Euscelis Incisus Research Articles

Identification and ecology of alternative insect vectors of \u2018Candidatus Phytoplasma solani\u2019 to grapevine

Bois noir, a disease of the grapevine yellows complex, is associated with ‘Candidatus Phytoplasma solani’ and transmitted to grapevines in open fields by the cixiids Hyalesthes obsoletus and Reptalus panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing by stamp gene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized by stamp gene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi, Dicranotropis hamata, Dictyophara europaea, Euscelis incisus, Euscelidius variegatus, Laodelphax striatella, Philaenus spumarius, and Psammotettix alienus/confinis) are alternative vectors of ‘Candidatus Phytoplasma solani’ to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management.

Diversity of phytoplasmas identified in the polyphagous leafhopper Euscelis incisus (Cicadellidae, Deltocephalinae) in Serbia: pathogen inventory, epidemiological significance and vectoring potential

Euscelis incisus is polyphagous leafhopper that is widely distributed and abundant in diverse agroecosystems. Two generations per year allows this species to come into contact with a plethora of different phytoplasmas. We first investigated the interactions of E. incisus with phytoplasmas in different ecosystems as a bioindicator of phytoplasma diversity. Among the 510 analyzed E. incisus specimens collected from eight localities in Serbia, 53 (10%) tested positive for diverse ‘Ca. Phytoplasma’ taxa. Restriction fragment length polymorphism (RFLP) analysis and sequence comparison based on the F2nR2 fragment of the 16S rRNA gene revealed the presence of six 16S phytoplasma ribosomal groups and eight subgroups in E. incisus (16SrI-R, 16SrI-F, 16SrII-E, 16SrIII-B, 16SrIX-C, 16SrIX-E, 16SrXI-G and 16SrXII-A). The most prevalent was the 16SrXII-A phytoplasma group, represented by two multilocus stolbur phytoplasma genotypes: STOLg and Rqg31g. While the 16SrXII-A phytoplasmas were identical to the previously recorded isolates from Serbia, the 16SrI-F and 16SrI-R subgroups closely related to the previously documented 16SrI-B and 16SrI-C subgroups were recorded for the first time in Serbia. In addition, records of the 16SrIX and 16SrXI groups represent the first findings of these phytoplasma groups in Serbia. Transmission trials of ‘Ca. P. solani’ were performed with naturally infected E. incisus adults. The leafhoppers successfully transmitted stolbur phytoplasma to exposed Catharanthus roseus plants, indicating their role as a natural vector.

Diversity of symbiotic microbiota in Deltocephalinae leafhoppers (Insecta, Hemiptera, Cicadellidae)

Symbiotic microorganisms associated with thirteen species of the subfamily Deltocephalinae were examined using microscopic and molecular techniques. Athysanus argentarius, Euscelis incisus, Doratura stylata, Arthaldeus pascuellus, Errastunus ocellaris, Jassargus flori, Jassargus pseudocellaris, Psammotettix alienus, Psammotettix confinis, Turrutus socialis and Verdanus abdominalis harbor two types of ancient bacteriome-associated microorganisms: bacteria Sulcia (phylum Bacteroidetes) and bacteria Nasuia (phylum Proteobacteria, class Betaproteobacteria). In Balclutha calamagrostis and Balclutha punctata, the bacterium Nasuia has not been detected. In the bacteriomes of both species of Balclutha examined, only bacteria Sulcia occur, whereas Sodalis-like symbionts (phylum Proteobacteria, class Gammaproteobacteria) are localized in the fat body cells, in close vicinity of the bacteriomes. To our knowledge, this is the first report of the co-existence in Deltocephalinae leafhoppers of the ancient symbiont Sulcia and the more recently acquired Sodalis-like bacterium. The obtained results provide further evidence indicating that Deltocephalinae leafhoppers are characterized by a large diversity of symbiotic systems, which results from symbiont acquisition and replacement. The obtained results are additionally discussed in phylogenetic context.

Diverse phytoplasmas associated with leguminous crops in Russia

A large scale survey of diseased legume plants (mainly clover and alfalfa in the Fabaceae family) was conducted from 2009 to 2013 in four Economic Regions of Russia, Northern (Arkhangelsk and Vologda oblast), Central (Moscow oblast), Volga (Samara oblast) and West Siberian (Novosibirsk oblast). The majority of infected clover plants exhibited symptoms typical of clover phyllody (CPh), clover yellow edge (CYE), or clover proliferation (CP), and infected alfalfa plants exhibited symptoms typical of alfalfa witches’-broom (AWB). Of 161 symptomatic plants from 22 different legume species, 103 tested positive for phytoplasma infection. Phytoplasmas belonging to four groups and six subgroups were detected, of which 31.1% were group 16SrI, with the majority belonging to subgroup 16SrI-C- (causal agent of CPh disease), two belonging to 16SrI-B and two group 16SrI phytoplasmas not identified to the subgroup level;47.6% were group 16SrIII, with the majority belonging to subgroup 16SrIII-B or 16SrIII-B variant (causal agent of CYE disease), and one strain belonging to16SrIII-F; 8.7% were subgroup 16SrVI-A (causal agent of CP and AWB diseases); 9.7% were subgroup 16SrXII-A (causal agent of AWB disease); and 2.9% were mixed infected with subgroups 16SrIII-B and 16SrI-C. The predominant phytoplasma species detected varied by region. In the Northern and Central Regions, the majority of the phytoplasmas detected belonged to subgroups 16SrI-C and 16SrIII-B. In the West Siberian and the Volga Regions, the phytoplasmas predominately detected belonged to subgroups 16SrVI-A and 16SrXII-A, respectively. Subgroup 16SrIII-F was detected in a single plant in the West Siberian Region and a mixed infection of 16SrIII-B and 16SrI-C was detected in three plants, one in the Northern Region and two in the Central Region. Eleven species of insects of the order Hemiptera, suborder Auchenorrhyncha, were collected from leguminous plants in the Moscow oblast of the Central Region. Euscelis incisus and Aphrodes bicinctus were the most prevalent species and may be potential phytoplasma vectors in the Central Region.

Diverse phytoplasmas associated with potato stolbur and other related potato diseases in Russia

A large scale survey on diseased potato plants that exhibited symptoms similar to those of stolbur, potato purple top wilt, potato witches’-broom, and potato round-leaf in eight Economical Regions of Russia was conducted from 2006 to 2012. A total of 1228 potatoes were collected and the associated phytoplasmas were identified. Phytoplasmas belonging to five 16S rRNA (16Sr) phytoplasma groups (16SrI, 16SrII, 16SrIII, 16SrVI, and 16SrXII) and at least eight subgroups (16SrI-B, 16SrI-C, 16SrI-P, 16SrII-A, 16SrIII-B, 16SrVI-A, 16SrVI-C, and 16SrXII-A) were identified. To our knowledge, subgroup 16SrIII-B and 16SrVI-C phytoplasmas have not been reported to infect potatoes. Based on the results of the survey, the percentages of infection caused by various phytoplasmas were: 40.2 % (16SrXII), 25.5 % (16SrIII), 20.9 % (16SrI), 12.4 % (16SrVI), and 1.2 % (16SrII). The results also indicated potato diseases exhibiting stolbur or similar symptoms could be caused by stolbur phytoplasma (16SrXII-A) alone or by other phytoplasma groups (e.g., 16SrI and 16SrIII). A survey on potential insect vectors was conducted from 2009 to 2012 in the Moscow oblast of the Central Region. A total of 23 different species of Hemiptera insects were screened for the presence of phytoplasmas. Eight leafhopper and three froghopper species carried phytoplasmas. Euscelis incisus and Macrosteles laevis were the most prevalent. The other species were present in very low populations.

Euscelis incisus(Cicadellidae, Deltocephalinae), a natural vector of 16SrIII-B phytoplasma causing multiple inflorescence disease ofCirsium arvense

We investigated multiple inflorescence disease of Cirsium arvense (CMI) and its association with phytoplasmas of the 16SrIII-B subgroup, potential natural vector(s) and reservoir plant(s). From five locations in northern Serbia, 27 plants of C. arvense, 1 C. vulgare and 3 Carduus acanthoides with symptoms of multiple inflorescences (MIs) were collected and tested for 16SrIII group phytoplasmas. All symptomatic plants were found to be infected. Tentative reservoir plants and insect vectors were collected at a Dobanovci site where the continuous presence of CMI disease was recorded. Among the 19 most abundant plant species submitted to phytoplasma testing, all symptomless, the presence of the 16SrIII group was detected only in two legumes: Lathyrus tuberosus (2/5) and L. aphaca (1/5). Among 19 insect species from six families of Auchenorrhyncha, the deltocephalid leafhopper Euscelis incisus was the only insect carrying a 16SrIII phytoplasma (10% of analysed individuals). Transmission trials were performed with naturally infected E. incisus adults of the summer generation and with a laboratory population reared on red clover. After an acquisition period of 48 h on C. arvense symptomatic for MIs and a latent period of 28 days, 83% of the E. incisus adults (300/360) were infected with CMI phytoplasma. In two transmission tests, the leafhoppers successfully transmitted the phytoplasma to exposed plants (C. arvense and periwinkle), proving its role as a natural vector. Test plants of C. arvense infected with the 16SrIII-B phytoplasma expressed typical symptoms similar to those observed in the field, such as MIs or the absence of flowering, shortened internodes and plant desiccation. Typical symptoms in infected periwinkles were virescence and phyllody. The molecular characterisation of the CMI phytoplasma isolates from diseased and asymptomatic field-collected plants, vectors, and test plants was performed by sequence analyses of the 16S rRNA, rpl22-rps3 and rpl15-secY genes. Phylogenetic analyses of other members of the 16SrIII group of phytoplasmas indicated closest relatedness with clover yellow edge phytoplasma (CYE) of the 16SrIII-B subgroup.

The molecular epidemiology of bois noir grapevine yellows caused by ‘Candidatus Phytoplasma solani’ in the Republic of Macedonia

Bois noir (BN), which is induced by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), is an important grapevine yellows disease that causes severe damage in viticultural regions throughout the Euro-Mediterranean basin. An epidemiological survey to determine potential insect vectors and the primary reservoir plants of BN phytoplasma in Macedonian vineyards was undertaken between 2012 and 2013 in the southeastern part of the country. A study on the species diversity from the suborder Auchenorrhycha revealed the prevalence of the principal vector of ‘Ca. P. solani’, which is the planthopper Hyalesthes obsoletus. Reptalus panzeri, which is the second-most documented BN vector, was not recorded in Macedonian vineyards. Three leafhopper species, namely Psammotettix alienus, Artianus manderstjernii and Euscelis incisus, were also widespread in the BN-affected vineyards, but only H. obsoletus tested positive for ‘Ca. P. solani’. Molecular characterizations were performed by the sequencing and/or RFLP typing of tuf, vmp1 and stamp genes, and the results were used to gain detailed insight into the molecular diversity of the ‘Ca. P. solani’ strains associated with grapevines, tentative reservoir plants (Urtica dioica and Convolvulus arvensis) and the H. obsoletus associated with these plants. Among the 91 ‘Ca. P. solani’ strains detected in diverse plant and insect hosts, three tuf, five vmp1 and 11 distinct stamp genotypes were identified. Twelve comprehensive genotypes of ‘Ca. P. solani’ were detected according to the tuf/vmp1/stamp genotyping. The highest diversity of genotypes was detected among the strains from H. obsoletus individuals associated with U. dioica, of which the most frequent genotype was tuf-ab/V18/M1 (43 %). The tuf-b/V2-TA/STOL comprehensive genotype was found in 33 % of naturally infected grapevines. Two ‘Ca. P. solani’ genotypes were associated with U. dioica, namely (i) tuf-ab/V18/M1 (60 %) and tuf-a/V3/M4 (40 %), and only one genotype (tuf-b/V2-TA/Rqg50) was associated with C. arvensis.

Potential Hemipteran vectors of “stolbur” phytoplasma in potato fields in Serbia

During 2014 composition of planthoppers and leafhoppers populations was studied in potato fields showing symptoms of ’’stolbur” phytoplasma infection in several regions in Serbia. Overall, 18 localitites have been inspected for the hemipteran specimens presence, and 19 species belonging to eight families could be identified. Nine species belong to the family Cicadellidae, three to Cixiidae and two to Delphacidae, whereas the families Aphrophoridae, Tettigometridae, Issidae, Cercopidae and Membracidae were present with only a single species. The most common was Psammotettix alienus (Dahlbom) collected on 16 sites, followed by Hyalesthes obsoletus Signoret, Reptalus quinquecostatus (Dufour), Reptalus panzeri (Low) and Euscelis incisus (Kirschbaum). All five dominant species captured in potato fields are known to harbour or vectoring “stolbur” phytoplasma, which gives new lights to possible pathways of potato “stolbur” transmission and propagation.

Lietuvoje aptikti vabzdžiai, galimi fitoplazmų ‘Candidatus Phytoplasma asteris’ ir ‘Ca. Phytoplasma pruni’ giminingų kamienų pernešėjai

Phytoplasma strains affiliated with groups 16SrI, 16SrIII, 16SrV and 16SrXII have been found in Lithuania, but their insect vectors in the country have not been determined. ‘Candidatus Phytoplasma asteris’ and ‘Ca. Phytoplasma pruni’-related phytoplasma strains were identified in five leafhopper species and three spittlebug species occurring in Lithuania. The occurrence in Lithuania of Anaceratagallia ribauti, reported as a vector of stolbur phytoplasma (‘Ca. Phytoplasma solani’, subgroup 16SrXII-A) elsewhere in Europe, provokes the question of whether this phytoplasma may be present but yet undetected in Lithuania. The finding of subgroup 16SrI-C strains in A. ribauti, Aphrodes sp., Macrosteles sexnotatus and Euscelis incisus; 16SrI-B strains – in Aphrophora alni, Aphrodes sp. and Cicadella viridis; 16SrIII-B strains – in A. ribauti; and 16SrIII-P – in Aphrodes sp. and E. incisus is consistent with the hypothesis that these leafhopper species are vectors of the respective phytoplasmas in Lithuania. Transmission trials were initiated based on these results. Results thus far have revealed that Lithuanian biotype of E. incisus is capable of vectoring subgroup 16SrI-C phytoplasma strains that are found in Lithuania. The presence of diverse phytoplasmas in three spittlebug species, Aphrophora alni, Lepyronia coleopterata and Philaenus spumarius, indicates that these xylem feeders actually ingested phloem sieve cell contents during feeding on phytoplasmainfected plants, bringing into focus the question of whether some of such xylem feeders might act as occasional vectors of phytoplasmas.

Variation in vector competency depends on chrysanthemum yellows phytoplasma distribution within Euscelidius variegatus

AbstractPhytoplasmas are plant‐pathogenic Mollicutes transmitted by leafhoppers, planthoppers, and psyllids in a persistent propagative manner. Chrysanthemum yellows phytoplasma (CY) is a member of ‘Candidatus Phytoplasma asteris’, 16Sr‐IB, and is transmitted by at least three leafhopper species, Macrosteles quadripunctulatus Kirschbaum, Euscelidius variegatus Kirschbaum, and Euscelis incisus Kirschbaum (all Homoptera: Cicadellidae: Deltocephalinae). Although M. quadripunctulatus transmits CY with very high efficiency (near 100%), 25% of E. variegatus repeatedly fail to transmit CY. The aims of this work were to correlate vector ability with different pathogen distribution in the insect body and to investigate the role of midgut and salivary glands as barriers to CY transmission. Euscelidius variegatus individuals acquired CY by feeding on infected plants or by abdominal microinjection of a phytoplasma‐enriched suspension. Insects were individually tested for transmission on daisy seedlings [Chrysanthemum carinatum Schousboe (Asteraceae)], and thereafter analysed by real‐time polymerase chain reaction (PCR) for CY concentration on whole insects or separately on heads and the rest of the body. Hoppers were classified as early and late transmitters or non‐transmitters, according to the time inoculated plants required for expression of CY symptoms. Similar transmission efficiencies were achieved following feeding or abdominal microinjection, suggesting that salivary glands may be a major barrier to transmission. Following acquisition from infected plants, all transmitters tested positive by PCR, and 60% of non‐transmitters also tested positive although with a significantly lower CY concentration. This indicates that a minimum number of phytoplasma cells may be required for successful transmission. The midgut may have prevented phytoplasma entry into the haemocoel of PCR‐negative non‐transmitters. Results suggest that both midgut and salivary glands may act as barriers. To assess the effect on CY transmission of a specific parasitic bacterium of E. variegatus, tentatively named BEV (Bacterium Euscelidius variegatus), we established a BEV‐infected population by abdominal microinjection of BEV bacteria. The presence of BEV did not significantly alter the efficiency of CY transmission.

Transmission of a Stolbur Phytoplasma by the Agalliinae Leafhopper Anaceratagallia ribauti (Hemiptera, Auchenorrhyncha, Cicadellidae)

AbstractSeventeen Auchenorrhyncha species were studied for their ability to transmit stolbur phytoplasma. Adult insects were collected alive in Bois Noir infected vineyards, transferred to Vicia faba seedlings (two leaf stage) in cylindrical pot cages and kept in a growth chamber at 23°C (L16:D8). After 5 weeks, plants were inspected visually and sampled for PCR analysis. Anaceratagallia ribauti, Dryodurgades reticulatus and Euscelis incisus were also reared in the laboratory. Insects trapped in the vineyard were allowed to oviposit on V. faba seedlings. Hatching nymphs were transferred to cubical breeding cages containing potted V. faba seedlings and stolbur‐infected Convolvulus arvensis collected in the vineyard. Plants in these cubical cages were routinely tested by PCR for stolbur infections. Adults raised in these cubical cages were subjected to further pot cage experiments with V. faba seedlings. In five of 40 experiments with field‐trapped A. ribauti, stolbur transmission was observed. Laboratory‐reared A. ribauti transmitted stolbur phytoplasma to seven of 23 broad beans in the cubical breeding cages. Laboratory‐reared A. ribauti adults transmitted stolbur phytoplasma in one of 13 pot cage experiments. No transmission of stolbur phytoplasma with any other Auchenorrhyncha species was observed. Furthermore, we characterized Austrian stolbur isolates by Restriction Fragment Length Polymorphism analysis of the tuf gene. Plant samples were collected in 17 symptomatic vineyards all over eastern Austria. Vicia faba plants from transmission experiments were also analysed. All isolates belonged to tuf‐type II.

Insect Injection and Artificial Feeding Bioassays to Test the Vector Specificity of Flavescence Dorée Phytoplasma

ABSTRACT The specificity of vector transmission of Flavescence dorée phytoplasma (FDP) was tested by injecting FDP, extracted from laboratory-reared infective Euscelidius variegatus, into specimens of 15 other hemipteran insect species collected in European vineyards. Concentrations of viable phytoplasma extracts and latency in vectors were monitored by injection of healthy-reared E. variegatus leafhoppers. Based on these preliminary results, insects were injected by using phytoplasma extracts that ensured the highest rate of FDP acquisition and transmission by E. variegatus. Transmission into an artificial diet through a Parafilm membrane about 3 weeks after insect injection was attempted. FDP-injected insects that belonged to 15 hemipteran species were confined in cages and fed through the membrane for a 4- to 5-day inoculation access period. FDP DNA was detected by polymerase chain reaction (PCR) in the feeding buffer fed upon by Anoplotettix fuscovenosus, Aphrodes makarovi,E. variegatus, and Euscelis incisus. PCR amplification with specific primers detected FDP DNA in injected insects of all test insect species. Band intensity was positively correlated with the transmissibility of FDP. Transmission of FDP to plants by feeding was confirmed for Anoplotettix fuscovenosus, E. variegatus, and Euscelis incisus, but not for Aphrodes makarovi. Our results suggest that vector competency of FDP is restricted to specimens belonging to the family Cicadellidae, subfamily Deltocephalinae.

TRANSMISSION OF CHRYSANTHEMUM YELLOWS, A SUBGROUP 16SrI-B PHYTOPLASMA, TO GRAPEVINE BY FOUR LEAFHOPPER SPECIES

SUMMARY Chrysanthemum yellows phytoplasma (CY), subgroup 16SrI-B, was experimentally transmitted to seedlings of grapevine (Vitis vinifera), cv. ‘Barbera’ and ‘White Muscat’ with four leafhopper species: Euscelidius variegatus, Euscelis incisus, Macrosteles quadripunctulatus and Scaphoideus titanus. Acquisition was accomplished in three days by 3 rd instar nymphs on CY-infected marguerites (Chrysanthemum carinatum). After three weeks, insects were moved to new test plants of the same species and allowed to feed for seven days. They were then transferred to grapevine seedlings for another seven-day-period, and so on as long as they survived. The four leafhopper species transmitted CY to both marguerite and grapevine with the following respective rates: M. quadripunctulatus, 100 (25/25) and 33.3 (9/27); E. variegatus , 59.6 (31/52) and 47.5 (28/59); E. incisus, 95.6 (22/23) and 33.3 (6/18); S. titanus 12.5 (7/56) and 17.0% (8/47). Symptoms on C. carinatum were typical of those already described, while on V. vinifera they consisted of growth reduction and abnormal production of axillary shoots bearing small chlorotic leaves. Downward rolling and reddening of basal leaves was occasionally observed on cv. ‘Barbera’. RFLP analysis of ribosomal DNA amplified in direct and/or nested PCRs driven by universal or 16SrI group-specific primers confirmed that the phytoplasma present in symptomatic plants, either used as inoculum or obtained by transmission, and in inoculative S. titanus did belong to subgroup 16SrI-B and was identical to the CY reference isolate.

Cyclamen (Cyclamen persicum L.): a dead-end host species for 16Sr-IB and -IC subgroup phytoplasmas

Phytoplasmas belonging to the 16S rDNA subgroups IB and IC were found in five cyclamen (Cyclamen persicum L.) plants showing virescence and yellow stunted leaves and one plant showing phyllody, rolled and thickened leaves, respectively. Two cyclamens, representing the two syndromes, were chosen as source plants for transmission trials in which three leafhopper species, known as vectors of IB and IC subgroup phytoplasmas, were used to inoculate cyclamen and periwinkle [Catharanthus roseus (L.) G. Don] test plants. Out of 366 tested plants only one periwinkle exposed to Euscelis incisus was found harbouring a 16Sr-IB phytoplasma. Out of 60 tested vector insects, only one adult of Macrosteles quadripunctulatus and two of E. incisus fed on 16Sr-IB source cyclamen gave a positive amplification signal in nested PCR. This extremely low level of transmission to both cyclamen and the very susceptible periwinkle strongly suggests that cyclamen, commonly found infected in crops, is an unsuitable species for phytoplasma acquisition and can be regarded as a dead-end host plant for phytoplasmas belonging to both IB and IC subgroups. Indications for glasshouse management are drawn from these findings. Among the leafhoppers investigated E. incisus falls most under suspicion since it feeds better than the others on cyclamen, was able to transmit the disease to one periwinkle plant, and IB phytoplasmas were detected in two individuals.

Direct PCR detection of phytoplasmas in experimentally infected insects

Summary.Phytoplasmas in leafhoppers have been detected by PCR using chrysanthemum yellows (CY)‐infected chrysanthemum as source plants, and two cicadellid Deltocephalinae species, Macrosteles quadripunctulatus and Euscelis incisus, as vectors. Three different primer pairs were used; two of these are universal and have been designed on conserved sequences of the 16S rRNA gene of phytoplasmas, and one was designed on extrachromosomal DNA of a severe strain of western aster yellows phytoplasma. They were used to amplify CY DNA obtained by two different extraction procedures; one was extraction with cetyl‐trimethyl‐ammonium‐bromide (CTAB), and the other was boiling in Tris‐EDTA buffer. The chromosomal primers amplified phytoplasma‐specific bands only from “CTAB” samples, while the plasmid primers were successful with both procedures. Amplification of phytoplasma DNA was possible from as little as 1/10000 of total DNA extracted from a single hopper. Failure to amplify phytoplasma DNA from insects stored at –20oC for 2 yr suggested that some kind of inhibition develops during long term tissue storage. Direct PCR appeared a very specific, sensitive and rapid method to detect phytoplasmas in fresh leafhoppers, provided that a proper combination of extraction and amplification procedures was used.

Differential acquisition of chrysanthemum yellows phytoplasma by three leafhopper species

AbstractChrysanthemum yellows (CY) phytoplasma has been transmitted with three leafhopper species: Euscelidius variegatus (Kirschbaum), Macrosteles quadripunctulatus (Kirschbaum) and Euscelis incisus (Kirschbaum): the first two species are reported as CY phytoplasma vectors for the first time. Leafhoppers were allowed to acquire the pathogen from the following source plants: Apium graveolens L., Catharanthus roseus L., Chrysanthemum carinatum Schousboe L. and C. frutescens L. DNA extracted from healthy or inoculative leafhoppers‐exposed plants were analyzed by dot‐blot and Southern hybridizations with a molecular probe constructed onto a fragment of European aster yellows phytoplasma DNA. The three leafhopper species were able to transmit CY phytoplasma after acquisition on chrysanthemum, but only M. quadripunctulatus and E. variegatus transmitted after feeding on periwinkle, and none acquired it from celery. All plant species tested were susceptible to CY, but while chrysanthemum and periwinkle were suitable for both inoculation and acquisition, celery did not seem to be a good source of phytoplasma for further inoculations. It is concluded that host plants influence leafhoppers' vectoring ability, possibly due to the different feeding behaviour of the insects on diverse plant species. Since CY, like several other phytoplasmas, can be transmitted by different insect species, it is likely that a close transmission specificity probably does not exist between phytoplasmas and their leafhopper vectors.

The Hemiptera of Two Sown Calcareous Grasslands. II. Differences Between Treatments

(1) Hemiptera were sampled by vacuum insect net from replicated sown swards of two contrasting types, a fine-leaved grass mixture and a coarse one. Samples were taken in late autumn and winter 1973 and throughout 1974 and 1975. (2) Species richness of grassland Heteroptera was low and few differences between treatments were observed. Significantly more plant bugs overwintered in the coarse grassland than in the fine, with consistent, significant differences between treatments for Notostira elongate and Nabisferus. (3) Auchenorhyncha were more abundant on the coarse grassland in 1974, but on the fine grassland in 1975. Species richness, equitability, and especially diversity, were higher on the coarse sward. The fauna of the coarse grassland tended to be more diverse at any one time, but cumulatively the fauna of the fine sward was slightly more diverse. (4) Species predominant on the coarse grassland were Neophilaenus lineatus, Conosanus obsoletus, Mocydia crocea, Cicadula persimilis, Stenocranus minutes, Zyginidia scutellaris and Javesella pellucida (in the second generation only). The last four species were abundant. (5) Species predominant on the fine grassland were Deltocephalus pulicaris, Turrutus socialis, Euscelis incisus, Paluda adumbrate, Mocydiopsis attenuate, Dikraneura variata and Criomorphus albomarginatus. None of these species was abundant, and significant differences between treatments were observed only in 1975 for three of them. (6) A few species showed a response to the two grassland types which varied in time. Arthaldeus pascuellus was significantly more numerous on the coarse sward in summer 1974, but much more abundant on the fine sward in summer 1975. (7) The results are discussed in relation to the known feeding requirements of the species and to the establishment of grassland swards for nature conservation. It is concluded that mixtures of many grass species should be used to establish patches of grassland of variable structure and composition.

Experimental studies on the mycetome symbiosis in the leafhopper Euscelis incisus

The leafhopper Euscelis incisus contains two morphologically-distinct symbiotic microorganisms, a-symbionts and t-symbionts, in a pair of discrete organs, the mycetomes. Pure preparations of t-symbionts were obtained by lysis of insect cells with 0.05% digitonin, followed by density-gradient centrifugation, but the a-symbionts could not be isolated. Symbiont-free E. incisus were obtained by ligation of newly deposited eggs. The embryos developed normally to hatching, but the larvae did not grow and died in 2 weeks. The mycetome symbionts were not disrupted in larvae feeding on plants with the antibiotic chlortetracycline at 60 μg g −1 shoot but the guts of these insects lacked microorganisms and the larvae grew slowly and died in the second or third instar. Control insects had a substantial gut microflora. It is concluded that both the mycetome symbionts and the gut flora are required for normal growth and development of larval E. incisus. The incorporation of [ 14C]leucine into protein by isolated mycetomes of E. incisus was insensitive to chloramphenicol, an inhibitor of prokaryotic protein synthesis. It is suggested that the mycetome symbionts synthesise protein at very low rates and this may account for their insensitivity to chlortetracycline.

Endocytobiosis: General principles

The leafhopper Euscelis incisus K. (Homoptera) hosts two types of obligate intracellular symbionts (endocytobionts). The DNA molecular weight (∼10 8) of endocytobionts corresponds to that of the mitochondria and plastids. They are transmitted to the next host generation by incorporation between the egg coat and egg cell in the form of an infection mass. Excision of the infection mass results in cephalothorax embryos which lack the abdomen. Endocytobionts synthesize metabolites such as vitamins, amino acids for the hosts using their waste products such as urea and uric acid. The endocytobionts regulate pH, osmotic pressure and certain endogenous rhythms of their hosts. This implies that the leafhopper endocytobionts represent for the host cell not only nutrition but also genomic supplement. According to this hypothesis, the structure, function and information of endocytobionts and eukaryotic DNA-containing cell organelles are analogous; these analogies indicate that endocytobionts may provide a model for molecular analysis of eukaryotic cell system. “Endocytobiology” consequently represents a modern field of research between symbiosis and cell biology.