Laboratory and field studies on the integrated pest management of Helicoverpa armigera (Hübner) in cotton, based on pheromone trap catch threshold level

Abstract: With a view to using parasitoids and predators in integrated pest management of the target pest Helicoverpa armigera in cotton fields, basic studies on the egg parasitism, toxicity of insecticides to parasitoids and predators and compatibility of nuclear polyhedrosis virus (NPV) of H. armigera with other insecticides were conducted in the laboratory. Results revealed that egg parasitism in the laboratory by Trichogramma chilonis was 75.6%. Among the insecticides tested against T. chilonis and the predator Chrysoperla carnea , nimbecidine (neem product) and dipel resulted in zero mortality, with only a low level of mortality by dimethoate, cypermethrin, fenvalerate, alphamethrin and monocrotophos. Combinations of nimbecidine 2% + NPV at 250 larval equivalents (LE)/ha and dipel 8 l + NPV @250 LE/ha were the most effective treatments against H. armigera . The integrated pest management components ( T . chilonis , C . carnea , NPV, nimbecidine, dipel and synthetic chemicals) were imposed at different intervals on the basis of pheromone trap threshold level (7 moths/trap per night) on a consolidated block of 40 ha cotton (MCU-1) fields at two locations, Shankarabanda and Korlagundi. The results demonstrated a significant superiority of the IPM strategy in terms of both cost versus benefit and environmental safety over that used in the farmer's fields where only conventional control methods were followed.     

Successes and challenges of managing resistance in Helicoverpa armigera to Bt cotton in Australia

Bt cotton has been gradually released and adopted by Australian growers since 1996. It was initially deployed in Australia primarily to control the polyphagous pest Helicoverpa armigera (Hübner), which in the 1990s became increasingly difficult to control due to widespread resistance to synthetic chemical insecticides. Bt-cotton has become a key tool in a program of integrated pest management for the production system that reduces pesticide dependence and the problems associated with its use. Herein we overview the deployment of Bt cotton in Australia including its performance and the approaches used to prolong the evolution of resistance to it by H. armigera. An integral component of this approach is monitoring resistance in this pest. We outline resistance screening methods, as well as the characteristics of resistant strains of H. armigera that have been isolated from field populations, or selected in the laboratory. We then highlight the successes and challenges for Bt cotton in Australia by way of discussing adaptive resistance management in light of potential changes in resistance.     

Efficacy of new insecticides for management of Helicoverpa spp. (Lepidoptera: Noctuidae) in Australian grain crops

Abstract  Insecticide resistance in Helicoverpa armigera (Hübner) has led to the reduced efficacy of some older insecticide groups (pyrethroids and carbamates) and serious crop losses. Eight small-plot experiments were conducted to evaluate new insecticides for the management of H. armigera in grain crops. Several products showed efficacy equivalent to or better than the commercial standard, thiodicarb. Indoxacarb and spinosad at rates 50% or less of the registered rates for cotton were consistently superior to other tested products across the range of crops treated and provided residual protection for up to 14 d. The insect growth regulator compound, methoxyfenozide, was slower acting than other products tested, but demonstrated potential for H. armigera management. Pyridalyl performed well and warrants further evaluation in grain crops. We discuss the positioning of new compounds in an Insecticide Resistance Management Strategy (IRMS) in relation to a farming system that incorporates both grain and cotton crops. Use guidelines are recommended for indoxacarb, the first new compound to be registered in selected grain crops and cotton in Australia. These guidelines include restricted-use periods and limits on the number of applications per crop. It is anticipated that additional new compounds will be registered in grain crops, leading to the reduced selection pressure on the limited number of efficacious products. Coordinated insecticide use across farming systems and compatibility with developing integrated pest management programs should be fundamental considerations for the future IRMS.     

Cotton pests and their natural enemies in Madagascar

An update on the cotton pest complex and its associated natural enemies in Madagascar is provided. Since the end of the 1970s, when the previous reports had been published, the population dynamics of the principal pests in Malagasy cotton have undergone considerable changes. The American bollworm Helicoverpa armigera Hübner (Lep., Noctuidae) is still a limiting factor for production and can be considered the key pest, whereas the Egyptian leaf worm Spodoptera littoralis Boisduval (Lep., Noctuidae) and the cotton aphid Aphis gossypii Glover (Hom., Aphididae) have become significant pests as a result of indiscriminate use of synthetic pyrethroids in the 1980s. New records of beneficials, in particular the discovery of the aphidopathogenic fungus Neozygites fresenii (Nowakowski) (Entomophthorales) in cotton aphid populations, are reported. Strategies to preserve predator populations in view of reducing disruptive insecticide treatments are discussed.     

Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide

The Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) has been registered and is commercially produced in China as a biopesticide to control the bollworm in cotton. However, the virus has a relatively slow speed of action. To improve its efficacy, recombinant HearNPVs were generated by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene (HaCXW1 and HaLM2) or by inserting the insect-specific toxin gene AaIT in the egt locus (HaCXW2) of HearNPV using conventional recombination strategies in insect cell culture. The various recombinants remained genetically stable when cultured in HzAM1 insect cells. Bioassay data showed a significant reduction in the time required for all HearNPV recombinants to kill second instar H. armigera larvae. The LT(50) of the egt deletion recombinants HaCXW1 and HaLM2 was about 27% faster than that of wild-type HearNPV. The largest reduction in LT(50) was achieved by inserting the gene for the insect-specific neurotoxin, AaIT, in the egt locus, giving a reduction in LT(50) of 32% compared to wild-type HearNPV. The ability to genetically improve the properties of HearNPV as a biopesticide provides a further opportunity to develop this virus into a commercially viable product to control the bollworm in China.     

Frequency of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an important lepidopteran pest of cotton (Gossypium spp.) in Australia and the Old World. From 2002, F2 screens were used to examine the frequency of resistance alleles in Australian populations of H. armigera to Bacillus thuringiensis (Bt) CrylAc and Cry2Ab, the two insecticidal proteins present in the transgenic cotton Bollgard II. At that time, Ingard (expressing Cry1Ac) cotton had been grown in Australia for seven seasons, and Bollgard II was about to be commercially released. The principal objective of our study was to determine whether sustained exposure caused an elevated frequency of alleles conferring resistance to Cry1Ac in a species with a track record of evolving resistance to conventional insecticides. No major alleles conferring resistance to Cry1Ac were found. The frequency of resistance alleles for Cry1Ac was <0.0003, with a 95% credibility interval between 0 and 0.0009. In contrast, alleles conferring resistance to Cry2Ab were found at a frequency of 0.0033 (0.0017, 0.0055). The first isolation of this allele was found before the widespread deployment of Bollgard II. For both toxins the experiment-wise detection probability was 94.4%. Our results suggest that alleles conferring resistance to Cry1Ac are rare and that a relatively high baseline frequency of alleles conferring resistance to Cry2Ab existed before the introduction of Bt cotton containing this toxin.     

棉铃虫核型多角体病毒ORF33基因的原核表达和在宿主细胞中亚细胞定位

对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆抗体检测了HearSNPV感染的宿主细胞(HzAMI)中ORF33基因的表达,表达产物的分子量为31kDa。并通过共聚焦荧光显微镜方法,用多克隆抗体检测编码的蛋白在宿主细胞(HzAM1)中的亚细胞定位,发现ha33编码的蛋白存在于宿主细胞的细胞质中,并持续到感染后期。     

Monitoring and adaptive resistance management in Australia for Bt-cotton: current status and future challenges

In the mid-1990 s the Australian Cotton industry adopted an insect-resistant variety of cotton (Ingard) which expresses the Bt toxin Cry1Ac that is specific to a group of insects including the target Helicoverpa armigera. A conservative resistance management plan (RMP), that restricted the area planted to Ingard, was implemented to preserve the efficacy of Cry1Ac until two-gene transgenic cotton was available. In 2004/05 Bollgard II replaced Ingard as the transgenic cotton available in Australia. It improves on Ingard by incorporating an additional insecticidal protein (Cry2Ab). If an appropriate refuge is grown, there is no restriction on the area planted to Bollgard II. In 2004/05 and 2005/06 the Bollgard II acreage represented approximately 80 of the total area planted to cotton in Australia. The sensitivity of field-collected populations of H. armigera to Bt products was assayed before and subsequent to the widespread deployment of Ingard cotton. In 2002 screens against Cry2Ab were developed in preparation for replacement of Ingard with Bollgard II. There have been no reported field failures of Bollgard II due to resistance. However, while alleles that confer resistance to H. armigera in the field are rare for Cry1Ac, they are surprisingly common for Cry2Ab. We present an overview of the current approach adopted in Australia to monitor and adaptively manage resistance to Bt-cotton in field populations of H. armigera and discuss the implications of our findings to date. We also highlight future challenges for resistance management in Australia, many of which extend to other Bt-crop and pest systems.     

Collection of Trichogramma Westwood (Hymenoptera: Trichogrammatidae) from tropical northern Australia: a survey of egg parasitoids for potential pest insect biological control in regions of proposed agricultural expansion

Abstract  Trichogramma limit pest damage to crops by killing the developing embryo of their insect host at the egg stage. Their impact on the potentially insecticide resistant species, Helicoverpa armigera (Hübner), is considered integral to the resistance management plan for transgenic cotton production in the Ord River Irrigation Area in northern Western Australia. The dominant species of egg parasitoid in Ord River Irrigation Area cotton crops is the introduced Trichogramma pretiosum (Riley). Surveys of similar northern Australian regions earmarked for agricultural expansion revealed that T. pretiosum has been introduced, or has adventitiously dispersed, to all developing agricultural regions of northern Australia. Several previously unsequenced species were collected during surveys in more pristine habitats. Trichogramma pretiosum demonstrates an apparent ability to supersede native Trichogramma species and is perhaps favoured by agricultural expansion.     

Yield, biological activity, and field performance of a wild-type Helicoverpa nucleopolyhedrovirus produced in H. zea cell cultures

This paper reports studies of the in vitro production of a virus from Helicoverpa armigera (HaSNPV) and its possible use as a specific Helicoverpa/Heliothis larvicide. Growth kinetics of Helicoverpa zea (H. zea) cells and virus occlusion body yields were compared in three SF900II-based media, namely, SF900II (serum-free), SF900II + 1% serum, or SF900II + 10% serum. Viable cell densities were usually higher in the media supplemented with serum than in the serum-free medium; however, in the serum-free medium, cell diameters were 1.7 times greater (i.e., individual cell volumes were five times larger). Both volumetric production of virus occlusion bodies and production per cell were higher in the serum-free medium than in the media supplemented with serum. However, the infectivity of the occlusion bodies from the serum-free medium was less than that with those from the medium supplemented with 10% serum, when compared in bioassays employing newly hatched larvae. The infectivity of the in vitro produced occlusion bodies was also less than that of in vivo produced occlusion bodies in a commercially available virus product, GemStar. High levels of infection of H. armigera larvae obtained in a preliminary field assessment on preflowering tomatoes using the in vitro produced occlusion bodies indicated the suitability of the in vitro process for biopesticide production.     

Helicoverpa armigera granulovirus interference with progression of H. zea nucleopolyhedrovirus disease in H. zea larvae

Capsular proteins from Helicoverpa armigera granulovirus (HaGV) have previously been shown to enhance H. armigera nucleopolyhedrovirus (HaSNPV) infection in H. armigera larvae. Yet, HaGV and HaS-NPV, as viable viruses, interfered with one another. In our study, we have examined the effects of co-infection of the slow-killing virus HaGV with the fast-killing virus Helicoverpa zea NPV (HzSNPV) on H. zea larvae. The mortality parameter measured was survival time. Virus stocks had 50% lethal concentrations of 3.2x10(-9) g HaGV-infected cadavers (GVC) (HaGV) and 32 occlusion bodies (HzSNPV) per cup. Average survival times were 16.8 and 5.5 days for larvae treated with HaGV and HzSNPV, respectively; death of HzSNPV-treated larvae was as early as 72 h posttreatment. In co-infection experiments in which larvae were treated concurrently with both viruses, the viruses competed in typical fashion for host resources. However, interference with disease progression in HzSNPV-fed larvae occurred even when HaGV was fed to larvae up to 36 h after the NPV, a time at which NPV infection should have been well established in host larvae. At death, co-infected larvae were observed microscopically to be filled with HaGV granules rather than HzSNPV polyhedra. The time study results imply that HaGV might be outcompeting HzSNPV by inhibiting its replication. We also observed that H. zea larvae treated with high dosages of HaGV (> or =3x10(-5) g GVC) were initially stunted but had survival times similar to those of larvae treated with lower dosages.     

Evaluating legume species as alternative trap crops to chickpea for management of Helicoverpa spp. (Lepidoptera: Noctuidae) in central Queensland cotton cropping systems

Mounting levels of insecticide resistance within Australian Helicoverpa spp. populations have resulted in the adoption of non-chemical IPM control practices such as trap cropping with chickpea, Cicer arietinum (L.). However, a new leaf blight disease affecting chickpea in Australia has the potential to limit its use as a trap crop. Therefore this paper evaluates the potential of a variety of winter-active legume crops for use as an alternative spring trap crop to chickpea as part of an effort to improve the area-wide management strategy for Helicoverpa spp. in central Queensland's cotton production region. The densities of Helicoverpa eggs and larvae were compared over three seasons on replicated plantings of chickpea, Cicer arietinum (L.), field pea Pisum sativum (L), vetch, Vicia sativa (L.) and faba bean, Vicia faba (L.). Of these treatments, field pea was found to harbour the highest densities of eggs. A partial life table study of the fate of eggs oviposited on field pea and chickpea suggested that large proportions of the eggs laid on field pea suffered mortality due to dislodgment from the plants after oviposition. Plantings of field pea as a replacement trap crop for chickpea under commercial conditions confirmed the high level of attractiveness of this crop to ovipositing moths. The use of field pea as a trap crop as part of an area-wide management programme for Helicoverpa spp. is discussed.     

Development of a viral insecticide: concept to commercialization

About 18 months ago, on December 8, 1970, the Food and Drug Administration officially granted the Heliothis nucleo polyhedrosis virus (NPV) the status of temporary exemption from requirement of a tolerance. This NPV, registered for use against Heliothis on cotton, has also been extensively tested on Heliothis species attacking corn, sorghum, tobacco and truck crops. This was the first time an insect virus had been officially registered by a federal agency for use as an insecticide. More recently Spain has registered this virus, and other governments also are in the process of registering the virus.The historical and technical development of this virus from concept to commercialization is the subject of this presentation. The virus, first isolated from diseased Heliothis attacking cotton in the Rio Grande Valley in Texas, was successfully developed through laboratory, pilot-plant, and commercial phases. At each phase the virus was evaluated on the basis of: (1) its safety to man, animals, and plants; (2) its production feasibility and costs; and (3) its effectiveness against the specific target pest. Currently, two companies (International Minerals and Chemical Corporation, Libertyville, Ill., and Nutrilite Products, Inc., Buena Vista, Calif.) are producing the Heliothis NPV under their trade names Viron/H and Biotrol VHZ, respectively, for experimental programs and for limited sales.     

Ants as egg predators of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Australian cotton crops

Abstract Helicoverpa armigera is a major pest of Australian cotton crops. To assess the impact of ant predation on H. armigera populations, the behaviour of four common ant taxa was observed in cotton crops in northern New South Wales over the 1999−2000 and 2001−02 seasons. Areas of cotton were artificially stocked with H. armigera eggs prior to observation. Pheidole spp. were the most frequently observed ants within the crop canopy in 1999−2000 and took the most H. armigera eggs. Iridomyrmex spp. were more frequently observed than Pheidole spp. in 2001−02 and also took some H. armigera eggs. Neither Paratrechina spp. nor Rhytidoponera metallica (Smith) took any H. armigera eggs, although both were seen in the crop canopy. Irrigation, cultivation and insecticide application disrupted foraging ants and limited their impact on H. armigera populations.     

Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae)

In Australia, transgenic cotton plants expressing the cry1Ac gene from Bacillus thuringiensis Berliner variety kurstaki are less toxic to first-instar Helicoverpa armigera (Hübner) after the plant is producing fruit. We developed two bioassay methods (leaf mush, leaf disk) to test if the physiological state of the plants explained changes in toxicity and a third method (diet incorporation) was developed to quantify the toxicity of Bt leaves when mixed in chickpea diet. Cry1Ac protein was less toxic to H. armigera larvae when the protein was mixed with leaves from fruiting versus presquare conventional cotton. Differences in LC50 varied from 2.4- to 726-fold, depending on the source of toxin and conventional plant material. These results suggest that plant-toxin interactions in fruiting cotton are reducing the toxicity of the Cry1Ac protein. The possible role of tannins in these changes is discussed.     

Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest

Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.     

Evolution, ecology and management of resistance in Helicoverpa spp. to Bt cotton in Australia

Prior to the widespread adoption of two-gene Bt cotton (Bollgard II?) in Australia, the frequency of resistance alleles to one of the deployed proteins (Cry2Ab) was at least 0.001 in the pests targeted namely, Helicoverpa armigera and Helicoverpa punctigera. In the 7 years hence, there has been a statistically significant increase in the frequency of alleles conferring Cry2Ab resistance in field populations of H. punctigera. This paper reviews the history of deploying Bt cotton in Australia, the characteristics of the isolated Cry2Ab resistance that likely impact on resistance evolution, aspects of the efficacy of Bollgard IIχ, and the behavioural ecology of Helicoverpa spp. larvae as it pertains to resistance management. It also presents up-to-date frequencies of resistant alleles for H. punctigera and reviews the same information for H. armigera. This is followed by a discussion of current resistance management strategies. The consequences of the imminent release of a third generation product that utilizes the novel vegetative insecticidal protein Vip3A are then considered. The area planted to Bt-crops is anticipated to continue to rise worldwide and many biotechnical companies intend to add Vip3A to existing products; therefore the information reviewed herein for Australia is likely to be pertinent to other situations.     

Group Ⅰ but not Group Ⅱ NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus (NPV) is divided into Group Ⅰ and Group Ⅱ based on the phylogenetic analysis. It has been reported that Group Ⅰ NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group Ⅱ NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group Ⅱ had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.     

Exploitation of wild Cicer reticulatum germplasm for resistance to Helicoverpa armigera

In the absence of high levels of resistance to Helicoverpa armigera (Hübner) in the cultivated germplasm of chickpea, we evaluated accessions of Cicer spp. mostly Cicer reticulatum Ladzinsky, for resistance to this important pest. Under multichoice conditions in the field, 10 accessions showed lower leaf damage and lower numbers of eggs, larvae, or both of H. armigera. Of these, IG 69960, IG 72934, and IG 72936 showed significantly lower leaf feeding than the cultivated genotypes or other accessions at the vegetative and reproductive stages. Larval weight was lower or comparable with that on C. bijugum (IG 70019) and C. judaicum (IG 70032) in C. reticulatum accessions IG 72933, IG 72934, IG 72936, and IG 72953 at the seedling stage and on IG 69960 and IG 72934 at the flowering stage. The accessions showing resistance to H. armigera in the field and laboratory conditions were placed in different groups, indicating the presence of diversity in C. reticulatum accessions for resistance to this pest. Less than seven larvae survived on IG 70020, IG 72940, IG 72948, and IG 72949, and IG 72964 compared with 12 on ICC 506. Larval and total developmental periods were prolonged by 6-15 and 3-8 d, respectively, on C. reticultatum accessions compared with those on ICCC 37. Less than five larvae pupated on the C. reticulatum accessions (except IG 72958 and ICC 17163) compared with 11 in ICCC 37. Accessions showing lower leaf feeding and adverse effects on the survival and development can be used in increasing the levels and diversifying the basis of resistance to H. armigera in chickpea.     

Group I but not Group II NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus(NPV) is divided into Group I and Group II based on the phy-logenetic analysis.It has been reported that Group I NPVs such as Autographa californica multiple NPV(AcMNPV) can transduce mammalian cells,while Group II NPVs such as Helicoverpa armigera single NPV(HaSNPV) cannot.Here we report that AcMNPV was capable of stimulating antiviral ac-tivity in human hepatoma cells(SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus(VSV) replication.In contrast,the HaSNPV and the Spodoptera exigua multiple NPV(SeMNPV) of group II had no inhibitory effect on VSV.Recombinant AcMNPV was shown to induce interferons al-pha/beta even in the absence of transgene expression in human SMMC-7721 cells,while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.