马铃薯卷叶病毒PLRV RT-LAMP检测方法优化

马铃薯卷叶病毒Potato leafroll virus(PLRV)是目前严重影响马铃薯产量与品质的主要病毒之一,给马铃薯产业造成巨大损失。本研究采用环介导等温核酸扩增(loop-mediated isothermal amplification, LAMP)技术建立PLRV的RT-LAMP检测方法。采取单因素变化试验,对RT-LAMP反应体系中多个因素包括引物组合、温度条件及Mg~(2+)、betaine、Bst 3.0 DNA聚合酶、dNTPs、UNG、SYBR GreenⅠ和引物组合的浓度进行一系列试验和优化。采用RT-PCR检测方法进行平行比对试验,对优化后的RT-LAMP反应体系进行了验证。结果表明,最佳引物组合为P3,最适反应温度62℃,25μL反应体系中,Mg~(2+)、betaine、Bst 3.0 DNA聚合酶和UNG的最佳终浓度分别为4 mmol/L、0 mmol/L、0.64 U/μL和0.08 U/μL,dNTPs的最佳用量为1μL(dATP、dGTP、dCTP各0.4 mmol/L,dUTP 1.2 mmol/L),SYBR GreenⅠ(20×)的最佳用量1μL,primer mix的最佳用量2.5μL(PLRV-FIP/BIP、PLRV-F3/B3和PLRV-LF/LB的浓度分别为0.8、0.2μmol/L和0.6μmol/L),RNA模板1μL(2 ng/μL),加DEPC-H_2O至25μL,反应时间50 min。优化后的RT-LAMP检测结果与RT-PCR一致,且可视化判读结果。因此,建立的PLRV RT-LAMP检测方法为进一步开发RT-LAMP检测试剂盒及其实际应用奠定了基础。     

以18S rRNA为内参照的多重RT-PCR检测3种百合病毒

 本研究对多重PCR体系各成分和循环参数进行了摸索和优化,建立了以18SrRNA为内参照的同时检测3种百合病毒的多重RT-PCR体系,所检测的3种病毒是黄瓜花叶病毒(CMV)、百合斑驳病毒(LMoV)和百合无症病毒(LSV)。它们为侵染我国百合的主要病毒。在RT反应体系中加入3种病毒和18S rRNA的特异性反向引物的混合物,使反应体系中各反向引物终浓度均为0.5μmol/L,反转录酶(AMV)反转录合成各病毒和18SrRNA的互补第一链cDNAs。多重RT-PCR条件实验显示:将标准RT-PCR体系中的Taq HS DNA聚合酶量改为0.100U/μL,Mg²⁺浓度改为4.0mmol/L,各引物浓度选择0.2μmol/L,那么25个循环反应以上就能在一个反应管中以18SrRNA为内参照同时检测百合中的3种常见病毒。用该体系检测了10个百合样品,同时与³²P同位素标记膜杂交检测作对照,结果显示,该检测体系检测灵敏度更高。     

白背飞虱RAPD-PCR反应体系的正交设计优化

白背飞虱Sogatella furcifera(Horváth)是水稻重要害虫之一。研究采用L16(45)正交设计优化试验,构建了白背飞虱RAPD最优反应体系,并对引物退火温度和反应循环次数进行了优化。结果表明:白背飞虱RAPDPCR最优反应体系为2μL 10×PCR Buffer(10 mmol/L Tris-HCl;50 mmol/L KCl),2.4 mmol/L Mg2+,0.15 mmol/L dNTPs,0.7μmol/L引物,0.8U Taq DNA Polymerase和30 ng模板DNA,ddH2O补充至20μL。本研究采用的10条引物的最佳退火温度在35-39℃间,以引物相应最优退火温度和40次PCR反应循环可获得较好的白背飞虱RAPD-PCR结果。     

小菜蛾ISSR-PCR反应体系的建立与正交设计优化

以小菜蛾(Plutella xylostella)基因组DNA为模板,采用L16(45)正交试验设计方法,建立小菜蛾的ISSR最佳反应体系。通过梯度退火试验,确定不同引物的最适退火温度。优化得到的反应体系(20μL)为:Mg2+浓度1.5mmol/L、Taq DNA聚合酶2.5U、dNTPs浓度0.2mmol/L、引物浓度1.25μmol/L、模板DNA量20ng。反应程序为:94℃预变性5.0min;94℃变性45.0s,40～61℃(不同引物退火温度各异)退火1.0min,72℃延伸1.5min,40个循环;72℃延伸10.0min;10℃保存。利用所建立的ISSR-PCR反应体系,获得了清晰、重复性好的DNA谱带。     

基于环介导等温扩增技术检测由强雄腐霉引起的玉米茎腐病

为建立强雄腐霉Pythium arrhenomanes的快速分子检测技术,基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)以β-tubulin基因为靶标序列,设计强雄腐霉的特异性引物,建立了一种准确快速的LAMP检测方法,并对该检测方法的特异性、灵敏度和实际应用效果进行评估。25μL LAMP最终反应体系为:10×ThermoPol Buffer 2.5μL、10 mmol/L dNTPs 3.5μL、6 mmol/L MgSO_4 2μL、5 mol/L甜菜碱4μL、40μmol/L FIP-1/BIP-1各1μL、5μmol/L F3-1和B3-1各1μL、40μmol/L LB-1 1μL、Bst DNA polymerase 1μL、2.5 mmol/L HNB 1.9μL、模板DNA 2μL、灭菌水3.1μL。LAMP体系在等温64℃条件下反应60 min,HNB显色反应显示天蓝色即为阳性反应;扩增产物经2%琼脂糖凝胶电泳验证为梯形条带,也可判定为阳性反应。特异性检测结果显示,LAMP体系能特异性地检测出强雄腐霉,而其它卵菌近缘种和常见植物病原真菌均未检测出。灵敏度检测结果显示,LAMP体系的最低检测灵敏度为10 pg/μL,是普通PCR反应灵敏度的1 000倍。在实际应用中,LAMP体系能够快速检测出人工接种和实际发病玉米组织中的病原菌。表明本研究建立的快速、准确、可视化的LAMP检测方法能在60 min内检测出强雄腐霉。     

褐飞虱SRAP-PCR体系的优化与确立

采用L16（4 5）正交试验设计,对褐飞虱SRAP PCR反应体系中的Mg2+、dNTPs、TaqDNA聚合酶、引物浓度和DNA模板浓度5个因素进行优化,确立了褐飞虱SRAP PCR最佳的反应体系。结果表明：褐飞虱SRAP-PCR最佳反应体系为：总体积10 μL,Mg2+浓度3 mmol/L、dNTPs为150 μmol/L、TaqDNA聚合酶2 U、正反向引物各0.3 μmol/L、DNA用量25 ng以及1×PCR Buffer。使用生物型1雌虫与生物型2雄虫单对杂交F2群体对优化后的褐飞虱SRAP-PCR反应体系进行验证,获得了条带清晰、多态性丰富的图谱,并发现共显性条带,表明确定的反应体系稳定可靠。     

牛筋草ISSR-PCR反应体系的建立与优化

以牛筋草地上组织为材料,采用正交优化和单因子试验两种方法对影响牛筋草ISSR-PCR体系的Mg2+浓度、dNTP浓度、Taq DNA聚合酶浓度、引物浓度和DNA模板浓度5个因素进行优化试验,建立适合牛筋草ISSR-PCR的反应体系.结果表明,牛筋草ISSR-PCR反应体系的最佳条件为:Mg2+1.75 mmol/L,dNTP 0.4 mmol/L,Taq DNA聚合酶1 U/25 μL,引物0.3μmol/L,模板DNA 80 ng/25μL,10×PCR Buffer 2.5 μL/25 μL.利用优化体系进行牛筋草的ISSR-PCR反应,可获得稳定性高、重复性好、背景清晰的电泳结果.     

白背飞虱ISSR-PCR反应体系构建的正交设计优化

白背飞虱是水稻重要害虫之一。研究采用L16(45)正交设计优化试验,构建了白背飞虱ISSR最优反应体系,并对引物退火温度和反应循环次数进行了优化。结果表明：白背飞虱ISSR PCR最优反应体系为2 μL 10×PCR Buffer（10 mmol/L Tris HCl;50 mmol/L KCl）,1.5 mmol/L Mg2+,0.25 mmol/L dNTPs,0.9 μmol/L引物,1U TaqPolymerase和50 ng 模板DNA,ddH2O补充至20 μL。本研究采用的10条引物的最佳退火温度在51.0~54.0 ℃间,以引物相应最优退火温度和40次反应循环可获得较好的白背飞虱ISSR-PCR结果。     

三重PCR检测茄子青枯病菌、黄萎病菌和白绢病菌

为快速、准确对田间茄子发病植株和土壤中3种土传病害的病原菌青枯病菌Ralstonia solanacearum、黄萎病菌Verticillium dahliae和白绢病菌Sclerotium rolfsii进行检测,筛选这3种病菌的特异性引物,建立这3种病菌的三重PCR检测体系,对其退火温度、引物浓度、10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶进行优化,对优化后体系的特异性和灵敏度进行检测,并利用优化体系对田间采集的病害样品和土壤样品进行检测。结果表明,在优化后的50μL三重PCR检测体系中,RS-1-F/RS-3-R、dllz1/dllz2和SRITSF/SRITSR引物的最佳浓度分别为0.16、0.16和0.28μmol/L,10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶体积分别为6、5和1μL,检测体系的最佳退火温度为54℃。按照优化后的反应条件能分别扩增出青枯病菌、黄萎病菌和白绢病菌3种病菌的特异条带,分别为716、350和500 bp,其他对照病菌无条带。该体系对病菌DNA检测灵敏度达到0.1 ng/μL。该...     

抗草甘膦转基因大豆Cp4-epsps基因快速简便的LAMP检测方法

针对抗草甘膦转基因大豆的外源基因Cp4-epsps,建立了一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术的抗草甘膦转基因大豆的检测体系,其扩增产物既可利用常规琼脂糖凝胶电泳检测,还可通过SYBR Green I染色进行快速检测。LAMP检测体系中dNTPs浓度为0.8 mmol/L、Mg2+浓度为3mmol/L、反应时间为45min时扩增效果最佳,其检测灵敏度为5μg/L,比常规PCR灵敏100倍。田间实际检测结果表明,LAMP检测结果和PCR检测结果完全一致,准确率为100%。本研究所建立的抗草甘膦转基因大豆LAMP检测方法具有简便快速、特异性强、灵敏度高等特征,是一种能够用于抗草甘膦转基因大豆检测、田间基因漂移监测和环境安全研究的有力工具。     

Dermestid beetles in ‘evolution Canyon’, lower Nahal Oren, Mt. Carmel, including new records for Israel

Fifteen species of dermestid beetles were recorded at ‘Evolution Canyon’ (EC), Lower Nahal Oren, Mt. Carmel, Israel. They represent ~35% of known Israeli dermestid species. The following three species were recorded for the first time in Israel:Trogoderma svriaca Dalla Torre, 1911;Ctesias svriaca Ganglbauer, 1904; andAnthrenus (s.str.) jordaniens Pic, 1934. Adults of 13 species were collected on the more solar radiated, warmer and climatically more fluctuating south-facing slope (SFS); ten species were collected on the opposite, north-facing slope (NFS), which was cooler and climatically more stable. The abundance of adult dermestid beetles was 1.9 times higher on the SFS than on the NFS (86 and 47, respectively). Species richness and abundance distribution at EC (three collecting stations on each slope and one at the valley bottom) were significantly negatively correlated with the plant cover that consisted of trees and bushes (Spearmanr _s ,P=0.007 and 0.039, respectively) and perennials (Spearmanr _s ,P=0.039 and 0.077, respectively), indicating that non-woody plants were preferred by adult dermestid beetles.     

Diagnostics,genetic diversity and pathogenic variation of ascochyta blight of cool season food and feed legumes

Molecular diagnostic techniques have been developed to differentiate the Ascochyta pathogens that infect cool season food and feed legumes, as well as to improve the sensitivity of detecting latent infection in plant tissues. A seed sampling technique was developed to detect a 1% level of infection by Ascochyta rabiei in commercial chickpea seed. The Ascochyta pathogens were shown to be genetically diverse in countries where the pathogen and host have coexisted for a long time. However, where the pathogen was recently introduced, such as A. rabiei to Australia, the level of diversity remained relatively low, even as the pathogen spread to all chickpea-growing areas. Pathogenic variability of A. rabiei and Ascochyta pinodes pathogens in chickpea and field pea respectively, appears to be quantitative, where measures of disease severity were based on aggressiveness (quantitative level of infection) rather than on true qualitative virulence. In contrast, qualitative differences in pathogenicity in lentil and faba bean genotypes indicated the existence of pathotypes of Ascochyta lentis and Ascochyta fabae. Therefore, reports of pathotype discrimination based on quantitative differences in pathogenicity in a set of specific genotypes is questionable for several of the ascochyta-legume pathosystems such as A. rabiei and A. pinodes. This is not surprising since host resistance to these pathogens has been reported to be mainly quantitative, making it difficult for the pathogen to overcome specific resistance genes and form pathotypes. For robust pathogenicity assessment, there needs to be consistency in selection of differential host genotypes, screening conditions and disease evaluation techniques for each of the Ascochyta sp. in legume-growing countries throughout the world. Nevertheless, knowledge of pathotype diversity and aggressiveness within populations is important in the selection of resistant genotypes.     

Epidemiology of Toxigenic Fungi and their Associated Mycotoxins for Some Mediterranean Crops

Recent data on the epidemiology of the common mycotoxigenic species of Fusarium, Alternaria, Aspergillus and Penicillium in infected or colonized plants, and in stored or processed plant products from the Mediterranean area are reviewed. Emphasis is placed on the toxigenicity of the causal fungal species and the natural occurrence of well known mycotoxins (aflatoxins, ochratoxins, fumonisins, trichothecenes, zearalenone, patulin, Alternaria-toxins and moniliformin), as well as some more recently described compounds (fusaproliferin, beauvericin) whose toxigenic potential is not yet well understood. Several Fusarium species reported from throughout the Mediterranean area are responsible of the formation of mycotoxins in infected plants and in plant products, including: Fusarium graminearum, F. culmorum, F. cerealis, F. avenaceum, F. sporotrichioides and F. poae, which produce deoxynivalenol, nivalenol, fusarenone, zearalenone, moniliformin, and T-2 toxin derivatives in wheat and other small grains affected by head blight or scab, and in maize affected by red ear rot. Moreover, strains of F. verticillioides, F. proliferatum, and F. subglutinans, that form fumonisins, beauvericin, fusaproliferin, and moniliformin, are commonly associated with maize affected by ear rot. Fumonisins, were also associated with Fusarium crown and root rot of asparagus and Fusarium endosepsis of figs, caused primarily by F. proliferatum. Toxigenic A. alternata strains and associated tenuazonic acid and alternariols were commonly found in black mould of tomato, black rot of olive and citrus, black point of small cereals, and black mould of several vegetables. Toxigenic strains of A. carbonarius and ochratoxin A were often found associated with black rot of grapes, whereas toxigenic strains of A. flavus and/or P. verrucosum, forming aflatoxins and ochratoxin A, respectively, were found in moulded plant products from small cereals, peanuts, figs, pea, oilseed rape, sunflower seeds, sesame seeds, pistachios, and almonds. Finally, toxigenic strains of P. expansum and patulin were frequently found in apple, pear and other fresh fruits affected by blue mould rot, as well as in derived juices and jams.     

Hymenopteran Parasitoids on Fruit-infesting Tephritidae (Diptera) in Latin America and the Southern United States: Diversity, Distribution, Taxonomic Status and their use in Fruit Fly Biological Control

We first discuss the diversity of fruit fly (Diptera: Tephritidae) parasitoids (Hymenoptera) of the Neotropics. Even though the emphasis is on Anastrepha parasitoids, we also review all the information available on parasitoids attacking flies in the genera Ceratitis, Rhagoletis, Rhagoletotrypeta, Toxotrypana and Zonosemata. We center our analysis in parasitoid guilds, parasitoid assemblage size and fly host profiles. We also discuss distribution patterns and the taxonomic status of all known Anastrepha parasitoids. We follow by providing a historical overview of biological control of pestiferous tephritids in Latin American and Florida (U.S.A.) and by analyzing the success or failure of classical and augmentative biological control programs implemented to date in these regions. We also discuss the lack of success of introductions of exotic fruit fly parasitoids in various Latin American countries. We finish by discussing the most pressing needs related to fruit fly biological control (classical, augmentative, and conservation modalities) in areas of the Neotropics where fruit fly populations severely restrict the development of commercial fruit growing. We also address the need for much more intensive research on the bioecology of native fruit fly parasitoids.     

A survey of the genera of<Emphasis Type="Italic">Microgaster</Emphasis> latreille and<Emphasis Type="Italic">Hygroplitis</Emphasis> thomson (Hymenoptera: Braconidae: Microgastrinae) from China

The genera ofMicrogaster Latreille 1804 andHygroplitis Thomson 1895 from China are presented systematically in this paper. Thirty-two species ofMicrogaster and three species ofHygroplitis are known in China. Diagnosis, character variation, distribution and host of each species among the two genera are presented, including its host and distribution. Keys to the species ofMicrogaster andHygroplitis are given. http://www.phytoparasitica.org posting Dec. 19, 2006.     

Plant Viruses Transmitted by Whiteflies

One-hundred and fourteen virus species are transmitted by whiteflies (family Aleyrodidae). Bemisia tabaci transmits 111 of these species while Trialeurodes vaporariorum and T. abutilonia transmit three species each. B. tabaci and T. vaporariorum are present in the European–Mediterranean region, though the former is restricted in its distribution. Of the whitefly-transmitted virus species, 90% belong to the Begomovirus genus, 6% to the Crinivirus genus and the remaining 4% are in the Closterovirus, Ipomovirus or Carlavirus genera. Other named, whitefly-transmitted viruses that have not yet been ranked as species are also documented. The names, abbreviations and synonyms of the whitefly-transmitted viruses are presented in tabulated form together with details of their whitefly vectors, natural hosts and distribution. Entries are also annotated with references. Whitefly-transmitted viruses affecting plants in the European–Mediterranean region have been highlighted in the text.     

The use of Solacol® (validamycin) as a growth retardant in the isolation of soil fungi

Solacol^®, a formulation of the antibiotic validamycin, at 0.33% in 2% malt extract agar, reduced the spread of fungi on dilution plates drastically and allowed twice as much incubation time before subculturing; this resulted in an elevated number of species isolated. Using pure cultures of 62 common soil fungi, it was shown that all fast-growing species (exceptPythium ultimum) were efficiently inhibited but not completely suppressed. Inhibition was comparable to that by 0.5% oxgall, though, while this substance completely suppressed several species, Solacol very strongly inhibited onlyGaeumannomyces graminis, Gerlachia nivalis, Harzia acremonioides, Verticillium biguttatum andRhizoctonia solani. In a further experiment each separate constituent of Solacol was tested against 22 fungi at equivalent concentrations. Validamycin strongly inhibitedChaetomium globosum and two Basidiomycetes, though hardly more than the non-ionic detergent which mainly inhibited the other fungi. A few species were, however, more inhibited by Solacol than by the detergent alone. Solacol at 0.33% is a suitable aid in dilution plating of soil fungi, by increasing the number of colonies and species observed.Samenvatting Solacol^®, een formulering van het antibioticum validamycine, remde de groei van schimmels in verdunningsplaten met een concentratie van 0.33% in 2% moutagar en maakte het mogelijk de periode tot afenten met een factor 2 te verlengen; daardoor was het aantal geïsoleerde soorten duidelijk toegenomen. Met reincultures van 62 algemene grondschimmelsoorten werd aangetoond, dat alle snelgroeiende soorten (met uitzondering vanPythium ultimum) voldoende geremd, maar niet volkomen onderdrukt werden. Het remmingspercentage was vergelijkbaar met dat van 0.5% ossegal, hoewel dit laatste sommige soorten volkomen onderdrukte; Solacol remde alleenGaeumannomyces graminis, Gerlachia nivalis, Harzia acremonioides, Verticillium biguttatum enRhizoctonia solani zeer sterk. In een volgend experiment werden de componenten van Solacol t.o.v. 22 fungi apart getoetst in concentraties equivalent aan 0.33% Solacol. Validamycine remde alleenChaetomium globosum en twee basidiomyceten behoorlijk, maar nauwelijks meer dan de niet-ionische uitvloeier, die in hoofdzaak de overige remeffecten veroorzaakte. Enkele soorten werden echter door het complete Solacol veel sterker geremd dan door de uitvloeier alleen. Solacol in een verdunning van 0,33% wordt aanbevolen bij verdunningsplaten voor het isoleren van grondschimmels ten einde het aantal kolonies en soorten te verhogen.     

Brief Guide for Authors

正Journal of Arid Land(JAL)is an international journal(ISSN 1674-6767;CN 65-1278/K)for the natural sciences,sponsored by the Xinjiang Institute of Ecology and Geography,Chinese Academy of Sciences and Science Press.It is published by Science Press and Springer-Verlag Berlin Heidelberg bimonthly.JAL publishes original,innovative,and integrative research from arid and semiarid regions,ad     

Broad bean mottle virus in Morocco; curculionid vectors,and natural occurrence in food legumes other than faba bean (Vicia faba)

Broad bean mottle virus (BBMV) was transmitted from infected to healthy faba-bean plants by the curculionid weevilsApion radiolus Kirby,Hypera variabilis Herbst,Pachytychius strumarius Gyll,Smicronyx cyaneus Gyll, andSitona lineatus L. The latter appeared to be an efficient vector: acquisition and inoculation occurred at the first bite, the rate of transmission was c. 41%, and virus retention lasted for at least seven days.S. lineatus transmitted the virus from faba bean to lentil and pea, but not to the three genotypes of chickpea tested. This is the first report on the generaHypera, Pachytychius, andSmicronyx as virus vectors, and onA. radiolus, H. variabilis, P. strumarius, andS. cyaneus as vectors of BBMV.Out of 351 samples of food legumes with symptoms suggestive of virus infection, 16, 11, 19, and 17% of the samples of chickpea, lentil, pea, and common bean, respectively, were found infected when tested for BBMV in DAS-ELISA. This is the first report on the natural occurrence of BBMV in chickpea, lentil, pea, and common bean. The virus should be regarded as a food-legume virus rather than a faba-bean virus solely, and is considered an actual threat to food legume improvement programmes.     

Characterization of potato potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates

A collection of 38 PVY isolates from seed potato batches, originating from several Western European countries, was characterized by using current biological, serological and molecular tools differentiating PVY strains and groups. The correlation between the three kinds of tests was good but not absolute. No single serological or PCR method was able to discriminate among the five isolate groups found. Twenty-nine isolates belonged to the PVY^N strain and six to the PVY^O strain. No PVY^C was found. Two other isolates reacted serologically like PVY^O, but were unable to elicit a hypersensitive response from the Ny_tbr gene and probably represent the PVY^Z group. At the molecular level, these two isolates showed a combination of both PVY^O and PVY^N and could be recombinants of these strains. Another isolate reacted serologically like PVY^O, but induced vein necrosis in tobacco, like PVY^N-Wilga. Some PVY^N isolates caused tuber ring necrosis in glasshouse conditions. These might belong to the PVY^NTN group. The PVY^NTN, PVY^N-Wilga and PVY^Z groups probably represent pathotypes within strains PVY^N and PVY^O, respectively. The present study also confirms previous reports showing a high genetic variation at the 5 end within the PVYN strain.