The predicted impact of possible climatic change on virus-vector nematodes in Great Britain

Data extracted from surveys of plant-parasitic nematodes in Great Britain allowed relatively detailed maps of the geographical distribution of various longidorid and trichodorid virus-vector nematode species to be produced. These distributions are related to long-term monthly mean temperature. Recently published figures for climate change were applied to the distribution data. A potential increase in nematode associated problems due to climate change using examples of existing published data are examined and discussed.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.     

Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root-knot nematode (Meloidogyne spp.)

A molecular protocol is presented for distinguishing seven of the most common and economically important Meloidogyne spp. DNA was extracted from individual second-stage juvenile (J2) nematodes of Meloidogyne spp. and amplified by PCR (polymerase chain reaction). Fifteen PCRs including amplification of rDNA, specific SCAR (sequence characterized amplified region) and RAPD (random amplified polymorphic DNA) fragments were possible from the extracted DNA. This enabled a molecular diagnostic key for M. incognita , M. javanica , M. arenaria , M. mayaguensis , M. hapla , M. chitwoodi and M. fallax to be designed. The key unifies published methods into a single logical schematic using primer combinations that were previously validated and shown to work reliably and specifically. The protocol can be used with single juvenile or adult nematodes and the schematic can readily be expanded to accommodate more species. The use of RAPD amplification to assist with identification of samples which do not yield diagnostic amplification products after the first three steps of the molecular key is also described.     

Suitability of weed species prevailing in Spanish vineyards as hosts for root-knot nematodes

Commercial vineyards in southern Spain were surveyed and sampled during October to December 2004 to determine the extent to which common weeds present were suitable hosts of root-knot nematodes infesting soils of those vineyards. Seven weed species commonly growing in grapevine soils in southern Spain were found infected by either Meloidogyne incognita or M. javanica: Amaranthus retroflexus (redroot pigweed), Anchusa azurea (ox-tongue), Chenopodium album (goosefoot), Erodium moschatum (musk stork’s bill), Malva rotundifolia (low mallow), Sinapis alba (white mustard), and Solanum nigrum (black nightshade). The host suitability of the weeds to root-knot nematodes was evaluated on the basis of root galling severity and nematode population densities in soil and roots. Also, the host–parasite relationship in these naturally Meloidogyne-infected weeds was examined. All the weed species in the study were considered suitable hosts for M. incognita and M. javanica because: (a) high Meloidogyne spp. populations occurred in roots and surrounding soil of the weed species; (b) the severity of root galling was high, and (c) well-established permanent feeding sites were observed in the histopathological studies of infected root tissues. In addition, this study presents the first reports of S. alba and A. azurea as hosts for M. incognita, and of E. moschatum as a new host for M. javanica, thus increasing the list of reported weed hosts for Meloidogyne spp. These results indicate that noticeable population densities of M. incognita and M. javanica can be maintained or increased in these weeds, at population levels higher than those previously reported for the same nematodes infecting grapevine roots. The weeds infesting vineyards thus represent an important source of inoculum of Meloidogyne spp., and furthermore may act as reservoirs of these nematodes which can be disseminated within or among vineyards by agricultural operations.     

Evaluation ofAmaranthus sp. andVernonia amygdalina, and soil amendments with poultry manure for the management of root-knot nematodes on eggplant

The suppressive effect of vernonia (Vernonia amygdalina), amaranth (Amarathus sp.) and poultry manure on root-knot nematodes (RKNs) (Meloidogyne spp.) infecting eggplant (Solanum macrocarpon) was studied at two sites in southern Benin naturally infested with these nematodes. After 3 months, soil and root-inhabiting RKN populations were significantly less (P≦0.05) in the plots cropped with vernonia, amaranth, and eggplant amended with poultry manure (PM) at the rate of 40 t ha⁻¹ as compared with the rate of 20 t ha⁻¹ and with the control. Poultry manure was more effective after 2 months than after 3 months. Overall, vernonia was the most effective treatment affecting RKN populations in the roots and the soil. The use of these treatments in nematode management through rotation and co-planted crops is discussed. http://www.phytoparasitica.org posting August 6, 2008.     

Development of a PCR-based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium-aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small-subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii , a plasmodiophorid pathogen of watercress ( Rorippa nasturtium-aquaticum ). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration-based method for obtaining pathogen DNA for PCR from watercress-bed water was developed.     

First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.     

A novel approach to spider mite control based on expression of sarcotoxin IA peptidevia a virus-vector system in plants

Control of the spider miteTetranychus cinnabarinus Boisduval is problematic, and there is a pressing need for efficient, non-hazardous and inexpensive strategies for limiting the damage it causes. The gene for the anti-bacterial peptide sarcotoxin IA of the flesh flySarcophaga peregrina was cloned into the nonpathogenic potyvirus-based vector system ZYMV-AGII (Zucchini yellow mosaic virus-AGII). Expression of this peptidevia the AGII vector was detected in infected squash leaves and was not deleterious to the host plant. Leaf discs of squash infected with the recombinant virus AGII-sarcotoxin IA were tested for spider mite control under laboratory conditions. Spider mite egg production on plants expressing the sarcotoxin IA gene was decreased by a factor of two or three compared with that on AGII-infected plants or healthy leaf discs, respectively. In contrast to its effect on oviposition, sarcotoxin IA expressing squash did not significantly affect the mortality and the ability to repel spider mites. Crude extract from squash leaves infected with AGII-sarcotoxin IA was also found to cause a significant decrease of mite fecundity compared with extracts from AGII-treated or healthy plants and also caused a rise in mite mortality. Our results demonstrate that sarcotoxin IA affects mite fecundity and, to a lesser degree, mortality, and shows potential for controlling spider mites in the field.     

三种重要皮蠹科害虫的幼虫形态与分子鉴定

为寻求白腹皮蠹和赤毛皮蠹幼虫简便科学的鉴定方法,利用形态学结合分子生物学对其做了鉴定研究,首先对其触角、上内唇、臀突和胸足胫节的特征进行了形态描述;再分别提取白腹皮蠹、赤毛皮蠹和外群黑毛皮蠹幼虫的虫体DNA进行PCR扩增,通过测序比对构建了系统进化树。结果表明:白腹皮蠹幼虫上内唇的中间2根缘刚毛呈蘑菇状,粗短,而赤毛皮蠹幼虫的则呈矛状,细长,二者上内唇的形态特征差异非常明显,可用于二者形态上的鉴定;对3种皮蠹幼虫的序列进行比对,白腹皮蠹与赤毛皮蠹的碱基相似率为89.97%,黑毛皮蠹与白腹皮蠹和赤毛皮蠹的碱基相似率分别为77.54%和77.05%,差异均明显,可用于3种皮蠹幼虫种间鉴定的线粒体COⅠ基因标记序列。     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

昆虫病原线虫对草地贪夜蛾生理生化和组织病理学的影响

为明确昆虫病原线虫侵染对草地贪夜蛾Spodoptera frugiperda生理生化和组织病理学的影响,选择草地贪夜蛾6龄幼虫分别注入樱桃异小杆线虫Heterorhabditis beicherriana和小卷蛾斯式线虫Steinernema carpocapsae,测定草地贪夜蛾幼虫体内抗氧化酶和解毒酶活性以及能源物质含量的变化,并于显微镜下观察线虫侵染后草地贪夜蛾幼虫中肠和脂肪体组织的病理变化。结果显示,供试 2 种线虫对草地贪夜蛾6龄幼虫均有致死作用,注射剂量为2~3条侵染期线虫（infective juve-niles,IJs）/μL 的樱桃异小杆线虫和小卷蛾斯式线虫 24 h 后,草地贪夜蛾幼虫的死亡率分别为16.67%和96.67%。注射2~3 IJs/μL线虫后,草地贪夜蛾幼虫体内超氧化物歧化酶、过氧化物酶、过氧化氢酶、谷胱甘肽S-转移酶和羧酸酯酶的活性基本呈先上升后下降的变化趋势,注射樱桃异小杆线虫和小卷蛾斯式线虫后各酶活性达到峰值的时间分别为注射后24 h和12 h;血浆中总蛋白含量呈波动变化,海藻糖和游离脂肪酸的含量均逐渐降低。注射线虫后草地贪夜蛾6龄幼虫的中肠和脂肪体结构均被破坏,且小卷蛾斯式线虫引起的病变更快也更严重。表明相较于樱桃异小杆线虫,小卷蛾斯式线虫对草地贪夜蛾的生防潜能更大。     

Recent progress in understanding relationships betweenVerticillium species and subspecific groups

Improved understanding of the genetic diversity within fungi in the genusVerticillium has resulted from recent studies based on vegetative compatibility analysis and several techniques of molecular biology. Although the method used to identify vegetative compatibility groups (VCGs) does affect the results, vegetative compatibility appears to be a stable characteristic among isolates. Fairly low VCG diversity has been detected withinV. dahliae andV. albo-atrum using nitrate non-utilizing mutants. VCGs do not appear to be related to pathogenicity to particular host species, with the exception ofV. albo-atrum on alfalfa. However, there is some correlation with virulence on certain hosts and with the ability ofV. dahliae to interact with root-lesion nematodes. Studies based on DNA analysis indicate thatV. dahliae andV. albo-atrum are closely related but separate species. Restriction fragment length polymorphism (RFLP) studies have identified several subspecific groups withinV. dahliae, including two non-host-adapted groups and two that are host-adapted. They also have confirmed that alfalfa strains ofV. albo-atrum are a distinct subgroup that is probably a separate population of clonal origin. Using polymerase chain reaction (PCR), a second non-host-adapted subgroup withinV. albo-atrum was identified that was previously unknown.     

两种分子技术检测松木中松材线虫的效果评价

为更好地解决当前松材线虫Bursaphelenchus xylophilus检测工作中频现的假阴性问题,通过不同分子技术检测效果的比较评价,摸索出了一套可提高松材线虫检测准确性的技术体系。针对早期感病松木的低线虫量,建立了线虫的最大量分离方法和基于rDNA ITS序列的分子检测体系,该体系可高效检测出单条松材线虫,准确率达93.75%。以来自浙江省不同疫区的96份松木样品为检测材料,比较了SCAR标记和ITS序列2种分子检测技术的阳性率。结果显示,通过首次PCR,ITS_Ⅰ和ITS_Ⅱ序列的检测阳性率分别为52.08%和55.21%,SCAR标记的检测阳性率为30.21%;通过第二次或巢式PCR,ITS_Ⅰ和ITS_Ⅱ序列的检测阳性率提高到了97.92%和100.00%,SCAR标记的检测阳性率提高到了59.38%,表明基于ITS序列的检测阳性率明显高于SCAR标记,且通过第二次或巢式PCR方法可进一步提高检测灵敏度,降低假阴性率。因此,通过线虫的最大量分离并基于rDNA ITS序列的分子检测可明显提高检测准确性,更适用于松材线虫的常规检测。     

Powdered neem seed and cake for management of the banana weevil,cosmopolites sordidus, and parasitic nematodes

Field trials were conducted in Kenya with ‘Nakyetengu’, an East African highland banana cultivar (AAA-EA), highly susceptible to banana pests. Regardless of soil fertility levels, incorporation around the plant base of powdered neem(Azadirachta indica A. Juss.) seed or cake at 60-100 g/mat at 4-month intervals, gave better control of the banana weevil,Cosmopolites sordidus (Germar), and of parasitic nematodes, than that achieved with soil application of Furadan 5G (carbofuran) at 60 g/mat at 6-month intervals. Compared with untreated control, fruit yield in most of the neem treatments was significantly higher, particularly during the second cycle of crop production. Neem application conferred a net economic gain, whereas Furadan application proved uneconomical. Application of powdered neem seed or cake at higher rates (200–400 g/mat) at 6-month intervals caused phytotoxicity, resulting in drying up of banana plants before fruiting, or in ‘chokethroat’,i.e., inflorescence emergence failure.     

Identification and management of virulence genes in potato cyst nematodes

Field inoculation of leek with zoospores ofPhytophthora prorri resulted in high infection within a short time. Inoculation with infected leaf tissue resulted in a more gradual increase of disease incidence. Inoculation with oospores was relatively unsuccessful. Zoospores were produced in Petri-dishes by treating fast-growing, young mycelium with a diluted soil extract for at least 2 days, followed by a cold treatment in sterile demineralized water. The successful methods can be used for evaluation of resistance or fungicide performance, and for epidemiological experiments.     

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。     

Field Response to Pratylenchus Thornei of a Wheat Line with the CreAet Gene for Resistance to Heterodera Avenae

The gene CreAet for resistance to Heterodera avenae, transferred from Aegilops triuncialis to Triticum aestivum introgression line TR-353, was assessed for responses to Pratylenchus thornei under field conditions. After 2.5 months, P. thornei infestation on TR-353 was similar to those on its progenitors (T. aestivum H10-15, T. turgidum H1-1 and A. triuncialis A-1) and on the cultivar Capa. After 5 months, TR-353 hosted significantly more P. thornei per plant than A. triuncialis, T. aestivum H10-15 or cultivar Rinconada, being very close to the maximum value obtained from T. turgidum. The final infestation of line TR-353 by H. avenae was significantly lower than on the other plants, except for A. triuncialis. In addition, at 2.5 months, the abundance of P. thornei on TR-353 was intermediate between the introgression wheat line H93-8 (Cre2 gene) and the cultivar Loros (Cre1 gene), but the final number of P. thornei per plant on TR-353 was significantly greater than on Loros. Line TR-353 was not able to control P. thornei in the field, but it confirmed its resistance to H. avenae, which was significantly lower than those due to Cre1 and Cre2 genes. No competition was observed between P. thornei and H. avenae populations on line TR-353 or A. triuncialis.     

Identification and management of virulence genes in potato cyst nematodes

Efficient and accurate diagnostic assays are essential for the design and evaluation of control measures of the potato cyst nematodesGlobodera rostochiensis andG. pallida by means of resistance. The hybridoma technology and the polymerase chain reaction (PCR) offer in potential various possibilities to design such diagnostic tests for routine purposes. We set out to devise a refined advisory system based on biochemical assays by using the following stepwise approach.In the early 80's a research program was started to develop an immunoassay to differentiate the two sibling species of potato cyst nematodes. Species specific monoclonal antibodies were raised against nematode proteins which are thermostable, abundant and homologous, and which enable reliable species identification using single eggs.     

Phylogenetic and morphological analyses of Pythium graminicola and related species

Isolates of Pythium graminicola and related species were differentiated using restriction fragment length polymorphism (RFLP) analyses of the internal transcribed spacer (ITS) regions of rDNA and the cytochrome c oxidase subunit II (COX II) gene. These sequences were used in subsequent phylogenetic analyses. Finally, the phylogenetic placement of species was compared to that determined from morphological characteristics. The 62 isolates tested were divided into seven groups, A–G, based on RFLP analysis of the rDNA-ITS region. In the RFLP analysis of the COX II gene, isolates were divided into groups similar to those based on ITS-RFLP. Groups A and B were each separated into two additional subgroups. Grouping of isolates based on RFLP analyses agreed with the morphological differentiation. Groups A, B, D, E, F, and G were identified as P. graminicola, P. arrhenomanes, P. aphanidermatum, P. myriotylum, P. torulosum, and P. vanterpoolii, respectively. Group C was closely related to group B based on phylogenetic analysis of the rDNA-ITS region and the COX II gene and is similar to P. arrhenomanes. Each of the other species occupied their own individual clades. Although P. arrhenomanes is morphologically similar to P. graminicola, our phylogenetic analyses revealed that it was evolutionarily distant from P. graminicola and more closely related to P. vanterpoolii. Our analysis also revealed that P. torulosum with smaller oogonia is more closely related to P. myriotylum with large oogonia than to P. vanterpoolii, which forms smaller oogonia and is morphologically similar to P. torulosum. P. aphanidermatum with large oogonia and aplerotic oospores was not related to the morphologically similar species P. myriotylum. Results suggest that P. graminicola and related species are phylogenetically distinct, and molecular analyses, in addition to morphological analyses, are necessary for the accurate taxonomic placement of species in this complex.     

Effect of solarization and fumigant applications on soilborne pathogens and root-knot nematodes in greenhouse-grown tomato in Turkey

A study was conducted in two greenhouses with a history of Fusarium crown and root rot (Fusarium oxysporum f.sp.radicis-lycopersici, Forl) and root-knot nematodes (Meloidogyne javanica andM. incognita). During the 2005–06 growing season, the effectiveness of soil disinfestation by solarization in combination with low doses of metham-sodium (500, 750, 1000 and 1250l ha⁻¹) or dazomet (400 g ha⁻¹), was tested against soilborne pathogens and nematodes in an attempt to find a suitable alternative to methyl bromide, which is soon to be phased out. Solarization alone was not effective in the greenhouse with a high incidence ofForl. In the greenhouse with a low level ofForl, all the treatments tested reduced disease incidence, and were therefore considered to be applicable for soil disinfestation. In addition, root-knot nematode density decreased with all the treatments tested in both of the greenhouses.