转二价核酶基因马铃薯及抗病性研究

 用克隆的特异性切割马铃薯卷叶病毒(Potato leaf roll virus,PLRV)复制酶基因负链RNA的二价核酶基因,构建植物表达载体pROKⅡ/DR,经土壤农杆菌(Agrobacterium tumefaciens)介导叶盘法转化马铃薯外植体,获得再生植株。PCR和Southern-blot检测,证明目的基因已成功地导入马铃薯再生植株,其转化率约为14.5%,并能够在无性繁殖后代植株中稳定存在。RT-PCR检测表明,再生马铃薯植株中的二价核酶基因可以转录表达。经病毒接种的转基因马铃薯株系L5、L7、L8和J-1的无性繁殖后代在继发感染中仍表现出较高的抗病性,为最终获得抗PLRV马铃薯新品系打下了基础。     

Invasive weed facilitates incidence of Colorado potato beetle on potato crop

We examined whether the invasive silverleaf nightshade, Solanum elaeagnifolium (Cavanilles) can facilitate the infestation of potato (Solanum tuberosum (L.)) by the Colorado Potato Beetle, CPB, Leptinotarsa decemlineata (Say) in Greece, which would have important financial and pest management implications for the growing of potato crops. In laboratory tests, CPB from Lesvos could utilise S. elaeagnifolium if supplied with whole plants. In the field, however, CPB was only found on S. elaeagnifolium after the start of the spring potato harvest and the resulting loss of potato foliage, and no eggs were laid. This suggests that S. elaeagnifolium provides only a temporary food resource for adult beetles. One of 10 surveyed summer potato fields near the study population of S. elaeagnifolium was infested with CPB. It is likely that the presence of S. elaeagnifolium in the vicinity of spring and summer potato fields can help maintain CPB population viability in the immediate post harvest period of the spring potato crop, which may in turn facilitate the infestation of summer potato fields with CPB.     

马铃薯转GhABF2转录因子苗耐盐性研究

以转GhABF2转录因子马铃薯试管苗为材料,进行苗期耐盐性研究,以期了解该作物对逆境的适应性。实验结果表明:在不同浓度NaCl胁迫条件下,转GhABF2基因株系材料(T1、T2)与对照未转基因材料(WT)植株的干鲜重、生理生化指标的变化趋势基本一致,就各性状值的变化来看,转基因植株表现出了明显的抗逆性。随着NaCl胁迫浓度的增加,与对照非转基因植物WT相比,转GhABF2基因材料T1、T2的生物量显著增加;WT、T1、T2的叶绿素含量均随着盐浓度的增加而降低;可溶性糖、丙二醛、脯氨酸,SOD酶活性、POD酶活性均随着盐胁迫浓度的增加而升高;可溶性蛋白含量随着胁迫程度的加重略有下降,但变化趋势不显著。从植株的生长状态、生理生化等性状指标方面来看,转基因植株比对照非转基因植株表现出了更强的抗逆性。     

Effect of strip-intercropping potato and annual alfalfa on populations of Leptinotarsa decemlineata Say and its predators

Intercropping systems are practiced to reducing pest density, enhancing predator's diversity and stabling crop yield. We evaluated the effect of strip-intercropping potato and annual alfalfa on populations of Leptinotarsa decemlineata Say, predator's biodiversity and potato yield over two seasons (2014 and 2015). Our results revealed that the densities of egg masses, eggs, larvae and adults of Colorado potato beetle (CPB) were significantly lower in intercrops than in monoculture. The main CPB predators recorded on potato plants (ladybirds and the green lacewing) showed a significant increase in the density at each of the three intercrops than in monoculture. The percentage of dry tubers weight loss was 40.9%–42.6% in monoculture, 16.3%–18.7% in 6P: 2A and <10% in 2P: 2A and 4P: 2A in two studied years. Our results suggest that strip-intercropping potato with annual alfalfa, particularly in 2P: 2A and 4P: 2A patterns may be an effective way in integrated management of CPB for reducing pest density, enhancing the presence of predators in potato fields and improving potato yield.     

Efficacy of Bacillus thuringiensis Cry3Aa protoxin and protease inhibitors against coleopteran storage pests

BACKGROUND: Environmental impacts and resistance to insecticides pose serious challenges to stored‐product insect and other types of pest control. Insect‐resistant transgenic grain is a potential alternative to fumigants, but candidate control proteins are needed, especially for coleopterans. Therefore, we evaluated the efficacy of a coleopteran‐active toxin, Bacillus thuringiensis Cry3Aa, with or without protease inhibitors, in laboratory feeding assays against coleopteran storage pests. RESULTS: In a comparison of the toxicity of Cry3Aa protoxin towards three species of coleopteran storage pests, Tenebrio molitor L. was found to be most sensitive, Tribolium castaneum (Herbst.) was most refractory and Rhyzopertha dominica F. displayed an intermediate response. For R. dominica, Cry3Aa combined with 3500 mg potato carboxypeptidase inhibitor or 5000 mg aprotinin kg^?1 diet resulted in both delayed development and increased mortality. Potato carboxypeptidase inhibitor and bovine aprotinin reduced the LC₅₀ of Cry3Aa for R. dominica two‐ and threefold respectively. Cry3Aa treatment resulted in fewer progeny from R. dominica, and progeny was further reduced when the protoxin was combined with potato carboxypeptidase inhibitor. CONCLUSIONS: These data support the hypothesis that a combination of Cry3Aa protoxin and protease inhibitors, particularly a potato carboxypeptidase inhibitor, may have applications in control strategies for preventing damage to stored products and grains by coleopteran pests. Published 2011 by John Wiley & Sons, Ltd.     

Prohibitin,an essential protein for Colorado potato beetle larval viability,is relevant to Bacillus thuringiensis Cry3Aa toxicity

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30 kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828 pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5 days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.     

甘薯病毒病害SPVD抗性鉴定方法及产量损失估计

为了建立规范、有效的甘薯病毒病害（sweet potato virus disease,SPVD）抗性鉴定方法,于2011—2012连续两年,利用田间人工嫁接病毒接穗的方法对12个甘薯品种进行抗性鉴定和产量损失测定。结果显示,嫁接接种后,接穗成活率接近100%,12个品种都有不同程度发病,病情指数在51.0~95.2之间;感染SPVD的甘薯植株叶绿素含量降低、蔓长缩短;单株薯块产量损失范围在55.1%~97.8%之间。研究表明,供试的12个甘薯主栽品种感染SPVD后均可引起严重的产量损失,且田间人工嫁接病毒接穗是一个有效的SPVD抗性鉴定方法。     

Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata

BACKGROUND: RNA interference (RNAi) is a breakthrough technology for conducting functional genomics studies and also as a potential tool for crop protection against insect pests. The major challenge for efficient pest control using RNAi in the field is the development of efficient and reliable methods for production and delivery of double‐stranded RNA (dsRNA). In this paper, the potential of feeding dsRNA expressed in bacteria or synthesized in vitro to manage populations of Colorado potato beetle, Leptinotarsa decemlineata (Say) (CPB), was investigated. RESULTS: Feeding RNAi successfully triggered the silencing of all five target genes tested and caused significant mortality and reduced body weight gain in the treated beetles. This study provides the first example of an effective RNAi response in insects after feeding dsRNA produced in bacteria. CONCLUSION: These results suggest that the efficient induction of RNAi using bacteria to deliver dsRNA is a possible method for management of CPB. This could be also a promising bioassay approach for genome‐wide screens to identify effective target genes for use as novel RNAi‐based insecticides. Copyright © 2010 Society of Chemical Industry     

Spatial dispersion and binomial sequential sampling for the potato psyllid (Hemiptera: Triozidae) on potato

BACKGROUND: The potato psyllid is a serious pest of potatoes. Sampling plans on potatoes for the potato psyllid have yet to be developed, thus the authors' objectives were (1) to determine the most efficient within‐plant sampling unit, (2) to determine the spatial dispersion of potato psyllids in potato fields and (3) to develop a binomial sequential sampling plan for this pest. RESULTS: Significantly more potato plants were infested with potato psyllids on the edges of the field, and significantly more plants were infested with psyllids on the ‘top’ and ‘middle’ of the potato plant. Significantly more psyllids were also found on the undersides of leaves. The potato psyllid has an aggregated distribution in potato fields. Binomial sequential sampling plans were developed for three action thresholds representing 0.5, 1 and 5 psyllids per plant. The average sample numbers for these action thresholds were between 12 and 16 samples, depending on the action thresholds. However, based on the shape of the operating characteristic curve, the 0.5 and 1 sampling plans were more reliable than the 5 psyllids per plant plan. CONCLUSION: The binomial sequential sampling plans are useful for detecting potato psyllids at low levels of infestation, which will be useful for pest management purposes. Copyright © 2011 Society of Chemical Industry     

29个马铃薯品种(品系)抗晚疫病基因组成与抗病相关性分析

由致病疫霉引起的马铃薯晚疫病是马铃薯生产中最具危害性的病害,明确现有马铃薯品种(品系)中抗病基因组成情况对于指导抗病品种合理布局及抗病育种具有重要意义。本研究利用农杆菌介导的无毒基因瞬时表达技术分析了致病疫霉8个无毒基因在29个马铃薯品种(品系)中的分布情况。结果表明,品种间抗病基因组成各异,平均每个品种含有4个抗病基因。同时,不同抗病基因在品种中的分布频率也不同,抗病基因R1(76%)、Rpi-blb1(66%)和Rpi-blb2(66%)在品种中分布较普遍;而含抗病基因R3b(45%)、R4(38%)和Rpi-vnt1(34%)的品种较少。此外,品种中所含抗性基因的数量和品种室内抗性程度呈显著正相关(r=0.915 6,P=0.029 1)。     

Characteristics of a resistance-breaking isolate of potato virus Y causing potato tuber necrotic ringspot disease

An Austrian isolate of potato virus Y^NTN, the causal agent of potato tuber necrotic ringspot disease (PTNRD), was serologically compared with seven Dutch PVY^N isolates. Using polyclonal and monoclonal antibodies, it was found indistinguishable from PVY^N. Determination of the nucleotide sequence of the coat protein cistron and comparison of the deduced amino acid sequence with coat protein sequences of other potyviruses revealed a high level of homology with PVY^N coat protein sequences. This confirmed the close taxonomic relationship of PVY^NTN with the PVY^N subgroup of potato virus Y. PVY^NTN is able to overcome all resistance genes known so far in commercial potato cultivars. Remarkably, transgenic PVY-protected tobacco plants are also resistant to PVY^NTN infection upon mechanical and aphid-mediated inoculation. These experiments indicate that genetically engineered resistance offers great potential in protection of potato to new aggressive strains of PVY^N.     

Exploiting host resistance to reduce the use of fungicides to control potato late blight

Field trials in 1996, 1997 and 1998 with six potato cultivars differing in levels of foliar and tuber race-nonspecific resistance to late blight were treated with 100, 50 and 33% of the recommended dose of the fungicide fluazinam at application intervals of 7, 14 and 21 days. Using a mixed inoculum of six or seven indigenous isolates of Phytophthora infestans small potato plots were inoculated via infector plants. A foliar blight model for the relationship between the effects of resistance, fungicide application and disease pressure was developed using multiple regression analysis. Cultivars with a high level of quantitative resistance offered the greatest potential for fungicide reduction. The model showed that the effect of resistance on integrated control increased exponentially with increasing cultivar resistance. Reducing fungicide input by lowering the dose resulted in less foliar disease than extending application intervals. The higher the disease pressure, the greater the risk associated with reducing fungicide input by extension of application intervals. The field resistance of cultivars to tuber blight mainly determined the frequency of tuber infection. Exploiting high foliar resistance to reduce fungicide input carried a high risk when cultivar resistance to tuber blight was low. When field resistance to tuber blight was high, a medium level of resistance in the foliage could be exploited to reduce fungicide dose to c . 50%, provided application was at the right time. At a high level of field resistance to both foliar and tuber blight, application intervals could be extended.     

Seed transmission of Sweet potato leaf curl virus in sweet potato (Ipomoea batatas)

Sweet potato leaf curl virus (SPLCV) infects sweet potato and is a member of the family Geminiviridae (genus Begomovirus). SPLCV transmission occurs from plant to plant mostly via vegetative propagation as well as by the insect vector Bemisia tabaci. When sweet potato seeds were planted and cultivated in a whitefly‐free greenhouse, some sweet potato plants started to show SPLCV‐specific symptoms. SPLCV was detected by PCR from all leaves and floral tissues that showed leaf curl disease symptoms. More than 70% of the seeds harvested from SPLCV‐infected sweet potato plants tested positive for SPLCV. SPLCV was also identified from dissected endosperm and embryos. The transmission level of SPLCV from seeds to seedlings was up to 15%. Southern blot hybridization showed SPLCV‐specific single‐ and double‐stranded DNAs in seedlings germinated from SPLCV‐infected seeds. Taken altogether, the results show that SPLCV in plants of the tested sweet potato cultivars can be transmitted via seeds and SPLCV DNA can replicate in developing seedlings. This is the first seed transmission report of SPLCV in sweet potato plants and also, to the authors' knowledge, the first report of seed transmission for any geminivirus.     

The Red Queen in a potato field: integrated pest management versus chemical dependency in Colorado potato beetle control

Originally designed to reconcile insecticide applications with biological control, the concept of integrated pest management (IPM) developed into the systems‐based judicious and coordinated use of multiple control techniques aimed at reducing pest damage to economically tolerable levels. Chemical control, with scheduled treatments, was the starting point for most management systems in the 1950s. Although chemical control is philosophically compatible with IPM practices as a whole, reduction in pesticide use has been historically one of the main goals of IPM practitioners. In the absence of IPM, excessive reliance on pesticides has led to repeated control failures due to the evolution of resistance by pest populations. This creates the need for constant replacement of failed chemicals with new compounds, known as the ‘insecticide treadmill’. In evolutionary biology, a similar phenomenon is known as the Red Queen principle – continuing change is needed for a population to persevere because its competitors undergo constant evolutionary adaptation. The Colorado potato beetle, Leptinotarsa decemlineata (Say), is an insect defoliator of potatoes that is notorious for its ability to develop insecticide resistance. In the present article, a review is given of four case studies from across the United States to demonstrate the importance of using IPM for sustainable management of a highly adaptable insect pest. Excessive reliance on often indiscriminate insecticide applications and inadequate use of alternative control methods, such as crop rotation, appear to expedite evolution of insecticide resistance in its populations. Resistance to IPM would involve synchronized adaptations to multiple unfavorable factors, requiring statistically unlikely genetic changes. Therefore, integrating different techniques is likely to reduce the need for constant replacement of failed chemicals with new ones. © 2014 Society of Chemical Industry     

Attract‐and‐kill: a new strategy for the management of the potato tuber moths Phthorimaea operculella (Zeller) and Symmetrischema tangolias (Gyen) in potato: laboratory experiments towards optimising pheromone and insecticide concentration

BACKGROUND: Sex pheromones of the potato tuber moths Phthorimaea operculella (Zeller) and Symmetrischema tangolias (Gyen) are ideal tools to monitor pest flight activity but are not used as means of control. The aim of the present study was to test the suitability of an attract‐and‐kill strategy consisting of pure pheromones and the contact insecticide cyfluthrin as the active ingredient, formulated with plant oils and ultraviolet absorbers, and applied in droplet sizes of 100 µL. RESULTS: Cyfluthrin at a concentration of 5 g L^?1 resulted in the highest and fastest killing of males after 48 h, with a 100% mortality after 3–4 days. In contrast, control males survived for 13 days. In olfactometer experiments, the pheromone concentration of 0.5 g L^?1 was significantly most attractive against eight virgin females. At controlled conditions (20 °C), no reduction in efficacy of the attract‐and‐kill formulation was observed for a minimum period of 36 days, whereas under natural environmental conditions the efficacy reduced gradually after day 6 of exposure. The longer the droplet was exposed, the longer was the time to reach 100% mortality of males. CONCLUSIONS: Compared with attract‐and‐kill studies for other pest species, the results are promising as a means of achieving highly effective control of potato tuber moths under field conditions. Copyright © 2010 Society of Chemical Industry     

利用CRISPR/Cas13a编辑技术获得马铃薯抗PVX植株

为探究利用CRISPR/Cas13a系统获得抗马铃薯X病毒（potato virus X,PVX）马铃薯的可行性,通过设计靶向PVX中TGBp1基因的小向导RNA（small guide RNA,sgRNA）,构建CRISPR/Cas13a基因编辑载体,以马铃薯栽培种Désirée为受体材料进行稳定遗传转化,并通过机械摩擦接种法鉴定转基因植株对PVX的抗性。结果显示,成功构建了靶向PVX的PVX-Cas13a载体,并获得了表达Cas13a的转基因马铃薯植株,对其中3个高表达Cas13a的株系进行PVX抗性鉴定,发现在接种PVX 10~20 d后,转基因植株系统叶上无明显发病症状,且PVX积累量均显著低于未接种PVX对照。表明靶向PVX的CRISPR/Cas13a系统能够有效抑制该病毒的积累,这为马铃薯抗PVX育种提供了一条有效策略。     

Marker‐free,tissue‐specific expression of Cry1Ab as a safe transgenic strategy for insect resistance in rice plants

BACKGROUND: Rice is the major food resource for nearly half of the global population; however, insect infestation could severely affect the production of this staple food. To improve rice insect resistance and reduce the levels of Bt toxin released into the environment, the Cry1Ab gene was conjugated to the rice rbcS promoter to express Bt toxin in specific tissues of transgenic plants. RESULTS: Eight marker‐free, T₂ lines were separated from the T₀ cotransformants. Using RT‐PCR, high levels of Cry1Ab expression were detected in the leaf but not in the seed. The Cry1Ab protein level ranged from 1.66 to 3.31 µg g^?1 in the leaves of four transgenic lines, but was barely detectable in their seeds by ELISA. Bioassays showed that the mortality rate of silkworm larvae feeding on mulberry leaves dipped in transgenic rice flour and pollen was less than that of the positive control (KMD), and that their average weight was higher than that of KMD, suggesting that the Cry1Ab protein was not expressed in the seed and pollen. CONCLUSION: The transgene conferred a high level of resistance to insects and biosafety to the rice plants, which could be directly used in rice breeding. Copyright © 2012 Society of Chemical Industry     

Ralstonia solanacearum causing potato bacterial wilt: host range and cultivars’ susceptibility in Rwanda

Members of the Ralstonia solanacearum species complex (RSSC), causing potato bacterial wilt or brown rot, are highly contagious and there are no known cultivars with durable resistance to the pathogen. This study hypothesized (a) that crops intercropped or rotated with potato, plants in the same family, and plants grown in the neighbouring fields can host the pathogen and they can be potential sources of primary inoculum, and (b) that potato cultivars currently multiplied by the public tissue culture laboratory in Rwanda are less susceptible to the pathogen. Fourteen plant species and potato, and nine potato cultivars were tested for susceptibility to an RSSC phylotype II strain under greenhouse conditions. The bacteria induced symptoms on potato, tomato, tree tomato, sweet pepper, and eggplant only. Among the plant species with symptoms, potato, tomato, and tree tomato wilted completely. There was a significant difference in days to symptom expression and to complete wilting (p < .0001). While all tested potato cultivars were found to be susceptible, the number of days to first symptom expression, days to complete wilting, area under the disease progress curve (AUDPC), and the number and weight of harvested tubers varied considerably. Cultivars Cruza, Kinigi, and CIP-58 were less susceptible whereas the cultivars Gikungu, Kirundo, and Victoria were highly susceptible. There is a strong need to search for other sources of resistance. The results indicate that some plant species that might serve as a reservoir of the bacterium should be avoided in the vicinity of potato crops.