Insect Antimicrobial Peptides: Advancements, Enhancements and New Challenges.

Several insects are known as vectors of a wide range of animal and human pathogens causing various diseases. However, they are also a source of different substances, such as the Antimicrobial Peptides (AMPs), which can be employed in the development of natural bioactive compounds for medical, veterinary and agricultural applications. It is well known that AMP activity, in contrast to most classical antibiotics, does not lead to the development of natural bacterial resistance, or at least the frequency of resistance is considered to be low. Therefore, there is a strong interest in assessing the efficacy of the various peptides known to date, identifying new compounds and evaluating possible solutions in order to increase their production. Moreover, implementing AMP modulation in insect rearing could preserve insect health in large-scale production. This review describes the current knowledge on insect AMPs, presenting the validated ones for the different insect orders. A brief description of their mechanism of action is reported with focus on proposed applications. The possible effects of insect diet on AMP translation and synthesis have been discussed.

Impact of the Diet on the Mortality and on Gene Expression of the Antimicrobial Peptide Tenecin 3 in Tenebrio molitor Larvae Infected by Beauveria bassiana.

Large-scale insect rearing can be subjected to microbial infections, leading to serious economic losses. When possible, the use of antibiotics is to be avoided for insects intended as feed or food and new strategies to preserve the health of the farmed insects are required. The effectiveness of insect immune system depends on several factors, including the nutritional composition of the diet. The possibility to modulate immune responses through the diet is currently a topic of great interest from an application point of view. We evaluated the effect of two different diets on the survival rate and gene expression of the antimicrobial peptide Tenecin 3 in uninfected and Beauveria bassiana-infected Tenebrio molitor L. larvae. A wheat bran diet, mixed 50% with brewers' spent grains, could positively influence the expression of Tenecin 3 gene when uninfected T. molitor is allowed to develop on such a substrate from early larval stages. Even if, in our trial, the diet with added brewers' spent grains could not reduce the mortality of the larvae infected with B. bassiana, higher transcriptional levels of the antifungal peptide were observed in insects fed this diet, depending on the timing of diet administration.

Immune Responses of the Black Soldier Fly Hermetia illucens (L.) (Diptera: Stratiomyidae) Reared on Catering Waste.

The black soldier fly (BSF), Hermetia illucens L. (Diptera: Stratiomyidae), has a great bioconversion potential and ability to develop on diverse substrates. Although the use of catering waste and food by-products containing meat and fish would reduce the footprint of the insect sector, to date, in Europe, their use is still facing legal obstacles for insects as food and feed. Since a major request from the EU insect sector is to diversify the spectrum of allowed substrates, and considering that variations in diet composition could influence insect immune responses, we evaluated the impact of different catering wastes on BSF prepupae immunity. Insects were reared on five diets: one based on feed for laying hens and four based on catering waste containing (i) fruits and vegetables; (ii) fruits, vegetables and bread; (iii) fruit, vegetables, bread and dairy products; (iv) fruits, vegetables, bread, meat and fish. The gene expression of two antimicrobial peptides (AMPs), one defensin and one cecropin, was assessed. Moreover, the hemolymph inhibitory activity against Escherichia coli DH5α and Micrococcus yunnanensis HI55 was evaluated using diffusion assays in solid media. The up-regulation of both AMPs' encoding genes was observed in insects fed a bread-added and dairy product-added diet. All hemolymph samples showed inhibitory activity against both bacteria, affecting the colony size and number. The obtained results show how catering waste positively influences the H. illucens immune system. The possibility of modulating AMP expression levels through the diet opens up new perspectives in the management of insect health in mass rearings.

Effect of the rearing diet on gene expression of antimicrobial peptides in Hermetia illucens (Diptera: Stratiomyidae).

Insect proteins have been proposed for human and animal food production. Safeguarding the health status of insects in mass rearing allows to obtain high-quality products and to avoid severe economic losses due to entomopathogens. Therefore, new strategies for preserving insect health must be implemented. Modulation of the insect immune system through the diet is one such strategy. We evaluated gene expression of two antimicrobial peptides (one defensin and one cecropin) in Hermetia illucens (L.) (Diptera: Stratiomyidae) reared on different diets. Analyses were performed on prepupae and 10-day-old larvae reared on cereal- and municipal organic waste-based diets and on only prepupae reared on a cereal-based diet supplemented with sunflower, corn, or soybean oil. The inclusion of sunflower oil at different points in the cereal-based diet was also evaluated. Moreover, diet-driven differences in the inhibitory activity of the hemolymph were tested against Escherichia coli DH5α and Micrococcus yunnanensis HI55 using diffusion assays in solid media. Results showed that a municipal organic waste-based diet produced a significant overexpression of antimicrobial peptides only in prepupae. Inclusion of vegetable oils caused an upregulation of at least one peptide, except for the corn oil. Higher expression of both genes was observed when sunflower oil was added 5 days before pupation. All hemolymph samples showed an inhibitory activity against bacteria colonies. Our results suggest that municipal organic waste-based diet and vegetable oil-added diet may successfully impact the immune system of H. illucens. Such alternatives may also exist for other species of economic interest.

First Multi-Target Application of Exclusion Net in Nectarine Orchards: Effectiveness against Pests and Impact on Beneficial Arthropods, Postharvest Rots and Fruit Quality.

Over the past few years, there has been an increasing interest in the development of alternative pest control strategies to reduce environmental impact. In this contest, exclusion nets have been evaluated as a sustainable alternative to pesticides. In this study, the use of a photoselective exclusion net was investigated in semi-field conditions as a potential strategy to protect nectarine orchards from different pests (i.e., fruit moths, Halyomorpha halys and Drosophila suzukii) in NW Italy. The presence and abundance of pest populations inside and outside the net, as well as the damage they caused on fruits, were evaluated. Moreover, any possible effects of the net on beneficial arthropods, postharvest rots and fruit quality and nutraceutical parameters were considered. The exclusion net significantly reduced pest populations. At harvest, fruit damage caused by Grapholita molesta and H. halys in netted plots was reduced up to 90% and to 78%, respectively, compared with insecticide-treated plots. The exclusion net allowed the production of healthier fruits with a strong reduction of insecticide treatments (up to seven less) and of their related costs without any negative impact on postharvest rots, neither fruit quality nor nutraceutical properties.

Temporal Dynamics of 'Ca. Phytoplasma mali' Load in the Insect Vector Cacopsylla melanoneura.

The transmission of phytoplasmas is the result of an intricate interplay involving pathogens, insect vectors and host plants. Knowledge of the vector's competence during its lifespan allows us to define more sustainable well-timed control strategies targeted towards the most worrisome life stages. We investigated the temporal dynamics of 'Candidatus Phytoplasma mali' load in Cacopsylla melanoneura in the different developmental stages in Northwest Italy. The phytoplasma load in the vector was evaluated in overwintering adults, nymphs and newly emerged adults after different acquisition access periods. Moreover, we followed the multiplication of the phytoplasma during the aestivation and the overwintering period on conifers. Our results confirmed the ability of remigrants to retain the phytoplasma until the end of winter. We also highlighted the high acquisition efficiency and vector competence, based on phytoplasma load, of nymphs and newly emerged adults. Therefore, particular attention should be paid to the management of overwintered C. melanoneura as soon as they return to the orchards, but also to newly emerged adults, particularly in orchards with a high infection rate and when the migration to conifers is delayed.

Photoselective exclusion netting in apple orchards: effectiveness against pests and impact on beneficial arthropods, fungal diseases and fruit quality.

BACKGROUND: Frequent pesticide treatments in fruit orchards increase hazards for workers, consumers and the environment. Moreover, the indiscriminate and excessive use of pesticides often induces resistance in pests. In the past few years, physical exclusion strategies have been proposed as an alternative for the control of insect pests. The goal of this study was to evaluate the effectiveness of anti-hail photoselective netting in protecting apples against key and emerging pests, as well as the impact on beneficial arthropods, fungal diseases and fruit quality. RESULTS: In netted plots, a significant reduction in pest populations, i.e. fruit moths, Halyomorpha halys (Stål) and Drosophila suzukii (Matsumura), was recorded in comparison with un-netted controls. Moreover, the damage on fruits caused by H. halys was reduced up to 62% compared with insecticidal treatments. The net did not negatively affect the abundance of predators and the incidence of post-harvest rot. In addition, the incidence of bitter pit on apple was reduced up to 52%. Furthermore, fruit quality was unaffected by the net coverage (both at harvest and after 4 months of storage). CONCLUSION: Anti-hail photoselective pearl netting proved a promising exclusion system that can prevent attack by more than one insect pest at a time, allowing for a strong reduction in insecticide treatments and relative costs. At the same time, the netting did not negatively influence the presence of predators, the incidence of fungal disease or fruit quality. © 2019 Society of Chemical Industry.

Activation of Immune Genes in Leafhoppers by Phytoplasmas and Symbiotic Bacteria.

Insect immunity is a crucial process in interactions between host and microorganisms and the presence of pathogenic, commensal, or beneficial bacteria may result in different immune responses. In Hemiptera vectors of phytoplasmas, infected insects are amenable to carrying high loads of phytopathogens, besides hosting other bacterial affiliates, which have evolved different strategies to be retained; adaptation to host response and immunomodulation are key aspects of insect-symbiont interactions. Most of the analyses published to date has investigated insect immune response to pathogens, whereas few studies have focused on the role of host immunity in microbiota homeostasis and vectorial capacity. Here the expression of immune genes in the leafhopper vector of phytoplasmas Euscelidius variegatus was investigated following exposure to Asaia symbiotic bacteria, previously demonstrated to affect phytoplasma acquisition by leafhoppers. The expression of four genes related to major components of immunity was measured, i.e., defensin, phenoloxidase, kazal type 1 serine protease inhibitor and Raf, a component of the Ras/Raf pathway. The response was separately tested in whole insects, midguts and cultured hemocytes. Healthy individuals were assessed along with specimens undergoing early- and late-stage phytoplasma infection. In addition, the adhesion grade of Asaia strains was examined to assess whether symbionts could establish a physical barrier against phytoplasma colonization. Our results revealed a specific activation of Raf in midguts after double infection by Asaia and flavescence dorée phytoplasma. Increased expression was observed already in early stages of phytoplasma colonization. Gut-specific localization and timing of Raf activation are consistent with the role played by Asaia in limiting phytoplasma acquisition by E. variegatus, supporting the involvement of this gene in the anti-pathogen activity. However, limited attachment capability was found for Asaia under in vitro experimental conditions, suggesting a minor contribution of physical phytoplasma exclusion from the vector gut wall. By providing evidence of immune modulation played by Asaia, these results contribute to elucidating the molecular mechanisms regulating interference with phytoplasma infection in E. variegatus. The involvement of Raf suggests that in the presence of reduced immunity (reported in Hemipterans), immune genes can be differently regulated and recruited to play additional functions, generally played by genes lost by hemipterans.

The genus Cixius Latreille, 1804 (Hemiptera, Fulgoromorpha, Cixiidae) in Lebanon with the description of two new species.

A first list of Cixius fauna of Lebanon is here provided with the description of two new species: one within the subgenus Acanthocixius, Cixius bifidispinus sp. nov. and one within the subgenus Ceratocixius Cixius superremotus sp. nov. The standard morphological description is supplemented by sequence data of the mitochondrial cytochrome c oxidase subunit I (COI) gene fragment. Some notes on their distribution and economic importance are also reported.

'Candidatus Phytoplasma phoenicium' associated with almond witches'-broom disease: from draft genome to genetic diversity among strain populations.

BACKGROUND: Almond witches'-broom (AlmWB), a devastating disease of almond, peach and nectarine in Lebanon, is associated with 'Candidatus Phytoplasma phoenicium'. In the present study, we generated a draft genome sequence of 'Ca. P. phoenicium' strain SA213, representative of phytoplasma strain populations from different host plants, and determined the genetic diversity among phytoplasma strain populations by phylogenetic analyses of 16S rRNA, groEL, tufB and inmp gene sequences. RESULTS: Sequence-based typing and phylogenetic analysis of the gene inmp, coding an integral membrane protein, distinguished AlmWB-associated phytoplasma strains originating from diverse host plants, whereas their 16S rRNA, tufB and groEL genes shared 100 % sequence identity. Moreover, dN/dS analysis indicated positive selection acting on inmp gene. Additionally, the analysis of 'Ca. P. phoenicium' draft genome revealed the presence of integral membrane proteins and effector-like proteins and potential candidates for interaction with hosts. One of the integral membrane proteins was predicted as BI-1, an inhibitor of apoptosis-promoting Bax factor. Bioinformatics analyses revealed the presence of putative BI-1 in draft and complete genomes of other 'Ca. Phytoplasma' species. CONCLUSION: The genetic diversity within 'Ca. P. phoenicium' strain populations in Lebanon suggested that AlmWB disease could be associated with phytoplasma strains derived from the adaptation of an original strain to diverse hosts. Moreover, the identification of a putative inhibitor of apoptosis-promoting Bax factor (BI-1) in 'Ca. P. phoenicium' draft genome and within genomes of other 'Ca. Phytoplasma' species suggested its potential role as a phytoplasma fitness-increasing factor by modification of the host-defense response.

EvaGreen real-time PCR protocol for specific 'Candidatus Phytoplasma mali' detection and quantification in insects.

In this paper the validation and implementation of a Real-time PCR protocol based on ribosomal protein genes has been carried out for sensitive and specific quantification of 'Candidatus (Ca.) Phytoplasma mali' (apple proliferation phytoplasma, APP) in insects. The method combines the use of EvaGreen(®) dye as chemistry detection system and the specific primer pair rpAP15f-mod/rpAP15r3, which amplifies a fragment of 238 bp of the ribosomal protein rplV (rpl22) gene of APP. Primers specificity was demonstrated by running in the same Real-time PCR 'Ca. Phytoplasma mali' samples with phytoplasmas belonging to the same group (16SrX) as 'Ca. Phytoplasma pyri' and 'Ca. Phytoplasma prunorum', and also phytoplasmas from different groups, as 'Ca. Phytoplasma phoenicium' (16SrIX) and Flavescence dorée phytoplasma (16SrV). 'Ca. Phytoplasma mali' titre in insects was quantified using a specific approach, which relates the concentration of the phytoplasma to insect 18S rDNA. Absolute quantification of APP and insect 18S rDNA were calculated using standard curves prepared from serial dilutions of plasmids containing rplV-rpsC and a portion of 18S rDNA genes, respectively. APP titre in insects was expressed as genome units (GU) of phytoplasma per picogram (pg) of individual insect 18S rDNA. 'Ca. Phytoplasma mali' concentration in examined samples (Cacopsylla melanoneura overwintered adults) ranged from 5.94 × 10(2) to 2.51 × 10(4) GU/pg of insect 18S rDNA. Repeatability and reproducibility of the method were also evaluated by calculation of the coefficient of variation (CV%) of GU of phytoplasma and pg of 18S rDNA fragment for both assays. CV less than 14% and 9% (for reproducibility test) and less than 10 and 11% (for repeatability test) were obtained for phytoplasma and insect qPCR assays, respectively. Sensitivity of the method was also evaluated, in comparison with conventional 16S rDNA-based nested-PCR procedure. The method described has been demonstrated reliable, sensitive and specific for the quantification of 'Ca. Phytoplasma mali' in insects. The possibility to study the trend of phytoplasma titre in the vectors will allow a deepen investigation on the epidemiology of the disease.

Insect vector transmission assays.

Phytoplasmas are transmitted in a persistent propagative manner by phloem-feeding vectors belonging to the order Hemiptera, suborder Homoptera. Following acquisition from the infected source plant, there is a latent period before the vector can transmit, so transmission assays consist of three basic steps: acquisition, latency, and inoculation. More than 90 vector species (plant-, leafhoppers, and psyllids) have been discovered so far but many others are still undiscovered, and their role in spreading economically important crop diseases is neglected. Therefore, screening for vectors is an essential step in developing rational control strategies targeted against the actual vectors for phytoplasma-associated diseases. The mere detection of a phytoplasma in an insect does not imply that the insect is a vector; a transmission assay is required to provide conclusive evidence. Transmission experiments can be carried out using insects from phytoplasma-free laboratory colonies or field-collections. Moreover, transmission assays can be performed by feeding vectors on an artificial diet through Parafilm(®), after which phytoplasmas can be detected in the sucrose feeding medium by PCR. Transmission trials involve the use of different techniques according to the biology of the different vector species; planthoppers, leafhoppers, and psyllids.

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 (Förster) (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.

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois noir in Vitis vinifera.

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.

'Candidatus Liberibacter europaeus' sp. nov. that is associated with and transmitted by the psyllid Cacopsylla pyri apparently behaves as an endophyte rather than a pathogen.

'Candidatus Liberibacter spp.' cause serious plant diseases. 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus' and 'Ca. L. africanus' are the aetiological agents of citrus greening (Huanglongbing) in Asia, America and Africa. 'Candidatus Liberibacter solanacearum' causes diseases in Solanaceae in America and New Zealand. All four species are vectored by psyllid insects of different genera. Here, we show that the pear psyllid pest Cacopsylla pyri (L.) hosts a novel liberibacter species that we named 'Ca. Liberibacter europaeus'. It can bloom to high titres in the psyllid host, with more than 10(9) 16S rRNA gene copies per individual. Fluorescent in situ hybridization experiments showed that 'Ca. L. europaeus' is present in the host midgut lumen, salivary glands and Malpighian tubules. 'Candidatus L. europaeus' has a relatively high prevalence (> 51%) in C. pyri from different areas in the Piedmont and Valle d'Aosta regions in Italy and can be transmitted to pear plants in experimental transmission trials. However, even though high titres of the bacterium (more than 10(8) 16S rRNA gene copies g(-1) of pear plant tissue) could be detected, in the pear tissues no specific disease symptoms could be observed in the infected plants over a 6-month period. Despite liberibacters representing potential quarantine organisms, 'Ca. L. europaeus', first described in Italy and Europe, apparently behaves as an endophyte rather than a pathogen.

A novel Bacteroidetes symbiont is localized in Scaphoideus titanus, the insect vector of Flavescence dorée in Vitis vinifera.

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, "Candidatus Phytoplasma vitis," which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the "Candidatus Cardinium hertigii" group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of "Ca. Cardinium hertigii." This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of "Ca. Phytoplasma vitis" were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and "Ca. Phytoplasma vitis" have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Fieberiella florii (Homoptera: Auchenorrhyncha) as a Vector of "Candidatus Phytoplasma mali".

Laboratory trials were carried out to transmit "Candidatus Phytoplasma mali" to healthy apple seedlings with the leafhopper Fieberiella florii. Experiments on serial inoculation access period and molecular analyses performed on test plants and insects confirmed the ability of the leaf-hopper to carry and transmit the phytoplasma. Field surveys by means of yellow sticky traps were conducted in northwestern Italy to verify the abundance and the natural infectivity of F. florii in apple orchards and in wild vegetation in areas surrounding apple orchards. Despite the high percentages of infected specimens obtained in the apple orchards (5.7%) and in the wild vegetation areas (20.0%), the risk of apple tree infection by F. florii in nature is probably low because of the very low insect density recorded. In spite of the low number of specimens collected, the presence of the leafhopper in apple orchards in summer, when the main vector, the psyllid Cacopsylla melanoneura, feeds on alternative hosts, is meaningful. Moreover, the high degree of polyphagy of the leafhopper opens up new interesting prospects for the epidemiology of apple proliferation.

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.

DNA-based methods for the detection and the identification of phytoplasmas in insect vector extracts.

DNA extraction and storage methods have been evaluated with laboratory-reared leafhoppers and/or field-collected leafhoppers and psyllids. Detection of four different phytopathogenic phytoplasmas, belonging to three taxonomic groups, has been achieved by several direct or nested polymerase chain reaction (PCR) methods with such DNA extracts. Reactions differed in both the 16/23S ribosomal primer pairs used and the specific assay and cycling conditions. Merits and possible hindrances of the various primer pairs, in relation to insect DNA extracts, are discussed. However, identification of the phytoplasma(s) necessarily relied on comparison of the polymorphism in length of the amplified DNA fragments obtained by restriction with appropriate endonucleases. Endonuclease digestion is crucial for determining the identity (subgroup affiliation) of phytoplasmas of the same groups that can be carried by an individual vector.

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.