Chemical ecology and management of Lobesia botrana (Lepidoptera: Tortricidae)

The moth Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) feeds on grapes (Vitis vinifera L.), reducing yield and increasing susceptibility to fungal infections. L. botrana is among the most economically important insects in Europe and has recently been found in vineyards in Chile, Argentina, and California. Here, we review L. botrana biology and behavior in relation to its larval host (the grapevine) and its natural enemies. We also discuss current and future control strategies in light of our knowledge of chemical ecology, with an emphasis on the use of the sex pheromone-based strategies as an environmentally safe management approach. Pheromone-mediated mating disruption is the most promising technique available on grapes and is currently implemented on approximately 140,000 ha in Europe. Experience from several growing areas confirms the importance of collaboration between research, extension, growers, and pheromone-supply companies for the successful implementation of the mating disruption technique. In the vineyards where mating disruption has been successfully applied as an areawide strategy, the reduction in insecticide use has improved the quality of life for growers, consumers, as well as the public living near wine-growing areas and has thereby reduced the conflict between agricultural and urban communities.     

Physiological factors regulating polyandry in Lobesia botrana (Lepidoptera: Tortricidae)

Abstract. The effects of several physiological factors related to female multiple mating in the monandrous Lobesia botrana Denis and Schiffermuller (Lepidoptera: Tortricidae) were studied under laboratory conditions. Polyandry was assessed observing the induction of re-calling in previously once-mated females. Female age at first mating had no effect on induction of re-calling during practically the whole of the female lifetime, but in older females it was significantly lower. The percentage of re-calling was negatively correlated with the volume of the spermatophore received, ranging from ≅ 23% with the largest spermatophores to ≅ 75% with the smallest ones. Furthermore, the smaller the spermatophore volume, the earlier the re-calling was induced, significantly reducing die female refractory period after the first mating. Heavy females showed a significantly higher rate of re-calling (52.8%) than light ones (37.0%), but no differences were observed when females received only small spermatophores. This finding was explained by die allometric relationship between me female weight and the size of its reproductive system that affects relative replenishment by the spermatophore. Females with a supply of water displayed a significantly higher rate of re-calling (41.2%) than control females (22.8%), highlighting the effect of adult feeding (or drinking) status on the re-calling behaviour. The short-distance presence of virgin males with once-mated females promoted a re-calling rate (and subsequent matings) close to 37%, significantly higher than that of isolated females (20%). It is concluded mat re-calling and polyandry in L. botrana are controlled, as expected, by a number of mating-derived stimuli, but also to a great extent by other physiological stimuli unrelated to mating. The reproductive strategies in relation to polyandry and the mechanisms controlling female sexual inhibition are discussed.     

Control of Lobesia botrana (Lepidoptera: Tortricidae) by biodegradable ecodian sex pheromone dispensers

Mating disruption with a high density of sex pheromone dispensers is a new strategy recently developed for the control of the moth Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae). Ecodian LB dispensers, made of low-cost biodegradable material, were formulated with 10 mg of (E,Z) -7,9-dodecadienyl acetate and placed at a rate of 1,600 dispensers per ha. Seasonal dispenser performances were studied using different methods. The female attractiveness disruption and the efficacy of the method were evaluated in the field. The release rates of field-aged Ecodian LB dispensers, measured directly by solid phase microextraction, was comparable with that of the standard monitoring lure after 50-60 d of field exposure and significantly lower beyond 60 d; however, at the end of the season, it was approximately 46 times higher than that of a calling L. botrana female. Electroantennographic recordings showed that dispensers of different field age strongly stimulated male antennae. In a wind tunnel test, dispensers elicited close-range approaches and direct source contacts irrespective of their age. In fields treated with Ecodian dispensers the attractiveness of traps lured with calling females and monitoring baits was significantly reduced. Our data suggest that Ecodian dispensers are active sources of pheromone throughout the season. The efficacy of Ecodian strategy for L. botrana control was comparable with standard mating disruption and curative insecticides.     

Influence of generation and photoperiod on larval development of Lobesia botrana (Lepidoptera: Tortricidae)

The influence of generation (under field conditions) and photoperiod (under laboratory conditions) on Lobesia botrana larvae development was studied. Some larvae were collected during three annual generations in two grape-growing areas of northeastern Italy, and others were individually reared in the laboratory from egg to pupa on an artificial diet under two different photoperiod conditions (respectively, daylight 16 h/d [long day {LD}] and 14 h/d [short day {SD}]). The mandible lengths of collected larvae were measured and the data analyzed morphometrically to determine the number of larval instars. In the laboratory study, the number of larval moultings, the mandible length of each instar, the development time from hatching larva to pupa, and the pupal weight were considered. The measurement of mandible lengths of larvae collected in the field indicated the existence of five larval instars in all three annual generations, but the size of the two oldest larval instars was significantly higher for third-generation larvae than for the previous generations. Under laboratory conditions, the larvae usually exhibited five instars, but the mandible lengths of larvae and the pupa size were greater for individuals reared under SD. These also took a greater number of days to develop from hatching larvae to pupae. Because a larger size of the final larval instar occurs in individuals that produce diapausing pupae under SD in both the laboratory and the field, a positive association between larval size and the probability of surviving the winter can be inferred.     

Mating disruption of Lobesia botrana (Lepidoptera: Tortricidae): effect of pheromone formulations and concentrations

The reluctance of Israeli vine growers to adopt the mating disruption technique to control the moth Lobesia botrana Den. & Schiff. has been attributed to the high cost of this method compared with that of traditional insecticide control. In this study, we tested the possibility of reducing the cost, first by testing different pheromone formulations (and thus open the market for competition) and second by reducing the pheromone concentration used in vineyards. Comparisons were made between two pheromone formulations--Shin-Etsu (Tokyo, Japan) at 165 g/ha and Concep (Sutera, Bend, OR) at 150 g/ha--and between two concentrations of Shin-Etsu, 165 and 110 g/ha. Pheromone dispensers were placed at the onset of the second moth generation. Comparison of the numbers of clusters infested with eggs and larvae of L. botrana showed no significant differences in the performance, either between the two formulations, or between the two tested concentrations. The results suggest that 1) the two formulations are equally effective, and 2) a low pheromone concentration is sufficient to maintain good control of small populations of L. botrana. However, when the population is high, pest control efficacy is not improved by increasing the pheromone concentration. Therefore, in the interest of reducing the relatively high cost of mating disruption, we emphasize that increasing the pheromone concentration does not provide improved control of high populations of L. botrana. The cost of mating disruption can be diminished by reducing the applied pheromone concentration and by using the least expensive pheromone formulations     

Effects of lufenuron on Lobesia botrana (Lepidoptera: Tortricidae) egg, larval, and adult stages

The effect of the chitin synthesis inhibitor lufenuron was evaluated against different developmental stages of Lobesia botrana Den. & Schiff. (Lepidoptera: Tortricidae). Lufenuron fed to adults at 10 ppm reduced their fecundity and fertility, but it did not affect adult longevity. High activity was observed against L. botrana eggs with greater effect on 1-d-old eggs than on the other age classes and on eggs treated by direct contact rather than those laid on a previously treated surface. Eggs laid by treated adults showed the same effects during development as eggs treated by contact or those laid on a treated surface. Larvae that emerged from treated eggs could not perforate grape berries. Administered into the diet, lufenuron had a larvicidal effect, resulting in similar LC50 values for different instars: 0.07 ppm for first instars, 0.08 ppm for third instars, and 0.11 ppm for fifth instars. None of the larvae treated with sublethal concentrations throughout their life emerged as adults at the highest concentration (0.08 ppm), and only 70% emerged at the lowest concentration (0.0025 ppm).     

Delayed mating reduces reproductive output of female European grapevine moth,Lobesia botrana (Lepidoptera: Tortricidae)

Virgin females of Lobesia botrana Denis & Schiffermüller were mated at ages of 1, 3, 5, 8, 12 and 16 days and the effect of mating delay on reproductive output assessed. Delayed mating did not affect female mating success but fertilization was reduced in 16-day-old females. Increased mating delays substantially affected daily oviposition pattern and resulted in a significant reduction of both fecundity and fertility, so that mean number of viable eggs laid decreased from 150 to 22 in 1- and 16-day-old mated females, respectively. Heavier females were more constrained than lighter ones by mating delays and female reproductive efficiency (no. viable eggs/female body weight) was also significantly reduced. Female longevity significantly increased and oviposition period gradually declined with mating delay. The number of viable eggs was positively correlated with both female weight and oviposition period; female longevity and female weight were also significantly correlated. However, the significance of these correlations declined with increased mating delay. Results overall indicated that mating delay drastically reduces female L. botrana reproductive output. The implications of delayed mating of females are discussed from an ecological perspective in relation to L. botrana control using mating disruption.     

Modulation of Flight Activity in Lobesia botrana Den. & Schiff. (Lepidoptera: Tortricidae) Females Studied in a Wind Tunnel

We studied modulations of flight activity in European grapevine moth females (EGVM) by individual observations in a wind tunnel. The effect of different factors was analyzed: variation in atmospheric pressure prior to the experiments, time of day, first experience of flight, age, mating, and odor of tansy, which attracts females. The circadian flight activity showed a peak the hour preceding the onset of scotophase and sustained activity occurred during the 6 h around this peak. Females with a flight experience in the tunnel took off more quickly than naive ones (3.9 ± 7.4 vs 20.3 ± 22.8 s). Three-day-old unmated females subjected to negative variations of atmospheric pressure (10 hPa) during the 4 h prior to the experiments increased their duration of flight (12.1 ± 8.7 vs 5.3 ± 3.4 s) compared to those not subjected to variation. One-day-old females were less active than older ones; flight was shorter than in 2-day-old females (2.7 ± 6.7 vs 5.1 ± 9.5 s) and fewer of them took off (28 vs 63%). Mating also affected the flight activity of 2-day-old females; mated females flew longer than virgins (12 ± 16.8 vs 5.1 ± 9.5 s) and took off more quickly (6.5 ± 14.4 vs 19.3 ± 20.1 s). Tansy odor in the tunnel did not significantly affect the flight behavior of virgin females, but it increased the proportion of mated females that initiated flight (87 vs 70%) and duration of flight (11.2 ± 24.4 vs 7.2 ± 13.7 s), and it reduced the latency to takeoff (2.1 ± 7.4 vs 8.1 ± 19.1 s). Flight duration in tansy odor was inversely correlated with the total number of eggs laid during the female's whole life. Our experimental settings did not allow observation of movements directed toward the odor source.     

Effectiveness of insecticides in controlling the first and second generations of the Lobesia botrana (Lepidoptera: Tortricidae) in table grapes

The moth Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) is a key pest of table and wine grape (Vitis spp.) varieties in Cyprus. Many different insecticide combinations were applied for three consecutive years (2006-2008) in a Sultana seedless table grape vineyard, aimed at controlling the first and second generations of this pest under warm and dry Mediterranean climatic conditions. In Cyprus, Sultana is the main early maturing table grape variety grown in the country. L. botrana has two generations and a partial third on this export variety, of which the first two generations are the most destructive. Applications were made according to pheromone trap captures of males. One application was used against the first and two applications against the second generation of L. botrana. A high rate of bunch damage was observed in the untreated rows during all years, reaching 56.7, 62.5, and 69.2% in 2006, 2007, and 2008, respectively. Differences between insecticide treatments and the untreated control were statistically significant. The treatment combination of lufenuron, spinosad, and indoxacarb as well as the combination of chlorpyrifos, spinosa and indoxacarb, used against the first and second generations of L. botrana, were the most effective compared with the untreated control. Satisfactory control of the pest also was observed with other combinations such as lufenuron, cypermethrin, and Bacillus thuringiensis; chlorpyrifos, cypermethrin, and B. thuringiensis; and lufenuron, deltamethrin, and azadirachtin.     

Forecasting the flight activity of Lobesia botrana (Denis & Schiffermüller) (Lepidoptera,Tortricidae) in Southwestern Spain

This study aimed at elaborating a forecasting tool of the phenology of the serious pest Lobesia botrana in Southwestern Spanish vineyards, by analysing data on male catches in sex pheromone traps recorded over a 12‐year period. Our data confirmed the minor importance of the first generation which appears during flowering time, both in terms of male trap catches and damage of L. botrana to the inflorescences. Therefore, data related to the first flight were not further processed, although they were considered for the computation of degree‐days of the following generations. The outcome of the elaboration of temperature accumulations and data on male captures for the second and third flights was a statistically acceptable linear behaviour obtained by properly transforming the variables. The models established proved to be efficient and may represent a useful tool to improve the efficacy of integrated pest management strategies targeting L. botrana in the studied region.     

Grape variety affects larval performance and also female reproductive performance of the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae)

For insect herbivores, the quality of the larval host plant is a key determinant of fitness. Therefore, insect populations are supposed to be positively correlated with the nutritional quality of their host plant. This study aimed to determine if and how different varieties of grapes (including the wild grape Lambrusque) affect both larval and adult performance of the polyphagous European grapevine moth Lobesia botrana (Denis & Schiffermüller). Significant differences were found in larval development time, but not in pupal mass, adult emergence rate, or sex ratio. Although the fecundity of females is not different among varieties, females fed on some varieties produced eggs of different sizes which are correlated to their fertility. Thus, females adapt resource allocation to eggs depending on their diet as larvae. Using a fitness index, the average reproductive output was found to be highest for females reared on cv. Chardonnay. Females reared on wild grape produced a fitness index identical to the cultivated grapes. However, Lambrusque and Gewurztraminer separate themselves from the cultivated varieties according to our discriminant analyses. It is emphasized, through this study, that cultivars fed on by larvae should be considered in the population dynamics of L. botrana and that egg number is insufficient to determine host plant quality.     

Developing optimum sample size and multistage sampling plans for Lobesia botrana (Lepidoptera: Tortricidae) larval infestation and injury in northern Greece

The purpose of this research was to quantify the spatial pattern and develop a sampling program for larvae of Lobesia botrana Denis and Schiffermüller (Lepidoptera: Tortricidae), an important vineyard pest in northern Greece. Taylor's power law and Iwao's patchiness regression were used to model the relationship between the mean and the variance of larval counts. Analysis of covariance was carried out, separately for infestation and injury, with combined second and third generation data, for vine and half-vine sample units. Common regression coefficients were estimated to permit use of the sampling plan over a wide range of conditions. Optimum sample sizes for infestation and injury, at three levels of precision, were developed. An investigation of a multistage sampling plan with a nested analysis of variance showed that if the goal of sampling is focusing on larval infestation, three grape clusters should be sampled in a half-vine; if the goal of sampling is focusing on injury, then two grape clusters per half-vine are recommended.     

A bioassay to evaluate the activity of chemical stimuli from grape berries on the oviposition of Lobesia botrana (Lepidoptera: Tortricidae)

A two-choice bioassay was developed to evaluate the role of host-plant berry compounds on the oviposition site acceptance of the generalist moth Lobesia botrana (Denis & Shiffermüller). A key feature was the lining of the bioassay arena with felt which focused oviposition on the test substrates. Initial experiments comparing substrates with different physical features indicated that smooth textures and spherical shapes with interstices favour oviposition. Artificial oviposition substrates were thus constructed with glass spheres in order to test the behavioural activity of grapevine berry extracts. Only polar extracts obtained by soaking berries in methanol or water stimulated oviposition (more eggs were laid on the extract-treated substrate than on the control substrate), whereas more apolar ones obtained with chloroform or hexane had no significant effect. The prior removal of epicuticular waxes from grape berries before extraction did not enhance the stimulatory activity of the methanol extract. The oviposition response to this extract was dose-dependent. It is concluded that polar compounds present on grape berries act as oviposition stimulants for L. botrana.     

Potential of the Bacillus thuringiensis toxin reservoir for the control of Lobesia botrana (Lepidoptera: Tortricidae), a major pest of grape plants

The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 microg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 microg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.     

Biological control of Lobesia botrana (Lepidoptera: Tortricidae) larvae by using different formulations of Bacillus thuringiensis in 11 vine cultivars under field conditions

This work describes the results of an experiment that was conducted in the vineyard of the American Farm School, Thermi, Thessaloniki Greece, during 2001. Its aim was to study the efficacy of two formulations of Bacillus thuringiensis Berliner (wettable powder and dust) to control the larvae of Lobesia botrana Denis & Schiffermueler (Lepidoptera: Tortricidae). The experimental results showed that the two formulations of B. thuringiensis are significantly more effective than the control, the dusting being more effective in most cultivars (Asyrtico, Sauvignon blanc, Debina, Athiri, Agiorgitico, Limnio, Syrah, and Cabernet sauvignon), and the spraying in a few cultivars (Xinomavro, Roditis, and Chardonnay). Dusting in the blue-black cultivars was found to be more effective than spraying, this not being the case in the yellow-green cultivars. This fact should be taken into consideration when cultivars of different color were planted in the same field. Single dusting proved to be better in cultivars with loose, average and dense berry cluster compactness, whereas double spraying gave better results in compact cultivars.     

Potential of the Bacillus thuringiensis Toxin Reservoir for the Control of Lobesia botrana (Lepidoptera: Tortricidae), a Major Pest of Grape Plants

The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.     

Insecticidal resistance and cross-resistance in populations of Cydia pomonella (Lepidoptera: Tortricidae) in central Europe

Insecticide bioassays were used to investigate resistance of Cydia pomonella (L.) (Lepidoptera: Tortricidae) to insecticides with various types of active ingredients. The efficacy baselines of selected insect growth regulators (fenoxycarb), insect growth inhibitors (diflubenzuron and teflubenzuron), organophoshorous insecticides (phosalone), and neonicotinoids (thiacloprid) against the eggs and first and fifth instars of sensitive laboratory strains of codling moth were determined. According to concentration-mortality baseline, 50% lethality concentration values and 90% lethality concentration values were determined for all the tested insecticides. The lethal concentration ratio quantified the relation between the efficacy of selected insecticides against fifth instars found by topical application and against first instars found by diet-treated bioassay. No difference was detected when the efficacy of technical grade diflubenzuron diluted in tetrahydrofuran and diflubenzuron in the formulated product Dimilin 48 SC diluted in water was compared. However, just before the application of insecticide, the integument of larvae must be treated with acetone. Two bioassays were used to monitor the resistance of codling moths collected in 2003-2005 in two apple (Malus spp.) orchards with different intensities of chemical control. Resistance ratios (RRs) to the tested insecticides were determined for both field populations of codling moth. For the population of codling moth from a commercial apple orchard in Velké Bílovice, cross-resistance to fenoxycarb, teflubenzuron, and phosalone was detected after the topical application of insecticides to fifth instars. The population of codling moth from Prague-Ruzyne was slightly resistant to phosalone and teflubenzuron. No resistance to diflubenzuron was detected in either tested population.     

Non-host plant odor (Tanacetum vulgare; Asteracea) affects the reproductive behavior ofLobesia botrana Den. et Schiff (Lepidoptera: Tortricidae)

Females ofLobesia botrana Den. et Schiff. (Lepidoptera Tortricidae) are attracted in natural conditions by volatiles released by a nonhost plant: tansy (Tanacetum vulgare L.; Asteracea). We have shown that both tansy flowers and their odor inhibit oviposition behavior and mating behavior and reduce adult longevity. The mean number of eggs laid per female isolated with tansy flowers was reduced by up to 50% every 2 days during the 6 days of exposure. This reduction was maintained after the tansy was removed. In the presence of tansy essential oil, the egg-laying reduction ranged from about 30 to 80% according to the odor concentration. The number of spermatophores found in females isolated with tansy flowers was also reduced twofold compared to the control treatment, indicating that the presence of tansy reduced mating activity. This mating activity is strongly reduced, by two-thirds, when adults face the highest dose of essential oil compared to controls. The number of eggs laid by the controls cannot be explained by the number of spermatophores. Therefore, the reduction in oviposition has been attributed to the presence of tansy flowers or to the tansy odor. Tansy flowers and tansy odor increased male mortality during the exposure (10% in the control, 50% in the tansy treatment, and up to 98% in the odor treatment). The highest rates of male mortality occurred during the 4- to 6-day period of exposure to flowers or odor. Repellence resulting in sustained locomotor activity is a possible cause of such a mortality. Female mortality was increased only in response to the highest dose of odor. This increase might be due to egg retention, and not directly to a plant effect. We discuss the effects of tansy flower odor on different patterns relative to the reproductive behavior ofL. botrana and, especially, on oviposition behavior in the ecological context of plant selection and polyphagy.     

Characterization of a nucleopolyhedrovirus of Epinotia granitalis (Lepidoptera: Tortricidae)

A nucleopolyhedrovirus (NPV) from the cypress bark moth Epinotia granitalis Butler was partially characterized. Electron microscopy indicated that E. granitalis NPV (EpgrNPV) is a singly embedded nucleocapsid NPV with a relatively small polyhedron, ranging from 480 to 1100nm in diameter. Phylogenetic analysis revealed that EpgrNPV is a Group II NPV that is related to but distinct from Adoxophyes honmai NPV. The LC(50) against third instar E. granitalis larvae was 10(5.5) polyhedral occlusion bodies per gram (OBs/g diet), and the average time to death was 14 days. E. granitalis was the only EpgrNPV-susceptible species in a series of infection tests using insect species belonging to the family Tortricidae. Other test species, including Eucoenogenes ancyrota, A. honmai, Adoxophyes dubia, Pandemis heparana, Homona magnanima, and Archips insulanus, were not susceptible to the infection.