Biological Control of the Potato Tuber Moth (Phthorimaea operculella Zeller) in the Republic of Yemen Using Granulosis Virus: Biochemical Characterization, Pathogenicity and Stability of the Virus

A granulosis virus infecting the potato tuber moth, Phthorimaea operculella, has been identified, isolated and purified from diseased potato tuber moth larvae collected from potato fields in the highland basin Qa al-Boun of the Republic of Yemen. The granulosis virus was propagated by feeding potato tuber moth larvae with potatoes treated with a concentration of one granulosis virus-infected fourth instar larva to 5l of water (corresponding to an occlusion body (OB) concentration of 2 106 OB ml-1). Restriction enzyme analysis of viral DNA revealed that the isolated virus seemed to correspond to an isolate from the Lima region of Peru. A median LC 50 value of 7.3 104 OB ml-1 was calculated for a purified virus preparation. Different preparations of the granulosis virus were investigated for their persistence in the field on tubers and leaves. A purified virus preparation (PoGV) applied to potato tubers and exposed in the open had a half-life of 1.3 days. On leaves, the activity of granulosis virus spray deposits of an unpurified virus preparation (PoGV I) and of a glycerine-formulated preparation (PoGV II) also declined with exposure time. Mortalities of potato tuber moth larvae of 43% for PoGV I and 49% for PoGV II were recorded when first instar larvae were fed with potato leaves collected 2 days after treatment. After 8 days, 25.8% of the larvae had died from PoGV I treatment and 19.4% from PoGV II. Neither preparation displayed any effect 17 days after application.     

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.     

Evaluation of a granulovirus (PoGV) and Bacillus thuringiensis subsp. kurstaki for control of the potato tuberworm (Lepidoptera: Gelechiidae) in stored tubers

Liquid suspensions and dry formulations of a granulovirus (family Baculoviridae, genus Granulovirus, PoGV) derived from infected larvae and the bacterium Bacillus thuringiensis subsp. kurstaki (Berliner) (Btk) were evaluated for control of the potato tuberworm, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), in stored tubers. Laboratory bioassays at 25 degrees C showed that both PoGV and a wettable powder (WP) formulation of Btk incorporated with carriers (water, talc, sand, diatomaceous earth, and kaolin clay), were effective against neonate larvae. Depending on the technique, 100% larval mortality was achieved at concentrations as low as 0.025 larval equivalents (LE) PoGV per kg tuber and 150 mg Btk WP per kg tuber. However, 100% mortality was never achieved with tests on preinfested tubers, ostensibly due to the higher dosage required to kill older instars inside tubers. The most effective PoGV formulations were dipping (water) and talc, with dipping most effective for postinfestation treatments, causing up to 91.6% mortality at 0.4 LE per kg. There was no significant effect of formulation in the Btk treatments. The protective effects of residues were also evaluated under longer-term storage conditions. Batches of tubers treated with PoGV or Btk via dipping (up to 0.1 LE and 150 mg WP per kg tuber) were stored in cages containing an initial potato tuberworm infestation (10% of tubers). Although potato tuberworm populations were reduced by up to 98.4% after 2 mo at 25 degrees C, no treatments prevented the development and reproduction of the F1 generation. The sprouting of stored tubers seemed to be a limiting factor for sustained control. No significant treatment effects were detected in similar cages held at 12 degrees C for 4.5 mo. Improved strategies for the application of PoGV and Btk for long-term potato tuberworm control in tuber stores, including the use of chemical sprout suppressants, are discussed.     

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.     

颗粒体病毒在小菜蛾种群中的垂直传播方式的初步观察

在室内观察了小菜蛾颗粒体病毒PlutellaxylostellaGranulosisVirus(RxGV)在小菜蛾种群水平上可能的垂直传播途径。结果表明 :(1 )上代小菜蛾Plutellaxylostella(L .)幼虫期饲毒不能将小菜蛾颗粒体病垂直传递到下代 ;(2 )取食病毒的雌蛾产的卵所孵化的幼虫中 ,没病毒感染出现 ,说明病毒不能经小菜蛾成虫体内传播到下一代。 (3 )用小菜蛾颗粒体病毒处理卵卡时 ,其初孵幼虫有少量被病毒感染死亡 ,浓度高时感染率也较高 ,但感染率均在 1 0 %以下 ,说明小菜蛾颗粒体病毒能经卵表垂直传递给下代 ,但传递效率不高。但当用病毒处理叶片上的卵时 ,初孵幼虫死亡率高达 77 73 % ,而且孵化出的幼虫大多数染病且死于 3龄以前 ,因此田间病毒的施用时间可提早到卵高峰期。     

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.     

棉铃虫颗粒体病毒增效蛋白基因2.1kb片段在大肠杆菌中的表达

以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组DNA为模板,设计引物PCR扩增病毒增效蛋白(Enhancin)基因,然后经SacI/PstI双酶切消化,得到5′端截短的约2.1kb增效蛋白基因片段,再与pQE30质粒连接,构建了重组表达载体pQE/EnC,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为78×10³ D的融合蛋白并命名为P78,纯化的P78包涵体显示了明显的增效活性,可提高AcMNPV对小菜蛾幼虫的感染率27.88%～32.92%。     

Evaluation of potato tuber moth (Lepidoptera: Gelechiidae) resistance in tubers of Bt-cry5 transgenic potato lines

The potato tuber moth, Phthorimaea operculella (Zeller), in tropical and subtropical countries, is the most destructive pest of potato, Solanum tuberosum L. The larvae attack foliage and tubers in the field and in storage. The purpose of this study was to evaluate the efficacy of a Bt-cry5 transgene to control the potato tuber moth in tuber tissues. Tuber bioassays using stored (11-12 mo old) and newly harvested tubers of Bt-cry5-Lemhi Russet and Bt-cry5-Atlantic potato lines showed up to 100% mortality of 1st instars. Mortality was lowest in the newly harvested tubers of Bt-cry5-Atlantic lines (47.1-67.6%). Potato tuber moth mortality was 100% in the Bt-cry5-Spunta lines that were transformed with Bt-cry5 gene controlled by the CaMV 35S promoter (pBIML5 vector) and in 2 of 3 lines transformed with Bt-cry5 gene controlled by the Gelvin super promoter (pBIML1 vector). The transgenic Spunta lines expressing Bt-cry5 controlled by the patatin promoter (pBMIL2 vector) showed the lowest tuber moth mortality (25.6 and 31.1%). The Bt-cry5 transgenic lines with high tuber expression of B. thuringiensis have value in an integrated pest management system to control potato tuber moth.     

The effect of temperature and duration of exposure of potato tuber moth (Lepidoptera: Gelechiidae) in infested tubers to the biofumigant fungus Muscodor albus

The endophytic fungus, Muscodor albus produces several volatile compounds (alcohols, esters, ketones, acids and lipids) that are biocidal for a range of organisms including plant pathogenic bacteria and fungi, nematodes and insects. We studied the effects of these volatiles on 3-day-old potato tuber moth larvae within infested tubers inside sealed chambers. The length of exposure to M. albus significantly affected mortality of larvae, calculated as percentage of larvae failing to survive to the adult stage. Exposure durations of 3, 7, or 14 days at 24 degrees C followed by incubation in fresh air at 27 degrees C until emergence resulted in mortalities of 84.2, 95.5 and 99.6%, respectively. However, the longer exposures also resulted in increased levels of carbon dioxide (CO(2)) that are unacceptable for tuber storage. Effects of M. albus on larval survival was also monitored at 10, 15 and 24 degrees C, using an exposure duration of 7 days followed by incubation in clean air at 27 degrees C until emergence. Mortality of larvae was sharply reduced at the lower temperatures resulting in 50.8, 76.8, and 95.4% mortality, respectively. Tuber storage conditions, especially cooling rates, are discussed with respect to using M. albus as a fumigant without simultaneously producing unacceptable (for tuber storage) levels of CO(2).     

Fruit Bins Washed with Steinernema carpocapsae (Rhabditida: Steinernematidae) to Control Cydia pomonella (Lepidoptera: Tortricidae)

A bin washing apparatus (drencher) was used to treat late-instar codling moth, Cydia pomonella Linnaeus-infested wooden fruit bins with the entomopathogenic nematode Steinernema carpocapsae . Sentinel late-instar codling moth larvae were placed in strips of wood, made from dismantled bins, that were subsequently screwed into the corners of real bins before treatment. Pre-soaking the bins with water prior to treatment and covering the bins with a plastic tarp post-treatment, to maintain a high humidity, significantly increased sentinel codling moth mortality. Treatment for 1 min with 50 S. carpocapsae infective juveniles mL -1 water resulted in more than 80% mortality over 6 h in which the nematodes were continuously pumped through the drencher. The temperature of the water in the bin washer was ≤15°C and the chlorine in the city water was too low to harm the nematodes. The key disadvantage of washing the bins was the labour involved. It may be possible to reduce post-treatment holding time and increase codling moth mortality by holding the bins at a higher temperature post-treatment, however this parameter was not included in the study. The presence of the codling moth granulosis virus within the larvae did not influence the nematode-induced codling moth mortality over a range of concentrations and two temperatures in laboratory trials.     

Dosage-mortality and time-mortality studies of a granulosis virus in a laboratory strain of the codling moth,Laspeyresia pomonella

Different doses of a granulosis virus were administered to first- and fifth-instar larvae of the codling moth Laspeyresia pomonella. Virus was very pathogenic for both larval instars. The LD₅₀ values for first- and fifth-instar larvae were 5 and 49 capsules/larva, respectively. However, fifth-instar larvae were much more variable in their response to virus than first-instar larvae. Using probit methods it was calculated that 1 capsule could cause death in about 25% of both larval instars but 1578 capsules were required to cause 70% mortality of fifth-instar larvae as compared to 12 capsules for first-instar larvae. This is the first report of a decided difference in variability of response to virus by two larval instars of the same species. A bimodal response by both larval instars was observed in time-mortality studies. Apparently, about 20% of the larvae were very resistant to virus infections.     

Studies of transovum and transstadial transmission of a granulosis virus of the codling moth

Capsules of a granulosis virus of the codling moth were never found in codling moth eggs, although they were observed in the larval, pupal, and adult stages. However, eggs obtained from a British Columbia codling moth colony were found to have virus on the egg surface.Transstadial transmission of the virus from the larval to the pupal stage occasionally occurred after intact capsules had been inoculated per os into the larva. The virus was also occasionally transmitted from the pupal to the adult stage, but only after virions had been injected into the hemocoel of the pupa.Fertile eggs obtained from virus-injected insects were not shown to contain active virus. Eggs were tested by maceration in antiserum, by the fluorescent-antibody technique, and by bioassay of their contents.Only one larva of the F₁ and F₂ generations obtained from virus-injected insects succumbed to granulosis even when the larvae were exposed to various types of stressors.Circumstantial evidence supports the hypothesis of transovum transmission, but such transmission cannot be attributed to injections of virus into larvae and pupae in this study.     

Spatial and temporal dynamics of potato tuberworm (Lepidoptera: Gelechiidae) infestation in field-stored potatoes

The potato tuberworm, Phthorimaea operculella (Zeller), is a major pest of potatoes in fields and traditional storage. A common method of nonrefrigerated storage is to pile potatoes in straw-covered heaps in the field. Tubers may be stored up to 3-4 mo in this manner, until the next harvest. We studied the dynamics of potato tuber moth infestation associated with such field storage in a 12-wk experiment in Israel. We set up six potato heaps, and sampled them for potato tuber moth at different locations at weekly intervals. Potato tuber moth infestation was significantly higher at the perimeter of the heaps than at their center, but it did not differ between bottom, mid-height, and top of the heaps. The proportion of potato tuber moth-infested potato tubers increased from 10 to 65% over the course of the experiment, and the mean number of potato tuber moth larvae per tuber increased from 0.25 to 2.50. Potato tuberworm populations increased sharply after 3, 6, and 9 wk of study, possibly corresponding to successive generations that developed within the heaps. This interpretation is supported by calculations of potato tuberworm generation length based on temperature data. Catches in pheromone traps that were placed near the heaps were not correlated (spatially and temporally) with potato tuberworm densities within heaps, hinting that migration among heaps did not considerably affect within-heap population dynamics. Potato tuberworm levels were not correlated with ambient temperatures, perhaps because of the warm, humid, and constant microclimate within the heaps. We discuss the significance of our findings for control efforts of the potato tuberworm.     

用马铃薯人工饲养马铃薯块茎蛾的方法

介绍一种马铃薯块茎蛾Phthorimaeaoperculella的室内人工饲养方法。在人工养虫室内用 1 0 %的蜂蜜水喂养成虫 ,用滤纸收集卵块 ,幼虫在薯块上饲养 ,化蛹于牛皮纸卷成的纸筒中 ,饲养结果发现马铃薯块茎蛾完成 1代大约需要 35～ 4 6d ,室内连续饲养块茎蛾 3代 ,卵的孵化率、幼虫化蛹率、成虫羽化率均在 90 %以上。     

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.     

Susceptibility of potato plants grown from tubers irradiated with stimulation doses of gamma irradiation to potato tuber moth, Phthorimaea operculella Zeller (Lep., Gelechiidae)

The feeding behaviour of potato tuber moth, Phthorimaea operculella Zeller (Lep., Gelechiidae), reared on leaves and tubers of potato plants, which were irradiated as seed (tubers) with stimulation doses of gamma irradiation (1, 3, 5 and 10 Gy), was studied. Significant differences in the larvae and pupae developmental time, pupal weight, mortality, fecundity and percentage egg hatch, were observed between insects fed on plants which resulted from the irradiated seeds and those of the control. It appears that leaves of potato plants grown from irradiated seeds particularly those of 3 Gy, became more attractive to the larvae, although the resulting tubers, with the exception of those of 10 Gy, became more resistant to potato tuber moth. Storing the tubers at ambient temperature conditions affected the degree of larval sensitivity. The leaves and tubers of 10 Gy-irradiated seeds became more suitable for insect development, indicating that the later dose may inhibit the production of secondary plant metabolities and chemical compounds.     

Production and field evaluation of codling moth granulosis virus for control of Cydia pomonella in the United Kingdom

Codling moth granulosis virus (Cp GV) was produced in larvae of Cydia pomonella reared on artificial diet. The average yield of virus (9 × 10⁹capsules/larva) was increased by raising the larvae on diet containing methoprene, a juvenile hormone analogue. In field trials in 1978 and 1979, one or two high-volume applications of Cp GV at 7 × 10¹⁰ capsules/litre achieved reductions in numbers of mature larvae and damaged fruit that were little different from those obtained by two applications of azinphos-methyl. A field trial in 1980 showed that at concentrations of Cp GV ≥ 6 × 10⁸ capsules/litre, damaged diminished slowly with increasing virus concentration. Following virus application in 1980, Cp GV infectivity was reduced by half in 3 days, but some activity persisted at least 4–8 wk after spraying. The results indicate that codling moth GV is an effective control agent particularly in reducing numbers of larvae and the more severe forms of fruit damage.     

苹果蠹蛾老熟幼虫诱杀技术

本文对简易杀虫诱集带诱杀苹果蠹蛾Cydia pomonella(L.)老熟幼虫的效果进行了研究。发现在编织袋,旧衣物,瓦楞纸3种材料中,旧衣物对苹果蠹蛾的老熟幼虫捕获效果最好,其诱捕量占总诱捕量的68.42%;在诱集带中添加的农药中(包括乐斯本、敌敌畏、白僵菌、灭幼脲3号、高效氯氰菊酯等),乐斯本的杀虫效果最好,幼虫死亡率达到98.20%;在不同浓度的乐斯本溶液中,500倍乐斯本杀虫效果最好,幼虫死亡率达到91.61%,且对幼虫没有驱避作用;500倍乐斯本+50倍白僵菌药杀虫诱集带有效期长达40多天,37d后对幼虫的杀伤效果依然达到96%以上,作为老熟幼虫的防治措施十分理想。     

Susceptibility of potato tuber moth (Lepidoptera: Gelechiidae) to postharvest gamma irradiation

Gamma irradiation doses applied to inhibit potato sprouting were tested against potato tuber moth larval and pupal stages. Young larvae and pupae were more susceptible to irradiation injuries than older ones. When larvae of different ages were exposed to doses > or = 100 Gy, only 13-35% pupated, but no adult emergence was obtained. The exposure of 1-1.5- or 3-3.5-d-old pupae to 150 Gy induced high level of sterility and remarkable reduction in female mating ability and fecundity, whereas the reduction was less noticeable for 5-5.5-d-old pupae. Postharvest gamma irradiation of potatoes could be considered as an efficient control approach against potato tuber moth larval and pupal infestations.