Susceptibility of North American Native and Non-native Slugs (Mollusca: Gastropoda) to Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae)

Phasmarhabditis hermaphrodita caused significant mortality of the two native species, Deroceras laeve and Leidyula floridana, and of one introduced species D. reticulatum, but not of the other three introduced species, Arion hortensis, A. subfuscus and Limax maximus. Even the juvenile stages of A. subfuscus and L. maximus showed no mortality in nematode treatments. However, treatments with nematodes resulted in rapid and strong feeding inhibition in all six species. Surviving slugs resumed feeding when fresh food was provided. This study expands the host range of P. hermaphrodita to include a new family Vaginulidae and demonstrates the inability of P. hermaphrodita to cause mortality of A. hortensis, A. subfuscus and L. maximus. These three species use feeding suspension as an evasive behavior to escape nematode infection.     

The parasitic nematode Phasmarhabditis hermaphrodita defends its slug host from being predated or scavenged by manipulating host spatial behaviour

Infective stages of commercially used molluscicidal rhabditide nematodes Phasmarhabditis hermaphrodita contain bacterial symbionts which kill their host by septicaemia. The nematodes feed on the multiplying bacteria and entire host tissue, develop and repeatedly reproduce. Invertebrate cadavers are rapidly (from minutes to hours) removed by scavengers. However nematodes need days to complete their life cycle inside the host.

The post mortem locations of slugs killed by six different treatments (three types of molluscicides, a simulation of unsuccessful predation and two P. hermaphrodita nematode treatments) were compared.

In comparison to other pathogenic states, significantly more slugs killed by the nematodes died within the soil, where the scavenging pressure is weaker than on the soil surface (where most of the slugs died regardless treatment). We suggest that this is an outcome of behavioural manipulation, which prevent the parasites from being predated or scavenged together with their host until the nematodes complete development inside the host cadaver.     

Non-Susceptibility of Earthworm Eisenia fetida to the Rhabditid Nematode Phasmarhabditis hermaphrodita, a Biocontrol Agent of Slugs

The rhabditid nematode Phasmarhabditis hermaphrodita is a lethal parasite for slugs that is sold commercially in Europe under the trade name Nemaslug™. We evaluated the effects of P. hermaphrodita on the earthworm Eisenia fetida (Savigny). Adults of E. fetida were exposed in one-liter glass beakers to P. hermaphrodita at three concentrations (1×, 10× and 50× of the field recommended rate of 3×10⁹ billion nematodes/ha) during a 14-day period in an artificial soil substrate. The average body weight, burrowing behavior, mortality and other clinical signals of the earthworms were recorded at 0, 7, and 14 days after exposure to the treatments. In addition, injured earthworms (posterior ends removed) were exposed to the 10× field recommended rate of the commercial formulation. Neither intact nor injured E. fetida showed susceptibility to the slug-parasitic nematode P. hermaphrodita during the 14 days of exposure even at concentrations 10 and 50 times higher than the label dose. However, the worms in the attenuated control (autoclaved formulation) had higher mortality and lost less weight compared to the other treatments. Under the conditions of the test, we conclude that the use of the commercially available strain of P. hermaphrodita is safe to E. fetida.     

Susceptibility of indigenous UK earthworms and an invasive pest flatworm to the slug parasitic nematode Phasmarhabditis hermaphrodita

We exposed five earthworm species, one flatworm species and the susceptible slug Deroceras reticulatum to the recommended field application rate and five times this rate of Phasmarhabditis hermaphrodita. P. hermaphrodita caused significant mortality to D. reticulatum at the recommended rate and five times the recommended rate. Survival of the earthworms and the flatworm was not different from the untreated controls. P. hermaphrodita is a safe biological control agent causing no harm to earthworms and could not be used to kill Arthurdendyus triangulatus.     

Nematode Community Structure in Desert Soils: Nematode Recovery

The sugar-flotation-sieving (SFS) and Baermann-funnel (BF) methods were compared for nematode extraction efficiency. The SFS method recovered nematodes from more trophic groups whereas greater total numbers of individuals were recovered by BF. In a test to validate the efficiency of SFS, virtually 100% of the nematodes added to desert soil prior to extraction were recovered by four consecutive SFS washings of each soil sample. Estimations of nematode biomass in desert soils based on numbers of nematodes extracted by the two methods were similar unless there was large reserve of eggs in the soil. The biomass of nematodes from a Colorado desert soil was 0.9 g/m² as determined by both methods, whereas BF gave 0.17 g/m² for nematodes from a Mojave desert soil as compared to 0.9 g/m² with SFS.     

青海海北植物群落类型与土壤线虫群落相互关系

土壤线虫是良好的指示生物, 是植物群落演替的重要驱动力, 其生态功能影响着生态系统正常生态效应的发挥。该研究以海北矮生嵩草(Kobresia humilis)草甸、西藏嵩草(Kobresia tibetica)沼泽化草甸、暗褐薹草(Carex atrofusca)沼泽化草甸和金露梅(Potentilla fruticosa)灌丛4种不同植物群落类型的土壤线虫为研究对象, 研究不同植物群落类型下的土壤线虫群落组成、分布特征、物种多样性及其营养类群组成, 分析植物类群结构与土壤线虫群落之间的相关性。主要研究结果: (1)在4种植物群落土壤样本中共分离线虫3 800条, 分属于2纲5目15科30属, 线虫平均个体密度为每100 g干土580条, 随土壤深度增加而递减, 具有明显的表聚性。不同植物群落间的土壤线虫群落组成存在一定差异, 矮生嵩草草甸0-40 cm土壤线虫总数(1 811条·392.5 cm^-3)显著高于其他植物群落类型, 暗褐薹草沼泽化草甸的土壤线虫总数最少(324条·392.5 cm^-3)。4种植物群落下土壤线虫的优势属和营养类群组成存在差异, 这种差异在矮生嵩草草甸与暗褐薹草沼泽化草甸之间表现得尤为明显。 (2)不同植物群落下土壤线虫的多样性指数(H′)和均匀度指数(J′)均为金露梅灌丛最高, 暗褐薹草沼泽化草甸最低, 其两种植物群落间H′差异显著, 而优势度指数(λ)相反, 为暗褐薹草沼泽化草甸最高, 金露梅灌丛最低。表明金露梅灌丛土壤线虫群落多样性最高, 暗褐薹草沼泽化草甸土壤线虫群落多样性最低, 土壤线虫群落趋于单一化。4种植物群落土壤有机质的分解途径均以细菌通道为主。西藏嵩草沼泽化草甸的瓦斯乐斯卡指数(WI)显著高于矮生嵩草草甸, 表明从高寒沼泽化草甸过渡到高寒灌丛、高寒草甸, 土壤肥力不断降低, 沼泽化草甸有利于食微生物线虫的生长。暗褐薹草沼泽化草甸的植物寄生线虫指数(PPI)、成熟度指数(MI)均表现为最低, 表明其生态系统的成熟度较低, 这与暗褐薹草沼泽化草甸土壤含水量较高有关。不同植物群落下的富集指数(EI)、结构指数(SI)均为暗褐薹草沼泽化草甸最高, 由此可以看出暗褐薹草沼泽化草甸的食物网相对连通性较高, 食物链较长, 食物网的阻力相对较小。(3)主成分分析(PCA)结果显示4种植物群落最大贡献属不同。相关性分析表明: 食细菌性线虫数量与西藏嵩草沼泽化草甸有显著的正相关关系; 金露梅灌丛的植物多样性与线虫的H′、J′有显著的负相关关系, 与λ则有显著的正相关关系; WI与矮生嵩草草甸的植物多样性有显著的正相关关系, PPI与矮生嵩草草甸的物种多样性指数有显著的负相关关系。综上所述, 植物群落深刻地影响着土壤线虫群落的多样性。     

Interaction between a fungal endophyte and root herbivores of Ammophila arenaria

The effect of an endophytic fungus (Acremonium strictum) on plant-growth related parameters of marram grass (Ammophila arenaria), and its potential as a protective agent against root herbivores (Pratylenchus dunensis and Pratylenchus penetrans, root-lesion nematodes) was investigated in two inoculation experiments under different conditions. Acremonium strictum-inoculated plants showed increased plant development in terms of root biomass in the first experiment and increased number of tillers in the second experiment and biomass was less suppressed by nematodes than the Acremonium strictum-free plants. In neither experiment did Acremonium strictum reduce multiplication of root herbivores. On the contrary, Acremonium strictum-inoculated plants seemed to increase herbivore multiplication. Plants infected with P. penetrans benefitted more from the endophytic fungus than those with P. dunensis in terms of total biomass. The effect of Acremonium strictum on interspecific competition was also analyzed by plant inoculation with both nematode species. In Acremonium strictum-free plants with mixed nematode inoculum, the total number of nematodes, compared to numbers observed in one-species inoculation, was less than expected, suggesting that interspecific competition took place. In Acremonium strictum-inoculated plants no interspecific competition was observed. Plants inoculated with P. dunensis, P. penetrans and Acremonium strictum showed decreased total biomass compared to Acremonium strictum-free plants inoculated with the same nematodes. The implications of increased tillering and root growth of plants with Acremonium endophytes are discussed in relation to the sand stabilizing role of Ammophila arenaria in coastal dunes.     

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.     

Serological identification and quantification of potato cyst nematodes from clean cysts and processed soil samples

Two monoclonal antibodies which specifically recognise each of the two species of potato cyst nematodes (PCN) and do not cross react with other species of soil nematodes, were used successfully in an immunoassay to identify and quantify PCN species using clean cysts and mixed populations. These antibodies show reactivity only towards antigens prepared from live eggs and they recognise antigens which are easily released from the nematodes. The results presented in this paper show that serological identification and quantification of PCN, not only from clean cysts but also from processed soil samples is achievable. A simple procedure to recover nematodes from soil samples and then to release nematode antigens was devised. The use of these procedures and the immunoassay for quantification of PCN was validated in tests with soil samples from Northern Portugal. The flotation method proved to be as efficient as the Fen wick Can for cyst recovery from soil samples. A significant correlation was obtained between results from immunoassay estimates and the traditional method of cyst picking and egg counting. The amount of organic matter (OM) present in the soil samples affected the sensitivity of the immunoassay but quantification of nematodes extracted from soil samples was possible with soils containing up to 14% of OM. The challenge remains to optimise the extraction procedures and the immunoassay, in practical conditions with highly organic soils containing cysts of different sizes and ages.     

Habitat associations of two entomopathogenic nematodes: a quantitative study using real-time quantitative polymerase chain reactions

1. Despite nematodes being the most abundant animals on earth, very few animal ecologists study them, probably because of the difficulties of identifying them to species by morphological methods. 2. A group of nematodes that are important both ecologically and economically is the entomopathogenic nematodes, which play a key role in regulating soil food webs and are sold throughout the world as biological insecticides, yet for which very little is known of their population ecology. 3. A novel detection and quantification method was developed for soil nematodes using real-time polymerase chain reaction (PCR), and the technique was used to estimate numbers of two closely related species of entomopathogenic nematodes, Steinernema kraussei and S. affine in 50 soil samples from 10 sites in Scotland representing two distinct habitats (woodland and grassland). 4. There was a high degree of correlation between our molecular and traditional morphological estimates of population size and our data clearly showed that Steinernema affine occurred only in grassland areas, whereas S. kraussei was found in grassland and woodland samples to a similar degree. 5. Real-time PCR offers a rapid and accurate method of detecting individual nematode species from soil samples without the need for a specialist taxonomist, and has much potential for use in studies of nematode population ecology.     

Synergistic effects of antagonistic fungi and a plant growth promoting rhizobacterium, an arbuscular mycorrhizal fungus, or composted cow manure on populations of Meloidogyne incognita and growth of tomato

Effects of four antagonistic fungi (Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma harzianum and Gliocladium virens) alone and together with a plant growth promoting rhizobacterium Pseudomonas putida, an arbuscular mycorrhizal fungus Glomus intraradices or with composted cow manure (CCM) were assessed on the growth of tomato and on the reproduction of Meloidogyne incognita in glasshouse experiments. Application of all antagonistic fungi (except G. virens), P. putida, G. intraradices or CCM caused a significant increase in the growth of plants without nematodes. However, use of either of these fungi, P. putida, G. intraradices and CCM against plants with nematodes caused a significant increase in tomato growth. Paecilomyces lilacinus caused a 42% increase in the growth of nematode-inoculated plants followed by P. chlamydosporia (36%), T. harzianum (18%) and G. virens (15%). CCM caused about 57% increase in the growth of nematode-inoculated plants followed by P. putida (37%) and G. intraradices (31%). Maximum increase (71%) in the growth of nematode-inoculated plants was observed when CCM was used with P. lilacinus. Moreover, P. lilacinus caused a high reduction (55%) in galling and nematode multiplication, while G. virens the least (25%). Use of P. putida also caused a 39% reduction in galling and nematode multiplication followed by CCM (34%) and G. intraradices (32%). Combined use of CCM with P. lilacinus caused maximum reduction (79%) in galling and nematode multiplication. Re-isolation of antagonistic fungi from nematodes revealed that P. lilacinus parasitised more females and eggs than other antagonistic fungi. Root colonisation by P. putida was increased with P. lilacinus, while colonisation by G. intraradices was reduced in the presence of antagonistic fungi.     

Real-time quantitative PCR assay on bacterial DNA: In a model soil system and environmental samples

Real-time quantitative PCR (RTQ-PCR) was used to quantify the bacterial target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in presence of liquid nitrogen and hot detergent SDS based enzymatic lysis). For the purpose of our study, pure culture of Bacillus cereus (model organism), sterilized soil seeded with a known amount of B. cereus (model soil system) and samples from woodland and grassland (environmental samples) were chosen to extract DNA by three different protocols. The extracted DNA was then quantified by RTQ-PCR using 16S rDNA specific universal bacterial primers. The standard curve used for the quantification by RTQ-PCR was linear and revealed a strong linear relationship (r(2)=0.9968) with a higher amplification efficiency, e5=1.02. High resolution gel electrophoresis was also carried out to observe the effect of these extraction methods on diversity analysis. For the model soil system, the liquid nitrogen method showed the highest target DNA copy number (1.3 x 10(9) copies/microl). However, for both the environmental samples, the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (5.38 x 10(9) and 4.01 x 10(8) copies/ml for woodland and grassland respectively), high yield (6.4 microg/g and 1.76 microg/g of soil for woodland and grassland respectively) and different band patterns on high resolution gel electrophoresis suggesting an overall high extraction efficiency. This difference in the extraction efficiency between the model soil system and environmental samples may be attributed to different affinity of seeded and native DNA to soil particles.     

Extraction of nematodes from Dry Valley Antarctic soils

Nematode density and taxonomic composition from Dry Valley soil processed by the sugar centrifugation (SC) method in Antarctica was compared to those extracted from soils shipped frozen to the USA and processed by either the SC or Baermann Funnel (BF) (at 5°C and 10°C) techniques. Soil selected for the extraction comparisons represented a wide range of soil properties found in the Dry Valleys. More nematodes were recovered from freshly collected Antarctic soil and from stored frozen soil using the SC technique than from BF at either temperature (P<0.05). Temperature had no effect on nematode densities extracted by the BF. Scottnema lindsayae was the most abundant species recovered by all extraction methods, but recovery was significantly lower from stored soils. Thus, nematodes can be extracted qualitatively following frozen storage using SC, but quantitative studies of nematode populations should be based on soils extracted following field sampling.     

Seasonal Fluctuations of Globodera tabacum solanacearum as Estimated by Two Soil Extraction Techniques

Two soil extraction methods were compared to determine their efficiency in recovering cysts and juveniles of a tobacco cyst nematode, Globodera tabacum solanacearum. The methods were equally efficient when extracting nematodes from soil samples seeded in the laboratory; however, there was a significant extraction method × month interaction when the methods were used to estimate field soil populations over 2 years. The centrifugal sugar flotation method recovered greater numbers of cysts when densities were near 400 cysts/100 cm³ soil and greater numbers of juveniles in all samples. The sugar flotation method recovered greater numbers of cysts during months when densities were less than 400 cysts/100 cm³ soil. Numbers of cysts and juveniles were lowest in June and July following land tillage in May. A soil freeze in January 1982 may have been responsible for unusually high numbers of recovered cysts in February and March 1982, a pattern that did not occur in 1983.     

高通量测序技术在线虫多样性研究中的应用

土壤线虫多样性是土壤生态学研究的热点之一, 然而对土壤线虫群落组成及多样性的研究通常受到分类学和方法学的限制。当前, 分子生物学技术的快速发展丰富了我们对土壤线虫多样性的认识, 但也存在一定的局限性。本文综述了常用分子生物学技术如变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)、末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism, T-RFLP)、实时荧光定量PCR (quantitative real-time PCR, qPCR)和高通量测序(high-throughput sequencing, HTS)技术近年来在线虫多样性研究中的应用, 重点从土壤线虫DNA提取方法、引物和数据库的选择、高通量测序技术和形态学鉴定结果的比较等方面阐述了高通量测序技术在线虫多样性研究中的优势与不足, 并提出选择合适的线虫DNA提取方法结合特定引物和数据库进行注释分析, 仍是今后使用高通量测序技术开展线虫多样性研究的重点。当研究目标是土壤线虫多样性时, 优先推荐富集线虫悬液提取DNA的方法, 因此, 研究人员应根据具体目标选择最优组合开展实验研究。     

陇中黄土高原半干旱区不同种植年限紫花苜蓿土壤线虫群落分布特征

为明确长期种植紫花苜蓿对土壤线虫群落演变的影响,以不同种植年限紫花苜蓿为研究对象(2 a、9 a、18 a),并以农田为对照,采用 Illumina MiSeq测序技术研究了陇中黄土高原半干旱区紫花苜蓿土壤线虫群落结构及其多样性,探讨影响其群落变化的主控因子。结果表明: 所获土壤样品线虫种群隶属于2纲7目16科21属,其中,色矛纲为黄绵土优势线虫类群(44.6%～81.4%),相对丰度随苜蓿种植年限延长而降低。针线属、螺旋属、剑线属、Pristionchus、茎属、盆咽属、长针属、艾普鲁斯属、Isolaimium和野外垫刃属为苜蓿地特有线虫,其中,针线属(54.1%)、螺旋属(23.9%)、剑线属(21.9%)分别为2 a、9 a、18 a苜蓿地优势线虫属。紫花苜蓿土壤均以植物寄生线虫为优势类群(31.8%～67.1%),其相对丰度随苜蓿年限延长先降后升。冗余分析显示,土壤速效磷和全氮含量是影响紫花苜蓿土壤线虫群落结构的主导环境因子。     

应用AFLP分析梅根系与其根围土壤丛枝菌根真菌DNA多态性差异

应用巢式PCR并进行梅根系与根围土壤丛枝菌根真菌（arbuscular mycorrihizal fungi,AMF）DNA扩增片段长度多态性（amplified fragment length polymorphism,AFLP）的差异比较,研究梅共生AMF的作用机理。结果表明,从18个梅品种的30个根围土壤样品中有28个样品获得纯化的DNA片段,占样品数93.3%,样品平均多态性位点数为6.5个,Nei’s基因多样性为0.3559±0.1382,Shannon信息指数为0.5299±0.1676。与梅根系AMF DNA多态性比较,根围土壤的平均多态性位点数明显较多;且根系AMF的DNA多态性位点绝大多数存在于土壤AMF的DNA多态性位点中,表明根系内AMF是由土壤AMF发育而来;根系与根围土壤AMF DNA的聚类均与梅品种群、品种关联性不强,表明AMF对宿主梅品种或品种群没有特异的共生关系。     

Molecular detection of predation by soil micro-arthropods on nematodes

The relative importance of the factors driving change in the population dynamics of nematodes in the soil is almost completely unknown. Top-down control by micro-arthropod predators may have a significant impact on nematode population dynamics. We report experiments showing that mites and Collembola were capable of reducing nematode numbers in the laboratory and were feeding on a targeted nematode species in the field. A PCR-based approach was developed for the detection of predation on three species of slug- and insect-pathogenic nematodes: Phasmarhabditis hermaphrodita, Heterorhabditis megidis and Steinernema feltiae. The collembolan Folsomia candida and the mesostigmatid mite Stratiolaelaps miles were employed as model predators to calibrate post-ingestion prey DNA detection times. Fragments of cytochrome oxidase I (COI) mtDNA were sequenced and species-specific primers were designed, amplifying 154-, 154- and 203-bp fragments for each of the nematode species. Detection times for nematode DNA within the guts of Collembola were longer than in mites, with half-lives (50% of samples testing positive) of 08.75 h and 05.03 h, respectively. F. candida significantly reduced numbers of the nematode H. megidis, with rates of predation of approximately 0.4 nematode infective juveniles per collembolan per hour over 10 h. Four taxa of field-caught micro-arthropod that had been exposed to the nematode P. hermaphrodita for a period of 12 h were analysed and significant numbers of three taxa tested positive. This is the first application of PCR techniques for the study of nematophagy and the first time these techniques have been used to measure predation on nematodes in the field.     

Inter- and intra-specific cuticle variation between amphimictic and parthenogenetic species of root-knot nematode (Meloidogyne spp.) as revealed by a bacterial parasite (Pasteuria penetrans)

Specific host–parasite interactions exist between species and strains of plant parasitic root-knot nematodes and the Gram-positive bacterial hyperparasite Pasteuria penetrans. This bacterium produces endospores that adhere to the cuticle of migrating juveniles, germinate and colonise the developing female within roots. Endospore attachment of P. penetrans populations to second-stage juveniles of the root-knot nematode species Meloidogyne incognita and Meloidogyne hapla showed there were interactive differences between bacterial populations and nematode species. Infected females of M. incognita produced a few progeny which were used to establish two nematode lines from single infective juveniles encumbered with either three or 26 endospores. Single juvenile descent lines of each nematode species were produced to test whether cuticle variation was greater within M. hapla lines that reproduce by facultative meiotic parthenogenesis than within lines of M. incognita, which reproduces by obligate parthenogenesis. Assays revealed variability between broods of individual females derived from single second-stage juvenile descent lines of both M. incognita and M. hapla suggesting that progeny derived from a single individual can differ in spore adhesion in both sexual and asexual nematode species. These results suggest that special mechanisms that produced these functional differences in the cuticle surface may have evolved in both sexually and asexually reproducing nematodes as a strategy to circumvent infection by this specialised hyperparasite.     

Effects of Storage Temperature and Extraction Procedure on Recovery of Plant-parasitic Nematodes from Field Soils

Storage of nematodes in soil at -15 C for 1 to 16 weeks greatly increased nematode recovery by a sugar-flotation-sieving procedure. One week of exposure to -15 C killed all nematodes except Pratylenchus zeae and Tylenchorhynchus claytoni which were recoverable in decreasing numbers up to 10 weeks by the Baermann funnel method. Optimum storage temperature for survival of most nematode species was 13 C. The numbers of Meloidogyne incognita, T. claytoni, Belonolaimus Iongicaudatus, and P. zeae recoverable by either extraction method remained constant or increased when stored at 13-24 C for 16 weeks. This was also true for Helicotylenchtts dihystera and Xiphinema americanum extracted by the Baermann funnel technique, whereas the numbers retrieved by the sugar-flotation-sieving method decreased slightly. All species except T. claytoni decreased appreciably in soil stored at 36 C.