Field resistance of codling moth against Cydia pomonella granulovirus (CpGV) is autosomal and incompletely dominant inherited

The current appearance of local codling moth populations with resistance to Cydia pomonella granulovirus (CpGV) is an impediment to continuous CpGV application. Therefore, crossing experiments have been performed in order to gain information about the inheritance of resistance. Evidence is presented that the observed field resistance is stably inherited even under non-selective conditions in the laboratory. Offspring of reciprocal F(1) crosses between a susceptible ('S') and a resistant ('R') strain and backcrosses between F(1) and S were bioassayed at different CpGV concentrations. The resistant strain showed 100 times lower susceptibility in 7-day bioassays. The responses of the reciprocal crosses (male S x female R and female S x male R) did not differ significantly, indicating that resistance is autosomally inherited. The median lethal concentration for the F(1) progeny was intermediate between those of its parental strains. Mortality data obtained from the backcrosses suggested that inheritance of resistance is due to a non-additive, polygenic trait.     

Investigating the horizontal transmission of the Cydia pomonella granulovirus (CpGV) in a model system

Repeated applications of Cydia pomonella granulovirus (CpGV) can effectively control the codling moth (CM) in apple orchards. However, it is still unknown whether horizontal transmission of the virus from infected to uninfected larvae contributes to the efficacy of the virus insecticide. Horizontal transmission of CpGV was assayed using detached apples. In experiments using artificially applied virus dots on the apple’s surface or infected CM larvae as virus inoculum, it was found that the likelihood of infection of healthy CM larvae relied mainly on the larval behavior. The amount of virus inoculum, either applied artificially or produced by the infected larvae, impacted the infection rate only to a small degree. In the experiments, CM larvae exhibited a strong preference in entry sites, increasing the chance for horizontal transmission. Depending on the experimental design, horizontal transmission rates of about 40% were observed in laboratory assays.     

Overcoming the resistance of codling moth against conventional Cydia pomonella granulovirus (CpGV-M) by a new isolate CpGV-I12

Recently, codling moth (CM, Cydia pomonella L.) populations with a significantly reduced susceptibility to C. pomonella granulovirus (CpGV) products have been observed in Germany. A novel CpGV isolate, designated CpGV-I12, is able to overcome the CpGV resistance. CpGV-I12 originated from Iran and showed superior efficacy in laboratory bioassays against a resistant CM strain (CpR), which has a 100-fold reduced susceptibility to commercially used isolate CpGV-M. Determination of the median lethal concentration (LC₅₀) indicated that CpGV-I12 is nearly as efficient in resistant CpR as CpGV-M in a susceptible CM strain (CpS). Beyond, CpGV-I12 caused superior mortality in CpS. Infection experiments showed that the resistance breaking effect can be observed in all instars of CpR. CpGV-I12 is a promising alternative control agent of CM in orchards where conventional CpGV products fail. In addition, we demonstrate in bioassays with recombinant expressed Cry1Ab that cross-resistance to CpGV and Bacillus thuringiensis products is not likely.     

Activity of Bacillus thuringiensis delta-endotoxins against codling moth (Cydia pomonella L.) larvae

Solubilized protoxins of nine Cry1 and one hybrid Cry1 delta-endotoxin from Bacillus thuringiensis were tested for their activity against larvae of the codling moth (Cydia pomonella L). Cry1Da was the most toxic, followed by Cry1Ab, Cry1Ba, and Cry1Ac, while Cry1Aa, Cry1Fa, Cry1Ia, and SN19 were still less active. Cry1Ca and Cry1Cb showed no activity. In vitro trypsin activation increased activity of all eight active delta-endotoxins, and dramatically enhanced toxicity of hybrid SN19, Cry1Aa, Cry1Ac, and Cry1Fa. The differences between toxicity of proteins before and after trypsin digestion suggests that proteolytic activation in the C. pomonella digestive tract plays a critical role for the activity of Cry proteins against this insect.     

Comparative activity of baculoviruses against the codling moth Cydia pomonella and three other tortricid pests of tree fruit

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54-93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC(50) values for each virus. The LC(50)s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm(2), 1.77 x 10(3) occlusion bodies (OBs)/mm(2), and 3.05 x 10(3)OBs/mm(2), respectively. The LT(50) determined for AfMNPV using an approximate LC(95) of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.     

Attachment ability of the codling moth Cydia pomonella L. to rough substrates

Host plant surfaces of the codling moth, Cydia pomonella L. (Lepidoptera, Tortricidae), vary in microtopography, which can affect its attachment, locomotion, and oviposition behaviour. This study was performed to investigate the effect of surface roughness on the attachment ability of adult insects. Using a centrifugal force device, friction forces of both sexes were assessed on six epoxy resin substrates differing only in the dimensions of their surface asperities, ranging from 0 μm to 12 μm. Surface topography significantly affected friction forces. Maximal force was measured on the smooth substrate whereas minimal force was assessed on microrough substrates with 0.3 μm and 1.0 μm size of asperities. On the remaining rough substrates, friction forces were significantly higher but still lower than on the smooth substrate. Both sexes generated similar forces on the same substrate, in spite of the considerable difference in their body mass. Thus, it is expected that both sexes can attach effectively to differently structured plant substrates in their habitat. However, since smooth surfaces have been reported previously to be the most favorable substrates for ovipositing females of C. pomonella, it is possible that they use their attachment system to sense the substrate texture and prefer those substrates to which their arolia attach the best.     

New method for testing solar sensitivity of commercial formulations of the granulovirus of codling moth (Cydia pomonella, Tortricidae: Lepidoptera)

A method for screening codling moth granulovirus (CpGV) formulation sensitivity to sunlight using specially prepared half apples and a solar simulator is described. The half apple preparation allows an even coverage of virus over the surface of the fruit that would not be possible using whole apples. Leaves and artificial medium were not usable for extended periods of exposure in the solar simulator due to excess drying. Fruit was sprayed with 10⁻³ and 10⁻⁵ dilutions of three commercial formulations of CpGV (Carpovirusine, Cyd-X, and Virosoft) and infested with codling moth neonates. Half of the sprayed fruit was exposed to 650 W/m² for 4 h in an Atlas Suntest CPS solar simulator resulting in an accumulated radiant energy of 9.36 × 10⁶ J/m² before they were infested with neonate codling moth larvae. Spraying non-irradiated fruit with the 10⁻³ dilution of Cyd-X and Virosoft resulted in nearly 100% mortality of neonate larvae. Irradiation reduced viral activity by 71-98% at the 10⁻³ dilution and by up to 32% at the 10⁻⁵ dilution relative to non-irradiated fruit. The procedures utilized enabled good preservation of the fruit throughout the incubation period and minimized invasion of the fruit by plant pathogens and saprophytic organisms. This laboratory method for screening candidate formulations and potential UV protectants could conserve time and resources by eliminating adjuvants with less potential in laboratory tests and field testing only the most promising candidates. It also enables year-round testing.     

Incidence and transmission of a granulovirus in a large codling moth [Cydia pomonella L. (Lepidoptera: Tortricidae)] rearing facility

Incidences of potential per os Cydia pomonella granulovirus (CpGV) transmission within a large codling moth colony were identified. CpGV was detected in the water which is used to wash egg sheets. When pre-neonates were extracted from eggs prior to emergence and tested for the presence of CpGV, 40% were found to carry amounts of CpGV detectable by a polymerase chain reaction (PCR) assay, suggesting possible transovarial transmission of the virus. Although symptoms typical of virus infection and larval death were found infrequently within communal rearing trays, the frequency with which CpGV DNA was detected by PCR assays increased from a mean of 31% of 10-day-old larvae to 94% of 25-day-old larvae. CpGV in codling moth cadavers remained virulent after being held at 60 degrees C for 3 days under conditions similar to the treatment of spent diet at the rearing facility before its disposal. PCR tests of surface samples taken from air filters and rearing rooms of the rearing facility were found to contain CpGV. Bioassays of surface samples from the diet trash bin and a filter through which outside air is passed before entering the rearing chambers resulted in significant codling moth neonate mortality. The virulence of CpGV in dust from the spent diet and the original inadvertent positioning of the diet trash bin directly below one of the air intake ducts are suggested as a possible additional source of CpGV contamination within the facility.     

Development of a direct test of baculovirus resistance in wild codling moth populations

The occurrence of resistance of codling moth (CM, Cydia pomonella L.) to Cydia pomonella granulovirus (CpGV) used as biological control agent revealed the need for fast and reliable resistance monitoring methods. Here, we describe the development of a laboratory resistance test that is directly performed on larvae extracted from infested apples. This test is based on a 14‐day bioassay at a discriminative CpGV concentration of 2 × 10⁵ occlusion bodies/ml diet and can be applied to L1–L4 larvae. Information on virus resistance can be obtained within <4 weeks. In a survey, CM larvae were isolated and tested from 6698 apples from 10 different orchards in Germany, Austria, Switzerland, Italy and the Netherlands. We identified seven CM populations resistant or partly resistant to CpGV isolates. Although some of the orchards were treated with commercial CpGV products, this method allowed us to obtain reliable information about the resistance status of the examined populations.     

Field evaluation of commercial formulations of the codling moth granulovirus: persistence of activity and success of seasonal applications against natural infestations of codling moth in Pacific Northwest apple orchards

Inundative applications of the codling moth (CM), Cydia pomonella L., granulovirus (CpGV), which target neonate larvae before or during initial entry into fruit, offer potential for selective control of this key pest. In field tests on apple we compared the persistence and efficacy of single applications of three CpGV products approved for organic orchards in North America. In addition, the success of repeated (2–14) applications of one product (Cyd-X) as a principal control measure for CM in apple orchards was monitored following operational use by cooperating growers at four separate locations. In the first study, an early season application of all products at label rates remained highly effective for the first 24 h (averaging 94% larval mortality relative to controls) and moderately effective after 72 h (averaging 71% mortality) during dry sunny conditions. Significant activity remained up to 14 days, suggesting prolonged survival of the virus in UV-protected locations, such as the calyx of fruit. A second application later in the season was slightly less effective. Data obtained from commercial sites provide circumstantial evidence for the effectiveness of well-timed CpGV applications against CM outbreaks. In all cases where first generation larvae were targeted beginning at egg hatch (≈250 degree days) and treated areas monitored (0.3–1.6 ha plots), fruit damage during the second larval generation was reduced or eliminated. Based on the number of live larvae recovered throughout the season, mortality rates remained high (80.3–100% across sites). The cumulative number of moths caught in pheromone-baited traps was reduced (66–94%) in the second flight. Data from tree bands placed to catch diapause-destined larvae indicated overwintering generations remained low in treated sites (0.18 larvae/band).     

Evaluation of spray-dried lignin-based formulations and adjuvants as solar protectants for the granulovirus of the codling moth, Cydia pomonella (L)

Commercial formulations of the codling moth, Cydia pomonella L., granulovirus (CpGV) are limited by their short residual activity under orchard conditions in the Pacific Northwest. We evaluated spray-dried lignin-encapsulated formulations of CpGV for improved solar stability based on laboratory bioassays with a solar simulator and in field tests in an infested apple orchard. In laboratory tests, aqueous lignin formulations containing a high dosage of 3 x 10(10) occlusion bodies (OB)/L, with and without the additives titanium dioxide (TiO(2)) and sugar, provided significant solar protection of virus, i.e., mortality of codling moth exposed to lignin formulations that had been irradiated with 9.36 x 10(6) joules/m(2) was 92-94%, compared with 66-67% from a glycerin-stabilized product (Cyd-X) or suspension of pure unformulated virus at the same rates. By comparison, a lower dosage of the lignin formulation (3 x 10(8)OB/L) did not provide significant solar protection. Equivalent dosage-dependent patterns in solar protection were observed in further tests with the lignin formulation, when an intermediate (3 x 10(9)OB/L) as well as the low dosage provided no solar protection. Equivalent rates of a blank lignin formulation (containing no virus) did not affect larval mortality, suggesting a protective effect of the lignin on the virus at the high rate. The use of several spray adjuvants, 'NuFilm-17' and 'Organic Biolink' (sticker-spreaders at 0.06% v/v), 'Raynox' (sunburn protectant at 5% v/v), and 'Trilogy'(neem oil at 1% v/v) did not provide solar protection of a commercial CpGV preparation in laboratory tests. In season long orchard tests (Golden Delicious), the lignin formulation of CpGV applied at 6.57 x 10(12)OB/ha did not significantly improve control of codling moth or protection of fruit compared with Cyd-X at equivalent rates. Our studies show that lignin-based CpGV formulations provided solar protection at relatively high virus dosages. The testing of lignin formulations containing reduced virus concentrations may allow virus solar protection to be achieved at more economical rates.     

Death of Cydia pomonella larvae and damage to apple fruit, after field application of codling moth granulosis virus

In field experiments, larvae of codling moth Cydia pomonella (L.) rarely acquired granulosis virus on hatching from the egg, but picked up most later, on the tree surface. Deposits of virus sprayed in 1.0% w/v skimmed milk did not affect neonate larval behaviour. Larvae died, usually in the first instar, after entering treated fruit, but they frequently entered via the calyx or near the base of the stalk or through cracks in the skin, where little feeding damage by first-and sometimes second-instar larvae was seen.

---

Résumé En verger, la pulvérisation d'oeufs de carpocapse avec du virus de la granulose en suspension dans l'eau (additionnée de lait écrémé dilué à 1%) n'a pas modifié la survie des chenilles avant pénétration dans le fruit; par contre la pulvérisation des arbres a provoqué une forte mortalité. Bien que des chenilles consommant des poils et la surface des feuilles aient été observées avant leur pénétration dans le fruit, ce qui aurait pu provoquer leur contamination par le virus, il semble que la contamination létale provienne des fruits seuls.La présence de produit n'a modifié ni le comportement larvaire, ni le taux de pénétration dans les fruits; la mortalité y a lieu ensuite, généralement au premier stade. Dans 74 à 78% des cas, les chenilles ont pénétré dans le fruit par le calice ou près de la base du pédoncule — aucun dégât provenant de larves du premier stade n'y était visible, de même que dans le calice pour les larves du deuxième stade. Par contre, toute pénétration par la surface du fruit était repérable dès le premier stade. Il est possible que la répartition des lieux de pénétration dans le fruit influe sur la létalité due au virus et explique les variations d'efficacité observées en verger. Un système de classification des dégâts, provoqués lors de la pénétration dans le fruit, de chenilles du premier au troisième stade est proposé pour évaluer l'efficacité des essais en verger.

     

Control of diapause in codling moth larvae

Diapause in a New Zealand strain of codling moth (Cydia pomonella Linnaeus [Lepidoptera: Olethreutidae]) was induced in larvae by photoperiods of 15 h or less. Once diapause had been initiated, it could not be terminated by any combination of conditions tested for at least 20 days after cocooning. In diapausing larvae a low rate of pupation occurred at 25 °C under a long day (18 h) photoperiod. A high rate of pupation was achieved under a long day regime when larvae were decocooned, and provided with apple as nourishment. Diapause could be terminated predictably in 94–100% of larvae by 1) conditioning at 15 °C and constant darkness for periods of 40–100 days, then 2) chilling at 2±2 °C and constant darkness for 20–50 days followed by 3) any post-chill condition periods at 25 °C, 18 h photoperiod. Complete diapause termination was achieved when 100 days conditioning was followed by 30 days or 50 days post-chill period. Under these conditions, 76% termination occurred in the post-chill period after 10 days, and 93% after 25 days.To terminate diapause in codling moth larvae, we recommend that a 100 days conditioning followed by 30 days chilling and 50 days post chilling periods be used.     

Vertical distribution of codling moth adults in pheromone-treated and untreated plots

The vertical distribution of codling moth,Cydia pomonella (L.) within pheromone-treated and untreated apple and pear orchard canopies was determined using tethered virgin females, unbaited sticky traps, and blacklight observation of released moths. Mating of virgin females tethered at various heights in untreated orchard canopies increased with placement height from 1–4 m. Application of pheromone dispensers for mating disruption at 2 and 4 m above the ground greatly decreased mating. Greatest capture of males and females on unbaited sticky traps occurred at mid- and upper-canopy heights. Total capture of males and females in pheromone-treated plots was not statistically different than in untreated plots. The percentage of mated females captured on sticky traps did not vary with trap height or pheromone treatment. Released moths marked with flourescent powder and observed at dark with a blacklight indicated that moths are primarily distributed high in the canopy. However, males shifted to a position lower in the canopy when pheromone dispensers were placed 2.1 m above the ground. Results suggest that pheromone dispensers be placed in the upper canopy for optimal disruption of codling moth mating.     

Differential ovicidal and larvicidal resistance to benzoylureas in the codling moth, Cydia pomonella

French populations of the codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae), a major pest in apple and pear orchards, have developed resistance to different classes of insecticides including the benzoylurea diflubenzuron, a chitin synthesis inhibitor. Ovicidal tests performed on two susceptible strains and one strain selected for its resistance to diflubenzuron revealed the same order of magnitude in resistance ratios to this compound (30-fold) and two other benzoylureas teflubenzuron and flufenoxuron (22- and 11-fold, respectively). Field rates of these three compounds induced a 45–55% decrease in hatching in the resistant strain, compared to over 90% in the susceptible insects. Despite a 52-fold ovicidal resistance ratio to the juvenile hormone analog fenoxycarb, this compound induced a 85% decrease in hatching in the resistant strain. Conversely the newly hatched larvae of the resistant strain exhibited a 45 000-, 33- and 2.1-fold resistance ratio to diflubenzuron, teflubenzuron and flufenoxuron, respectively. The latter value was not significant, and the field rate of flufenoxuron killed over 97% of the resistant larvae while diflubenzuron had no effect. This lack of relationship between ovicidal and larvicidal resistance may be due to different transport properties together with differential enzymatic metabolization. Our results may limit the validity of substitution instars, which approach is frequently used for resistance monitoring. More importantly for resistance management, the resistance of different target instars to each compound has to be considered when establishing control strategies.     

Tarsal morphology and attachment ability of the codling moth Cydia pomonella L. (Lepidoptera, Tortricidae) to smooth surfaces

Despite several studies on the attachment ability of different insect taxa, little is known about this phenomenon in adult Lepidoptera. In this study we combined morphological and experimental analyses of tarsal adhesive devices and the attachment ability of the codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae) to smooth surfaces. Pretarsi of C. pomonella attach to smooth substrates by means of their smooth, flexible and well developed arolia. Using the centrifugal force measurement technique, friction forces of males and females were assessed on hydrophobic and hydrophilic glass surfaces. Adults of both sexes generated similar forces in spite of the noticeable difference in their body masses. That is why males showed significantly higher safety factors (attachment force divided by body weight) compared to those of females. Hydrophobicity of the substrate had no considerable effect on friction forces. For females, friction forces (sliding parallel to the substrate plane) were compared with adhesive forces (pulling off perpendicularly from the substrate plane) measured on Plexiglas surfaces. It can be concluded that the attachment system of C. pomonella is rather robust against physico-chemical properties of the substrate and is able to achieve a very good attachment on vertical and horizontal substrata.     

Haemolymph protein reserves of diapausing and nondiapausing codling moth larvae, Cydia pomonella (L) (Lepidoptera: Tortricidae)

Haemolymph from diapausing codling moth larvae contain a dominant soluble protein, which is of minor significance in the haemolymph of nondiapausing larvae. This haemolymph protein is accumulated during the ultimate larval instar and its concentration is maintained throughout diapause development. Larvae induced into a diapause-like condition following topical treatment with a juvenile hormone mimie stored the haemolymph protein.     

Susceptibility of Cydia pomonella to Bacillus thuringiensis under laboratory and field conditions

The delta-endotoxin of 12 strains in 10 subspecies of Bacillus thuringiensis Berliner was highly active against Cydia pomonella (L.) when assayed under laboratory conditions on artificial diet. These results could not be confirmed in the field.The disappointing results obtained under field conditions are due to the behaviour of the target insect. C. pomonella larvae do not ingest food during penetration of the fruit. The larva bites pieces of the epidermis and deposits them without ingestion on top of the entry hole.

---

Zusammenfassung Das delta-Endotoxin von B.t. war in Laborversuch auf Kunstmedium gegenüber den Larven des Apfelwicklers, C. pomonella, sehr aktiv. Die hohe Aktivität konnte aber unter Feldbedingungen nicht mehr bestätigt werden.Es wurde nachgewiesen, dass die unbefriedigenden Resultate von B. thuringiensis unter Feldbedingungen auf das Verhalten der Junglarven zurückzuführen sind: Die Larven nehmen während dem Eindringen in den Apfel keine Nahrung auf, sondern deponieren die herausgebissenen Epidermistücke über der Einbohrstelle.

     

Diapause induction in the codling moth, Cydia pomonella: effect of prediapause temperatures

The effect of four prediapause temperatures (18, 22, 26 and 30°C) on the photoperiodic response of the codling moth, Cydia pomonella (L.), was studied under controlled conditions. The highest rates of diapause were recorded, for all day-lengths, at temperatures of 22 and 26°C while relatively lower rates of diapause were elicited at 18 and 30°C. The same trend was demonstrated by projecting the values of the critical photoperiod which induces 50% diapause (=CPhP₅₀) over the prediapause temperature. The change in diapause incidence as a function of photoperiod, at all prediapause temperatures, exhibited a response characteristic of long-day insects, i.e. high rates of diapause at short days (12–13.5 h) and a decrease in diapause incidence at long days (14–15 h). The results for temperatures 22, 26 and 30°C support the view that lower prediapause temperatures enhance diapause induction, at a give photoperiod, while higher temperatures tend to avert or diminish the process. On the other hand, the low rates of diapause obtained at 18°C contradict this view. Nevertheless, high correlation was found between the laboratory evidence and field data, indicating the adaptability of the Israeli codling moth to subtropical climate.     

Diapause induction in the codling moth, Cydia pomonella: effect of larval diet

The effect of larval diet on diapause induction in the Israeli strain of the codling moth, Cydia pomonella (L.), was studied in a field trial using intact apple fruits of two varieties: Ana (early-ripening, in the end of June) and Granny Smith (late-ripening, in October). Diapause incidence increased as fruit age (determined as days from fruit-set) progressed. These results corroborate former studies on other strains of the codling moth, where excised fruits were used.The combination of 80-day-old, fully ripe, Ana fruit treatment with the longest days of the year, yielded 38% diapause. This result demonstrates that mature fruit (inducing diapause) cannot completely override the effect of long day (averting diapause), but does confirm that larval diet modifies the photoperiodic induction of diapause in the codling moth.Deceased, October 1988