烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析

为探讨UV-B胁迫对烟蚜Myzus persicae热激蛋白Hsp90基因表达量的影响,采用RT-PCR与RACE技术克隆了烟蚜热激蛋白Hsp90基因的全长,并对其进行生物信息学分析,利用实时荧光定量PCR技术研究了烟蚜Hsp90基因在不同时长UV-B胁迫下的表达量变化。结果表明,烟蚜Hsp90基因的cDNA全长为2 670 bp,编码728个氨基酸,编码蛋白质的相对分子量为82.6 kD,等电点为4.95,获得的氨基酸序列具有Hsp90蛋白家族的1个签名序列及C末端MEEVD基序,推测其属于胞质型热激蛋白。系统进化树结果显示,烟蚜Hsp90与其它昆虫Hsp90具有很高的相似性。实时荧光定量PCR结果表明,不同时长UV-B胁迫下烟蚜Hsp90均有表达,随着照射时间延长,Hsp90表达量表现为先上升后下降的趋势;与对照相比,照射时间为15、30、60、90和120 min时,Hsp90表达量均显著升高,且在60 min时Hsp90表达量达最大,是对照组的2.05倍。表明Hsp90基因在不同时长UV-B胁迫下差异表达,在烟蚜适应紫外胁迫的分子机制中具有重要作用。     

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.     

Potential impacts of climate change on the threat of potato cyst nematode species in Great Britain

Potato cyst nematode (PCN) species have different temperature optima for various life cycle stages, therefore a risk assessment of the threat of PCN species under future climates is essential to guide adaptation strategies. Data defining the spatial coverage of potato crops in Great Britain were combined with probabilistic climate change data and a newly developed PCN life cycle model to project the future risk to potato crops from PCN. The model was based on the results of controlled environment experiments to investigate the effect of temperature on survival to female maturity using three PCN populations: Globodera pallida (Lindley) and G. rostochiensis from the James Hutton Institute PCN collection, and a field population of G. pallida (S‐Fife). It was found that projected increases in soil temperature could result in increased survival to female maturity for all three PCN populations, with greater increases expected for Scotland, followed by Wales then England. The largest projected increases in Scotland were for G. pallida, whereas G. rostochiensis showed the largest increases in Wales and England. The potential impact of several agronomic adaptation strategies on projected PCN risk were also investigated. The results from the model suggest that soil infestation levels would have to be reduced by up to 40% in order to negate projected increases in PCN risk, and that advancing the start date of the growing season or modifying planting patterns could be successful strategies to reduce future PCN risk.     

番茄枯萎病菌和颈腐根腐病菌KASP-SNP检测技术的建立

为快速、准确地对番茄枯萎病菌Fusarium oxysporum f. sp. lycopersici(FOL)和番茄颈腐根腐病菌F. oxysporum f. sp. radicis-lycopersici(FORL)进行检测,基于尖孢镰刀菌F. oxysporum多聚半乳糖醛酸外切酶基因pgx4的单核苷酸多态性（single nucleotide polymorphism,SNP）位点,设计FORL、FOL生理小种1(FOL-R1)、2(FOL-R2)和3(FOL-R3)的竞争性等位基因特异性PCR-SNP(kompetitive allele specific PCR-SNP,KASP-SNP)引物,建立番茄颈腐根腐病菌和番茄枯萎病菌KASP-SNP检测技术,并通过与常规PCR比对及ITS与pgx4序列分析对该检测技术的可靠性进行验证。结果显示,在FORL、FOL-R1、FOL-R2和FOL-R3中存在35个变异SNP位点,设计出18对KASP-SNP引物,筛选出FORL＿KASP、FOLrace1＿KASP、FOLrace2＿KASP和FOLrace3＿KASP共4对分型清晰的...     

Interactions Between the Potato Cyst Nematode Globodera rostochiensis and Diseases Caused by Rhizoctonia solani AG3 in Potatoes Under Field Conditions

Findings from 2 years of field experiments investigating the relationship between Globodera rostochiensis and Rhizoctonia solani on unique field sites are reported. In 2000, a field experiment was positioned on land that had previously been used for experimental work investigating integrated potato cyst nematode (PCN) management methods. This study had produced an ‘untypical’ mosaic of PCN population densities ranging from 5 to 221 eggs g⁻¹ soil. In 2001, the field experiment was conducted on a different field site and overlaid on a focus of G. rostochiensis population densities ranging from 11 to 108 eggs g⁻¹ soil. In each experiment, potatoes (cv. Désirée) were grown in plots with similar population densities of G. rostochiensis that were either uninoculated or inoculated with R. solani. A series of potato plant harvests were undertaken to investigate the effects of nematode infestation on the incidence and severity of R. solani diseases and the associated development of plants. In both experiments, a clear relationship was found between the density of G. rostochiensis juveniles present in potato roots and the incidence of stolons infected by R. solani, 6 weeks after planting. For the first time this interaction has been determined under field conditions. The results of the study suggest that the interaction between nematode and fungus is indirect and possible mechanisms are discussed.     

Managing potato cyst nematodes to minimize nematicide use1

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are widespread in the ware potato-growing areas of the UK. Traditionally they were controlled by rotation but more intensive production methods have increased the PCN threat, especially from G. pallida. G. rosrochiensis has become less important since cultivars fully resistant to it were introduced but, where both species are present in a mixture, the G. pallida portion comes to dominate. It is possible to control G. pallida as effectively as G. rostochiensis if cultivars partially resistant to G. pallida are grown with nematicide treatment. Where nematicide is not used, control of G. pallida is more variable. In some years, some cultivars achieve good control but the results are not consistent. In general, G. pallida has increased on untreated plots about 5-fold in trials in the last 3 years.     

Morphological and molecular identification of potato and cereal cyst nematode isolates from Algeria and their phylogenetic relationships with other populations from distant theirgeographical areas

A nematode survey conducted in 2013 in Algeria, revealed that potato cyst nematodes (PCN) and cereal cyst nematodes (CCN) are widely distributed in several potato and cereal growing regions of the country. Sixteen PCN populations from five localities and five CCN populations from four of these localities were collected and characterized at the morphological and molecular levels. The PCN populations were identified as Globodera rostochiensis and G. pallida occurring separately or in mixed populations. Two species of CCN were detected. Heterodera avenae was found in four localities, whereas H. hordecalis only in one locality in association with H. avenae. The morphological and morphometric identification of PCN and CCN was confirmed by diagnostic ITS-RFLP profiles and sequencing. Phylogenetic analysis of the ITS, D2-D3 expansion domains of the 28S rRNA gene and 18S rRNA gene was made for PCN and CCN populations. Globodera pallida and G. rostochiensis from Algeria show great similarity with European and South American populations. Because of the high divergence among Algerian populations of G. pallida and G. rostochiensis it can be assumed that they were multi-introduced in Algeria. The most divergent population of G. pallida, that formed a well-separated group with some populations from Chile and Peru, suggests a later or independent introduction of this population into Algeria. Heterodera avenae and H. hordecalis formed a well-supported cluster with the corresponding populations.     

猪殃殃对AHAS抑制剂靶标抗性的快速分子检测

为建立猪殃殃靶标抗性快速检测方法,并明确小麦田猪殃殃Galium aparine var.tenerum对AHAS抑制剂靶标的突变类型及分布,从河南、陕西、安徽、江苏和山东5省不同田块采集疑似对AHAS抑制剂产生抗性的猪殃殃植株,采用特异性引物PCR扩增靶标酶AHAS基因保守区片段,并以直接测序法检测采集样品,通过与拟南芥AHAS基因序列比对分析后明确其突变位点。结果显示,在5省25个农田的样品中共有19个农田检测到AHAS突变,分布在河南、安徽和江苏3省;在检测样品中发现突变发生在2个位点,共有3种突变类型,分别是197位脯氨酸(CCC)突变为丙氨酸(GCC)或丝氨酸(TCC),或者是574位色氨酸(TGG)突变为亮氨酸(TTG),检测结果与田间药效反应基本一致。这种用特异性引物扩增目的片段测序的方法,由于其可以在生长当季进行检测,适用于田间靶标突变抗性猪殃殃的快速检测与监测。     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.     

Sensitive detection of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andreanum by immunocapture-PCR (IC-PCR) using primers designed from sequence characterized amplified regions (SCAR) of the blight pathogen

One of the most devastating Xanthomonas diseases affecting the Anthurium cut flower industry worldwide is the bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). The disease can be spread through latently infected tissue-cultured plants that are used for the propagation of Anthurium worldwide. Current disease diagnostic techniques involve the use of semi-selective media and serological tests. This study describes the development of a PCR tool combined with a genus-specific monoclonal antibody for the sensitive detection of the pathogen directly from plants. It was demonstrated that the immunocapture PCR (IC-PCR) was more sensitive than the conventional PCR and even more sensitive than indirect ELISA for the detection of the pathogen. Latently infected plants could be positively screened for the presence of the pathogen. Three sets of primers were designed from DNA probes that were reported to show some specificity to the pathovar dieffenbachiae. The use of all three sets of primers in a single reaction successfully amplified the three individual loci when bacterial DNA was used as a template. The multiplex PCR generated PCR profiles that could differentiate between the reference strains of X. axonopodis pv. dieffenbachiae from other control bacteria. The new primers could therefore be used both for the diagnosis of Anthurium blight in single PCR reactions and also for the profiling of Xanthomonas. pv. dieffenbachiae strains using the multiplex PCR technique.     

进境玉米中玉米内州萎蔫病菌的PCR检测

 为了快速准确检测进境玉米样品中的玉米内州萎蔫病菌Clavibacter michiganensis subsp. nebraskensis(Cmn), 根据GenBank中Cmn的16S-23S序列设计引物CM1/CM4和引物PSM1/CM3。引物PSM1/CM3仅能从供试的4株Cmn菌株中扩增获得208 bp的预期产物, 而其他36株对照菌株均不能扩增出预期条带。灵敏度测试结果表明引物CM1/CM4和PSM1/CM3组合的巢式PCR方法的检测灵敏度高于常规PCR, 检测灵敏度可达40 fg DNA或6.8 CFU目标细菌。常规PCR和巢式PCR方法对进境美国玉米样品的阳性检出率分别为8%和24%, 试验结果表明所建立的PCR方法可用于玉米样品中Cmn的快速检测。     

Consistent action of two partially effective loci conferring resistance to Globodera pallida Pa2/3 across multiple nematode field populations

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are significant pests of potatoes worldwide. The most effective control methods are crop rotation and the deployment of resistant varieties. Complete resistance to G. rostochiensis based on a single resistance gene has successfully been integrated into many varieties. However, resistance to G. pallida has not been as successful to date, with current varieties only exhibiting partial resistance. Combining partially effective quantitative trait loci (QTLs) for resistance can increase the strength and breadth of the resistance. An additive effect on resistance has previously been demonstrated on combining two QTLs from Solanum tuberosum subsp. andigena (GpaIV^s_adg) and Solanum vernei (Gpa5). However, populations of G. pallida can be quite divergent and it was unclear whether the relative effects of the individual QTLs and the combined additive effect would be consistent across different G. pallida Pa2/3 populations. Using a mapping population segregating for both QTLs, the effect of the QTLs individually and combined was examined on four UK‐derived field populations of G. pallida pathotype Pa2/3, and the relative effects of the individual QTLs and the additive effect of the combination found to be consistent across all populations.     

Evaluation of PCR,IEF and ELISA techniques for the detection and identification of potato cyst nematodes from field soil samples in England and Wales

Effective management of potato cyst nematodes (PCNs) requires simple, rapid and accurate identification and quantification of field populations. Soil samples from a survey of 484 fields in potato rotations in England and Wales were used to compare the identification and quantification of PCNs using IEF, PCR, ELISA and bait plant tests. The cyst counts and bait plant test revealed that 64.3% of field samples contained PCNs. Bait plant tests increased the detection rate of PCNs in field samples by 4–6.4%. This means that some infestations are cryptic and would not normally be detected by standard counts. IEF, PCR and ELISA methods distinguished between Globodera rostochiensis and G pallida and were able to register mixed populations; however they were not in full agreement. All methods suggested that G pallida is the dominant species in the field samples tested. The PCR results indicated that 66% of field samples contained pure G pallida, 8% contained pure G rostochiensis and 26% contained mixtures of the two species. Estimates of the relative process times taken per sample in the PCR, IEF and ELISA techniques are given. © 2001 Society of Chemical Industry     

Application of RFLP analysis of recA, gyrA and rpoS gene fragments for rapid differentiation of Erwinia amylovora from Erwinia strains isolated in Korea and Japan

Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified fragments of recA, gyrA and rpoS genes was applied for the characterization of Erwinia amylovora and Erwinia strains, which cause fire blight and Asian pear blight in orchards. Primers, constructed on the basis of the published recA, gyrA and rpoS gene sequences of Erwinia carotovora, allowed us to amplify DNA fragments for RFLP differentiation of E. amylovora and E. pyrifoliae and finally to distinguish strains within these species and relate them to pear pathogens from Japan. Three to seven restriction endonucleases were applied for RFLP analysis of each gene fragment. The electrophoretic patterns generated after PCR–RFLP for each of the tested genes, were characteristic and specific for each species and allowed their differentiation. The data show that PCR–RFLP analysis of the recA, gyrA and rpoS gene fragments may be considered as a useful tool for the identification and differentiation of E. amylovora and E. pyrifoliae. Almost identical restriction patterns of the analyzed gene fragments indicated a high relationship of E. pyrifoliae strains from Korea and pear pathogens from Japan and a divergence to E. amylovora. For quick and effective differentiation of E. amylovora strains from Erwinia strains from Asia without nucleotide sequencing we recommend the amplification of recA and rpoS gene fragments and digestion of each of them with restriction endonuclease Hin6I.     

Differential hatching of the potato cyst nematodes Globodera rostochiensis and G. pallida in root diffusates and water of differing ionic composition

The hatching differences ofGlobodera rostochiensis andG. pallida were assessed in potato root diffusate (PRD) of cv. Bintje, cv. Elkana and clone ZB35-29.G. pallida hatched better in the PRDs thanG. rostochiensis. It was shown that the experimental test conditions strongly influenced the hatching results. The water type used in the hatching tests had a significant discriminating effect on the species;G. rostochiensis reached hatch percentages of 60 to 90% in demineralized and tap water, whereasG. pallida never exceeded the 15%. These differences were independent of the various batches that were used or the different years the tests were carried out. Silver sand percolate had a inhibiting effect on the hatching of both nematode species. Boron and a high electrical conductivity may be responsible for this. The results are discussed from an ecological point of view as well as for research consequences.     

猕猴桃溃疡病菌不致病菌株G230的鉴定及形成原因分析

为探索田间猕猴桃溃疡病菌Pseudomonas syringae pv. actinidiae(Psa)致病力丧失的分子机制,针对从猕猴桃果园中分离获得的1株不致病菌株G230,通过特异性引物检测和多基因序列分析明确其分类地位,并设计引物检测其是否由已知遗传变异引起,通过比较基因组学、基因表达、超敏反应和荧光素酶报告菌株检测确定引起菌株G230致病力丧失的原因。结果表明,不致病菌株G230为Psa生物型3(Psa3),其致病缺陷不是由已报道的遗传变异引起;基于基因组比较分析发现菌株G230中的hrpS基因被转座子ISPsy36插入破坏,导致Ⅲ型分泌系统（type Ⅲ secretion system,T3SS）不能正常表达;而在不致病菌株G230中表达hrpS基因后能恢复其T3SS功能,使其具备致病能力及激发非寄主超敏反应的能力。表明转座子ISPsy36插入hrpS基因内部可以破坏Psa的T3SS功能进而使其丧失致病力,这是自然条件下Psa3丧失致病力的一种新型机制。     

Quantification of Globodera rostochiensis and G. pallida in mixed populations using species-specific thermostable proteins

A method has been developed to quantify species ratios in mixed populations. The method is based on the separation of species-specific thermostable proteins by SDS-PAGE. Densitometric analyses of the 17 kD protein ofGlobodera pallida and the 18 kD protein ofG. rostochiensis revealed a high correlation (R ²=0.93) with the species ratio in the mixed samples. Within the limits of 10 to 90% of each species, one can estimate with 95% reliability the species composition with 3 to 6% deviation.Samenvatting Een methode is ontwikkeld om de samenstelling van soortenmengsels vanGlobodera rostochiensis enG. pallida te kwantificeren. Bij deze methode wordt gebruik gemaakt van de soort-specifieke thermostabiele eiwitten die met behulp van SDS-PAGE gescheiden worden. De kleurintensiteit van het 17 kD eiwit vanG. pallida en het 18 kD eiwit vanG. rostochiensis is per gel-laan bepaald m.b.v. een densitometer en heeft een lineair verband met de soortsverhouding in de mengsels (R ²=0.93). Binnen het bereik van 10 tot 90% van elke soort kan men met deze ijklijn met 95% betrouwbaarheid de soortsamenstelling bepalen op 3 tot 6% nauwkeurig.