Costa Rican soils contain highly insecticidal granulovirus strains against Phthorimaea operculella and Tecia solanivora

The aim of this work was to isolate and characterize novel Phthorimaea operculella granulovirus (PhopGV) strains from Costa Rican soils. Three novel strains, named PhopGV‐CR3, PhopGV‐CR4 and PhopGV‐CR5, were isolated from three locations in Costa Rica, Alvarado, Zarcero and Abangares, respectively, by means of soaking potato tubers with diluted soil samples. An additional strain, PhopGV‐CR2, was identified from diseased larvae from a Tecia solanivora laboratory culture. Restriction fragment length polymorphisms obtained for each isolate with six restriction endonucleases (RENs) allowed their identification as four distinct PhopGV strains. Both REN and Polymerase chain reaction analyses indicated the existence of an array of genotypic variants present in all isolates. Bioassays in P. operculella and T. solanivora showed that PhopGV‐CR3 was well adapted to the two coexisting hosts with high levels of pathogenicity against both pest species. The mean lethal dose values of this strain were 2.8 OBs/mm² for P. operculella and less than 0.5 OBs/mm² for T. solanivora. We conclude that PhopGV‐CR3 shows great promise for soil application against these pests in Costa Rican potato crops.     

Histopathology and effect on development of the PhopGV on larvae of the potato tubermoth, Phthorimaea operculella (Lepidoptera: Gelechiidae)

Larvae of the potato tubermoth (PTM), Phthorimaea operculella, feed on potato plants and tubers and are a major pest in the tropics and subtropics worldwide, causing up to 100% damage. The PTM granulovirus (PhopGV) provides significant potato protection, but little is known about its effect on larval development or its histopathology. Here we show that only 10% of larvae exited from PhopGV-treated tubers (1.4 × 10⁸ granule/ml), lagging significantly behind controls, and most of these died by 72 h after emergence. Histopathology studies showed the fat body and epidermis were the principal tissues infected. PhopGV morphogenesis was similar to other GVs, the exception being small vesicles between mature granules.     

Characterization of a Costa Rican granulovirus strain highly pathogenic against its indigenous hosts,Phthorimaea operculella and Tecia solanivora

A granulovirus isolate collected from diseased Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) larvae in Costa Rican potato [Solanum tuberosum L. (Solanaceae)] crops was characterized at the molecular and biological level. Restriction endonuclease analysis identified this isolate as a novel P. operculella granulovirus (PhopGV) (Baculoviridae: Betabaculovirus) strain and was designated as PhopGV‐CR1. In addition, PCR amplification of four specific variable genomic regions yielded multiple amplicons for two open reading frames, revealing the presence of different genotypic variants within the virus population. Biologically, PhopGV‐CR1 was highly pathogenic for its two indigenous hosts, although significant differences of up to four‐fold were detected against P. operculella [LD₅₀ = 17.9 occlusion bodies (OBs) mm^?2] and Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) (LD₅₀ = 69.1 OBs mm^?2). The two P. operculella colonies, from Costa Rica and France, were equally susceptible to PhopGV‐CR1. Serial passage of PhopGV‐CR1 over four generations in T. solanivora increased its pathogenicity by five‐fold in three generations, suggesting an ongoing adaptation to its alternate host.     

Semi‐field evaluation of a granulovirus and Bacillus thuringiensis ssp. kurstaki for season‐long control of the potato tuber moth,Phthorimaea operculella

There are few insecticidal options for potato tuber moth (PTM), Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae), control late in the growing season. We evaluated the PTM granulovirus (PoGV) and Bacillus thuringiensis Berliner ssp. kurstaki (Btk) for season‐long control of PTM on potato foliage in 2006 and 2007. Compared to untreated controls, 10 weekly applications of PoGV (10¹³ occlusion bodies/ha) reduced PTM populations in replicated 1‐m³ field cages by 86–96% on pre‐harvest foliage and 90–97% on tubers added to cages shortly before harvest. Infection rates of 82–95% of L4 larvae by PoGV were noted within individual larval cohorts. Equivalently timed Btk treatments (1.12 kg product/ha) were significantly less effective at population suppression, with a 36–76% reduction in larvae recovered from tubers added to cages. A PoGV/Btk alternation was significantly more effective than Btk alone and as effective as PoGV in 2007, but not in 2006. There was some evidence that reduced rate PoGV treatments (10% rate or 50% application frequency) were less effective than the standard program. There were no treatment effects on percentage of tubers growing in the ground that were infested at harvest, which remained comparatively low at ≤8.1%. Bioassays were conducted to evaluate the residual activities of foliar deposits. Early‐season applications were highly effective for the first 24 h (≥93% mortality) with a steady decline in activity over 10 days. A second application, applied later in the season, showed similar patterns, although in this case Btk was less persistent than PoGV, whereas both agents provided significant larval mortality compared with controls over 14 days. Both PoGV and Btk provide alternatives to manage field infestations of PTM prior to harvest, thus reducing the risk of tuber infestations in storage.     

Responses of different geographic populations of two potato tuber moth species to genetic variants of Phthorimaea operculella granulovirus

Phthorimaea operculella granulovirus (PhopGV) belongs to the genus Betabaculovirus of the arthropod‐infecting Baculoviridae. PhopGV is able to infect several gelechiid species. Among them are the potato tuber moths Phthorimaea operculella Zeller and Tecia solanivora Povolny (both Lepidoptera: Gelechiidae). In various South American countries, PhopGV‐based biopesticides are used to control either P. operculella or T. solanivora. Many trials have indicated that a particular viral isolate can exhibit very distinct pathogenicity when infecting different host species or different populations of one host species. In this study, we compared host–pathogen interactions using various PhopGV isolates and various populations of P. operculella and T. solanivora. Virus isolates from P. operculella were more pathogenic against their original host species than against T. solanivora. A PhopGV isolated from T. solanivora was less efficient against P. operculella. In addition, virus isolates differed in pathogenicity toward their hosts (i.e., lethal concentrations of isolates ranged from low to high). Unexpectedly, we also found that host populations of one species from distinct geographic origins did not differ significantly in susceptibility to the same PhopGV isolate. This was the case for both host species and for five PhopGV isolates. Comparative restriction fragment length polymorphism (RFLP) analyses of 11 isolates including those used in bio‐assays indicated three main regions of variation in the genome of PhopGV, corresponding to the regions of open reading frame PhopGV046, gene PhopGV129 (egt), and repeat 9 (located between open reading frames PhopGV083 and PhopGV084). Comparison of the nucleotide sequences of the insertions/deletions present in these regions were carried out for the most variable isolate, JLZ9f. The results are discussed in the context of the production and use of PhopGV as a biological agent against these two pest species.     

Susceptibility of Phthorimaea operculella Zeller (Lepidoptera; Gelechiidae) to its granulovirus PoGV with larval age

1 Granulovirus PoGV is a strong candidate to substitute for chemical insecticides in integrated pest management (IPM) of the potato tuber moth Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae). Generally, susceptibility to baculoviruses decreases with increasing larval age. For effective field applications, knowledge of the age‐related change in susceptibility is crucial. 2 The relative susceptibility of various instars of P. operculella larvae to PoGV was studied by leaf‐disc bioassays in the laboratory at 26 °C incubation temperature. The LC₅₀ increased from 4 × 10⁶ granules/mL for neonate larvae to 1.2 × 10⁷, 1.1 × 10⁸, 4.4 × 10⁸, and 4.2 × 10⁹ granules/mL, signifying resistance ratios of 3, 29, 110, and 1051, for 5, 6, 7, and 9‐day‐old larvae, respectively. 3 The relationship between log‐LC₅₀ values and log‐larval weight was significantly linear. A logistic function described significantly the relation between larval weights and physiological age measured in degree‐days (DD > 13.4 °C). Both functions allowed prediction of the activity ratio of PoGV for different larval ages by using temperature summation to express physiological age. 4 PoGV was found to be highly active against P. operculella larvae up to a physiological age of approximately 50 DD (>13.4 °C) (i.e. the first third of the total larval development time). Thereafter, the virus rapidly lost its activity against older larvae. Prospects for applying this knowledge in the field are discussed.     

The potential of novel African isolates of Phthorimaea operculella granulovirus for the control of Tuta absoluta

Phthorimaea operculella granulovirus (PhopGV) is infectious for larvae of different Gelechiidae insect species, including Tuta absoluta and Phthorimaea operculella. As these are major economic pests in North and sub‐Saharan Africa as well as in the Mediterranean area, the development of locally suitable biocontrol agents is essential. We have studied five isolates of PhopGV from Tunisia (Tns16, Tu1.11 and Tu2.11), Kenya (Ken13) and Yemen (Ym14) for their biological activity and the sequence polymorphism of their granulin and ecdysteroid UDP‐glucosyltransferase (egt) genes and allocated the isolates to two different egt types. Infection experiments with neonate larvae of T. absoluta and P. operculella demonstrated their pathogenicity to both host species. The isolate PhopGV Tu1.11 was the most virulent one for T. absoluta but had a relatively low infectivity to two P. operculella populations originating from Italy and Tunisia.     

Effect of a granulovirus on mortality and dispersal of potato tuber worm (Lepidoptera: Gelechiidae) in refrigerated storage warehouse conditions

Potato tuber worm (PTW), Phthorimaea operculella (Zeller), is a world-wide pest of potato. In rustic stores, PTW larvae can infest 100% of stored tubers. Treatment of tubers in rustic stores with the PTW granulovirus (PoGV) has been demonstrated to protect stored tubers. This is the first study to show the effects of PoGV for protection of tubers stored in refrigerated warehouse conditions. Tubers were treated by dipping in aqueous suspensions of PoGV or water. An estimated 0.0819 larval equivalents of virus or 1.88×10⁹ viral occlusion bodies were deposited on each kilogram of tubers. They were held at 16°C for 11 days before lowering the temperature by 0.5°C per day until 10°C was reached. The tubers were stored at this temperature for 53 days. Mean numbers of infested tubers at the end of the assay was affected by both pre-infestation rate and virus treatment. Mean numbers of infested tubers in the control treatment was 3 tubers per chamber higher than in the virus treatment providing strong evidence that PoGV controlled larvae and minimized spread into un-infested tubers. Of the larvae that were retrieved in virus-treated infested tubers, the mean mortality was 87% compared to 37% in controls.     

Virulence of a South African isolate of the <Emphasis Type="Italic">Cryptophlebia leucotreta</Emphasis> granulovirus to <Emphasis Type="Italic">Thaumatotibia leucotreta</Emphasis> neonate larvae

Surface inoculation dose–response and time–response bioassays and detached fruit bioassays were conducted with a novel South African isolate of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) against Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Noctuidae) neonate larvae. LC₅₀ and LC₉₀ values were estimated to be 4.095 × 10³ and 1.185 × 10⁵ OBs ml⁻¹, respectively. LT₅₀ and LT₉₀ values were estimated to be 4 days 22 h and 7 days 8 h, respectively, categorising the virus as a fast or type 2 granulovirus. There was a conspicuous difference in behaviour between larvae on inoculated diet and untreated diet, resulting in a significant reduction in penetration of diet. Bioassays on detached Navel oranges revealed LC₅₀ and LC₉₀ values of 9.310 × 10⁷ and 1.515 × 10⁹ OBs ml⁻¹, when using data on numbers of larvae per fruit rather than on numbers of infested fruit. Field trials will be conducted.     

Time-concentration-mortality response of potato tuber moth pupae to the biocontrol agent Cordyceps tenuipes

ABSTRACT

The potato tuber moth (PTM) Phthorimaea operculella is a critical potato pest. Larvae infest both foliage and tubers and mature larvae pupate in the soil or other safe places. Cordyceps tenuipes, an entomopathogenic fungus, infect lepidopteran pupae. To determine the effectiveness of this fungus as a biocontrol agent for PTM, we evaluated the time-concentration-mortality (TCM) response of PTM pupae to C. tenuipes using the following bioassays: (1) direct immersion in conidial suspensions, (2) incubation in sterilised or (3) unsterilised soilpremixed with conidia, and (4) incubation in unsterilised soil drenched with conidial suspensions to simulate field conditions. Fungal infection caused 100%, 83.3%, 73.3%, and 85.0% mortality of PTM pupae in assays 1–4, respectively. At 10⁸ conidia/mL or conidia/g concentration, assays 1 and 4 had short lethal times (LT₅₀) of 2.2 and 2.6 days compared with 3.7 and 4.8 days for assays 2 and 3, respectively. On day 7 after inoculation, assays 1 and 4 also had low lethal concentrations (LC₅₀) of 1.69 × 10³ conidia/mL and 1.10 × 10⁵ conidia/g compared with those of assays 2 and 3, which showed low virulence, with LC₅₀ of 3.50 × 10⁵ and 3.60 × 10⁶ conidia/mL, respectively. Our results demonstrate that C. tenuipes is a promising candidate for PTM biocontrol at the pupal stage. Drenching the soil surface with conidial suspensions may be the most effective method of field application.     

Comparison of temperature effects on the in vitro growth and disease development in potato tubers inoculated with bacteria Pectobacterium atrosepticum,P carotovorum subsp. carotovorum and Dickeya solani

The relationship between the rate of in vitro growth of bacterial isolates of Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum and Dickeya solani and their pathogenicity was investigated in tubers of two potato cultivars at four temperatures ranging from 18°C to 30°C. The rate of in vitro growth was highly positively correlated with the number of rotted tubers (r ranged from 0.91 to 0.93) and with the weight of macerated potato tissue, which, however, was only found for P. carotovorum and D. solani (r = 0.76; r = 0.91, respectively) and not for P. atrosepticum. The weight of macerated tissue increased with the temperature, but significant differences between species of bacteria were observed only at 26°C and above, at which temperatures D. solani was the most aggressive, followed by P. carotovorum and P. atrosepticum. Almost all potato tubers inoculated with bacteria showed symptoms of soft rot at 26°C and 30°C, but the number of rotting tubers at lower temperatures (22°C and 18°C) decreased significantly. The lowest disease incidence, 11% of tubers with symptoms, was observed for the D. solani and cultivar Sonda at 18°C, what was also confirmed in a separate experiment with tubers from four potato cultivars inoculated with the highly aggressive isolate of D. solani. At temperatures from 18°C to 30°C, the differences in disease severity between potato cultivars with various resistance to bacteria increased in line with temperature, while the differences in disease incidence decreased.     

Establishment of Beauveria bassiana as a fungal endophyte in potato plants and its virulence against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae)

The potato tuber moth, Phthorimaea operculella, is the most damaging potato pest in the world and is difficult to control as the larvae are internal feeders in the foliage and tubers. Entomopathogenic fungi that colonize plants as endophytes have lethal and sublethal pathological effects on insect pests. We show that Beauveria bassiana colonizes the aerial parts of potato plants endophytically after inoculation through soil drenching. Endophytic B. bassiana persisted in potato foliage for more than 50 days postinoculation. Bioassays indicated that foliage of B. bassiana-inoculated potato plants were pathogenic against larvae of P. operculella. Sublethal experiments indicated that B. bassiana negatively affected the growth, development, and reproduction of P. operculella. Development experiments showed that the weight of P. operculella pupae reared on B. bassiana-colonized potato plants (4.25 mg) was significantly less than that of those reared on uninoculated control plants (8.89 mg). Compared with newly eclosed larvae fed on control plants, those fed on B. bassiana-inoculated plants had significantly lower survivorship, with only 17.8% developing to the adult stage. Oviposition of P. operculella females reared on B. bassiana endophytically colonized plants was significantly lower (35 eggs/female) than of those reared on uninoculated plants (115 eggs/female). This study demonstrates that endophytic B. bassiana can be a potential biological control agent for the control and management of P. operculella. Comparing pupal weights of P. operculella reared on potato plants inoculated with the B. bassiana strain GZGY-1-3 and on untreated control plants, pupae from the control plants were significantly heavier than those from treated plants.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

Tuber production,dormancy and resistance against Phthorimaea operculella (Zeller) in wild potato species

The diversification of resistant potato varieties at a landscape level could slow adaptation by Phthorimaea operculella to potato resistance and promote sustainable crop protection. In this study, we assessed wild potato species as novel sources of foliage and tuber resistance against P. operculella. Tuber resistance was quantified for 136 and foliage resistance for 54 potato accessions representing 14 and nine potato species, respectively. Several accessions were highly resistant to moth damage in tubers and/or foliage. In particular, Solanum chiquidenum and Solanum sandemanii were highly resistant to damage in tubers. Several accessions of Solanum multiinterruptum and a small number of accessions of Solanum bukasovii, Solanum berthaultii, Solanum sparsipilum and Solanum wittmackii also had highly resistant tubers. Larval survival on foliage of S. bukasovii and S. chiquidenum was generally low. New resistance sources are listed, and insect performance on the plants is described with possible resistance mechanisms. The study also examined potential trade‐offs associated with resistance. Tuber resistance was negatively correlated with the number and weight of tubers produced per plant, but positively correlated with the length of dormancy across accessions, indicating that, although long dormancy is not a prerequisite for resistance, species and accessions with extended dormancy will have more resistant tubers. Tuber and foliage resistance were generally positively correlated across all accessions; however, among accessions from within a potato species, there were negative (S. berthaultii), positive (S. chiquidenum) and non‐significant (S. bukasovii) relations. These results indicate that, besides identifying novel resistance sources, an improved understanding of the mechanisms and inherent trade‐offs associated with tuber and foliage resistance will improve the efficiency of potato breeding programmes aimed at enhancing resistance against P. operculella.     

Carob pulp as raw material for production of the biocontrol agent <Emphasis Type="Italic">P. agglomerans</Emphasis> PBC-1

Large-scale production has been the major obstacle to the success of many biopesticides. The spreading of microbial biocontrol agents against postharvest disease, as a safe and environmentally friendly alternative to synthetic fungicides, is quite dependent on their industrial mass production from low-cost raw materials. Considerable interest has been shown in using agricultural waste products and by-products from food industry as nitrogen and carbon sources. In this work, carob pulp aqueous extracts were used as carbon source in the production of the biocontrol agent Pantoea agglomerans PBC-1. Optimal sugar extraction was achieved at a solid/liquid ratio of 1:10 (w/v), at 25°C, for 1 h. Batch experiments were performed in shake flasks, at different concentrations and in stirred reactors at two initial inoculums concentrations, 10⁶ and 10⁷ cfu ml⁻¹. The initial sugar concentration of 5 g l⁻¹ allowed rapid growth (0.16 h⁻¹) and high biomass productivity (0.28 g l⁻¹ h⁻¹) and was chosen as the value for use in stirred reactor experiments. After 22 and 32 h of fermentation the viable population reached was 3.2 × 10⁹ and 6.2 × 10⁹ cfu ml⁻¹ in the fermenter inoculated at 10⁶ cfu ml⁻¹ and 2.7 × 10⁹ and 6.7 × 10⁹ cfu ml⁻¹ in the bioreactor inoculated at 10⁷ cfu ml⁻¹. A 78% reduction of the pathogen incidence was achieved with PBC-1 at 1 × 10⁸ cfu ml⁻¹, grown in medium with carob extracts, on artificially wounded apples stored after 7 days at 25°C against P. expansum.     

Insecticidal Activity of the Granulosis Virus in Combination with Neem Products and Talc Powder Against the Potato Tuberworm Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

The potato tuberworm Phthorimaea operculella (Zeller) is an important agricultural pest that causes significant economic losses to potato growers worldwide. The addition of an effective method of biological control for the potato tuberworm is greatly needed, and is currently unavailable in Brazil. The granulosis virus (Baculoviridae) is a promising biological control agent to protect post-harvest potatoes and in storage from the potato tuberworm. However, the control measure must be economically feasible. Liquid suspensions of a granulosis virus applied alone or in mixture with two commercial neem oil-based products (DalNeem? and NeemAzal?), and a dry powder formulation of viral granules were evaluated for control of potato tuberworm larvae by treating potato tubers under laboratory conditions. High larval mortality (86.7%) was achieved when DalNeem and virus were applied together at 4?mg of azadirachtin/L and 10⁴ occlusion bodies (OBs)/mL, respectively. This combination resulted in ??50% efficacy in relation to their counterparts alone. Conversely, NeemAzal did not enhance virus effectiveness against larvae of the potato tuberworm. The talc-based virus formulation was used for dusting seed tubers at different concentrations and resulted in 100% larval mortality at 5?×?10⁸ OBs/g. Formulated and unformulated virus provided 50% mortality at 166 OBs/g and at 5.0?×?10⁵ OBs/mL, respectively. As a result, talc-based virus formulation had a better control efficiency on potato tuberworm than the aqueous virus suspension. The granulosis virus combined with DalNeem at low rates or formulated with talc powder is a viable option to control the potato tuberworm under storage conditions.     

An improved method to determine the biological activity (LC50) of the granulovirus PoGV in its host Phthorimaea operculella

Granulovirus (PoGV) is a promising candidate to substitute for chemical insecticides in integrated pest management (IPM) of the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae). A procedure to assess pathogenicity (LC₅₀) and its standardization is crucial for research into the use of pathogens as biocontrol agents. In the present study, an egg‐dip bioassay method was developed and its precision tested in a series of six bioassays over a time period of 18 months. Bioassays were carried out at 25 °C incubation temperature. The probit model as a statistical analysis method for the interpretation of concentration responses proved better than the exponential model. LC₅₀ values ranged from 2.3 × 10⁶ to 10⁷ granules ml^?1 with a mean of 5 × 10⁶ granules ml^?1 and regression slopes varied between 0.73 and 1.05 with a mean of 0.84. LC₅₀ values and slopes displayed no significant differences according to their 95% confidence limits. The rate of harvested infected larvae showed a linear increase with increasing concentration on a log‐log scale. The mean natural mortality was 15% (SE ± 1.8%) and can be considered as a main factor producing variability among bioassays. The advantages of this bioassay method and its application in PoGV research are discussed.     

Estimating temperature‐dependent developmental rates of potato tuberworm,Phthorimaea operculella (Lepidoptera: Gelechiidae)

Abstract The potato tuberworm, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), is the most destructive pest of potato, Solanum tuberosum L. (Solanaceae), in tropical and subtropical regions in both field and storeroom situations. The modeling of temperature‐dependent development can be useful in forecasting occurrence and population dynamics of the pests. Published developmental parameters for this pest vary greatly for many reasons. We determined temperature‐dependent development of P. operculella at seven constant temperatures (16, 20, 24, 28, 32, 34 and 36 °C). Developmental period of whole immature stage (egg to the end of the pupal stage) varied from 75.5 days at 16 °C to 17 days at 32 °C. The population failed to survive at 36 °C. The observed data was modeled to determine mathematical functions for simulating P. operculella development in each stage of development and overall. Two linear models, ordinary linear regression and the Ikemoto linear model were used to describe the relationship between temperature and development rate of the different stages of P. operculella and estimating the thermal constant and lower temperature threshold. The lower temperature threshold (t) and thermal constant (k) of whole immature stage were estimated to be 11.6 °C and 338.5 DD by Ikemoto linear model, and the estimated parameters were not substantially different with those estimated by ordinary linear models. Different models provided a better fit to the various developmental stages. Of the eleven nonlinear models fitted, the Beriere‐1, Logan‐6 and Lactin‐1 model was found to be the best for modeling development rate of egg, larva and pupa of P. operculella, respectively. Phenological models based on these findings can be part of a decision‐support tool to improve the efficiency of pest management programs.     

High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

The effect of soil depth and exposure to winter conditions on survival of the potato tuberworm,Phthorimaea operculella

The potato tuberworm, Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae), is an important pest of solanaceous crops including potato [Solanum tuberosum L. (Solanaceae)]. Recent evidence of survival of the pest in the Columbia Basin of Oregon and Washington, USA, is surprising as potato tuberworm is most commonly a pest in tropical and subtropical regions. Potato tuberworm was studied in a manipulative experiment to determine how stage (egg, larva, and pupa) and soil depth affected the potential for winter survival. In early January 2007, eggs, tubers infested with larvae, and pupae were placed in the soil at 6, 10, and 20 cm depths, or left on the soil surface. Each potato tuberworm life stage was sampled at 7–17‐day intervals for 90 days and survival was estimated at each time period. Eggs survived up to 54 days with the greatest survival observed at 6‐cm soil depth and the lowest survival on the soil surface. However, egg survival was significantly reduced after 1 month of exposure to winter conditions. Larvae were able to survive up to 30 days with the greatest survival observed at 20‐cm soil depth. Tubers at the surface and buried at 6 cm were frozen; thus, no larval survival was recorded. The pupal stage showed a greater tolerance to winter conditions than the egg or larval stages, surviving up to 91 days of exposure. These results suggest that the pupal stage is probably the stage most likely to survive winter conditions in the northwestern USA.