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.     

应用TaqMan探针进行马铃薯金线虫实时荧光PCR检测技术研究

马铃薯孢囊线虫是全球性的植物检疫性线虫,亦是我国重点关注的有害生物。针对马铃薯金线虫ITS序列,设计了引物和TaqMan 探针,使用15个马铃薯孢囊线虫群体和4个其他孢囊线虫样品进行验证,可高度灵敏地检测单个马铃薯金线虫的孢囊或幼虫,并可进行定量;最高检测灵敏度达到10fg;同时开展了混合样品和未知样品的检测,证明了引物的专化性和TaqMan探针的特异性。该检测方法可自动化检测马铃薯金线虫并进行定量,适合进行标准化的常规检测。     

实时荧光PCR检测马铃薯白线虫技术研究

马铃薯胞囊线虫是马铃薯上最重要的有害生物之一,也是我国特别关注的重要植物检疫性线虫.针对马铃薯白线虫ITS序列,我们设计了引物和TaqMan探针,使用15种马铃薯胞囊线虫群体和4种其它胞囊线虫样品进行验证,可高度灵敏地检测单个马铃薯白线虫的胞囊或幼虫,最高检测灵敏度达到10fs;同时开展了混合样品和未知样品的检测,证明了引物的专化性和TaqMan探针特异性.该检测方法可自动化检测马铃薯白线虫并进行定量,适合进行标准化的常规检测.     

The Basis of Predictive Modelling for Estimating Yield Loss and Planning Potato Cyst Nematode Management

The basis of modelling yield loss and population dynamics relations of potato cyst nematodes is that both are strongly density-dependent. Potato cyst nematodes (PCN; Globodera pallida and G. rostochiensis) are particularly suitable for analysing such relationships because they have only one generation per year, potato is their only field host, the juvenile nematodes within the egg are very durable (up to 20 years persistence), and they hatch mainly in response to specific chemicals exuded from host roots. Small populations increase the most, up to 50-fold, when a potato crop is grown. Multiplication rates decrease as the population density increases because damage decreases root system size and increases competition so that very large populations may actually be decreased when potatoes are grown. The newly formed eggs have a ‘half-life’ of c.two years when non-host, rotational crops are being grown. Control is achieved largely by the use of rotation, the application of nematicidal chemicals, and growing resistant cultivars. As rotations are shortened, so PCN populations will be increased, and crop damage becomes more likely. Thresholds for damage vary with both soil type (greatest on sandy soils) and potato cultivar. Cultivars differ in their tolerance of PCN damage depending on how vigorously they grow, on their root sensitivity to damage from the PCN juveniles which invade close to root tips, on cultivar resistance which decreases the parasitic effect by reducing the numbers of developing PCN females, and on various environmental factors such as the amounts of fertiliser applied. The dependence of the yield-loss relationship on population density, soil type and cultivar effects has been described in a simple equation and assessed using field-trial data. Effects on yield are described in proportion to the PCN-free yield but the addition of information on expected yield (in tonnes ha⁻¹) in the absence of PCN renders this equation predictive. Nematicides are widely applied to infestations of potato cyst nematodes, both to prevent the crop from being damaged and to prevent population increase which could hazard the next potato crop in the rotation, but they are generally more effective at preventing the former than the latter. A complex equation has also been developed to model the population dynamics of PCN. This equation incorporates a factor for host-crop growth and tolerance (from the yield-loss equation) and also the effects of host resistance. This latter is particularly relevant to G. pallida, where all the resistance currently available is determined by minor genes and hence is ‘quantitative’ or ‘partial’. Effects of rotation and of nematicides can also be incorporated into this model. To provide a realistic prediction also requires accurate information on PCN population densities, species composition and distributions, and rates of PCN population decline between potato crops.     

More accurate measurement of temperature in potato drills increases predicted potato cyst nematode population growth

Potato cyst nematodes (PCN) are economically important pests of potato plants worldwide that can result in yield losses exceeding 80%, with an estimated cost of £50 million annually in the United Kingdom alone. The life cycle of PCN, and in particular the hatching of eggs and development and survival of juveniles, is sensitive to temperatures in the soil. Despite this fact, relatively little is known about temperatures inside potato drills and how these may differ to temperatures in the surrounding soil. We monitored the temperature inside potato drills at 19 sites across the United Kingdom and used a published temperature-driven process-based PCN life cycle model to compare predictions of PCN population dynamics driven by these temperatures to predictions based on modelled soil temperatures derived from the HadUK-Grid climate. We found that mean soil temperatures derived from the climate model were lower than those observed inside potato drills; thus, using temperatures measured directly in potato drills resulted in increased estimates of population growth and shorter estimated generation times than when using modelled soil temperatures. This finding suggests that greater understanding of the temperature inside potato drills is required if we are to make accurate predictions about the effects of temperature on PCN populations.     

Effect of sample size on cyst recovery by flotation methods: Recommendations for sample processing during EU monitoring of potato cyst nematodes (Globodera spp.)

Under EC Council Directive 2007/33/EC member states of the European Union are recommended to take soil samples ranging from 100 mL to >1500 mL to monitor populations of potato cyst nematodes [PCN] ( Globodera spp.) in targeted fields. This study examines the effects of varying sample size on sample processing and cyst extraction using two widely used laboratory methods. Cyst recovery was stable using the Fenwick can from 100 mL up to the physical limits of the can. Recovery was significantly lower for low density 50 mL samples; however, this was partly due to a statistical artefact since higher numbers of cysts were lost from successively larger samples with constant cyst numbers (i.e. decreasing densities). The Schuiling centrifuge had functional limits of <100 mL and >500 mL. Outside this range, cyst recovery from low density samples was significantly reduced. Cyst recovery from samples of varying size, but with equal numbers of cysts was only constant above 100 mL. However, samples ranging from 200–500 mL were optimal for the Schuiling centrifuge. Cyst extraction efficiency was similar using both methods for samples ranging from 150 mL to 1500 mL, where larger samples were divided into <500 mL portions. However, processing times (i.e. cyst extraction and microscopic examination) and water consumption increased significantly with increasing sample size and were significantly higher when using the Fenwick can.     

Assessing reproduction of potato cyst nematodes (Globodera rostochiensis and G. pallida) on potato cultivars for National Listing1

To investigate testing procedures for assessing the reproduction of potato cyst nematodes (PCN) on potato cultivars, and to minimize variation in results, trials were done jointly for 3 years at two UK National Listing test centres (DANI in Northern Ireland, DAFS in Scotland). Test clones comprised susceptible, resistant and ‘partially resistant’ cultivars. The factors under investigation included the effect of season, site, location, temperature, PCN population, inoculation method, pot size, hatch rates and methods of assessment of reproduction. Standard procedures are recommended for testing potato cultivars for resistance to PCN.     

警惕检疫性有害生物马铃薯孢囊线虫(Globodera rostochiensis和G. pallida)入侵我国

马铃薯孢囊线虫包括马铃薯金线虫Globodera rostochiensis和马铃薯白线虫G. pallida, 是马铃薯生产上危害最为严重的植物寄生线虫, 一般造成30%的产量损失, 在热带发病严重地区, 产量损失高达80%~90%, 甚至绝收?由于其危害严重性, 包括我国在内的100多个国家将其列为重要检疫性有害生物?我国目前尚无马铃薯金线虫和白线虫的发生报道, 但随着贸易全球化, 马铃薯孢囊线虫传入我国的风险日趋增高?本文主要对马铃薯孢囊线虫的发生分布?危害症状?经济损失?生物学特性?传入我国的潜在风险和预防控制措施进行综述, 旨在为防止马铃薯孢囊线虫入侵我国提供参考?     

Hsp90 gene, an additional target for discrimination between the potato cyst nematodes, Globodera rostochiensis and G. pallida, and the related species, G. tabacum tabacum

The heat-shock gene, Hsp90, was targeted as a new variable genomic region to supplement other DNA-based tests for identification and discrimination of Globodera pallida, G. rostochiensis and G. tabacum tabacum. Populations of the potato cyst nematodes, G. pallida and G. rostochiensis (PCN), originating from Canada, France, Belgium and USA, together with two populations of G. tabacum tabacum from the USA and France were used for the amplification of a fragment of the Hsp90 gene. General and specific primers and probes for each species were derived from the consensus and non-consensus regions of the aligned sequences, respectively. A triplex conventional PCR assay, using a general forward and reverse or three specific reverse primers, as well as a real-time PCR using general primers and specific TaqMan probes, were developed. Melting curve analysis and restriction fragment polymorphisms using high resolution electrophoresis were explored for identifying PCR amplicons that characterized and discriminated the three Globodera species in both pure and mixed samples. Results from the different molecular assay strategies confirmed the usefulness of Hsp90 as a new additional gene target and showed that several different test options could be used for discrimination of PCN.     

Problems of potato cyst nematode pathotypes in the Czech Republic

During 1983–1989 about 1,200 infested plots in the Czech republic were investigated for the occurrence of new pathotypes of the potato cyst nematodes (PCN) Globoderu rostochiensis and G. pallida. Only eight populations of the pest were suspected of containing another pathotype than Ro1. Glasshouse pot trials were performed with those eight natural populations, two populations of Ro1, 10 potato cultivars resistant to Ro1 and 10 cultivars resistant to more pathotypes, G. pallida included. The multiplication rates (Pf/Pi) showed that four of the PCN populations were probably mixtures, with pathotype Ro1 prevailing. Three populations were suspected of containing G. pallida , but their morphological characters corresponded only with G. rostochiensis. One population corresponded with pathotype Ro5. The extent of the occurrence of pathotypes or virulence groups different from Ro1 can be estimated at 0.3–0.4% of infested plots.     

Isolation, Selection, and Efficacy of Pochonia chlamydosporia for Control of Rotylenchulus reniformis on Cotton

ABSTRACT The reniform nematode, Rotylenchulus reniformis, is a serious threat to cotton (Gossypium hirsutum) production in the United States, causing an annual loss of about $80 million. The objective of this study was to isolate fungi from eggs of R. reniformis and select potential biocontrol agents for R. reniformis on cotton. We focused on the fungus Pochonia chlamydosporia because it suppresses root-knot and cyst nematodes and because preliminary data indicated that it was present in Arkansas cotton fields. Soil samples were collected from six cotton fields in Jefferson County, Arkansas. A total of 117 isolates of the nematophagous fungus P. chlamydosporia were obtained. In an in vitro test, 105 of the 117 isolates parasitized fewer than 15% of R. reniformis eggs, but 12 isolates parasitized between 16 and 35% of the eggs. These 12 isolates produced from 6.8 x 10(4) to 6.9 x 10(5) chlamydospores per gram of medium in vitro, and chlamydospore production was similar on rice grain and corn grain media. In two greenhouse experiments, a single application of isolate 37 (5,000 chlamydospores per gram of soil) significantly reduced the numbers of R. reniformis on cotton roots and in soil. The three isolates (37, 26, and 14) that parasitized the most eggs in vitro were also the most effective in suppressing numbers of R. reniformis and in increasing cotton growth in the greenhouse.     

山西省中南部小麦上孢囊线虫的发生、分布及种群特征

本研究于2011-2017年对山西省主要市(县)小麦上孢囊线虫的发生与分布进行了较系统的调查研究,从运城、晋城、临汾、长治、晋中、太原等市小麦产区百余个乡镇采集和分离小麦根及根围土壤样品1 131份,其中336份检出有孢囊线虫,检出率为29.7%;运城市和临汾市小麦根及根围土壤样品孢囊检出率高,分别达到42.3%和32.5%,晋中市、长治市、太原市以及晋城市等地区也发现有不同程度的分布。因地区、栽培制度和土壤质地等的不同,土壤中线虫的群体密度也存在较大的差异,白雌虫密度最高的群体分布在运城市的一些地区(每100mL土壤中白雌虫数平均达到101个)。通过形态学鉴定明确山西省小麦上孢囊线虫群体种类较为单一,均为禾谷孢囊线虫Heterodera avenae,未发现我国小麦上另一重要的孢囊线虫种类——菲利普孢囊线虫H.filipjevi。小麦孢囊线虫在山西不同地区的发生和分布情况为山西省针对性地开展小麦上孢囊线虫的综合治理提供了有价值的信息。     

河南省禾谷类作物孢囊线虫的发生分布与种类鉴定

为探究河南省主要禾谷类作物孢囊线虫的发生分布,明确孢囊线虫对不同作物的危害情况,于2017—2020年对河南省18个市50个县（区）的小麦、玉米和水稻作物的孢囊线虫种类和发生分布进行随机取样调查,采用形态学特征、分子生物学鉴定和rDNA-ITS序列进化树分析技术鉴定不同作物孢囊线虫的种类,并根据土壤中孢囊基数和单孢囊卵数明确不同作物孢囊线虫的发病严重度。结果显示,共采集土壤样品308份,其中224份样品检测到孢囊,孢囊检出率为72.7%。小麦孢囊线虫发生分布范围覆盖40个县（区）,其中15个县（区）为禾谷孢囊线虫Heterodera avenae侵染,23个县（区）为菲利普孢囊线虫H. filipjevi侵染,南阳市西峡县西坪镇和开封市尉氏县张市镇为禾谷孢囊线虫和菲利普孢囊线虫混合侵染发生区;玉米孢囊线虫H. zeae在濮阳市清丰县韩村镇、许昌市长葛市董村镇和禹州市范坡镇检测点首次被发现;旱稻孢囊线虫H. elachista在信阳市潢川县魏岗乡、来龙乡和新乡市获嘉县亢村镇检测点首次被发现。孢囊线虫发病严重度数据表明,小麦田平均孢囊含量高达17.3个/100 mL;玉米田平均孢囊量为11.0个/100 mL;水稻田平均孢囊量为4.4个/100 mL。表明河南省孢囊线虫高发地块主要集中在豫北、豫东和豫中平原区。     

Identification of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> and <Emphasis Type="Italic">G. pallida</Emphasis> in the Ukraine by PCR

Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples from the Ukraine. The results show the occurrence of Globodera pallida in the Uzhhorod region (Zakarpatska oblast), where only G. rostochiensis had been previously reported. In the mixed potato cyst nematode (PCN) populations, G. pallida was less prevalent (2–5%) than G. rostochiensis (95–98%). A phylogenetic analysis based on ribosomal DNA internal transcribed spacer sequences showed that the Ukrainian population of G. pallida had >99% sequence identity with other G. pallida pa2/3 isolates from Europe. This study has demonstrated that polymerase chain reaction-mediated amplification of specific regions of the potato cyst nematode genome is not only highly effective as a species diagnostic tool but is also a sensitive method which can be used for taxonomic purposes with cyst collections which vary in age.     

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.     

Serologically Based Diagnostic and Quantification Tests for Nematodes

Plant parasitic nematodes are important plant pests which are difficult to identify and quantify. Serologically based techniques offer new ways to identify and quantify these parasites. Several factors need to be considered when developing an immunoassay for plant parasitic nematodes, including the application of the assay and the extraction procedure which together determine the likely organisms with which antibodies may cross-react. The four major factors: taxon specificity, nematode stage specificity, antibody specificity, and the level of management which is possible, need to be addressed interdependently and the best possible compromise made to develop a practical assay. Progress has been made in the immuno-diagnosis and quantification of potato cyst nematodes (Globodera spp) and root-knot nematodes (Meloidogyne spp) and these two groups of nematodes present different problems and therefore different challenges. The research reported here compares and contrasts work on these two groups of nematodes and highlights some of the important factors to be considered in the development of immunoassays for plant parasitic nematodes.     

甘肃省小麦禾谷孢囊线虫的发生及分布

为了解甘肃省小麦孢囊线虫病发生程度和分布情况,2009-2012年于小麦抽穗至灌浆期,采用Z字形取样法,取根际0~20cm土壤,用漂浮法分离孢囊,统计100g土样的孢囊数量,用形态学方法鉴定线虫。从甘肃省12个地区50个县区,134个乡镇共采集到463个小麦田间土样。结果表明,孢囊平均检出率为47.9%,其中冬麦区孢囊平均检出率为61.5%,春麦区平均检出率为34.2%,冬麦和春麦混播区为52.3%,冬麦、春麦及冬春麦混播区平均孢囊数分别为3.5、6.8和3.9个/100g土样。其中平凉市灵台县和兰州市永登县孢囊线虫发生最为严重,个别地块每100g土样中孢囊数超过40个。根据孢囊形态学特征的观察,将甘肃省采集到的孢囊线虫鉴定为禾谷孢囊线虫(Heterodera avenae)。研究结果可为有效防治甘肃省麦类孢囊线虫病提供依据。     

Detection of viable potato cyst nematodes (Globodera rostochiensis and G. pallida) by reverse transcription PCR

Journal of Plant Diseases and Protection - Currently plant infectivity assays (bioassays) seem most reliable to determine viability of potato cyst nematodes (Globodera rostochiensis and G....     

The influence of soil disinfection with DD,certain components of DD and some other compounds with nematocidal activity on the growth of white clover

In pots containing soil fumigated with DD before planting the growth of white clover was considerably better than in untreated pots. A similar effect was obtained both with cis- or trans-1,3-dichloropropene. Some other components of DD, including 1,2-dichloropropane, had no influence on clover growth in the concentration at which they occur in DD. Of the compounds not associated with DD, EN 18,133 and sugar showed a favourable effect on clover growth, but X 323 had no influence. In the untreated pots with poorly growing clover many roots had died. On the places where dead lateral roots had been attached many fungi were present in the tissue, especiallyRhizoctonia sp. andFusarium spp. These pots contained also more clover cyst nematodes and more springtails (Collembola). In one of the pot experiments poor growth could probably be attributed to an attack by the clover cyst nematode (Heterodera trifolii Goffart). The nematode level above which damage to the plants occurred was calculated at 1000–3000 eggs (larvae inclusive) per 100 ml of soil. In the other pot experiment, however, poor growth could not be explained by cyst nematodes only. In field experiments clover growth was promoted by soil fumigation with DD, also when only a few cyst nematodes were present. These observations increase the evidence that attack of white clover by pathogens is a complex phenomenon in which nematodes as well as fungi and perhaps insects too are involved. Apart from a small increase in growth at the beginning, soil fumigation with DD did not raise the yield of perennial rye grass.In this article the name DD is used as abbreviation for the trade name Shell D-D.     

<Emphasis Type="Italic">Solanum sisymbriifolium</Emphasis> - a new approach for the management of plant-parasitic nematodes

Plant-parasitic nematodes are serious pests causing important crop losses worldwide. After extensive screening of non-tuber-bearing Solanaceae, a resistant trap crop, Solanum sisymbriifolium, with a high production level of hatching agents, seemed an ideal control method for potato cyst nematodes (PCN), Globodera spp. Recently, root-knot nematodes (RKN), Meloidogyne spp., were found coexisting with PCN. Therefore, it is important to find alternative methods to control both nematode genera. The chemical properties of S. sisymbriifolium turns this plant into an excellent candidate for further nematicidal studies and to develop new crop production models. Studies concerning the effects of this plant on plant-parasitic nematodes are presented. Pathogenicity studies with four S. sisymbriifolium cvs (Domino, Pion, Sis 4004 and Sharp) and five Meloidogyne species showed that all cultivars of S. sisymbriifolium studied were resistant to M. chitwoodi and hypersusceptible to M. arenaria and M. hapla. For M. hispanica only cv Pion was susceptible. M. javanica induced different responses: cvs Pion and Sharp were susceptible; cv Domino resistant and Sis 4004 hypersusceptible. The studies of the hatching effects of root exudates from these cvs showed that they had an influence on the hatching inhibition of second stage juveniles of the five Meloidogyne species tested.