A molecular approach to unravel trophic interactions between parasitoids and hyperparasitoids associated with pecan aphids.

Advances in molecular ecology can overcome many challenges in understanding host-parasitoid interactions. Genetic characterization of the key-players in systems helps to confirm species and identify trophic linkages essential for ecological service delivery by biological control agents; however, relatively few agroecosystems have been explored using this approach. Pecan production consists of a large tree perennial system containing an assortment of seasonal pests and natural enemies. As a first step to characterizing host-parasitoid associations in pecan food webs, we focus on aphid species and their parasitoids. Based on DNA barcoding of field-collected and reared specimens, we confirmed the presence of 3 species of aphid, one family of primary parasitoids, and 5 species of hyperparasitoids. By applying metabarcoding to field-collected aphid mummies, we were able to identify multiple species within each aphid mummy to unravel a complex food web of 3 aphids, 2 primary parasitoids, and upward of 8 hyperparasitoid species. The results of this study demonstrate that multiple hyperparasitoid species attack a single primary parasitoid of pecan aphids, which may have negative consequences for successful aphid biological control. Although further research is needed on a broader spatial scale, our results suggest multiple species exist in this system and may suggest a complex set of interactions between parasitoids, hyperparasitoids, and the 3 aphid species. This was the first time that many of these species have been characterized and demonstrates the application of novel approaches to analyze the aphid-parasitoid food webs in pecans and other tree crop systems.

Protective Geometry and Reproductive Anatomy as Candidate Determinants of Clutch Size Variation in Pentatomid Bugs.

AbstractMany animals lay their eggs in clusters. Eggs on the periphery of clusters can be at higher risk of mortality. We asked whether the most commonly occurring clutch sizes in pentatomid bugs could result from geometrical arrangements that maximize the proportion of eggs in the cluster's interior. Although the most common clutch sizes do not correspond with geometric optimality, stink bugs do tend to lay clusters of eggs in shapes that protect increasing proportions of their offspring as clutch sizes increase. We also considered whether ovariole number, an aspect of reproductive anatomy that may be a fixed trait across many pentatomids, could explain observed distributions of clutch sizes. The most common clutch sizes across many species correspond with multiples of ovariole number. However, there are species with the same number of ovarioles that lay clutches of widely varying size, among which multiples of ovariole number are not overrepresented. In pentatomid bugs, reproductive anatomy appears to be more important than egg mass geometry in determining clutch size uniformity. In addition, our analysis demonstrates that groups of animals with little variation in ovariole number may nonetheless lay a broad range of clutch shapes and sizes.

Partial refuges from biological control due to intraspecific variation in protective host traits.

Predicting how much of a host or prey population may be attacked by their natural enemies is fundamental to several subfields of applied ecology, particularly biological control of pest organisms. Hosts or prey can occupy refuges that prevent them from being killed by natural enemies, but habitat or ecological refuges are challenging or impossible to predict in a laboratory setting-which is often where efficacy and specificity testing of candidate biological control agents is done. Here we explore how intraspecific variation in continuous traits of individuals or groups that confer some protection from natural enemy attack-even after the natural enemy has encountered the prey-could provide partial refuges. The size of these trait-based refuges (i.e., the proportion of prey that survive natural enemy encounters due to protective traits) should depend on the relationship between trait values and host/prey susceptibility to natural enemy attack and on how common different trait values are within a host/prey population. These can be readily estimated in laboratory testing of natural enemy impact on target or nontarget prey or hosts as long as sufficient host material is available. We provide a general framework for how intraspecific variation in protective host traits could be integrated into biological control research, specifically with reference to nontarget testing as part of classical biological control programs. As a case study, we exposed different host clutch sizes of target (pest) and nontarget (native species) stink bug (Hemiptera: Pentatomidae) species to a well-studied exotic biocontrol agent, the egg parasitoid Trissolcus japonicus (Hymenoptera: Scelionidae). We predicted that the smallest and largest clutches would occupy trait-based refuges from parasitism. Although we observed several behavioral and reproductive responses to variation in host egg mass size by T. japonicus, they did not translate to increases in host survival large enough to change the conclusions of nontarget testing. We encourage researchers to investigate intraspecific variation in a wider variety of protective host and prey traits and their consequences for refuge size.

First Documented Wild Population of the "Hunter Fly", Coenosia attenuata Stein (Diptera: Muscidae) in North America.

Coenosia attenuata is a member of the tigrina-group of Coenosia (sensu Hennig 1964) and is a capable generalist predator in its larval and adult stages. C. attenuata is common in greenhouses worldwide, however, there are few documented cases of its presence in the wild. Here, we estimated C. attenuata presence in the southeastern USA peach orchards using pan traps. Over two years, a total of 717 specimens were collected from both commercially managed and fungicide-only managed peach orchards. C. attenuata is a known biological control agent in artificial greenhouse settings, but its impact on pest species in the wild is still unknown. For the first time in North America, we document an established wild population of C. attenuata, provide an overview of basic identification, and review potential benefits for biological control.

DNA barcoding of Sclerogibbidae and confirmation of the opposite sexes of Sclerogibba berlandi Benoit (Hymenoptera: Sclerogibbidae).

The female and male of Sclerogibba berlandi Benoit, 1963 (Hymenoptera: Sclerogibbidae), collected in Abu Dhabi (United Arab Emirates) and characterized by a pronounced sexual dimorphism, were associated for the first time by mitochondrial COI sequences. A comparison with COI sequences of S. crassifemorata Riggio De Stefani-Perez, 1888 and S. talpiformis Benoit, 1950 from Abu Dhabi proved the diversity of S. berlandi. The COI sequence of a female of S. rossi Olmi, 2005 from Japan, Okinawa-jima, was analyzed for the first time and compared with the other sequences.

Parasitoid Communities in the Variable Agricultural Environments of Blueberry Production in the Southeastern United States.

In blueberry crops, there are multiple pest species, and some of those can be suppressed by natural enemies including parasitoid wasps and predators. Parasitoid wasps occur within the environment often tracking pest species for food resources to complete their lifecycle. These small wasps are also sensitive to agricultural environments including agrichemicals, habitat availability, and climate. We investigated how the structure of parasitoid communities varied between organic and conventional blueberry systems, and how the communities of these parasitoids varied within field spatial scales (forested border vs edge vs interior). With the lower intensity of agricultural interventions occurring in organic systems and forested borders, we predicted more stable parasitoid numbers that would be insulated from predicted climate variability. In our study, parasitoids were observed in low abundance in each cropping system, with community structure dependent on both management practice and field position. Unmanaged blueberry fields and forested field borders contained more parasitoid families, and in conventional systems, we saw fewer families present in the field interior as compared to field borders. In this first study to characterize Southern parasitoid communities in blueberry production systems, we observed over 50 genera of parasitoids, with a few dominant families (Braconidae and Ichneumonidae) that would contribute to biological control in blueberry systems. Overall, we captured few parasitoids, which indicates a potential vulnerability in biological control, and the need for further research using other sampling techniques to better understand these parasitoid communities.

Cold tolerance, water balance, energetics, gas exchange, and diapause in overwintering brown marmorated stink bugs.

Halyomorpha halys (Hemiptera: Pentatomidae) is an emerging pest which established in Ontario, Canada, in 2012. Halyomporpha halys overwinters in anthropogenic structures as an adult. We investigated seasonal variation in the cold tolerance, water balance, and energetics of H. halys in southwestern Ontario. We also induced diapause in laboratory-reared animals with short daylength at permissive temperatures and compared cold tolerance, water balance, energetics, and metabolism and gas exchange between diapausing and non-diapausing individuals. Halyomorpha halys that overwintered outside in Ontario all died, but most of those that overwintered in sheltered habitats survived. We confirm that overwintering H. halys are chill-susceptible. Over winter, Ontario H. halys depressed their supercooling point to c. -15.4 °C, and 50% survived a 1 h exposure to -17.5 °C. They reduce water loss rates over winter, and do not appear to significantly consume lipid or carbohydrate reserves to a level that might cause starvation. Overall, it appears that H. halys is dependent on built structures and other buffered microhabitats to successfully overwinter in Ontario. Laboratory-reared diapausing H. halys have lower supercooling points than their non-diapausing counterparts, but LT50 is not enhanced by diapause induction. Diapausing H. halys survive desiccating conditions for 3-4 times longer than those not in diapause, through decreases in both respiratory and cuticular water loss. Diapausing H. halys do not appear to accumulate any more lipid or carbohydrate than those not in diapause, but do have lower metabolic rates, and are slightly more likely to exhibit discontinuous gas exchange.

A temporal trophic shift from primary parasitism to facultative hyperparasitism during interspecific competition between two coevolved scelionid egg parasitoids.

Understanding competition between scelionid parasitoids that exploit the same host may provide insight into strategies that allow coexistence on a shared resource. Competition studies typically focus on interactions between native and exotic parasitoids that do not share an evolutionary history; however, coevolved parasitoids may be more likely to demonstrate strategies to avoid or exploit a shared resource. We examined intrinsic and extrinsic competition between Asian Trissolcus japonicus (Ashmead) and T. cultratus (Mayr) (Hymenoptera: Scelionidae) associated with Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) that share an evolutionary history. Interspecific interactions were assessed by providing parasitized egg masses to each species at various intervals post-parasitism, and measuring host acceptance, developmental suitability, and guarding behaviour. Trissolcus japonicus showed high acceptance of parasitized hosts up to 72 h following oviposition by T. cultratus, despite a very poor developmental outcome. In contrast, T. cultratus generally avoided ovipositing in H. halys eggs containing T. japonicus early-instar larvae but did not avoid parasitizing H. halys that contained eggs and third instar larvae. The adaptive value of this behaviour was supported by developmental outcome: T. cultratus outcompeted T. japonicus eggs but not early-instar larvae, and a trophic shift occurred wherein T. cultratus developed as a facultative hyperparasitoid on third instar T. japonicus larvae. Trissolcus japonicus guarded egg masses 8-12× longer and displayed more aggressive interactions than T. cultratus, suggesting T. japonicus is the superior extrinsic competitor. Development as a facultative hyperparasitoid provided a competitive niche for Asian T. cultratus and confirms its instrinsic competitive superiority. This also occurs in a biologically distinct European population of T. cultratus, suggesting that facultative hyperparasitism as a competitive strategy is retained in geographically separated populations that have not coevolved with H. halys or T. japonicus.

Hidden Host Mortality from an Introduced Parasitoid: Conventional and Molecular Evaluation of Non-Target Risk.

Hidden trophic interactions are important in understanding food web ecology and evaluating the ecological risks and benefits associated with the introduction of exotic natural enemies in classical biological control programs. Although non-target risk is typically evaluated based on evidence of successful parasitism, parasitoid-induced host mortality not resulting in visible evidence of parasitism (i.e., nonreproductive effects) is often overlooked. The adventive establishment of Trissolcus japonicus, an exotic parasitoid of the introduced stink bug Halyomorpha halys, provides an opportunity to investigate the total impact of this parasitoid on target and non-target hosts in the field. We developed a new methodology to measure nonreproductive effects in this system, involving a species-specific diagnostic PCR assay for T. japonicus. We applied this methodology to field-deployed eggs of four pentatomid species, coupled with traditional rearing techniques. Nonreproductive effects were responsible for the mortality of an additional 5.6% of H. halys eggs due to T. japonicus, and were even more substantial in some of the non-target species (5.4-43.2%). The observed hidden mortality of native non-target species from an introduced parasitoid could change predictions about direct and indirect ecological interactions and the efficacy of biological control of the target pest.

Examining population structure of a bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), outbreak in western North America: Implications for gene flow and dispersal.

The bertha armyworm (BAW), Mamestra configurata, is a significant pest of canola (Brassica napus L. and B. rapa L.) in western North America that undergoes cyclical outbreaks every 6-8 years. During peak outbreaks millions of dollars are spent on insecticidal control and, even with control efforts, subsequent damage can result in losses worth millions of dollars. Despite the importance of this pest insect, information is lacking on the dispersal ability of BAW and the genetic variation of populations from across its geographic range which may underlie potential differences in their susceptibility to insecticides or pathogens. Here, we examined the genetic diversity of BAW populations during an outbreak across its geographic range in western North America. First, mitochondrial cytochrome oxidase 1 (CO1) barcode sequences were used to confirm species identification of insects captured in a network of pheromone traps across the range, followed by haplotype analyses. We then sequenced the BAW genome and used double-digest restriction site associated DNA sequencing, mapped to the genome, to identify 1000s of single nucleotide polymorphisms (SNP) markers. CO1 haplotype analysis identified 9 haplotypes distributed across 28 sample locations and three laboratory-reared colonies. Analysis of genotypic data from both the CO1 and SNP markers revealed little population structure across BAW's vast range. The CO1 haplotype pattern showed a star-like phylogeny which is often associated with species whose population abundance and range has recently expanded and combined with pheromone trap data, indicates the outbreak may have originated from a single focal point in central Saskatchewan. The relatively recent introduction of canola and rapid expansion of the canola growing region across western North America, combined with the cyclical outbreaks of BAW caused by precipitous population crashes, has likely selected for a genetically homogenous BAW population adapted to this crop.

Discovery of Aphis ruborum (Hemiptera: Aphididae) and Aphelinus varipes (Hymenoptera: Aphelinidae) on Cultivated Strawberry in Mississippi, USA.

An adventive aphid and novel host-parasitoid association from cultivated strawberry (Fragaria × ananessa Duch. cv. Chandler; Fragaria × ananessa Duch. cv. Camarosa) in Mississippi, USA are reported herein. The aphid, first detected in high tunnel cultivation, was found predominately on newly emerged, not fully developed leaflets of daughter plants in the Fall of 2016. By 2017, aphids and their associated mummies were observed on fully developed leaflets on mother plants of both cultivars. The aphid was identified as Aphis ruborum (Börner & Schilder) using morphology and DNA barcoding studies. In addition, DNA barcoding identified parasitoid adults emerging from aphid mummies as two cryptic species, Aphelinus varipes (Foerster) and Aphelinus albipodus Hayat and Fatima. Occurrence of A. ruborum in Mississippi represents a new state record and the eastern-most established record in the United States. The A. ruborum - A. varipes or A. albipodus host-parasitoid association is reported for the first time anywhere in the world.

A modified DNA barcode approach to define trophic interactions between native and exotic pentatomids and their parasitoids.

The establishment of invasive Halyomorpha halys (Stål) outside of its native range may impact native species assemblages, including other pentatomids and their scelionid parasitoids. This has generated interest in defining species diversity and host-parasitoid associations in this system to better understand the impact of invasive alien species on trophic interactions in invaded regions. Information on scelionid-pentatomid associations in natural habitats is lacking, and species-level identification of these associations can be tenuous using rearing and dissection techniques. Naturally occurring pentatomid eggs were collected in areas where H. halys has established in Canada and were analysed using a modified DNA barcoding approach to define species-level trophic interactions. Identification was possible for >90% of egg masses. Eleven pentatomid and five scelionid species were identified, and trophic links were established. Approximately 70% of egg masses were parasitized; parasitism and parasitoid species composition were described for each species. Telenomus podisi Ashmead was the dominant parasitoid and was detected in all host species. Trissolcus euschisti Ashmead was detected in several host species, but was significantly more prevalent in Chinavia hilaris (Say) and Brochymena quadripustulata (Fabricius). Trissolcus brochymenae Ashmead and Tr. thyantae Ashmead were recorded sporadically. Parasitism of H. halys was 55%, and this species was significantly less likely to be parasitized than native pentatomids. The scelionid species composition of H. halys consisted of Te. podisi, Tr. euschisti and Tr. thyantae. Although these species cannot develop in fresh H. halys eggs, we demonstrate that parasitoids attempt to exploit this host under field conditions.

Understanding the mismatch between behaviour and development in a novel host-parasitoid association.

Foraging parasitoid females should preferentially oviposit on hosts most suitable for progeny development to maximize their fitness. However, the introduction of a new host species may disrupt the link between the reliability of the cues and the expected adaptive outcome of female choice, leading to an evolutionary trap. This mismatch between behavioural acceptance and lack of development exists for North American and European egg parasitoids (Scelionidae) that encounter invasive Halyomorpha halys in areas where this exotic host has recently established. To explain this mismatch, we utilized an L9 orthogonal array design to assess and rank the influence of several critical factors characterizing host resource (host species, egg age, egg status, and surface wash) on behaviour (acceptance, patch residence and patch exploitation) and development of North American native Trissolcus euschisti egg parasitoid. Our results indicate that the host egg age is most important for behaviour, but is least influential for development of the progeny. This study suggests that the maladaptive decision to oviposit in an unsuitable host is due to a mismatch between the cues that females use, and the subsequent expected outcome of this choice. Therefore, it is the relative importance of individual factors when assessed simultaneously that influences the decision-making of female parasitoids.

Seasonal parasitism and host specificity of Trissolcus japonicus in northern China.

The brown marmorated stink bug, Halyomorpha halys (Stål), native to China, Japan, and Korea, has emerged as a harmful invasive pest of a variety of crops in North America and Europe. The Asian egg parasitoid Trissolcus japonicus has been identified as the most promising agent for classical biological control of invasive H. halys populations. A 4-year study evaluated the fundamental and ecological host ranges of T. japonicus as well as its phenology and impact on H. halys populations in fruit orchards in its native range in northern China. In laboratory no-choice tests, developmental suitability of eight non-target host species for T. japonicus was demonstrated by the successful production of progeny on the majority (>85%) of non-target host species tested. In field-collected, naturally laid egg masses, T. japonicus was the most abundant parasitoid associated with H. halys and Dolycoris baccarum, but was also sporadically found in Plautia crossota. Furthermore, it was regularly reared from sentinel egg masses of Menida violacea, Arma chinensis, and Carbula eoa. The only species that did not support development in the laboratory and field was Cappaea tibialis. Besides the benefit of having a high impact on H. halys populations in Northern China, the risk assessment conducted in the area of origin indicates that native Pentatomidae in North America and Europe could be negatively impacted by T. japonicus. Whether the benefits of T. japonicus outweigh the possible risks will have to be evaluated based on the outcome of additional host range studies in the two invaded regions.

An exotic parasitoid provides an invasional lifeline for native parasitoids.

The introduction of an exotic species may alter food webs within the ecosystem and significantly affect the biodiversity of indigenous species at different trophic levels. It has been postulated that recent introduction of the brown marmorated stinkbug (Halyomorpha halys (Stål)) represents an evolutionary trap for native parasitoids, as they accept H. halys egg masses as a host but produce no viable progeny. Interspecific interactions between European egg parasitoid, Trissolcus cultratus (Mayr), and an Asian parasitoid, Trissolcus japonicus (Ashmead), were assessed by providing egg masses to T. cultratus at various time intervals following the initial parasitization by T. japonicus. The suitability of the host for the parasitoid development was re-assessed by providing T. cultratus with fresh and frozen egg masses of various ages. The likelihood of T. cultratus being able to attack previously parasitized egg masses was determined by assessing the duration of egg mass guarding behavior by T. japonicus following parasitization. The results of experiments examining the interspecific interactions between a native European egg parasitoid, T. cultratus, and an Asian parasitoid, T. japonicus (a candidate for the biological control of H. halys), showed that the native species can act as facultative hyperparasitoid of the exotic one. Although this is only possible during certain stages of T. japonicus development, the presence of the introduced parasitoid may reduce the impact of the evolutionary trap for indigenous parasitoid species. There is a possibility that the occurrence of facultative hyperparasitism between scelionid parasitoids associated with stinkbugs is common. This resulting intraguild predation could promote conservation and stabilization of natural communities by impacting the diversity and population dynamics of native stinkbugs and their parasitoids (e.g., by allowing native parasitoids to avoid wasting reproductive effort on unsuitable hosts), or reduce success of biological control programs (e.g., by reducing the population size of the exotic parasitoids).

Cold tolerance of third-instar Drosophila suzukii larvae.

Drosophila suzukii is an emerging global pest of soft fruit; although it likely overwinters as an adult, larval cold tolerance is important both for determining performance during spring and autumn, and for the development of temperature-based control methods aimed at larvae. We examined the low temperature biology of third instar feeding and wandering larvae in and out of food. We induced phenotypic plasticity of thermal biology by rearing under short days and fluctuating temperatures (5.5-19°C). Rearing under fluctuating temperatures led to much slower development (42.1days egg-adult) compared to control conditions (constant 21.5°C; 15.7days), and yielded larger adults of both sexes. D. suzukii larvae were chill-susceptible, being killed by low temperatures not associated with freezing, and freezing survival was not improved when ice formation was inoculated externally via food or silver iodide. Feeding larvae were more cold tolerant than wandering larvae, especially after rearing under fluctuating temperatures, and rearing under fluctuating temperatures improved survival of prolonged cold (0°C) to beyond 72h in both larval stages. There was no evidence that acute cold tolerance could be improved by rapid cold-hardening. We conclude that D. suzukii has the capacity to develop at low temperatures under fluctuating temperatures, but that they have limited cold tolerance. However, phenotypic plasticity of prolonged cold tolerance must be taken into account when developing low temperature treatments for sanitation of this species.

Reproductive arrest and stress resistance in winter-acclimated Drosophila suzukii.

Overwintering insects must survive the multiple-stress environment of winter, which includes low temperatures, reduced food and water availability, and cold-active pathogens. Many insects overwinter in diapause, a developmental arrest associated with high stress tolerance. Drosophila suzukii (Diptera: Drosophilidae), spotted wing drosophila, is an invasive agricultural pest worldwide. Its ability to overwinter and therefore establish in temperate regions could have severe implications for fruit crop industries. We demonstrate here that laboratory populations of Canadian D. suzukii larvae reared under short-day, low temperature, conditions develop into dark 'winter morph' adults similar to those reported globally from field captures, and observed by us in southern Ontario, Canada. These winter-acclimated adults have delayed reproductive maturity, enhanced cold tolerance, and can remain active at low temperatures, although they do not have the increased desiccation tolerance or survival of fungal pathogen challenges that might be expected from a more heavily melanised cuticle. Winter-acclimated female D. suzukii have underdeveloped ovaries and altered transcript levels of several genes associated with reproduction and stress. While superficially indicative of reproductive diapause, the delayed reproductive maturity of winter-acclimated D. suzukii appears to be temperature-dependent, not regulated by photoperiod, and is thus unlikely to be 'true' diapause. The traits of this 'winter morph', however, likely facilitate overwintering in southern Canada, and have probably contributed to the global success of this fly as an invasive species.

Adult plasticity of cold tolerance in a continental-temperate population of Drosophila suzukii.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a worldwide emerging pest of soft fruits, but its cold tolerance has not been thoroughly explored. We determined the cold tolerance strategy, low temperature thermal limits, and plasticity of cold tolerance in both male and female adult D. suzukii. We reared flies under common conditions (long days, 21°C; control) and induced plasticity by rapid cold-hardening (RCH, 1h at 0°C followed by 1h recovery), cold acclimation (CA, 5 days at 6°C) or acclimation under fluctuating temperatures (FA). D. suzukii had supercooling points (SCPs) between -16 and -23°C, and were chill-susceptible. 80% of control flies were killed after 1h at -7.2°C (males) or -7.5°C (females); CA and FA improved survival of this temperature in both sexes, but RCH did not. 80% of control flies were killed after 70 h (male) or 92 h (female) at 0°C, and FA shifted this to 112 h (males) and 165 h (females). FA flies entered chill coma (CTmin) at approximately -1.7°C, which was ca. 0.5°C colder than control flies; RCH and CA increased the CTmin compared to controls. Control and RCH flies exposed to 0°C for 8h took 30-40 min to recover movement, but this was reduced to <10 min in CA and FA. Flies placed outside in a field cage in London, Ontario, were all killed by a transient cold snap in December. We conclude that adult phenotypic plasticity is not sufficient to allow D. suzukii to overwinter in temperate habitats, and suggest that flies could overwinter in association with built structures, or that there may be additional cold tolerance imparted by developmental plasticity.

Species specific identification of the Neofabraea pathogen complex associated with pome fruits using PCR and multiplex DNA amplification.

Five species of pathogenic fungi belong to Neofabraea. One of these, N. krawtzewii (syn. N. populi), is responsible for bark lesions on poplar (Populus) trees. The other four species cause post-harvest bull's eye rot of pome fruits, and at least two of these also cause bark cankers on pome fruit trees. Morphological variation among these species is slight, and overlap in geographic range sometimes occurs. As a consequence, identification based on conventional criteria can be tenuous. PCR primers with putative species specificity were developed following genetic analysis of the beta-tubulin gene for isolates of each of the five species of Neofabraea. PCR conditions required to achieve specificity of the primer sets were determined, and a multiplex PCR protocol was developed to optimize their diagnostic utility on apple fruits. A protocol with higher annealing temperatures in the initial PCR cycles followed by lower temperatures in later cycles gave complete species-specificity when the primer sets were used individually and in multiplex, resulting in successful detection of the pathogens from axenic culture and infected apple fruits.