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.     

Population dynamics of Cacopsylla melanoneura (Hemiptera: Psyllidae) in northeast Italy and its role in the apple proliferation epidemiology in apple orchards

In the current study, incidence of 'Candidatus Phytoplasma mali' in an experimental apple orchard in northeast Italy, in addition to abundance and phytoplasma infectivity of Cacopsylla melanoneura (F?rster) (Hemiptera: Psyllidae) was determined and the role of this psyllid as a vector of 'Ca. P. mali' in this region was reviewed. Insect samples collected in the orchard by the beating method indicated high abundance of C. melanoneura (up to 7.92 specimens/branch); however, the psyllid C. picta was not observed. Molecular analyses revealed presence of 'Ca. P. mali' in 6.25% of overwintered psyllids. This infection rate is quite high in comparison to other localities where C. melanoneura is known as the main vector of the phytoplasma. This finding supports the assumption that C. melanoneura also is paramount in the epidemiology of the apple proliferation disease also in northeast Italy. Moreover, we correlated immigration dynamics to the temperatures registered in the apple orchard, and defined an immigration index to predict the progressive arrival of the overwintered adults from winter sites. Psyllids start to reach the apple orchards when either the average of the maximum temperature of the 7 d is above 9.5 degrees C or the immigration index has a positive value. This index will be a useful tool for the growers to prevent apple proliferation phytoplasma spread with well-timed insecticide treatments targeted against C. melanoneura. However, further research is needed to validate or adjust the index to other apple growing regions, which may affect more efficacious management of this disease and psyllid vector.     

Transmission of apple proliferation phytoplasma by Cacopsylla melanoneura (Homoptera: Psyllidae)

Transmission trials of apple proliferation (AP) phytoplasma to healthy apple plants were carried out with Cacopsylla melanoneura (Forster). Both field naturally infected and experimentally infected psyllids were evaluated. The capacity of the different life stages of the insect in transmitting AP was tested. Overwintered adults collected in the orchards were able to transmit AP with a variable efficiency, depending on the infectivity rate of source plants. Experimentally infected nymphs and springtime adults succeeded in the transmission of AP, but the lower number of insect tested and the shorter inoculation period, due to difficulties in rearing the whole cycle of the insect in the laboratory, affected the efficiency. Considering the life history of C. melanoneura, the overwintered adults are the most responsible of the diffusion of AP in apple orchards.     

Epidemiology of apple proliferation (AP) in northwestern Italy: evaluation of the frequency of AP-positive psyllids in naturally infected populations of Cacopsylla melanoneura (Homoptera: Psyllidae)

Adults of Cacopsylla melanoneura, vector of the apple proliferation (AP) phytoplasma, were collected every 2 weeks from January until May in 2000 and 2001 by the beating tray method in eight apple orchards of the Aosta Valley (northwestern Italy). Total DNA was extracted from batches of five insects and amplified with the universal phytoplasma primers P1/P7 in direct PCR. A nested PCR assay was then performed on P1/P7 amplicons using the primers fO1/rO1, specific for the AP‐ phytoplasma group. The digestion of fO1/rO1 amplicons with Ssp I restriction endonuclease confirmed that C. melanoneura adults harboured the AP phytoplasma. The data obtained with PCR were used to estimate the proportion of AP‐positive insects in over wintered and offspring adults. Percentages of AP‐positive insects of 3.6% and 0.8% were estimated in 2000 among over wintered and offspring psyllids respectively. In 2001 only the over wintered insects were found infected, with an estimated proportion of 2.8%. The seasonal abundance of the vector was measured using yellow sticky traps. C. melanoneura was always present at a low population level, and the highest density was recorded from mid‐February until mid‐March in both years. The results show that the overwintered population is higher and spends a longer period in apple orchards, suggesting the crucial role of the overwintered adults in vectoring AP.     

Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts

Phytoplasmas ("Candidatus Phytoplasma," class Mollicutes) cause disease in hundreds of economically important plants and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes for specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and therefore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and the presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of approximately 250 kb located between the lplA and glnQ genes are syntenic between the two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is approximately 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M.     

Seasonal infectivity of Cacopsylla pruni, vector of European stone fruit yellows phytoplasma

Epidemiology of European stone fruit yellows was studied by focussing on the life cycle and transmission characteristics of the vector Cacopsyllapruni. The proportion of both phytoplasma positive and inoculative insects was determined for the first C. pruni adults back colonising the stone fruit trees in spring and for the new generations of the vector, hatched at the beginning of summer. We showed that in spring, as soon as the insects moved to stone fruit trees from shelter plants, they were infective. After the vector fed on infected stone fruit trees, the proportion of phytoplasma positive insects increased. The new generation colonising Prunus species also acquired the phytoplasma from their hosts although several of these insects completed the latency period on secondary hosts. Results showed that the risk of natural transmission of European stone fruit yellows-phytoplasma by C. pruni within orchards is high when the vector is present. These results have implications for the control of European stone fruit yellows.     

Integrated management of phytoplasma diseases in pome fruit: an overview of efficacy results of IPM insecticides against pear Psylla (Cacopsylla pyri)

Phytoplasmas are plant pathogenic mollicutes that cause devastating diseases in various crops worldwide. The closely related pome fruit tree phytoplasmas Candidatus Phytoplasma mali and Candidatus Phytoplasma pyri are the causal agents of apple proliferation and pear decline, respectively. They can be transmitted from tree to tree by Psyllidae. As pear suckers (Cacopsylla pyri) are widely considered to be the most important pest in pear orchards, a good control of this insect vector is a key element for limiting the natural spread of pear decline. Efficient control relies on a perfect tuning of treatment schedules, taking into account efficacies of (at preferably) low-impact insecticides and side-(repellent)-effects of alternative products (e.g. kaolin, mineral oils and fungicides), the optimal positioning of these crop protection agents, and the best possible presence of beneficial predators. The department of Zoology of the pcfruit vzw research institute (Belgium) has a long tradition of executing insecticide field trials according to EPPO guidelines. Here, we present an overview of the results of a selection of IPM-compatible insecticides (abamectin, spirotetramat, thiacloprid, spinosad, spirodiclofen), tested in efficacy trials against pear Psylla on different life stages during the last decade. Based on these results and monitoring data of pest and beneficial biology, we additionally propose optimal pear Psylla control schedules which allow to reduce the number of (phytoplasma harbouring) psyllids in integrated systems to a minimum.     

Can Flowering Plants Enhance Numbers of Beneficial Arthropods in UK Apple and Pear Orchards?

A replicated experiment with fourteen flowering plant species was used to determine the effectiveness of flowering plants as attractants for beneficial arthropods in orchards. The greatest numbers of anthocorids were found on cornflower (Centaurea cyanus) and corn chamomile (Anthemis arvensis) and hymenopteran parasitoids on corn marigold (Chrysanthemum segetum) and corn chamomile. The impact on pest numbers of manipulating the orchard environment by the use of flowering plants was investigated in two ways. In an apple and a pear orchard, a proportion of the trees was undersown with a mixture of cornflower, corn marigold and corn chamomile. No significant differences were found in numbers of Panonychus ulmi (in apple) or Cacopsylla pyricola (in pear) on trees grown in the undersown areas compared with trees grown in conventional bare herbicide strips. When potted pear trees infested with pear psyllids were placed either into plots sown with the flower mixture or on bare earth, psyllid numbers declined rapidly on trees in both treatments. When beneficials were excluded from the potted control trees, numbers of psyllid larvae, but not eggs, declined more quickly on the trees surrounded by flowering plants. The importance of habitat diversity on biocontrol in orchards is discussed.     

A study of the effects of ‘Candidatus Phytoplasma mali’ on the psyllid Cacopsylla melanoneura (Hemiptera: Psyllidae)

Cacopsylla melanoneura is a univoltine psyllid vector of ‘Candidatus Phytoplasma mali’, the etiological agent of apple proliferation (AP), a severe disease in European apple orchards. The influence of ‘Ca. P. mali’ on the fitness of C. melanoneura was studied. In the spring of 2007, male-female pairs of field-collected adults were exposed to ‘Ca. P. mali’-infected or healthy ‘Golden Delicious’ apple shoots. Exposure to these diseased shoots did not affect the life span of the adult psyllids. However, significantly fewer eggs were laid on the diseased shoots. Furthermore, fewer of the eggs that were laid on the infected plants hatched. Data suggest a detrimental effect of AP phytoplasma on the fitness of C. melanoneura.     

Seasonal abundance of Draeculacephala minerva and other Xylella fastidiosa vectors in California almond orchards and vineyards

Almond leaf scorch (ALS) disease is caused by the bacterium Xylella fastidiosa and transmitted by xylem-feeding insects. Reports of increased incidence of ALS-diseased trees in California prompted surveys in three almond [Prunus dulcis (Mill.) D. A. Webb]-growing regions, from June 2003 to September 2005, to determine insect vector species composition and abundance. For comparison, sampling in and near vineyards in the San Joaquin Valley, California, also was completed. Sampling in or near almond orchards collected >42,000 Cicadomorpha of which 4.8% were xylem feeders, including 1912 grass sharpshooter, Draeculacephala minerva Ball; five Xyphon fulgida Nottingham; and a single spittlebug, Philaenus spumarius L. The most abundant vector was D. minerva. Season-long sampling indicated that D. minerva was a year-round resident in and/or near almonds in the Sacramento Valley, but not in the San Joaquin Valley. Similarly, D. minerca was rare in vineyards in the San Joaquin Valley, but was abundant in irrigated pastures near vineyards. D. minerva was most frequently collected along orchard margins, and peak densities were observed in summer, the period of time when bacterial titers are reported to increase in infected trees. Screening of D. minerva for presence of X.fastidiosa found that 1.1% of insects collected near almond orchards and 4.5% of insects collected from pastures tested positive. The X. fastidiosa subspecies and genotype detected in insects collected from orchards matched those collected from ALS-diseased almond trees in the same orchard. Of the few X. fulgida and P. spumarius collected, none tested positive for X. fastidiosa. Results are discussed with respect to X. fastidiosa vector control and detection methods.     

Occurrence of European Stone Fruit Yellows Phytoplasma (ESFYP) Infection in Peach Orchards in Northern-Central Italy

During field surveys in 1999 and 2000 of peach orchards in Northern-Central Italy, plants of different cultivars were observed with symptoms of early leaf reddening, abnormal thickening of midribs and primary veins, autumnal growth of latent buds which produce tiny chlorotic leaves and sometimes flowers, and early phylloptosis; such symptoms, rarely seen previously on peach, are associated with European stone fruit yellows phytoplasma (ESFYP). In the orchards inspected, 1–4% of trees were affected and most were grafted on cv. G.F. 677. In most of the symptomatic samples (130 of 157 tested), only ESFYP was detected using different diagnostic methods [4',6'-diamidino-2-phenilindole, 2HCl (DAPI), polymerase chain reaction (PCR) with ribosomal and non-ribosomal primer pairs, PCR-enzyme-linked immunosorbent assay, nested-PCR]. The immunoenzymatic detection of PCR products with a pathogen-specific probe ensured fast, sensitive and specific detection of ESFYP. This is the first survey to assess the occurrence of phytoplasmas in peach orchards in Northern-Central Italy.     

Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors

Three real‐time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr‐V, 16Sr‐X and 16Sr‐XII have been developed. Ribosomal‐based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real‐time PCR. For group‐specific detection of FD phytoplasma, ribosomal‐based primers fAY/rEY, specific for 16Sr‐V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non‐ribosomal and nitroreductase DNA sequences, respectively. SYBR^® Green I detection coupled with melting curve analysis was used in each group‐specific protocol. Field‐collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real‐time PCR assays. The diagnostic efficiency of each group‐specific protocol was compared with well‐established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real‐time PCR from DNAs of European aster yellows (16Sr‐I), elm yellows (16Sr‐V), stolbur (16Sr‐XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group‐specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real‐time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group‐specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.     

Histopathology and polyphenol content in plants infected by phytoplasmas

The alterations of cell walls and the localization of several compounds such as polyphenols, suberin, lignin, in plum and apple plants infected with plum leptonecrosis (PLN) and apple proliferation (AP) phytoplasmas respectively, were investigated. Catharanthus roseus plants, infected with AP or PLN were also studied. The 4,6-diamidino-2-phenylindole (DAPI) test and transmission electron microscopy showed the presence of phytoplasmas in all infected plants. Specific histological stainings for cutinized/suberinized cell walls, tannin deposits and vacuolar polyphenol inclusions, performed on leaf and stem tissues, revealed an increase of these substances in infected plum and apple plants. No differences occurred in C. roseus. Total polyphenol analysis confirmed a strong increase (3-fold) in the polyphenol content in infected tissues, particularly in plum leaves. From the data obtained it appears that polyphenols can be considered as defence-related metabolites in plum and apple plants infected by phytoplasmas. Further investigations are necessary to determine whether these compounds play a specific role in the development of all phytoplasma/host interactions and in the defence-related processes.     

Population dynamics of apple maggot (Diptera: Tephritidae) in south central Pennsylvania

The apple maggot, Rhagoletis pomonella (Walsh), was monitored with baited yellow panels and red spheres in commercial orchards, abandoned orchards, and unsprayed backyard apple trees in 1998 and 1999. Apple maggot adults were captured in all apple habitats, but the capture levels in the abandoned orchards and unsprayed backyard trees tended to be higher than in the commercial orchards. Peak capture occurred between mid-July and late August in both years. Emergence cages seeded with infested fruits were used to investigate bivoltinism, which was observed in both years.     

Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in Iran

During 2010–14 surveys in the major sesame growing areas of Fars, Yazd and Isfahan provinces (Iran), genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody were studied. Virtual RFLP, phylogenetic, and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to three ribosomal subgroups, 16SrII-D, 16SrVI-A, and 16SrIX-C. The same analyses using 16S rDNA sequences from sesame phyllody-associated phytoplasmas retrieved from GenBank database showed the presence of phytoplasmas clustering with strains in the same subgroups in other Iranian provinces including Bushehr and Khorasan Razavi. Circulifer haematoceps and Orosius albicinctus, known vectors of the disease in Iran, were tested for transmission of the strains identified in this study. C. haematoceps transmitted 16SrII-D, 16SrVI-A, and 16SrIX-C phytoplasmas, while O. albicinctus only transmitted 16SrII-D strains. Based on the results of the present study and considering the reported presence of phytoplasmas belonging to the same ribosomal subgroups in other crops, sesame fields probably play an important role in the epidemiology of other diseases associated with these phytoplasmas in Iran.     

Effect of Antibiotics on the Symptoms of Stunting Disease of Magnolia liliiflora Plants

Treatment of diseased magnolia plants with Oxytetracycline, Baytril or Tylan did not reduce the number of symptomatic plants, but promoted shoot growth, development of symptomless leaves and flower buds. The most efficient were 500 ppm Baytril, 200 ppm Tylan and 500 or 1000 ppm Oxytetracycline. Lower concentrations of Baytril and Oxytetracycline were less effective and higher concentrations of Tylan decreased the growth of magnolia shoots. All the tested antibiotic treated and untreated magnolias were shown by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) to contain the AY (16SrI) phytoplasma and two also to contain a phytoplasma related to apple proliferation phytoplasma group (16SrX). The results indicate that Magnolia is a natural host of phytoplasmas belonging to the aster yellows and apple proliferation phytoplasma groups, and support the suggestion that phytoplasmas are the cause of magnolia stunting disease.     

Host plant determines the phytoplasma transmission competence of Empoasca decipiens (Hemiptera: Cicadellidae)

Phytoplasmas are phloem-restricted plant pathogens transmitted by leafhoppers, planthoppers, and psyllids (Hemiptera). Most known phytoplasma vectors belong to the Cicadellidae, but many are still unknown. Within this family, Empoasca spp. (Typhlocybinae) have tested positive for the presence of some phytoplasmas, and phytoplasma transmission has been proven for one species. The aim of this work was to investigate the ability of Empoasca decipiens Paoli in transmitting chrysanthemum yellows phytoplasma (CYP, "Candidatus Phytoplasma asteris", 16SrI-B) and Flavescence dorée phytoplasma (FDP, 16SrV-C) to Chrysanthemum carinatum Schousboe (tricolor daisy) and Viciafaba (L.) (broad bean). Euscelidius variegatus Kirschbaum, a known vector of CYP and FDP, was caged together with Em. decipiens on the same source plants as a positive control of acquisition. Em. decipiens acquired CYP from daisies, but not from broad beans, and inoculated the pathogen to daisies with alow efficiency, but not to broad beans. Em. decipiens did not acquire FDP from the broad bean source. Consistent with the low transmission rate, CYP was found in the salivary glands of very few phytoplasma-infected Em. decipiens, indicating these organs represent a barrier to phytoplasma colonization. In the same experiments, the vector Eu. variegatus efficiently acquired both phytoplasmas, and consistently CYP was detected in the salivary glands of most samples of this species. The identity of the CYP strain in leafhoppers and plants was confirmed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. The CYP titer in Em. decipiens was monitored over time by real-time PCR. The damage caused by Em. decipiens feeding punctures was depicted. Differences in feeding behavior on different plant species may explain the different phytoplasma transmission capability. Em. decipiens proved to be an experimental vector of CYP.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

Transmission of Tomato Ringspot Virus by Xiphinema americanum and X. rivesi from New York Apple Orchards

Populations of Xiphinema americanum and X. rivesi were collected from apple orchards in eastern and western New York and tested in the laboratory for ability to transmit tomato ringspot virus (TmRSV) to cucumber and dandelion. Populations varied in the frequency with which they transmitted TmRSV, but this variation did not correspond to variation in disease prevalence in the orchard. The lower prevalence of TmRSV-incited disease in apple trees in western New York cannot be attributed to inability of the local Xiphinema spp. to transmit TmRSV.