Evaluation of lignins and particle films as solar protectants for the granulovirus of the codling moth,Cydia pomonella

The identification of effective solar protectants for field application of the codling moth, Cydia pomonella (L.), granulovirus (CpGV) is of interest to improve its efficacy and commercial viability as a biological pesticide. We evaluated several materials as potential adjuvants to protect CpGV from ultraviolet degradation. In laboratory tests with a solar simulator (9.36×10⁶ J/m²), the addition of kaolin clay (Surround® WP at 3 and 6%, w/v), a paraffin wax-based emulsion (SPLAT? at 5%, v/v), and a bark extract trans-cinnamaldehyde combined with a film agent (both at 1%, v/v) did not significantly reduce larval entries or increase larval mortality in irradiated apples that were treated with a commercial CpGV product (Cyd-X). In semi-field tests in an apple orchard, a spray-dried lignin formulation containing CpGV (6.57×10¹² OBs/ha) and a lignin-based adjuvant used with Cyd-X (both applied at 4.7–5.6 kg lignin/ha) significantly improved residual activity of CpGV compared with Cyd-X alone applied at the same rate. However, the benefits were short lived and could not be detected after 7 days. In orchard tests, we evaluated two additional refined lignin-products (Lignosulfonate and Vanisperse? CB at 5.61 kg/ha) and two particle film materials (kaolin clay, ‘Cocoon?’, and calcium with boron ‘Eclipse?’) as adjuvants for UV protection of Cyd-X (6.57×10¹² OBs/ha) in tests against a dense codling moth infestation. Although all virus treatments were highly effective (causing ≥90% larval mortality), no significant effects of the adjuvant treatments could be detected. In these latter tests, the use of a silicone based wetting agent at 0.025% (v/v) may have been beneficial at increasing mortality among older larvae inside the fruit.     

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.     

Effect of temperature on long-term storage of codling moth granulovirus formulations

Codling moth, Cydia pomonella (L.), is the major pest of apple (Malus spp.) in the western United States and many other regions of the world. The codling moth granulovirus (CpGV) provides a selective and safe means of its control. We assessed the long-term stability and storage potential of two commercial formulations of CpGV, Cyd-X, and Virosoft. All assays were performed with individual C. pomonella neonate larvae in 2-ml vials on 1 ml of artificial larval diet that was surface inoculated with 10 microl of the test virus suspension. Baseline quantitative assays for the two formulations revealed that the LC50 and LC95 values (occlusion bodies per vial) did not differ significantly between the formulations. For year-long studies on Cyd-X stability, the product was stored at -20, 2, 25, and 35 degrees C, and quantitative bioassays were conducted after 0, 3, 6, and 12 mo of storage. Cyd-X retained good larvicidal activity from -20 to 25 degrees C, and it was the least negatively affected at the lowest temperature. Storage of Cyd-X at 35 degrees C was detrimental to its larvicidal activity within 3 mo of storage. For longer term storage studies, Cyd-X and Virosoft formulations were stored at 2, 25, and 35 degrees C, and assayed for larvicidal activity over a 3-yr period. For recently produced product, a 10-microl sample of a 10(-5) dilution of both formulations resulted in 95-100% mortality in neonate larvae. Larvicidal activity for the Cyd-X formulation remained essentially unaffected for 156 wk when stored at 2 and 25 degrees C, but it began to decline significantly after 20 wk of storage at 35 degrees C. The Virosoft formulation stored at 2 degrees C also remained active throughout the 3-yr study, but it began to decline in larvicidal activity after 144 wk at 25 degrees C and 40 wk at 35 degrees C. The information reported in this study should be useful to growers and commercial suppliers for avoiding decreases in CpGV potency due to improper storage conditions.     

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.     

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.     

The Role of Formulation Additives in Increasing the Potency of Cydia pomonella Granulovirus for Codling Moth Larvae,in Laboratory and Field Experiments

Studies were undertaken to improve the biological efficacy of the granulovirus (CpGV) of the codling moth, Cydia pomonella , by evaluating the performance of some formulation additives that might improve virus persistence and/or virus uptake by first instar larvae. Laboratory studies, using a leaf disc bioassay, demonstrated that 15% cane molasses incorporated within a formulation of purified CpGV dramatically reduced the median lethal exposure time (LET 50 ) to CpGV for neonate larvae at a CpGV dosage rate of 10 7 occlusion bodies (OBs) ml -1 . Screening of a range of other compounds showed that sucrose, fructose and sorbitol (at 10% concentrations) and extracts of apple flesh and skin also gave significant reductions in the LET 50 of CpGV formulations containing these ingredients. Pectin, malic acid and &#102 -farnesene did not significantly reduce the LET 50 . In a field trial, molasses included at 15% (v/v) in a CpGV formulation, containing a dosage rate of 10 12 OBs ha -1 , gave as good control of codling moth damage as virus formulations containing the 'sticker' 0.2% skimmed milk at higher dosage rates of 10 13 and 10 14 OBs ha -1 . Studies of CpGV persistence on foliage revealed no significant improvement of virus persistence on apple foliage using 10% or 15% molasses formulations. A second field trial demonstrated that 10% molasses, 10% sorbitol or 0.08% &#102 -farnesene significantly reduced codling moth deep damage to fruit when these ingredients were added to formulations of pure CpGV. Substantial sooty-mould growth ( Cladosporium spp.) was observed on apple foliage treated with formulations containing molasses, indicating that this formulation additive has secondary consequences that would need to be taken into account if molasses was to be used in commercial CpGV formulations. Nonetheless, these studies clearly demonstrate that major biological improvements in CpGV performance can be achieved by the incorporation of formulation additives, including molasses and several other compounds, that probably function as attractants and/or feeding stimulants for codling moth larvae.     

Uptake of Granulovirus from the Surface of Apples and Leaves by First Instar Larvae of the Codling Moth Cydia pomonella L. (Lepidoptera: Olethreutidae)

Cydia pomonella granulovirus (CpGV) has received considerable attention as a potential microbial insecticide for the control of the codling moth (Cydia pomonella L.) , a worldwide pest of apples. Laboratory experiments were established to investigate virus uptake by first instar larvae, using a novel leaf disc bioassay technique. Virus uptake was found to be independent of active feeding and larvae became infected simply by walking or browsing on sprayed leaf disc surfaces in as little time as 3.5 min. Infection increased as a function of time spent on the leaf disc surface and a linear log time/probit mortality relationship could be fitted. The bioassay technique used has potential for the realistic laboratory testing of virus spray formulations. A field experiment showed that virus infection could be contracted by newly hatched codling moth larvae both from the surface of sprayed leaves and sprayed fruit. The potential for exploiting this knowledge for improving spray formulations is discussed.     

Evaluation of the pear ester kairomone as a formulation additive for the granulovirus of codling moth (Lepidoptera: Tortricidae) in pome fruit

(E,Z) -2,4-decadienoate (pear ester) is a larval kairomone for the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Orchard studies were conducted in 2005 and 2006 in apple, Malus domestica Borkhausen, and pear, Pyrus communis L., to evaluate a 5% active ingredient (AI), microencapsulated formulation of pear ester (PE-MEC) as an insecticidal additive for the codling moth granulovirus (CpGV). Although CpGV applied at 5-15-d intervals at commercial rates (2.2 X 10(12)-10(13) granules per ha) killed the majority (82-94%) of larvae found inside infested fruit, it did not eliminate significant damage, i.e., 30-92% fruit injury at harvest versus 51-82% in controls. PE-MEC treatments had significant but inconsistent results in our tests. In apple (mixed cultivars), PE-MEC (3.7-4.7 g [AI] /ha) plus CpGV reduced the percentage of fruit injured during the second but not the first larval generation, compared with CpGV alone, but there no was no additional population reduction (live larvae collected from infested fruit and tree bands). In 'Bartlett' pear, PE-MEC (3.7 g [AI] /ha) plus CpGV significantly increased larval mortality and reduced deep fruit entries at harvest over CpGV alone in 2006, but similar improvements were not observed in 2005 when a lower rate (1.5 g [AI] /ha) was tested. Surprisingly, compared with untreated controls, the PE-MEC formulation alone also reduced fruit injury (mid-season in Bartlett) and larval survivorship inside infested fruit at harvest (2006 apple tests and both years in Bartlett). Although pear ester seems amenable as a kairomonal adjuvant for use with insecticides, our inconsistent data with CpGV in apple and pear suggest practical improvements in formulation and application strategies (e.g., to optimize and maintain attractive release rates) are needed.     

Parasitoid exploitation of the seasonal variation in host plant volatile emission for herbivore location

Volatile compounds from the apple, Malus domestica Borkh. (Rosaceae), change considerably as the season progresses, and this is successfully exploited by the female codling moth Cydia pomonella L. (Lepidoptera, Tortricidae), as it searches for oviposition sites. In this study, we investigated the effect of seasonal emissions of apple fruit volatiles on the host location behaviour of a parasitoid of the codling moth larvae, Hyssopus pallidus (Askew) (Hymenoptera: Eulophidae). In dual choice olfactory bioassays, the behaviour of the parasitoid in response to apple cues was observed over the complete 2003 growing season. Our results show that codling moth infested apples evoked a strong response from the parasitoid at the beginning of the season, until July. Then, attraction dropped drastically, increasing again at the beginning of August. At the end of the growing season, just prior to harvest, infested apples hardly evoked any behavioural response. Interestingly enough, mid‐season emissions of healthy apples were per se attractive to the parasitoid, and even preferred over volatiles from infested apples. Simultaneous volatile collections from healthy apples on twigs in the field were analysed throughout the season, showing that the overall quantity of headspace volatiles peaks at the beginning of June and mid‐August. The seasonal volatile emission is correlated with the behaviour of the parasitoid during the fruit ripening stage. The results are discussed in relation to the use of H. pallidus as a potential biocontrol agent, in order to enhance current integrated pest management (IPM) programs.     

Ultraviolet protection of the Cydia pomonella granulovirus using zinc oxide and titanium dioxide

Cydia pomonella granulovirus (CpGV) is a specific pathogen of codling moth, the most serious pest of apple worldwide and has recently been isolated in China. However, its use for codling moth control is limited by ultraviolet (UV) solar radiation, which is a major factor affecting the field persistence of this virus. The virion is occluded in the granulin matrix of occlusion bodies. Many substances have been tested as sunscreen agents, but little has been published on the use of reflectors with the occluded bodies (OBs) of CpGV. This work investigates the susceptibility of a native GV, CpGV-ZY, to UVB radiation over different time periods and evaluates the protective effect of two sunscreen agents, zinc oxide (ZnO) and titanium dioxide (TiO₂). Laboratory tests showed 10⁴ OB/ml of CpGV-ZY exposed to UVB light (3.5 W/m²) for 3.75 h caused 50% inactivity. At 15 mg/ml ZnO and 10 mg/ml TiO₂, the mortality was highest after 4-h exposure to UVB light. Semi-field tests indicated both compounds are effective as UV protectants at low concentrations. These are the first results confirming that ZnO and TiO₂ hold promise as UV protectants for this CpGV-ZY isolate. Moreover, it is apparently safe and effective to use within the range of concentrations needed for codling moth control.     

Codling moth granulosis virus: effects of its use on some other orchard arthropods

In four field trials from 1978 to 1980, sprays of codling moth granulosis virus (CpGV) plus 1·0% skimmed milk powder did not significantly affect damage to fruit by leaf rollers (tortrix moths). In laboratory tests, survival of larvae of the leaf roller Archips podana fed on leaves sprayed with CpGV plus milk was unaffected and they grew faster than on unsprayed leaves, because of the milk deposits. This might increase damage by A. podana if CpGV plus milk were applied during the feeding period of this species. In one field trial an unusual infestation of fruit by larvae of pith moth Blastodacna atra was not affected by CpGV. Azinphos-methyl significantly reduced damage by B. atru and, in one field trial where sprays were correctly timed, that by leaf rollers. CpGV had no consistently significant effects on numbers of fruit tree red spider mite Panonychus ulmi or its predators, whereas azinphos-methyl induced outbreaks of P. ulmi by killing its predators.     

The susceptibility of the pea moth, Cydia nigricana, to infection by the granulosis virus of the codling moth, Cydia pomonella

Laboratory studies demonstrated that neonate larvae of the pea moth, Cydia nigricana, are susceptible to infection with a granulosis virus (CpGV) isolated from the codling moth, Cydia pomonella. Comparative LC₅₀ values for C. nigricana and C. pomonella are 1.90 × 10⁵ and 1.54 × 10⁴ capsules/ml of diet, respectively. The virus extracted from CpGV-infected pea moth larvae is serologically related, and probably identical, to CpGV.     

Characterisation of Cydia pomonella granulovirus from codling moths in a laboratory colony and in orchards of British Columbia*

Sequence analyses indicate that a granulovirus isolated from a laboratory colony of codling moth in British Columbia, Canada is identical or very similar to the Mexican isolate of Cydia pomonella granulovirus (CpGV-M). In addition to codling moth, the vims was found to cause mortality of secondary leafroller pests of North American orchards, including the fruit tree leafroller and the obliquebanded leafroller. A survey in apple growing regions of British Columbia indicates that 23% of the wild population of codling moth larvae carry CpGV.     

A model of the effect of codling moth granulosis virus on Cydia pomonella

A model was developed based on an initial ingestion phase, followed by a period of virus multiplication and host death, to describe the effects of codling moth granulosis virus (CpGV) concentration and time from initial infection on the percentage of codling moth larvae (Cydia pornonella) dying from virus infection in the laboratory. With modification, this model also described the effect of CpGV concentration on the reduction of damage to fruit by C. pomonella larvae in the field. In both cases, the effect of CpGV increased with the l/10th power of virus concentration. LT₅₀ (time to death of 50% of larvae) was inversely related to virus concentration in the laboratory. The model predicts the threshold concentration for death of larvae, as well as LD₅₀(C₅₀ and LD₉₀(C ₉₀,) for all ages of larvae. The model also predicts that if the majority of larvae entered through the sides of apple fruits, rather than through the calyx or near the stalk (as they probably did in the field), then the efficacy of CpGV in reducing fruit damage would be lowered. This effect of site of entry is especially marked at low virus concentrations.     

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.     

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).     

Sex linkage of CpGV resistance in a heterogeneous field strain of the codling moth Cydia pomonella (L.)

The occurrence of codling moth populations in European apple orchards that were not controlled by Cydia pomonella granulovirus (CpGV) is the first reported case of field resistance against a baculovirus control agent. A monogenic dominant sex-linked mode of inheritance was previously demonstrated in single-pair crosses between a homogeneous resistant (CpRR1) and a susceptible (CpS) laboratory strain of codling moth. However, resistant field populations (CpR) are more heterogeneous in their levels of resistance, and the possibility that they could harbor different resistance genes to CpRR1 had not been directly addressed. Here we report single pair crossing experiments using a resistant codling moth strain collected from an apple orchard in the southwest of Germany. Single-pair crosses within the field strain revealed a genetic basis to the heterogeneity of CpR concerning CpGV resistance. Hybrid crosses to a susceptible laboratory strain and backcrosses of the F1 generation to the resistant CpR strain confirmed that the homogeneous CpRR1 and the heterogeneous field strain CpR share the same mode of inheritance. Thus the variable levels of CpGV resistance in field populations is likely due to frequency differences of the same resistance-conferring gene, rather than different genes, which will facilitate future efforts to monitor and manage resistance.     

The occurrence and distribution of resistance of codling moth to Cydia pomonella granulovirus in Europe

Thirty‐five codling moth (CM, Cydia pomonella L., Lepidoptera, Tortricidae) populations collected in different commercial orchards in six European countries were tested for their susceptibility to Cydia pomonella granulovirus (CpGV‐M). Including previously published data on CpGV‐M resistance, a total of 38 CM colonies showed considerably elevated LC₅₀ values, independent of the country origin. When only few test individuals are available, determination of mortality of neonate larvae at a discriminating concentration range of 10⁴ to 10⁶ OB/ml (>log4) as a direct measure of percentage susceptible individuals in a CM population is more advisable than calculation of LC₅₀ values. The >log4 mortality alone or in combination with the LC₅₀ value can be used for identification of resistance in a population. Results indicated a locally separated but widely spread occurrence of CM populations with low susceptibility to CpGV‐M. The most plausible hypothesis for the emergence of CpGV‐M resistance is its selection by repeated use of CpGV products.     

Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K_m) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) for the substrate ACC, catalytic constants (K_cat) of 6.6 × 10⁻² (MD-ACO1), 3.44 × 10⁻² (Md-ACO2) and 9.14 × 10⁻² (MD-ACO3) and K_cat/K_m (μM s⁻¹) values of 7.38 × 10⁻⁴ μM s⁻¹ (MD-ACO1), 0.86 × 10⁻⁴ M s⁻¹ (MD-ACO2) and 3.8 × 10⁻⁴ μM s⁻¹ (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.     

Key elements in the successful control of diapausing codling moth,Cydia pomonella (Lepidoptera: Tortricidae) in wooden fruit bins with a South African isolate of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae)

The non-insecticidal control strategies currently being implemented in South African orchards for the control of codling moth, Cydia pomonella (L.) may be hampered by wooden fruit bins being infested with diapausing codling moth larvae, acting as a potential source of re-infestation. Key factors contributing to the success or failure of an entomopathogenic nematode application were investigated using the SF 41 isolate of Heterorhabditis zealandica in laboratory bioassays with wooden minibins. Under operational conditions, an application rate of 100 IJs/mL (LD₉₀₌102 IJs/mL) effectively controlled codling moth larvae in these bins, and for further laboratory bioassays, the LD₅₀ value of 18 IJs/mL (?25 IJs/mL) was identified as the discriminating dosage. Maximum mortality was attained when bins were pre-wet for at least 1 min (>90% RH) and maintained at maximum humidity (>95% RH) post-treatment for at least 3 days (LT₉₀=73 h), to ensure nematode survival and subsequent satisfactory infection of diapausing codling moth larvae. Tarping bins achieved the desired high level of humidity required. Furthermore, adjuvants (specifically Reverseal 10?) also improved an application. The study conclusively illustrated that if all the above-mentioned conditions are met, H. zealandica has the potential to successfully disinfest wooden fruit bins of codling moth.