Nine polymorphic microsatellite loci from the psyllid Cacopsylla pruni (Scopoli), the vector of European stone fruit yellows

Cacopsylla pruni is the vector of European stone fruit yellows, a quarantine disease of Prunus trees. Nine polymorphic microsatellite markers were developed from enriched DNA libraries. Allelic variability was assessed in a collection of 149 females obtained from five localities covering a large geographical area in France. The number of detected alleles ranged from 8 to 37. Within the localities, observed and expected heterozygosities averaged across loci ranged from 0.39 to 0.55, and from 0.68 to 0.81, respectively. A heterozygote deficiency was detected for almost all loci, possibly due to a high null allele frequency. Other possible causes of the homozygote excess (mode of reproduction, inbreeding, assortative mating or Wahlund effect) are discussed. These variable microsatellite loci can provide tools to assess overall genetic variation in this important vector species. They will be used to search for population structure and migration patterns of C. pruni.     

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

Phytoplasmas 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.     

Cacopsylla costalis (Flor 1861), as a Vector of Apple Proliferation in Trentino

Recently, a epidemic of apple proliferation (AP) in an orchard area of Trentino (North Italy) occurred. The most affected cultivars were Golden Delicious, Florina and Renetta Canada grafted on different rootstocks. In this area the known or supposed vectors of the disease were not present. At the same time as the AP symptoms appeared, a notable increase of the presence of psyllids was observed on apple trees so a correlation between these insects and the AP was hypothesized. Four different psyllid species were found in the orchard: Cacopsylla melanoneura (Förster 1848), Cacopsylla costalis (Flor 1861), Cacopsylla mali (Schmidberber 1836) and Trioza urticae (Linnaeus 1758). The first two were more frequent in spring at the adult stage. In 1997 C. costalis was particularly numerous and was used to plan AP transmission trials. The transmission from infected AP apple trees to the insect in field and from the psyllids to non-infected Golden Delicious and Florina plants in the greenhouse were conducted. For the control of phytoplasma presence in the psyllids and in the apple plants polymerase chain reaction (PCR), nested PCR and restriction fragment length polymorphism analysis were used. In autumn 1997, some inoculated plants showed symptoms of AP. Molecular results were consistent with the presence of phytoplasma in C. costalis and in inoculated apple cultivars.     

Cage and field experiments as basis for the development of control strategies against Cacopsylla pruni,the vector of European Stone Fruit Yellows

The efficacy and the instant effect of 13 insecticides and antifeedants towards Cacopsylla pruni, the vector of ‘Candidatus Phytoplasma prunorum’ were examined in cage studies (no choice experiments with 10 field‐collected overwintered adults per experiment) on potted apricot trees (budding trees under outdoor conditions in early spring and foliated seedlings kept at 21°C). Cypermethrin caused 100% insect mortality within 2–4 h, thiacloprid 90–100% mortality within 24 h both on foliated and on budding trees. On budding trees spinosad led to 70–90% mortality within 24 h, thixotropic white trunk paint to 90% mortality within 48 h. On foliated seedlings flonicamid gave 70–100% mortality within 1 day, abamectin, spinosad, acetamiprid and spirotetramat 70–100% within 72 h. Field studies monitoring the effects of thiacloprid on remigrants of C. pruni by yellow sticky traps were carried out in two apricot orchards. Additionally the influence of the insecticide on insect dispersal was examined by mark, release and recapture trials. As compared to the control thiacloprid significantly reduced the catches of naturally occurring and released insects, decreased the number of trees on which released insects were recaptured (by 25–100%) and shortened the migration distances of the released insects by more than half. Our results suggest that appropriate insecticide treatments both reduce C. pruni populations and have a direct effect on pathogen transmission. Application of Cypermethrin before bloom and thiacloprid after bloom seem best suited to achieve these objectives. Thixotropic white trunk paint could, a formulation for spray application provided, eventually be a sustainable alternative or complement for treatments before bloom.     

Vector-pathogen-host plant relationships of chrysanthemum yellows (CY) phytoplasma and the vector leafhoppers Macrosteles quadripunctulatus and Euscelidius variegatus

Chrysanthemum yellows (CY) phytoplasma is a plant-pathogenic mollicutes belonging to the 16Sr-IB genetic group which infects a variety of dicotyledonous plants and is transmitted in nature by some species of Cicadellidae Deltocephalinae. The transmission characteristics of CY and the factors influencing the vector efficiencies of the leafhoppers Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum are described in the present study using transmission experiments and phytoplasma-specific polymerase chain reaction (PCR) assays. Vector insects were allowed to acquire CY under different experimental conditions and then transferred to healthy test plants for inoculation and/or sampled for DNA extraction and amplification. The transmission efficiency of CY was very high and almost all the leafhoppers became infective following acquisition on CY-infected daisies. The latent period in the vector ranged from 16 to 20 days after the start of the acquisition and infectivity lasted, in general, for life. The PCR assay was successful in detecting CY phytoplasmas in the insects well before they became infective (5 versus 16–18 days) and was used to estimate the proportion of infective insects. When analysed for CY presence by PCR, all the leafhoppers fed for 7–18 days on source daisy reacted positively while, following one day of acquisition, some insects failed to provide amplification. Host-plant species influenced CY acquisition, and daisy appeared a more efficient source for both leafhoppers compared to periwinkle. Life stage did not appear to be critical for CY acquisition, although newly-hatched nymphs of E. variegatus acquired CY less efficiently than fifth instar nymphs.     

Vector status of three leafhopper species for Australian lucerne yellows phytoplasma

Abstract   The leafhoppers Orosius argentatus (Evans), Austroagallia torrida (Evans) and Batracomorphus angustatus (Osborn) were used in transmission tests to determine their vector status for the phytoplasma associated with Australian lucerne yellows (ALuY). Caged, seed-grown lucerne plants were monitored for foliar symptom expression after feeding by leafhoppers transferred from ALuY symptomatic lucerne plants. Twelve of 25 plants developed phytoplasma disease-like symptoms including stunting and yellowing. The most pronounced foliar symptoms were displayed by five plants that had been fed on by O. argentatus and four plants that had been fed on by A. torrida. One plant, fed on by O. argentatus , showed the distinctive root symptoms of ALuY . A phytoplasma was identified by electron microscopy in two plants fed on by O. argentatus and one by A. torrida. For each group of plants that had been fed on by a single leafhopper species, one plant was phytoplasma positive as determined by the polymerase chain reaction (PCR) using universal primers. The phytoplasma detected by PCR in the plant fed on by A. torrida was identified by restriction fragment length polymorphism (RFLP) analysis as the tomato big bud (TBB) phytoplasma. The PCR product from two plants fed on by B. angustatus and O. argentatus were too faint for RFLP analysis. PCR assays were conducted on DNA extracted from the head and thorax of each leafhopper species from transmission tests and from field-collected insects, but no phytoplasma DNA was detected. These findings suggest O. argentatus is a vector of the ALuY pathogen and A. torrida is a vector of the TBB phytoplasma.     

Detection and variability of aster yellows phytoplasma titer in its insect vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae)

The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper, Macrosteles quadrilineatus Forbes, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual aster leafhoppers, we developed a quantitative real-time polymerase chain reaction assay to measure AYp concentration in insect DNA extracts. Absolute quantification of AYp DNA was achieved by comparing the amplification of unknown amounts of an AYp target gene sequence, elongation factor TU (tuf), from whole insect DNA extractions, to the amplification of a dilution series containing known quantities of the tuf gene sequence cloned into a plasmid. The capabilities and limitations of this method were assessed by conducting time course experiments that varied the incubation time of AYp in the aster leafhopper from 0 to 9 d after a 48 h acquisition access period on an AYp-infected plant. Average AYp titer was measured in 107 aster leafhoppers and, expressed as Log10 (copies/insect), ranged from 3.53 (+/- 0.07) to 6.26 (+/- 0.11) occurring at one and 7 d after the acquisition access period. AYp titers per insect and relative to an aster leafhopper chromosomal reference gene, cp6 wingless (cp6), increased approximately 100-fold in insects that acquired the AYp. High quantification cycle values obtained for aster leafhoppers not exposed to an AYp-infected plant were interpreted as background and used to define a limit of detection for the quantitative real-time polymerase chain reaction assay. This method will improve our ability to study biological factors governing AYp replication in the aster leafhopper and determine if AYp titer is associated with frequency of transmission.     

Detection, characterisation and transmission by Macrosteles leafhoppers of watercress yellows phytoplasma in Hawaii

A new yellows disease of watercress (Nasturtium officinale) in Hawaii has symptoms of reduced leaf size, leaf yellowing and crinkling, and occasionally witches’ brooms. This disease is found on all watercress farms on Oahu but has not yet been found on other Hawaiian islands. Watercress plants were tested for phytoplasma infection by polymerase chain reaction assays using phytoplasma‐specific primers. Amplicons of the expected sizes were produced from all symptomatic plants but not from healthy plants raised from seed. Phylogenetic analysis of the 16S rRNA gene indicated that watercress yellows was caused by a phytoplasma in the aster yellows group, with sequence similarity to onion yellows from Japan. Six weed species collected from the vicinity of affected watercress farms, Amaranth sp., Eclipta prostrata, Emilia sonchifolia, Plantago major, Myriophyllum aquaticum and Sonchus oleraceus, were also determined to be hosts of this phytoplasma. Leafhoppers, identified as Macrosteles sp. (Hemiptera, Cicadellidae), collected from symptomatic watercress transmitted this phytoplasma to watercress, plantain and lettuce (Lactuca sativa) in greenhouse experiments.     

Biological and molecular evidence for the transmission of aster yellows phytoplasma to French marigold (Tagetes patula) by the flatid planthopper Metcalfa pruinosa

Aster yellows (AY) phytoplasmas (Candidatus Phytoplasma asteris) are associated with a number of plant diseases throughout the world. Several insect vectors are responsible for spreading AY diseases resulting in wide distribution and low host specificity. Because the role of sucking insects as vectors of phytoplasmas is widely documented, and the citrus flatid planthopper Metcalfa pruinosa is a phloem feeder, it has been incriminated as a possible vector of phytoplasmas. However, its ability to transfer phytoplasma has not been confirmed. The present work shows that M. pruinosa (Hemiptera: Flatidae), a polyphagous planthopper, is able to vector Ca. P. asteris to French marigold (Tagetes patula). Transmission experiments were conducted in 2017 and 2018 in central Hungary by two approaches: (a) AY-infected M. pruinosa were collected from an area with severe incidence of the disease on T. patula and caged on test plants for an inoculation-access period of 2 weeks, and (b) presumably phytoplasma-free insects were collected from apparently healthy grapevines (Vitis vinifera L.) and fed on AY-infected T. patula plants for 2 weeks prior to being caged on test plants. AY disease symptoms developed on 4 out of 10 and 10 out of 15 test plants, respectively. All phytoplasma-positive marigold and M. pruinosa samples showed identical RFLP patterns and shared 100% 16S rDNA sequence identity with each other and with the aster yellows phytoplasma strain AJ33 (GenBank accession number MK992774). These results indicated that the phytoplasma belonged to the phytoplasma subgroup 16SrI-B Ca. P. asteris. Therefore, the work presented here provides experimental evidence that M. pruinosa is a vector of a 16SrI-B subgroup phytoplasma to T. patula.     

Low frequency of horizontal and vertical transmission of two begomoviruses through whiteflies exhibits little relevance to the vector infectivity

Transmissions of plant viruses between individuals of their vector insects through mating are rare events. Recently, three begomoviruses were found to be transmitted between males and females of the whitefly Bemisia tabaci through mating, and two viruses were shown to be transmitted transovarially to progeny. However, results between reports were not consistent. Here we examined the horizontal and vertical transmission of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl China virus (TYLCCNV) by the B and Q biotypes of B. tabaci, using virus isolates and whitefly colonies established recently in China. Both TYLCV DNA and TYLCCNV DNA were shown to be transmitted horizontally and vertically by each of the two biotypes of the whitefly, but frequency of transmission was usually low. In transovarial transmission, virus DNA was detected in eggs and nymphs but not in the adults of the first generation progeny, except in the combination of TYLCV and Q biotype whitefly where 2–3% of the offspring adults contained the virus DNA. We also showed that the first generation adults, which developed from eggs of viruliferous whiteflies, were not infective to plants. These results demonstrated that for the viruses and whiteflies tested here low frequency of horizontal and vertical transmission can be expected but these two modes of transmission are unlikely to have much epidemiological relevance in the field.     

Occurrence,identification and transmission of the phytoplasma associated with tomato big bud disease and identification of its vector and weed host in Pakistan

Abstract

Tomato (Solanum lycopersicum L.) plants showing stunting, big bud, leaves yellowing or reddening and witches’-broom symptoms were observed since 2009 in Pakistan. A weed Parthenium hysterophorus grown in and around tomato fields also exhibited witches’-broom like symptoms. Fluorescence light microscopy of hand-cut stem stalk sections treated with Dienes’ stain showed blue areas in the phloem region of both tomato and P. hysterophorus symptomatic plants that indicated the association of phytoplasma with the complex. Amplification of 1.2?kb 16S rDNA fragment in nested PCR confirmed that the symptomatic tomato and P. hysterophorus plants are infected by a phytoplasma. Partial sequencing of 16S rRNA (GenBank accession: LT671581 and LT671583) and virtual restriction fragment length polymorphism confirmed that the phytoplasma associated with both plant species had the greatest homology to 16SrII-D subgroup. Disease was successfully transmitted by grafting and leafhopper Orosius albicinctus in tomato plants. This is the first report of natural occurrence of 16SrII-D phytoplasma in tomatoes and a weed P. hysterophorus in Pakistan.     

Detection and molecular characterization of an aster yellows phytoplasma in poker statice and Queen Anne's lace in Alberta, Canada

Queen Anne's lace and poker statice plants were found with a yellows-type disease with typical phytoplasma symptoms in an experimental farm near Brooks, Alberta in 1996. Phytoplasma bodies were detected by transmission electron microscopy in phloem cells of symptomatic plants, but not in healthy plants. The presence of a phytoplasma was confirmed by analysis with the polymerase chain reaction. Using a pair of universal primer sequences derived from phytoplasma 16S rRNA, an amplified product of the expected size (1.2 kb) was observed in samples from infected plants, but not in asymptomatic plants. Sequence analysis of the PCR products from the 16S/23S rDNA intergenic spacer region indicated that the two phytoplasma isolates in Queen Anne's lace and poker statice are genetically closely related to the western aster yellows phytoplasma.     

Study of the transmission of stolbur phytoplasma to different crop species, by Macrosteles quadripunctulatus

In an epidemiological study conducted on commercial agricultural plots affected by stolbur phytoplasma in Northern and Central Spain, different species of leafhoppers and planthoppers were identified as potential vectors of the phytoplasma. They included individuals of Macrosteles quadripunctulatus infected by stolbur phtytoplasma in most of the locations. The potential of this species as a vector of stolbur was evaluated in this work. The transmission trials were carried out on healthy plants of Catharanthus roseus (periwinkle), Lycopersicon esculentum (tomato), Daucus carota (carrot), Lactuca sativa (lettuce) and Vitis vinifera (grapevine). The first symptoms of infection in these plants were observed 2 weeks after the inoculation period in tomato and periwinkle, and after 4 weeks in carrot. Only one of five grapevines showed phytoplasma symptoms. PCR analysis was used to verify the ability of M. quadripunctulatus in transmitting stolbur phytoplasma in the plant species tested. The phytoplasma was not detected in lettuce or in the healthy control plants. Studies of stolbur transmission to insect‐feeding medium were also conducted and indicated that M. quadripunctulatus acquires and was capable of transmitting the phytoplasma after it fed during a single day on infected plants followed by a 19‐day latent period on healthy plants.     

European Stone Fruit Yellows: A Mark,Release and Recapture Experiment Tracking the Dispersal of its Vector Cacopsylla pruni (Hemiptera: Psyllidae) in a Model Apricot Orchard and Epidemiological Studies in Lower Austria

During the last 15 years, European stone fruit yellows (ESFY) has become a major concern in Austrian fruit production. Therefore, presence and temporal dynamics of its vector Cacopsylla pruni were investigated using a beating tray method and yellow sticky traps on Prunus armeniaca, Prunus domestica, Prunus spinosa and P. cerasifera nigra. Infection rates of C. pruni and Prunus spp. trees were assessed by direct, nested and real‐time PCR. Movement of remigrants in a model apricot orchard was tracked by aid of a mark, release and recapture study. Insects were marked by fluorescent dyes. Movement of the marked insects and presence of naturally occurring insects were monitored by yellow sticky traps. In 2011, remigration of C. pruni to Prunus spp. started in calendar week 10 (8th of March) and in 2012, in calendar week 12 (18th of March). Remigrants were observed until calendar week 20 (middle of May), significant numbers of the springtime generation adults were present until week 26 (end of June). The phytoplasma was ascertained in 0–11.5% of the remigrants and in 0–3.44% of the springtime generation insects. About 9.8–63.3% of the apricot samples, 20–40% of the plum samples and single blackthorn samples were infected. The mark, release and recapture study proved a fast and frequent tree‐to‐tree movement of remigrated C. pruni adults. Insects easily covered distances from row to row or even farther (ca. 13 m) within 24 h after release and were present in a large part of the model orchard after 8 days (up to 24 m from release point).     

Euscelis incisus (Cicadellidae,Deltocephalinae), a natural vector of 16SrIII‐B phytoplasma causing multiple inflorescence disease of Cirsium 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.     

Seasonal variation in susceptibility of the pear psyllid,Cacopsylla permixta (Hemiptera: Psyllidae) to selected insecticides

Although Cacopsylla pyri Forster and Cacopsylla pyricola Linnaeus have long been considered as more significant pests of pear trees around the world, Cacopsylla permixta Burckhardt and Hodkinson is the most significant pest in some parts of Iran, especially in pear gardens of Karaj. Current control strategies against this pest in Iran generally involve five or six insecticide treatments each year, despite unsatisfactory results are reported at many localities. So, it is crucial to know the most susceptible generation of the pest to apply a good strategy for its control. The aim of this study was to explore the seasonal variation in susceptibility of C. permixta to four commonly used insecticides. The results showed that winter forms (February) were 2.71‐fold, 4.58‐fold, 3.26 fold and 3.38‐fold more tolerant to diazinon, imidacloprid, acetamiprid and abamectin, respectively, compared with summer forms. Also, Esterase, GST and P450 monooxygenase activity was highest during February. Moreover, the content of lipid, carbohydrate, glycogen and protein was significantly higher in February compared with other months. Based on these results, the best period for insecticide treatment for efficient control is treatment against the first generation, at the time when eggs are laid by females as well as during the egg hatching and the larvae appearance. At this time of year, psylla are more susceptible, which would likely lead to better results and the reduction in damage during the next summer. However, further studies are needed to test this in farm settings, and to whether this holds true for other psylla species.     

AFLP-PCR and RAPD-PCR evidences of the transmission of the pathogen Aphanomyces astaci (Oomycetes) to wild populations of European crayfish from the invasive crayfish species, Procambarus clarkii

Aphanomyces astaci (Oomycetes) is responsible for the crayfish plague disease. This species is endemic of North America and five genotypes have been described using RAPD-PCR. The red swamp crayfish, Procambarus clarkii, is one of the most widely spread North American species and invasive in the world. However, no outbreaks on its specific genotype, i.e., genotype D, have ever been described in nature. We investigated three major series of crayfish plague outbreaks in indigenous crayfish populations of Austropotamobius pallipes, located in the areas of influence of P. clarkii. All samples collected tested positive for A. astaci using a rnDNA ITS-PCR test. We also performed an AFLP-PCR analysis on 19 isolates, and found that all isolates belong to genotype D. These isolates exhibited similar properties, i.e., adaptation to warm temperatures. We demonstrate, for the first time, the transmission of A. astaci genotype D to indigenous European populations of crayfish, and confirm that the properties of adaptation to warm water temperatures seem to be a specific character of genotype D. The results of this work emphasize once more the need of controlling invasive species and its trade, since they can carry harmful pathogens with specific adaptations or increased virulence in new environments.     

The correlation between infectivity and incubation period of measles, estimated from households with two cases

The generation time of an infectious disease is the time between infection of a primary case and infection of a secondary case by the primary case. Its distribution plays a key role in understanding the dynamics of infectious diseases in populations, e.g. in estimating the basic reproduction number. Moreover, the generation time and incubation period distributions together characterize the effectiveness of control by isolation and quarantine. In modelling studies, a relation between the two is often not made specific, but a correlation is biologically plausible. However, it is difficult to establish such correlation, because of the unobservable nature of infection events. We have quantified a joint distribution of generation time and incubation period by a novel estimation method for household data with two susceptible individuals, consisting of time intervals between disease onsets of two measles cases. We used two such datasets, and a separate incubation period dataset. Results indicate that the mean incubation period and the generation time of measles are positively correlated, and that both lie in the range of 11-12 days, suggesting that infectiousness of measles cases increases significantly around the time of symptom onset. The correlation between times from infection to secondary transmission and to symptom onset could critically affect the predicted effectiveness of isolation and quarantine.     

Proteomic maps of subcellular protein fractions of the Asian citrus psyllid Diaphorina citri,the vector of citrus huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two‐dimensional gel electrophoresis (2‐DE). A total of 1170 spots are detected in the 2‐DE gels of the three subcellular fractions. One hundred and sixty‐four differentially expressed proteins are successfully identified using liquid chromatography‐dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.