Linoleic acid — The nematicidal principle of several nematophagous fungi and its production in trap-forming submerged cultures

Linoleic acid was shown to be the only detectable nematicidal agent in the mycelial extracts of several predacious fungi of the genus Arthrobotrys. Although the compound is present in saprophytic cultures, induction of trap formation by nematodes or phenylalnyl-valine caused a significant increase in its production. In submerged cultures, the number of traps formed by Arthrobotrys conoides and Arthrobotrys oligospora was directly correlated to the increase of the concentration of linoleic acid. In A. conoides, the ratio of ergosterol to linoleic acid decreased from 2.6 in saprophytic cultures to 1.1 in trap-forming cultures induced with nematodes. Linoleic acid exhibited nematicidal activities towards the free-living nematode Caenorhabditis elegans with an LD₅₀ value of 5 g/ml.     

Growth of the Fungus Duddingtonia flagrans in Soil Surrounding Feces Deposited by Cattle or Sheep Fed the Fungus to Control Nematode Parasites

The results reported in this paper represent work from two separate experiments, namely a plot trial using cattle feces conducted at Kungsãngen in Uppsala, Sweden and a plot trial using sheep feces undertaken at Tåstrup in Copenhagen, Denmark. In both trials, a technique was used to monitor the level of Duddingtonia flagrans propagules in soil surrounding feces. The feces were from animals fed or not fed D. flagrans fungal chlamydospores. Also presented are the numbers of soil nematodes in soil surrounding sheep feces. The results indicate that D. flagrans has little growth beyond the fecal environment into surrounding soil when chlamydospores are fed to either sheep or cattle. This is substantiated by the soil nematode data. No statistical differences in the number of nematode taxa identified, Shannon Weiner H′, proportion of various feeding groups, and B/B + F (B and F are the proportions of bacterial and fungal-feeding nematodes) were found when soil surrounding sheep feces containing chlamydospores and parasitic nematode eggs was compared to soil surrounding feces containing parasitic nematode eggs alone. It is unlikely that the application of D. flagrans as a biological control agent against the free-living stages of nematode parasites of these livestock will negatively affect populations of nontarget soil nematodes.     

Interaction of nematodes with nematophagus fungi: induction of trap formation, attraction and detection of attractants

Induction of trap formation in Arthrobotrys oligospora, A. conoides and Monacrosporium cystosporum was studied during a 24 h period in the presence of the free-living nematodes Panagrellus redivivus on various nutrient media. A definite pattern of trap formation was observed in these fungi. Low nutrient mineral salt medium had the most pronounced effect on trap formation. Attraction and repulsion of P. redivivus towards these three nematophagous and three non-nematophagous fungi was studied. The nematodes were attracted towards three nematophagous and one non-nematophagous fungi, the other two repelled them. Attractants of the nematophagous fungi were determined by thin-layer chromatography. In the case of A. oligospora and M. cystosporum, four spots were traced, whereas in A. conoides, five spots were detected.     

Genetic and QTL analysis of resistance to <Emphasis Type="Italic">Xiphinema index</Emphasis> in a grapevine cross

Resistance to the dagger nematode Xiphinema index has been an important objective in grape rootstock breeding programs. This nematode not only causes severe feeding damage to the root system, but it also vectors grapevine fanleaf virus (GFLV), the causal agent of fanleaf degeneration and one of the most severe viral diseases of grape. The established screening procedures for dagger nematode resistance are time consuming and can produce inconsistent results. A fast and reliable greenhouse-based system for screening resistance to X. index that is suitable for genetic studies and capable of evaluating breeding populations is needed. In this report, the dynamics of nematode numbers, gall formation, and root weight loss were investigated using a variety of soil mixes and pot sizes over a 52-week period. Results indicated that the number of galls formed was correlated with the size of the nematode population and with the degree of root weight loss. After inoculation with 100 nematodes, gall formation could be reliably evaluated in 4–8 weeks in most plant growth conditions and results were obtained 6 months more rapidly than past evaluation methods. This modified X. index resistance screening method was successfully applied to 185 of the 188 F₁ progeny from a cross of D8909-15 × F8909-17 (the 9621 population), which segregates for a form of X. index resistance originally derived from Vitis arizonica. Quantitative trait loci (QTL) analysis was carried out on both parental genetic maps of 255 markers using MapQTL 4.0. Results revealed that X. index resistance is controlled by a major QTL, designated Xiphinema index Resistance 1 (XiR1), near marker VMC5a10 on chromosome 19. The XiR1 QTL was supported by a LOD score of 36.9 and explained 59.9% of the resistance variance in the mapping population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of Sialyl and Galactosyl Residues on the Body Wall of Different Plant Parasitic Nematodes

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-β-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.     

Factors responsible for transition of the <Emphasis Type="Italic">Duddingtonia flagrans</Emphasis> carnivorous fungus from the saprotrophic to the zootrophic nutrition type

Quantitative investigation of the factors responsible for trap formation in the nematophagous fungus Duddingtonia flagrans F-882 in submerged liquid culture was carried out. The data obtained suggest a complex program for the regulation of zootrophic nutrition in D. flagrans. Optimal concentrations of such carbon and nitrogen sources as sucrose (0.4%), ammonium ions (0.2%), and tryptone (0.2%) promote trap formation in the case of contact with the nematodes Panagrellus redivivus. Increased concentrations of these compounds, however, inhibit trap formation. The sensitivity of the mycelium to nematode excreta depends on the state of the culture and is increased under limitation by certain nutrient components or in the course of prolonged starvation. A direct correlation was found between the number of caught nematodes and the number of chlamydospores formed on the mycelium. The nutrients obtained from the nematode biomass are used for formation of additional chlamydospores (on average, about 20 chlamydospores per nematode). Environmental and evolutionary aspects of the role of zootrophic nutrition in carnivorous fungi are discussed.     

Biological control of sheep parasite nematodes by nematode-trapping fungi: <Emphasis Type="Italic">in vitro</Emphasis> activity and after passage through the gastrointestinal tract

Summary The main method used for the control of gastrointestinal nematodes in sheep production is the application of chemotherapeutic agents, which often lead to the selection of parasites resistant to given active principles. Biological control can be considered a promising alternative, contributing to an increase in the efficacy of verminous control. We determined the in vitro activity and in situ survival of the predatory fungi Arthrobotrys musiformis and Arthrobotrys conoides during passage through the gastrointestinal tract of sheep after oral administration of conidia in microencapsulated form and as a liquid in natura. Initial in vitro tests showed that both fungi were efficient in the predation of trichostrongylid L3 larvae present in the faeces of sheep naturally infected with gastrointestinal nematodes. The fungi presented high nematophagous activity, which was 99.3% for A. conoides and 73.7% for A. musiformis. A. conoides did not survive passage through the gastrointestinal tract under the conditions of the present experiment. On the other hand, A. musiformis was reisolated after administration in either microencapsulated or liquid form, suggesting that this species is a promising alternative for the control of nematodes in sheep since it survives without any protection (in natura).     

Survival of Xiphinema index in clay loam soil

A sample of clay loam vineyard soil, containing the nematode Xiphinema index, was divided into equal portions and stored in plastic bags. Nematodes were extracted immediately or remained in fridge for a short time or at room temperature for longer periods. The number of extracted nematodes did not differ significantly between treatments, indicating that X. index in soil samples collected for diagnostic purposes could remain viable for a period up to six months. Four other samples, of similar soil type, were collected from different vineyards and kept stored in plastic bags at room temperature. Variable periods of nematode survival recorded, ranging from less than two years in one sample, up to five years in another one. It is concluded that a long fallow period of at least five years may be required between successive grapevine crops to eliminate the nematode from clay loam soils.     

Influence of Four Nematodes on Root and Shoot Growth Parameters in Grape

Two grape cultivars, susceptible French Colombard and tolerant Rubired, and four nematodes, Meloidogyne incognita, Pratylenchus vulnus, Tylenchulus semipenetrans, and Xiphinema index, were used to quantify the equilibrium between root (R) and shoot (S) growth. Root and shoot growth of French Colombard was retarded by M. incognita, P. vulnus, and X. index but not by T. semipenetrans. Although the root growth of Rubired was limited by all the nematodes, the shoot growth was limited only by X. index. The R:S ratios of Rubired were higher than those of French Colombard. The reduced R:S ratios of Rubired were primarily an expression of reduction in root systems without an equal reduction in shoot growth, whereas in French Colombard the reduced R:S ratios were due to a reduction in both shoot growth and root growth and to a greater reduction in root growth than shoot growth. All nematodes reproduced equally well on both cultivars. Both foliage and root growth of French Colombard were significantly reduced by M. incognita and P. vulnus. Nematodes reduced the shoot length by reducing the internode length. Accumulative R:S ratios in inoculated plants were significantly smaller than those in controls in all nematode treatments but not at individual harvest dates. Bud break was delayed by X. index and was initiated earlier by P. vulnus and M. incognita. All buds in nematode treatments were less vigorous than in controls.     

Application of a formulation of the nematophagous fungus <Emphasis Type="Italic">Duddingtonia</Emphasis><Emphasis Type="Italic">flagrans</Emphasis> in the control of cattle gastrointestinal nematodiosis

The viability of a formulation of Duddingtonia flagrans was assessed in the control of parasite gastrointestinal nematodes of cattle. Two groups (A and B) of eight crossbred Holstein × Zebu cattle, approximately one year old, were placed in Brachiaria decumbens pasture. Each animal in group B (treated) received orally 20 g sodium alginate pellets containing mycelial mass of the D. flagrans fungus, while the animals in the group A (control) received pellets without fungus for seven months, starting in March 2005. The egg per gram of feces counting the gastrointestinal nematodes showed a difference (P < 0.05) in the treated group in June, July and August, with reductions of 58% (June), 47% (July) and 51% (August) compared to the control group. The infective larvae recovered in the pastures collected up to 20 cm from distance of the fecal dung in group B differed (P < 0.01) from the larvae recovered in group A. At the end of the experimental period, the animals in group B presented a greater weight gain (P < 0.01) compared to the untreated group (A). The treatment of cattle with pellets containing the D. flagrans nematophagous fungus, at the dose and duration used was effective in controlling the infective larvae of gastrointestinal nematodes of cattle.     

Effects of Seasonal and Site Factors on Xiphinema index Populations in Two California Vineyards

Sampling of Xiphinema index for 2 years (1993-95) in two California vineyards indicated that a greater number of nematodes occurred during the winter months. The number of juveniles increased four-fold from December 1993 to January 1994, indicating a high reproductive rate during this time. Extremely high or low soil temperatures corresponded to low nematode numbers. Samples were taken from 0 to 31 cm and 31 to 62 cm deep both within and between the vine rows. Numbers of nematodes were greatest at the 0- to 31-cm depth in one vineyard with a loamy sand soil, and at a depth of 31 to 62 cm in the second vineyard, which had a silt loam soil. In both vineyards, X. index population densities were greater within the vine row.     

食线虫真菌基因组测序和分析研究进展

食线虫真菌作为线虫的天敌,是一种潜在的线虫病害的生防制剂。食线虫真菌通过形成特殊的捕食器官或产生粘性孢子和毒素等方式捕捉和侵染线虫。近年来,随着测序技术的进步和生物信息学的应用,越来越多真菌基因组已经被测定和报道。目前,已经有7种食线虫真菌的基因组被报道,包括捕食线虫真菌寡孢节丛孢(Arthrobotrys oligospora)、线虫卵寄生真菌厚垣普可尼亚菌(Pochonia chlamydosporia)和内寄生真菌明尼苏达被毛孢(Hirsutella minnesotensis)等。本文对食线虫真菌的基因组特点、毒力相关基因家族的扩张、捕食器官形成调控和进化机制进行了系统地总结,对组学时代食线虫真菌研究面临的关键问题进行了评述。     

Some effects of oxamyl on the virus-vector nematodes Longidorus elongatus and Xiphinema diversicaudatutn

Oxamyl applied to field soil prevented Longidorus elongatus from acquiring and transmitting tomato black ring virus for at least 6 wk, although numbers of nematodes were not greatly decreased compared with the untreated control. Glasshouse and laboratory tests examined the effects of oxamyl on viruliferous L. elongatus and Xiphinema diversicaudatum. In these tests oxamyl (1.0 ppm) in the soil water largely prevented L. elongatus transmitting virus to bait plants over a period of 1 month. X. diversicaudatum was equally affected by smaller concentrations, 0.1 ppm being sufficient to inhibit virus transmission in one test. Inhibition of virus transmission was associated with a decrease in the number of root tip galls produced by nematode feeding, especially that of X. diversicaudatum. Few nematodes were seriously affected by oxamyl, except at the greatest concentration tested (100 ppm), when numbers of L. elongatus, but not of X. diversicaudatum, were decreased. In vitro-treated viruliferous nematodes protracted their stylets, but their subsequent ability to transmit virus was unimpaired.     

An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus

This study aimed to present a protease produced by Duddingtonia flagrans fungus (AC001), and to evaluate its activity in the biological control of cyathostomin infective larvae (L₃). The crude extract from D. flagrans grown in liquid medium was applied first to a DEAE-Sepharose? and later to a CM-Sepharose? ion exchange column. Protease activity was determined under different pHs and temperatures. Subsequently, the effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) inhibitor on activity were evaluated. Next, the protease activity in the biological control of nematodes was tested. A new 38 kDa serine protease (Df1) was purified. Optimum activity was obtained at pH 8.0 and 60°C; CuSO₄, ZnSO₄ and PMSF strongly inhibited the activity. Df1 (AC001) showed an L₃ reduction rate of 58%. In conclusion, a serine protease produced by D. flagrans (AC001) has been isolated, which is effective in the in vitro destruction of cyathostomin L₃.     

Secondary Metabolites of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> CHA0 Drive Complex Non-Trophic Interactions with Bacterivorous Nematodes

Non-trophic interactions are increasingly recognised as a key parameter of predator–prey interactions. In soil, predation by bacterivorous nematodes is a major selective pressure shaping soil bacterial communities, and many bacteria have evolved defence mechanisms such as toxicity. In this study, we show that extracellular secondary metabolites produced by the model soil bacterium Pseudomonas fluorescens CHA0 function as a complex defence strategy against bacterivorous nematodes. Using a collection of functional mutants lacking genes for the biosynthesis of one or several extracellular metabolites, we evaluated the impact of bacterial secondary metabolites on the survival and chemotactic behaviour of the nematode Caenorhabditis elegans. Additionally, we followed up the stress status of the nematodes by measuring the activation of the abnormal DAuer Formation (DAF) stress cascade. All studied secondary metabolites contributed to the toxicity of the bacteria, with hydrogen cyanide efficiently repelling the nematodes, and both hydrogen cyanide and 2,4-DAPG functioning as nematicides. Moreover, these metabolites elicited the DAF stress response cascade of C. elegans, showing that they affect nematode physiology already at sublethal concentrations. The results suggest that bacterial secondary metabolites responsible for the suppression of plant pathogens strongly inhibit bacterivorous nematodes and thus likely contribute to the resistance of bacteria against predators in soil.     

悉生培养微缩体系食细菌线虫提高土壤激素含量的机制

研究土壤食细菌线虫与细菌的相互作用及其生态功能是土壤生态学的核心内容之一。食细菌线虫取食细菌可以促进土壤中氮素的矿化,提高氮素养分的供给,改善土壤的营养条件,从而促进植物的生长发育。土壤食细菌线虫促进植物根系生长的"养分作用机制"已得到确认,而"激素作用机制"还存在争议。从供试土壤中筛选获得一株高效产IAA细菌和两种不同cp值的食细菌线虫,通过设置简化的悉生培养系统,对这两种土著食细菌线虫与土著产IAA细菌之间的相互作用,及其对土壤中IAA含量变化的影响进行研究。结果表明:两种食细菌线虫的取食均能促进细菌数量和活性的增强,食细菌线虫与产IAA细菌相互作用也能显著增加土壤中IAA的含量;这些促进作用受到接种食细菌线虫的种类以及培养时间的影响:在培养第10天和第20天时,接种cp值为1的中杆属食细菌线虫显著增加了产IAA细菌的数量;在培养第10天和第30天时,相比较接种cp值为2的头叶属食细菌线虫,接种中杆属食细菌线虫显著提高了土壤中IAA的含量。     

Synergistic effect of soil pathogenic fungi and nematodes reducing bioprotection of Arbuscular mycorrhizal fungi on the grass Leymus arenarius

We examined the role ofarbuscular mycorrhizal fungi (AMF) in thebioprotection of the sand dune grass Leymus arenarius against soil fungi andnematodes. Six soil fungi (Fusariumnivale, Fusarium sp., Cladosporiumherbarum, Cladosporium sp., Phomasp., Sporothrix sp.) and four species ofnematodes (Pratylenchoidesmagnicauda, Paratylenchusmicrodorus, Rotylenchus goodeyi, Merlinius joctus) were isolated from a coastalsand dune in Iceland where a population of L. arenarius was declining in vigour. Acommercial AMF inoculum (Microbio Ltd. England)containing Glomus caledonium, G.fasciculatum, and G. mossae was used.Seedlings of L. arenarius were grownunder controlled conditions in sterile sand andsubjected to the following treatments: (1)control, (2) + AMF, (3) + AMF + soil fungi, (4)+ AMF + nematodes, (5) + AMF + nematodes + soilfungi, (6) + soil fungi, (7) + soil fungi +nematodes, (8) + nematodes. Mycorrhizal plantshad significantly the highest root dry weightof all treatments. Mycorrhizal plants hadsignificantly higher leaf dry weight thanplants in other treatments, with the exceptionof AMF inoculated plants exposed to nematodes. Mycorrhizal plants exposed to soil fungi andnematodes had significantly higher growthparameters except total number of leaves thanAMF inoculated plants exposed to both soilfungi and nematodes. Mycorrhizal plantssubjected to a dual application of soil fungiand nematodes did not vary significantly in anygrowth parameters from plants without AMF thatwere exposed to a dual application of soilfungi and nematodes. This suggests asynergistic effect of soil fungi and nematodesthat break down the protection of AMF againstpathogens. The results are discussed inrelation to plant dynamics of sand duneecosystems.