Age-specific bioassays and fecundity of Phthorimaea operculella (Lepidoptera: Gelechiidae) reared on cry1Ac -transgenic potato plants

Variation in the susceptibility of lepidopterous pest larvae of different ages to transgenic crops and the potential for survivors to reproduce could have important consequences for the development of resistance in such pests. Experiments were undertaken in the laboratory to determine if larvae of the potato tuber moth, Phthorimaea operculella, of different ages (0 (< 1 day old), 3, 5, 7 days) varied in their susceptibility to cry1Ac9–transgenic potato (Solanum tuberosum) foliage grown in the glasshouse or field. The survival and fecundity of larvae reared on transgenic tubers was also determined in the laboratory. There were no apparent differences in susceptibility of larvae of different ages to transgenic foliage. Larvae fed glasshouse or field‐grown non‐transgenic foliage had significantly larger relative growth indices and more larvae pupated, than those fed transgenic foliage, regardless of larval age. Eggs from a laboratory colony were placed on transgenic or non‐transgenic tubers to measure survival and fecundity. Between 6% and 15% of eggs placed on transgenic tubers developed into pupae for three of the four transgenic potato lines tested. On one transgenic line, only six adults emerged from 1300 eggs. In contrast, between 71% and 97% of the eggs placed on non‐transgenic tubers developed into pupae. Male and female pupae from transgenic lines weighed less than those from non‐transgenic lines. The fecundity of females from two of four transgenic lines was lower than from the non‐transgenic parent cultivar. Although larvae of different ages did not exhibit any overall age‐dependent pattern of increasing or decreasing susceptibility to transgenic foliage of glasshouse or field‐grown plants, the ability of larvae to survive and reproduce on transgenic tubers suggests this pest has the ability to evolve resistance to the transgenic plants used in the present study.     

An experimental approach to simulate transgene pyramiding for the deployment of cry genes to control potato tuber moth (Phthorimaea operculella)

Alternating the daily feeding of potato tuber moth (Phthorimaea operculella) larvae (PTM) between isogenic pairs of potato plants provides an effective experimental approach to simulate transgene pyramiding in a clonal crop. This involves an experimental design with all six possible pairwise combinations of two transgenic lines expressing different cry genes and the non‐transgenic control. In this manner, we have simulated the pyramiding of pairwise combinations of cry1Ac9, cry9Aa2 and cry1Ba1 genes in potato and evaluated how pairs of these three cry genes interact to influence the growth rate of PTM larvae. The results show that all combinations of the three cry genes were largely consistent with additive impacts on PTM larval growth, although results from the combination of the cry1Ac9 and cry9Aa2 genes were suggestive of slight synergistic effects. Pyramiding the cry1Ac9, cry9Aa2 and cry1Ba1 genes in potato could therefore provide a more effective strategy to control PTM compared to single cry gene transgenic plants.     

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.     

Minor modifications to the cry1Ac9 nucleotide sequence are sufficient to generate transgenic plants resistant to Phthorimaea operculella

Minor modifications were made sequentially to the nucleotide sequence of truncated cry1Ac9 to produce cry1Ac9^A (one nucleotide change) and then cry1Ac9^B (seven nucleotide changes). The derivative genes under the control of the CaMV 35S promoter were transformed into Nicotiana tabacum in order to determine whether these modified genes conferred resistance on the resulting transgenic tobacco plants to larvae of the potato tuber moth (Phthorimaea operculella). Over two trials with PTM larvae on the transgenic plants expressing the cry 1Ac9^b gene, lower larval growth, development and survival was evident for most of the lines compared to the control plants. In the second trial, for four of these lines (7, 25, 26 and 28) larval growth rates were very low (0.28, 0.3, 0.42 and 0.28, respectively) compared to the control growth rate (4.18) and leaf damage was minimal. Northern analysis and RT‐PCR analysis showed that higher levels of cry 1Ac9 mRNA were present in the transgenic tobacco lines containing cry 1Ac9^b than in the tobacco lines containing cry 1Ac9^a. These results suggest that certain minor modifications to the nucleotide sequence of cry 1 Ac9 are sufficient to improve the stability of its mRNA when expressed in tobacco and that this increase in steady state mRNA is sufficient to confer significant resistance to PTM larvae.     

The toxicity of some medicinal plant extracts to the potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae)

The potato tuber moth Phthorimaea operculella is a serious pest of potato both in the field and storage that makes great problem in storage. Farmers routinely rely on chemical insecticides that could be hazardous for human health and environment. Insecticide resistance of P. operculella has been reported all over the world, so botanical origin materials may serve as proper alternative materials for controlling the pest. Natural products are generally preferred because of they are less harmful to non-target organisms, eco-friendly and biodegradable. In the present study, ovicidal activity of essential oils (EOs) of basil, European pennyroyal, lavender, mint, oregano and savoury and oviposition-deterrent activity of methanolic extracts of fumitory, lavender, licorice and oregano were investigated on P. operculella. Probit analysis showed that EOs of Lavandula angustifolia revealed the highest activity (LC₅₀ value 0.4 μL/L air). Fumitory extract elicited the highest reduction in percentage of laying eggs. The results indicated that these medicinal plants could be effective for declining damage of P. operculella in storages.     

Efficacy of entomopathogenic nematodes against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory conditions

The susceptibility of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) to native and commercial strains of entomopathogenic nematodes (EPNs) was studied under laboratory conditions. Native strains of EPNs were collected from northeastern Iran and characterised as Steinernema feltiae and Heterorhabditis bacteriophora (FUM 7) using classic methods as well as analysis of internal transcribed spacer (ITS) and D2/D3 sequences of 28S genes. Plate assays were performed to evaluate the efficiency of five EPN strains belonging to four species including Steinernema carpocapsae (commercial strain), S. feltiae, Steinernem glaseri and H. bacteriophora (FUM 7 and commercial strains). This initial assessment with 0, 75, 150, 250, 375 and 500 IJs/ml concentrations showed that S. carpocapsae and H. bacteriophora caused the highest mortality in both larval and prepupal stages of P. operculella, PTM. Thereafter, these three strains (i.e. S. carpocapsae, H. bacteriophora FUM 7 and the commercial strains) were selected for complementary assays to determine the effects of soil type (loamy, loamy–sandy and sandy) on the virulence of EPNs against the second (L2) and fourth instar (L4) larvae as well as prepupa. A soil column assay was conducted using 500 and 2000 IJs in 2-ml distilled water. Mortality in the L2 larvae was not affected by the EPN strain or soil type, while there was a significant interactive effect of nematode strains and soil type on larval mortality. The results also showed that EPN strains have higher efficiency in lighter soils and caused higher mortality on early larvae than that in loamy soil. In L4 larvae, mortality of PTM was significantly influenced by nematode strain and applied concentrations of infective juveniles. The larval mortality induced by S. carpocapsae was higher than those caused either by a commercial or the FUM 7 strain of H. bacteriophora. Prepupa were the most susceptible stage.     

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.     

Resistance of transgenic rice events (rbcS:cry1Ac) against three lepidopteran rice pests

The transgenic rice expressing cry1Ac gene, which is linked to the rice rbcS promoter and its transit peptide sequence (tp), was highly resistant against all instars of Cnaphalocrocis medinalis (Guenetée) (Lepidoptera: Crambidae). In this study, we evaluated the larval mortality, behavior change, and field occurrence of three main rice pests, C. medinalis, Naranga aenescens (Moore) (Lepidoptera: Noctuidae), and Parnara guttata (Bremer & Grey) (Lepidoptera: Hesperiidae) in T4 generations of three Bt rice events (rbcS3:cry1Ac; 608102, 608104 and 608107) and non-Bt rice. All of the three Bt rice events were resistant to C. medinalis which showed significantly higher mortality for all instars compared to non-Bt rice. The resistance of Bt rice events against the larvae decreased gradually as the larvae developed. However, the survived larvae which ingested Bt rice events died eventually without further development. The resistance of three Bt rice events was investigated in the pot test, which was conducted with 3rd instars of C. medinalis, N. aenescens, and P. guttata, showed mortalities of over 70%. In behavioral assay, C. medinalis fed on the Bt rice events showed feeding avoidance and less leaf rolling behavior compared to that of the larvae fed on non-Bt rice. A 2-yr field survey conducted with larvae of C. medinalis and P. guttata also showed that the three Bt rice events significantly had lower damaged on leaves compared to that of non-Bt rice. Overall, the three Bt rice events were highly resistant to the larvae of lepidopteran target rice pests.     

Behavior of newly hatched potato tuber moth larvae,Phthorimaea operculella Zell. (Lepidoptera: Gelechiidae), in relation to their host plants

A study was made of neonate larvae of Phthorimaea operculella.Host finding from soil-laid eggs and dispersal from hosts and nonhosts were first examined. Of first-instar larvae hatching from soil-laid eggs, 80% found the potato plant while roughly 50% found each of the other three plants (datura, tobacco, and tomato). Dispersal from potato, datura, and tobacco was very low, while on tomato it was higher and a high mortality was observed in the 24 h period. Dispersal from nonhost plants was high. Behavior on leaves of hosts and non-hosts is described and some host-plant specific behaviors are identified. Effects on behavior of some of the physical and chemical factors are described. Methylene dichloride extracts of potato leaf wax reduced locomotion rates and the number of turns during locomotion. Also methylene dichloride extract and, to a lesser extent, methanol extract caused biting behavior.     

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.     

Biological effects of some natural and chemical compounds on the potato tuber moth,Phthorimaea operculella Zell. (Lepidoptera:Gelechiidae)

The olfactory reaction of larvae and moths was investigated towards 18 oils (6 natural oils and 12 commercial chemical oils). Some of these oils such as peppermint and camphor (natural oils) and eugenol and camphene (commercial oils) were repellent to both larvae and moths. Other oils such as strawberry and d-limonene were attractive to both larvae and moths.Some of the repellent oils were, therefore, tested for their effect on certain biological aspects of the insects.Eugenol and peppermint oils, each at the 0.01% conc., caused a significant depression in the fecundity of moth and decreased the percentage of egg hatchability. Eugenol oil was much more effective than peppermint oil at 1%. Dried (leaves, fruits or seeds) powder of 14 different plants species were tested in different concentrations with talcum powder (carrier material) against egg deposition. The results indicated that dried powders of Allium cepa, Curcuma longa, Colocasia antiqurum, Ocimum basilicum. Dodonaea viscose and Thuja orientalis played a highly significant role in reducing egg deposition. The most impressive effect was displayed by powders of D. viscose and A. cepa, which caused the highest depression in egg deposition as well as in the emerging offsprings. Ethanolic extracts of 11 plants indicated that extracts of Pithuranthos tortosus and Iphiona scabra caused the maximum inhibition of egg hatchability, followed by C. longa, Citrullus colocynthia and T. orientalis. Ethanolic extracts of Schinus terebenthiflius (leaves) and I. scabra caused the highest depression in the deposited eggs, as they played a remarkable role as ovipositor deterrents.The majority of the plant extracts at 1% conc. could protect potato tubers at different intervals according to the calculated tuber damage index as follows: Iphiopna > Pithuranthos > Curcuma > Schinus (fruits) Thuja > Schinus (leaves) > Dodonaea > Citrullus.     

Area‐wide monitoring of potato tuberworm (Phthorimaea operculella) by pheromone trapping in Northern Italy: phenology,spatial distribution and relationships between catches and tuber damage

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转sck+cry1Ac基因水稻对二化螟及二化螟绒茧蜂存活和生长发育的影响

在实验室研究了转sck+cry1Ac基因水稻（MSB）对二化螟Chilo suppressalis (Walker)生长、存活以及经寄主对二化螟绒茧蜂Apanteles chilonis (Munakata)生物学特性的影响。连续取食转sck+cry1Ac基因水稻的二化螟, 体重下降、死亡率上升,从第2天开始,其体重显著低于取食明恢86的对照组;从第6天开始,死亡率极显著高于对照组。二化螟取食MSB 36 h后移至对照水稻上继续取食3、6、9、12天后,死亡率与对照差异都不显著;但体重均低于对照,其中第3天的体重差异达显著水平。二化螟绒茧蜂分别以取食MSB一定时间的3、4、5龄二化螟幼虫为寄主时,寄生率均低于以对照组,其中对4龄幼虫的寄生率差异显著;结茧率与对照差异均不显著;寄生在取食MSB的5龄二化螟幼虫体内的蜂、蛹期显著长于对照,而所结茧的茧长显著短于对照;但卵 幼虫历期、每茧块茧数、羽化率、雌性率、成蜂寿命和前翅长与对照无显著差异。结果表明转sck+cry1Ac基因水稻不仅对二化螟生长和存活有显著影响, 而且可经寄主二化螟影响到二化螟绒茧蜂的一些生物学特性。     

转cry1Ab/cry1Ac基因玉米Cry1Ab/Cry1Ac融合蛋白表达及对亚洲玉米螟的室内杀虫效果

【目的】室内抗螟性评价是转Bt基因抗虫玉米研发和安全性评价的重要环节。【方法】采用酶联免疫吸附测定法(ELISA)测定了转cry1Ab/cry1Ac基因玉米ZZM030心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量;采用室内生测法测定了分别取食转基因玉米ZZM030和非转基因玉米X249心叶后亚洲玉米螟Ostrinia furnacalis敏感品系ACB-BtS、Cry1Ab抗性品系ACB-AbR和Cry1Ac抗性品系ACB-AcR初孵幼虫的存活率。【结果】转基因抗虫玉米ZZM030 4叶期和8叶期心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量分别是10.62和2.94 μg/g FW。敏感品系亚洲玉米螟初孵幼虫取食转基因玉米ZZM030心叶2 d的存活率仅为23.6%,4 d后存活率为0,而取食非转基因对照玉米X249心叶4 d的存活率高达93.1%。Cry1Ab抗性品系和Cry1Ac抗性品系初孵幼虫取食转基因玉米ZZM030心叶6 d后的存活率分别为11.1%和12.5%,而取食非转基因玉米X249心叶6 d后的存活率分别为81.9%和77.8%。【结论】转cry1Ab/cry1Ac基因玉米ZZM030心叶中高表达的Cry1Ab/Cry1Ac融合蛋白对亚洲玉米螟初孵幼虫具有极高的杀虫效果。     

The potential of the fungus, Muscodor albus, as a microbial control agent of potato tuber moth (Lepidoptera: Gelechiidae) in stored potatoes

Potato tuber moth (PTM), Phthorimaea operculella, is a serious pest of stored potato in most countries where potatoes are grown. Entomopathogens offer promise as alternatives to broad spectrum insecticides for management of this pest. The fungus Muscodor albus, which produces a mixture of antimicrobial volatile organic chemicals, was tested for its insecticidal activity against PTM. Adults and neonate larvae were exposed to volatiles generated by 15 or 30 g of M. albus rye grain culture plus water for 72 h in hermetically sealed 28.3 L chambers at 24 degrees C. Mean percent mortalities in adult moths exposed to 0, 15, and 30 g of fungal formulation were 0.9, 84.6, and 90.6%, respectively. Development to the pupal stage of PTM that were exposed as neonate larvae to 15 or 30 of M. albus culture was reduced by 61.8 and 72.8%, respectively, relative to controls.     

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest,Chilo suppressalis (Lepidoptera: Crambidae)

Two transgenic rice lines (T2A‐1 and T1C‐19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A‐1 > Huahui 1 > T1C‐19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C‐19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C‐19b providing an excellent performance at the maturing stage. By comparison, T1C‐19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A‐1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C‐19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.     

The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes

Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide. New, more virulent P. infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties. R genes (for single-gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato. The molecular basis of single-gene and quantitative resistance to late blight is unknown. We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach. The R1 gene is member of a gene family. It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa. The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine-rich repeat domain. The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato. R1 is located within a hot spot for pathogen resistance on potato chromosome V. In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.     

Cloning and characterisation of a novel holotype crystal protein gene,cry59Ba1, from Bacillus thuringiensis strain Bm59-2, toxic to Spodoptera exigua (Lepidoptera)

Abstract

A novel cry59-type gene, cry59Ba1, was obtained from isolate Bm59-2 and identified from an assembled plasmid genome sequence. This gene was found to encode a polypeptide of 674 amino acid residues with a predicted molecular mass of 75.2 kDa. This polypeptide was 62.1% identical to cry59Aa1. The Cry59Ba1 protein was expressed in the acrystalliferous mutant strain HD73^? and tested against Culex quinquefasciatus (Diptera), Spodoptera exigua (Lepidoptera) and Helicoverpa armigera (Lepidoptera). The bioassay showed Cry59Ba1 protein to be highly toxic to S. exigua (Lepidoptera) (LC50 =26.2 µg/ml, 95% confidence limit, 16.2-75.3 µg/ml). The cloning of cry59Ba1 gene may provide a novel type insecticidal resource for resolving the problem of lepidopteran insects developing resistance to the Cry1 proteins.