Population dynamics of Cacopsylla melanoneura (Homoptera: Psyllidae), a vector of apple proliferation phytoplasma in northwestern Italy

Apple proliferation is a phytoplasma-associated disease transmitted by insects causing serious damage and economic losses to apple orchards. Investigations were carried out in 1999 and 2000 in northwestern Italy to identify the vector of apple proliferation and to study its population dynamics. Yellow sticky traps and beat tray samples revealed the presence of the psyllid Cacopsylla melanoneura (Forster) in eight apple orchards in the Aosta Valley. The species completes one generation per year; the overwintered psyllids colonized apple trees beginning in late January, whereas the springtime generation was observed beginning in early May. The offspring adults remained in apple orchards until the end of June, when they began to move onto other hosts. During 1999 and 2000, all apple trees present in the investigated orchards were visually checked to assess the fluctuation of disease symptoms. Polymerase chain reaction and restriction fragment-length polymorphism confirmed the presence of the apple proliferation phytoplasmas in both overwintering and offspring insects as well as in symptomatic apple plants. The ability of C. melanoneura to vector the disease was assessed by preliminary transmission trials. Overwintered psyllids, collected in the most affected orchards, caged on healthy apple test plants transmitted apple proliferation phytoplasmas.     

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.     

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.     

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.     

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.     

Resistance of the codling moth Cydia pomonella (L.) (Lep., Tortricidae) to pesticides in Israel

Abstract:  Resistance of the codling moth Cydia pomonella (L.) (Lep., Tortricidae) to the organophosphorus compound (OP) azinphosmethyl was observed in apple orchards in Israel. The level of resistance varied with the pest control strategy. Compared with a sensitive laboratory population, the resistance level was highest in insects from the preventative pest control strategy, intermediate in integrated pest management (IPM) orchards, and relatively low in the organic orchards. The level of azinphosmethyl resistance in larvae (but not in adults) exposed for 17 generations in the laboratory to a pesticide-free diet was reduced by 50%. Codling moth larvae resistant to azinphosmethyl were also resistant to various insect growth regulators (IGRs). The IGRs include three chitin synthesis inhibitors (diflubenzuron, novaluron and teflubenzuron), two juvenile hormone mimics (pyriproxyfen and fenoxycarb) and one ecdysone agonist (methoxyfenozide). Codling moth resistant to azinphosmethyl was tolerant to methoxyfenozide and novaluron without previous history of application in apple orchards, indicating the possibility of cross-resistance. According to this study, managing resistance programs in apple orchards should be based on IPM principles with minimum use of conventional neuroactive pesticides.     

Apple Proliferation Epidemics Detected in Scab-Resistant Apple Trees

In the Friuli-Venezia Giulia, a region of Italy, where serious epidemics of apple proliferation (AP) are known to occur, varieties resistant to scab (Venturia inaequalis (Cke.) Wint.) are increasingly being used in new orchards. The most important cvs are Florina. Prima and Priscilla. These varieties were cultivated according to organic farming regimes with no insecticides used. The results obtained in two orchards during a 7-year period of investigation indicated that the three varieties resistant to scab are highly susceptible to AP. Florina was most susceptible (high infection rate) to AP while Priscilla was most sensitive (severely affected). The identification of the disease was based on symptom expression, DAPI (4–6-diamidino-2-phenylindole) fluorescence technique, electron microscopy observations and by polymerase chain reaction (PCR). Neither complete spontaneous recovery nor death of AP-infected plants was noticed. The pattern of natural diffusion of AP does not seem to be uniform, the affected trees may be in line or grouped in certain spots of the orchards. This may indicate the activity of a not very mobile vector. It can be concluded that planting of Florina, Prima and Priscilla should be discouraged in areas where AP is a problem, particularly when organic farming regimes are being applied.     

DL‐β‐Aminobutyric acid application negatively affects reproduction and larval development of the rosy apple aphid,Dysaphis plantaginea,on apple

The rosy apple aphid, Dysaphis plantaginea (Passerini) (Hemiptera: Aphididae), is one of the major pests of European apple orchards, commonly controlled by the use of synthetic insecticides. In the present work, the non‐protein amino acid DL‐β‐aminobutyric acid (BABA), known to induce plant resistance against a wide range of abiotic and biotic stresses, has been tested for its protective effect against this pest on apple. We first verified the lack of any contact effect of BABA on the insect itself. Next we applied BABA as a soil drench to apple and monitored its effect on the population development of aphids after artificial infestation. We demonstrated that BABA strongly reduced the population growth and that this compound severely affected various life‐history characteristics of the aphid such as female longevity and fecundity, nymph mortality, and larval development.     

Influence of pest control pressure on occurrence of ground beetles (Coleoptera: Carabidae) in apple orchards

Bioindicators that reflect the level of chemical control have not been developed for use in apple orchards in Japan. In Japan, many carabid beetles and spiders are beneficial predators of a wide range of agricultural pest insects. In apple orchards, spider numbers are reflective of the level of control pressure, but there is no information on carabid beetles. To establish whether carabid beetles could indeed be useful bioindicators, the number of carabid beetles and spiders in six apple orchards (A–F) under different levels of control pressure was investigated in Akita prefecture, northern Japan, in 2008 and 2009. The negative impact on beneficial arthropods was assumed to decrease in the order of pyrethroids (A) > organophosphates (B) > neonicotinoids (C) > insect growth regulators (D) > fungicides only (E) > no spraying (F). Sprays were applied at 2-week intervals from mid-May to early August. Twenty species of carabid beetles were found in orchard F; about 50% of adults were Anisodactylus punctatipennis, and 30–40% were Amara chalcites. The number of Am. chalcites significantly decreased with increasing control pressure: (A–C) < (E, F) in 2008, (A–E) < F in 2009, and (A, B) < E in 2009. Most spiders collected were juvenile Tetragnathidae and Araneidae. The numbers of spiders and Am. chalcites adults were significantly highly correlated at each control pressure in both years. Thus, it was considered that only Am. chalcites adults can be used as bioindicators reflecting the control pressure in apple orchards in northern Japan, whereas the main diet of this species is probably C3 plants.     

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.     

Movement of Anastrepha fraterculus from native breeding sites into apple orchards in Southern Brazil

We report movements exhibited by adults of Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) from patches of native forests into apple orchards in Southern Brazil. Two mark-release-recapture experiments were conducted using wild flies. Released flies behaved as wild unmarked flies, and periods of peak captures of marked adults coincided with those of unmarked ones. In the first experiment (December 94), out of 2154 released adults, 7.1% were recaptured from day 2 through day 20 after release. Captures peaked from day 7–9 after release. Most marked flies (94.7%) were trapped within 200 m of the release point but eight adults (seven females and one male) were captured in traps placed in an apple orchard, 400–800 m from the release point. The vegetation found between forest and orchard consisted of pastures and annual crops. In the second experiment (January 95), a total of 3284 flies was released in an area where native host plants were abundant and located at ca. 900 m from an apple orchard. In all, 37.1% of marked flies were captured, 99.0% of them at a distance of less than 200 m from the release point. Four adults were captured in an apple orchard, 7 to 24 days after release. They may have reached the orchard through a large and continuous area of native forest. Our results unequivocally demonstrate that A. fraterculus adults are able to disperse from native forests where they originate and invade apple orchards, probably foraging for food and oviposition sites.     

Comparison of different techniques for inoculation of "Candidatus Phytoplasma mali" on apple and periwinkle in biological indexing procedure

As phytoplasmas are non cultivable micro-organisms, the research on phytoplasmal diseases can only be achieved with infected hosts. Biological indexing (by grafting) is the simplest detection method for phytoplasmal diseases. We tested four different grafting techniques for inoculation of apple trees or periwinkles in greenhouse, including whip graft, bark graft, budding and chip-budding. All techniques were tested on apple trees (six trees per phytoplasma isolates) in insect-proof greenhouse. The whip and bark grafting were not feasible for periwinkle plants, because of fineness and fragility of their tissues: only the chip-budding was performed (four plants per isolate). In apple trees, the best and soonest positive results were obtained by chip and bark grafting. Except for seven transplants not-grown after grafting, 100% efficiency of inoculation was obtained by both methods. Nevertheless, the transmission of phytoplasma from transplant not-grown to rootstock was sometimes recorded (28.6%). The earliest phytoplasma symptoms after whip or bark grafting appeared after 3 months. Symptoms were obtained much later with budding and chip-budding. In case of periwinkles, infected apple and periwinkle materials were used as inoculum sources. Transmission of phytoplasma from periwinkle to periwinkle was successfully carried out by chip-budding grafting. The symptoms were observed during the second month after inoculation. The transmission of phytoplasma from infected apple material to periwinkle (by chip-budding) was achieved for 60 % of the tested samples. Moreover, the latency period before symptom observation was longer. Finally, we perceived the apple trees are more convenient and rapid than periwinkle plants for biological indexing of apple materials.     

Larval performance of the oriental fruit moth across fruits from primary and secondary hosts

A common characteristic of many invasive herbivorous insects is their ability to utilize a broad range of host plants. By using various hosts in phenological succession, multivoltine herbivores may increase the number of successful annual generations, at the same time as potentially increasing their overall fitness. To achieve such success, herbivores must be able to develop efficiently on the nutritional resources offered by their hosts. The oriental fruit moth Cydia (= Grapholita) molesta (Busck) (Lepidoptera: Tortricidae) is one of the most damaging invasive insect species. Peach (Prunus persica) is its primary host, whereas the pome fruits apple (Malus × domestica Borkh) and pear (Pyrus communis) are considered as secondary hosts. In many parts of its geographical range, including southern Europe, populations of the moth switch from peach to apple or pear orchards during the growing season. The present study tests whether this temporal switch is supported by the physiological capability of the larvae with respect to developing efficiently on fruits of these taxonomically‐related host plants. Larvae are reared on peach, apple or pear fruits; several life‐history traits are measured; and correlations between the traits are calculated. The results obtained show that larvae do not have the same physiological capability with respect to using apple or pear fruits as hosts compared with using peach fruit. Pear fruit in particular is a sub‐optimal diet. These findings suggest that, in the case of continuous geographical expansion, concomitantly with global warming, apple orchards might support oriental fruit moth populations better than pear orchards, and that the switch onto novel hosts might be accompanied by restricted population growth.     

Impact of trap architecture, adjacent habitats, abiotic factors, and host plant phenology on captures of plum curculio (Coleoptera: Curculionidae) adults

Pyramid traps, 2.44 m and 3.66 m in height, were compared with standard-sized pyramid traps, 1.22 m in height, to assess the impact of trap architecture on captures of adult plum curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), in two apple (Malus spp.) orchards and a blueberry (Vaccinium spp.) planting. The effects of adjacent habitat (organic orchard versus wooded areas), abiotic factors, and phenological stages of apple also were assessed to determine whether these variables influenced trap captures. Standard-sized pyramidal traps captured significantly more adults than larger trap variants. In the apple orchards, most adults (70-80%) were captured before petal fall with the exception of blocks adjacent to the organic orchard (25%). Significantly more adults were captured along the edge of an apple orchard (managed using an integrated pest management strategy) facing an organic apple orchard (76%) than along the edge facing wooded areas (24%). There was a significant positive correlation between daily trap captures and mean daily temperatures before petal fall in apple orchards.     

Plantago asiatica groundcover supports Amblyseius tsugawai (Acari: Phytoseiidae) populations in apple orchards

Amblyseius tsugawai Ehara (Acari: Phytoseiidae) is a major predator of spider mites in orchards in Japan. To support populations in apple orchards in Akita Prefecture, northern Japan, we investigated whether it can use Plantago asiatica L. as a food resource. In laboratory tests, survival did not differ significantly between female adults given water only and those given a piece of P. asiatica leaf and water. However, A. tsugawai reproduces by feeding on P. asiatica pollen, and significantly more mites were reared on P. asiatica pollen than on tea pollen, which is commonly used for rearing phytoseiid mites. In orchards in 2013, female adults were observed on leaves of P. asiatica from late May; numbers peaked in mid-June and gradually decreased until late July. Most adults were found along veins on the hidden sides of the leaves. Female adults were also collected in Phyto traps attached to plants between late May and early August. Pollen production of P. asiatica peaked from mid-June to early July, when numbers of adults peaked on the plants. These results suggest that conservation of P. asiatica in apple orchards would sustain A. tsugawai populations.     

Biological control of insect pests in apple orchards in China

Apple is one of the most important fruits in China, and both yield and quality are greatly affected by insect pests. According to surveys, there are more than 200 species of natural enemies in apple orchards. Few, however, have been closely studied. Major natural enemies including parasitoids, predators and pathogens are briefly described in this review, especially focusing on two parasitoids of Trichogramma dendrolimi Matsumura and Aphelinus mali Haldeman, predatory mites and a pathogenic fungus of Beauveria bassiana (Balsamo) Vuillemin as case studies. Augmentation, one important strategy of biological control, supplements the natural control provided by the existing natural enemy community in apple orchards, and greatly increases their efficiency in controlling pests. Conservation biological control is also widely applied in four major apple-producing areas. Based on habitat manipulation, the ground cover planting system helps regulate the microclimate and enhance the biodiversity of apple orchards, effectively conserving the richness and diversity of beneficial insect species. Certain achievements have been made in the main biological control strategies including successful introduction of some exotic natural enemies such as A. mali and Typhlodromus occidentalis Nesbitt, augmentative production and application of biological control agents such as T. dendrolimi, B. bassiana and Bacillus thuringiensis, and further research in conservation of establishing adaptive ground cover planting patterns to local environment. Challenges, however, still exist. Biological control of insect pests in apple orchards is an important part of integrated pest management programs, requiring more research and application in China.     

Endophytic bacterial community living in roots of healthy and ‘Candidatus Phytoplasma mali’-infected apple (Malus domestica, Borkh.) trees

‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and ‘Ca. Phytoplasma mali’-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g. Bacillus, Pseudomonas, and Burkholderia) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control ‘Ca. Phytoplasma mali’ in order to develop sustainable approaches for managing AP.     

茶翅蝽在生态苹果园的危害和防治策略

近几年在实施有机生态苹果园的过程中,发现茶翅蝽Halyomorpha halys(Stal)对苹果的危害日益严重,已成为生态苹果园发展的重要难题之一。作者于2006年在北京市昌平区流村镇王家园生态苹果园中,对茶翅蝽的危害进行调查,表明茶翅蝽对苹果危害严重,果园中早、中、晚熟3个品种苹果的为害率分别为28.8%±4.1%、23.4%±4.6%和30.8%±3.6%,在P>0.05水平上无显著差异。在防治策略上,应强调减少苹果园周围茶翅蝽的数量,从而减少迁入量。