. However, other insects seen quite frequently in corn crops might well be its vectors in Argentina To identify any leafhoppers species other than D. maidis that can transmit S. kunkelii, transmission assays were conducted, using individuals of Exitianus obscurinervis (St?l) collected in field and reared under controlled conditions. S. kunkelii was transmitted to corn plants by E. obscurinervis. The pathogen was transmitted to seven of the 11 plants, which showed characteristic corn stunt symptoms, and the presence of the pathogen was confirmed by DAS-ELISA. The presence of S. kunkelii in the E. obscurinervis individuals used in transmission experiments was confirmed by polymerase chain reaction and electron microscopy. The current study shows the existence of a new experimental vector of S. kunkelii, the leafhopper E. obscurinervis, which acquired spiroplasmas from infected plants and inoculated it to healthy plants.     

Predicted ATP-binding cassette systems in the phytopathogenic mollicute <Emphasis Type="Italic">Spiroplasma kunkelii</Emphasis>

Spiroplasma kunkelii is a cell wall-free, helical, and motile mycoplasma-like organism that causes corn stunt disease in maize. The bacterium has a compact genome with a gene set approaching the minimal complement necessary for cellular life and pathogenesis. A set of 21 ATP-binding cassette (ABC) domains was identified during the annotation of a draft S. kunkelii genome sequence. These 21 ABC domains are present in 18 predicted proteins, and are components of 16 functional systems, which account for 5% of the protein coding capacity of the S. kunkelii genome. Of the 16 systems, 11 are membrane-bound transporters, and two are cytosolic systems involved in DNA repair and the oxidative stress response; the genes for the remaining three hypothetical systems harbor nonsense and/or frameshift mutations, so their functional status is doubtful. Assembly of the 11 multicomponent transporters, and comparisons with other known systems permitted functional predictions for the S. kunkelii ABC transporter systems. These transporters convey a wide variety of substrates, and are critical for nutrient uptake, multidrug resistance, and perhaps virulence. Our findings provide a framework for functional characterization of the ABC systems in S. kunkelii.Communicated by W. Goebel     

Interspecific competition between the braconid endoparasitoids Glyptapanteles porthetriae and Glyptapanteles liparidis in Lymantria dispar larvae

The effect of interspecific competition between the solitary endoparasitoid Glyptapanteles porthetriae Muesebeck (Hymenoptera: Braconidae) and the gregarious Glyptapanteles liparidis Bouché (Hymenoptera: Braconidae), was investigated in larvae of Lymantria dispar L. (Lepidoptera: Lymantriidae). Host larvae were parasitized by both wasp species simultaneously in premolt to the 2^nd or the 3^rd host instar or in an additional approach with a 4-day delay in parasitization by the second wasp species. Host acceptance experiments revealed that both wasp species do not discriminate between unparasitized host larvae and larvae parasitized previously by the same or the other species. In more than 90% female wasps parasitized the larva they encountered first. During the period of endoparasitic development, larvae of the competing parasitoid species never attacked the egg stage of the other species. When host larvae were parasitized simultaneously by both wasp species, the rate of successful development of both species depended on the age of the host larva at the time of its parasitization; G. liparidis emerged successfully from 44% of host larvae parasitized during the premolt to 2^nd instar, G. porthetriae from 28%, and in 20% of the hosts both parasitoid species were able to develop in one gypsy moth larva. However, when host larvae were parasitized simultaneously during premolt to the 3^rd instar, G. liparidis was successful in 90% of the hosts, compared to 8% from which only G. porthetriae emerged. In the experiments with delayed oviposition, generally the species that oviposited first succeeded in completing its larval development. Larvae of the species ovipositing with four days delay were frequently attacked and killed by larvae of the first parasitizing species or suffered reduced growth. As the secondary parasitoid species, G. porthetriae-larvae were never able to complete their development, whereas G. liparidis developed successfully in at least 12,5% of the multiparasitized host larvae. Thus, multiparasitism of gypsy moth larvae by both Glyptapanteles species corresponds to the contest type; however, G. porthetriae is only able to develop successfully as the primary parasitoid of young host larvae.     

Interspecific competition between the braconid endoparasitoids Glyptapanteles porthetriae and Glyptapanteles liparidis in Lymantria dispar larvae

The effect of interspecific competition between the solitary endoparasitoid Glyptapanteles porthetriae Muesebeck (Hymenoptera: Braconidae) and the gregarious Glyptapanteles liparidis Bouché (Hymenoptera: Braconidae), was investigated in larvae of Lymantria dispar L. (Lepidoptera: Lymantriidae). Host larvae were parasitized by both wasp species simultaneously in premolt to the 2^nd or the 3^rd host instar or in an additional approach with a 4‐day delay in parasitization by the second wasp species. Host acceptance experiments revealed that both wasp species do not discriminate between unparasitized host larvae and larvae parasitized previously by the same or the other species. In more than 90% female wasps parasitized the larva they encountered first. During the period of endoparasitic development, larvae of the competing parasitoid species never attacked the egg stage of the other species. When host larvae were parasitized simultaneously by both wasp species, the rate of successful development of both species depended on the age of the host larva at the time of its parasitization; G. liparidis emerged successfully from 44% of host larvae parasitized during the premolt to 2^nd instar, G. porthetriae from 28%, and in 20% of the hosts both parasitoid species were able to develop in one gypsy moth larva. However, when host larvae were parasitized simultaneously during premolt to the 3^rd instar, G. liparidis was successful in 90% of the hosts, compared to 8% from which only G. porthetriae emerged. In the experiments with delayed oviposition, generally the species that oviposited first succeeded in completing its larval development. Larvae of the species ovipositing with four days delay were frequently attacked and killed by larvae of the first parasitizing species or suffered reduced growth. As the secondary parasitoid species, G. porthetriae‐larvae were never able to complete their development, whereas G. liparidis developed successfully in at least 12,5% of the multiparasitized host larvae. Thus, multiparasitism of gypsy moth larvae by both Glyptapanteles species corresponds to the contest type; however, G. porthetriae is only able to develop successfully as the primary parasitoid of young host larvae.     

Corn Stunt Spiroplasma in the Leafhopper Euscelidius variegatus

Corn stunt spiroplasma (CSS) multiplied in all leafhoppers Euscelidius variegatus injected with a culture of CSS, reaching titres of over 1x10⁶ colony forming units (cfu) per insect and 2x10⁴ cfu per salivary gland of each insect. CSS could be isolated from the haemolymph and the salivary glands at any time after injection. The growth of CSS in culture was inhibited by insect extract at concentrations greater than the equivalent of 0.1 insect/ml. Transmission of CSS to sterile feeding solution and to broad beans were compared using 24 h feeding periods. A porportion of 1.7 % of injected leafhoppers began to transmit to sterile feeding solution through membranes by the 4th day after injection, and reached a maximum of 30 % by day 14. Similar insects started transmitting to broad bean plants on day 12 (2 %), reaching a maximum of 7.5 % by day 14. The number of spiroplasmas transmitted by each insect to sterile feeding solution increased from 3, cfu on day 4 to a maximum of 80 cfu by day 14. Helices were seen in the haemolymph at any time after injection. However, partially deformed cells were not present until the 1st week and clumps of 3–4 cells and small aggregates until the 3rd week after injection. The salivary glands of injected insects contained membrane-bound “pockets” or “colonies” packed with pleomorphic, filamentous (helical and non-helical) cells and aggregates. Intracellular colonies were always at the periphery of the acini and were easily detectable by fluorescence microscopy after staining with a DNA-binding fluorescent stain. Pleomorphic and filamentous cells were also seen intercellularly in the salivary glands.     

Growth inhibition of phytopathogenic spiroplasmas by membrane-interactive antimicrobial peptides Novispirin T7 and Caerin 1.1

Spiroplasma kunkelii and Spiroplasma citri, both helical-shaped cell wall-less bacteria, are the causative agents of corn stunt disease and citrus stubborn disease, respectively. Plants exhibiting natural resistance to these phytopathogenic spiroplasmas are currently lacking. Engineering artificial plant resistance using antimicrobial peptides (AMPs) has been conceived as a new approach to control the agronomically important spiroplasmal diseases. In preparation for such task, the present study focused on screening of AMPs that have potentials to curb the growth of S. kunkelii and S. citri. Four AMPs, including Novispirin T7, Caerin 1.1, Tricholongin and Dhvar4, were selected for in vitro growth inhibition test. A liquid assay method was developed for quick qualitative and quantitative evaluations of the AMPs. Our results demonstrated that Novispirin T7 and Caerin 1.1 were able to inhibit the growth of both phytopathogenic spiroplasmas with the efficacy comparable to that of tetracycline. Cell deformations were observed in spiroplasma cultures treated with these two peptides, indicating interactions of the AMPs with the spiroplasma cell membranes. The minimum inhibitory concentrations (MICs) of the AMPs against S. kunkelii and S. citri were determined.