Quantitative bioassay for chemotaxis with plant parasitic nematodes

A simple, flexible, and quantifiable bioassay for the attraction or repulsion of plant parasitic nematodes to or from root fractions or pure substances is described. Accurate gradients of volatile and nonvolatile substances can be measured and established. The method entails placing the nematodes in narrow agarose tracks such that their movement is essentially linear and the distance a population has traveled away or toward a given substance can be monitored with time. Plastic plates, each containing 10 such tracks, are described. The method is illustrated with second-stage larvae ofMeloidogyne incognita and a volatile attractant and nonvolatile repellent fraction obtained from cucumber roots.     

The Plant Sesquiterpene Nootkatone Efficiently Reduces Heterodera schachtii Parasitism by Activating Plant Defense

Plant parasitic nematodes, including the beet cyst nematode Heterodera schachtii, constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents. Here, we evaluated the impact of eight plant terpenoids on the H. schachtii parasitism of Arabidopsis thaliana. None of the metabolites affected the plant development (5 or 10 ppm). Nootkatone decreased the number of adult nematodes on A. thaliana to 50%, with the female nematodes being smaller compared to the control. In contrast, three other terpenoids increased the parasitism and/or female size. We discovered that nootkatone considerably decreased the number of nematodes that penetrated A. thaliana roots, but neither affected the nematode viability or attraction to plant roots, nor triggered the production of plant reactive oxygen species or changed the plant’s sesquiterpene profile. However, we demonstrated that nootkatone led to a significant upregulation of defense-related genes involved in salicylic and jasmonic acid pathways. Our results indicate that nootkatone is a promising candidate to be developed into a novel plant protection agent acting as a stimulator of plant immunity against parasitic nematodes.     

Chemotaxis of the Pinewood Nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>, to Volatiles Associated with Host Pine, <Emphasis Type="Italic">Pinus massoniana</Emphasis>, and its Vector <Emphasis Type="Italic">Monochamus alternatus</Emphasis>

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the most important invasive species in pine forests of Asia, is transported to new pine hosts by beetles of the genus Monochamus. Third-stage dispersal juveniles (J_III) aggregate in pupal chambers around the vector as it matures. We demonstrated that the ratio of three terpenes (α-pinene, β-pinene, and longifolene at 1:2.7:1.1) released by larval Monochamus alternatus strongly attract J_III, whereas the different ratio (1:0.1:0.01) of these three terpenes found in healthy xylem of Pinus massoniana attracts only the propagative stage (J_n) of the nematode. We suggest that the volatiles produced by the host plants could be the basis of a chemoecological relationship between plant parasitic nematodes and their vector insects. Capture of J_III with terpene-baited trap tubes deployed for 2 hr in the field was demonstrated. This technique may lead to the development of rapid sampling methodologies for use at either ports-of-entry or in the field.     

Effects of Geldanamycin on Hatching and Juvenile Motility in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Heterodera glycines</Emphasis>

Several Streptomyces species are known to produce metabolites that inhibit plant pathogens. One such compound is geldanamycin (GA), a benzoquinone ansamycin originally isolated from Streptomyces hygroscopicus. We examined the effect of geldanamycin on egg hatch and juvenile motility in Caenorhabditis elegans and in two populations of the plant-parasitic nematode Heterodera glycines. When C. elegans eggs were exposed to geldanamycin, both hatch and motility were reduced by GA doses between 2 and 50 μg/ml. The H. glycines inbred populations TN17 and TN18 exhibited low dose stimulation of hatch and motility, whereas levels occurring at higher GA doses were at or below control levels. These experiments represent the first demonstration of geldanamycin effects in C. elegans and H. glycines and suggest that the heat shock chaperone Hsp90, the known molecular target of geldanamycin, may be involved in nematode egg hatch and motility. This study also indicates that geldanamycin-producing strains of Streptomyces may be useful as biocontrol agents for nematodes.     

Ozonated Nutrient Solution Treatment as an Alternative Method for the Control of Root-Knot Nematodes in Soilless Cultivation

ABSTRACT

Root-knot nematodes (Meloidogyne incognita) are plant parasitic worms that widely influence the agricultural production including soilless cultivation. In this study, we investigated the inactivating effect of ozonated nutrient solution on the root-knot nematode juveniles in both nutrient solution and substrate, as well as the in vitro egg hatching. The results showed that twice ozonation treatments with dissolved ozone concentration (DOC) at 0.5 mg/L resulted in a mortality at 90.8% for in vitro study. DOC of 4.0 mg/L with three treatment repeats in the substrate experiment resulted in a mortality at 88.3%. Our results imply that the application of ozonated nutrient solution might be a feasible method in soilless cultivation for the control of root-knot nematodes. In practical application, ozonation treatment should be conducted with appropriate DOC and treatment repeats.     

腐植酸类物质对温室黄瓜根际植物线虫数量的影响

本研究旨在明确3种腐植酸类物质(NaFA、KFA、CFA)及与新药肥(SY)复配物对温室黄瓜根际植物线虫数量是否有抑制作用.研究结果表明:(1)矮化线虫属Tylenchorhynchus和根结线虫属Meloidogyne是2种主要的植物线虫,(2)随着生长季后期植物线虫数量的迅速增长,5种腐植酸类物质(CFA、NaFA、KFA、OFA SY、NaFA SY)及SY防治区矮化线虫和根结线虫数量低于对照区,(3)CFA与新药肥SY复配效果强于OFA和SY的单一作用效果.     

Isolation and structure determination of new nematicidal triglyceride fromArgemone mexicana

The petroleum ether extract ofArgemone mexicana seeds was found to possess nematicidal activity against the plant parasitic nematodeMeloidogyne incognita. The active nematicidal compound has been isolated from the crude extract by column Chromatographic techniques and purified by TLC. Chemical structure has been determined by chemical and spectro-scopic methods to be that of a triglyceride, sn-glycerol-1-eicosa-9, 12-dien-oate-2-palmitoleate-3-linoleate.     

Evaluation of the Evolution of Increased Competitive Ability (EICA) Hypothesis: Loss of Defense Against Generalist but not Specialist Herbivores

The evolution of increased competitive ability (EICA) hypothesis predicts that invasive plant species may escape their specialized natural enemies in their introduced range and subsequently evolve with a decrease in investment in anti-herbivore chemical defenses relative to native conspecifics. We compared the chemical profile of 10 populations of US native and 20 populations of European invasive Solidago gigantea. To test for differences in inducibility between native and invasive populations, we measured secondary chemistry in both damaged and undamaged plants. We also performed bioassays with three specialist and two generalist insect herbivores from four different feeding guilds. There was no evidence that invasive populations had reduced concentrations of sesquiterpenes, diterpenes, or short-chain hydrocarbons (SCH), although significant variation among populations was detected. Sesquiterpene and diterpene concentrations were not influenced by damage to the host plant, whereas SCH concentrations were decreased by damage for both native and invasive plants. Performance of the three specialist insects was not affected by the continental origin of the host plant. However, larval mass of the generalist caterpillar Spodoptera exigua was 37% lower on native plants compared to invasive plants. The other generalist insect, a xylem-tapping spittlebug that occurs on both continents, performed equally well on native and invasive plants. These results offer partial support for the defense predictions of the EICA hypothesis: the better growth of Spodoptera caterpillars on European plants shows that some defenses have been lost in the introduced range, even though our measures of secondary chemistry did not detect differences between continents. Our results show significant variation in chemical defenses and herbivore performance across populations on both continents and emphasize the need for analysis across a broad spatial scale and the use of multiple herbivores.     

Host recognition by entomopathogenic nematodes: Behavioral response to contact with host feces

Host recognition by entomopathogenic nematodes may occur through contact with insects' excretory products, cuticle, or gut contents. We analyzed the behavioral responses of four species of entomopathogenic nematodes during contact with feces of natural or experimental hosts. Host recognition by nematodes was manifested in alterations in the frequency and/or duration of one or more search parameters including forward crawling, headwaving, body-waving, stopping, backward crawling, head-rubbing, and headthrusting.Heterorhabditis bacteriophora andSteinernema glaseri showed behavioral responses to contact with feces of their natural hosts,Spodoptera exigua (Lepidoptera) andPopillia japonica (Coleoptera), and to the experimental hosts,Acheata domesticus (Orthoptera) andBlatella germanica (Blatteria).Steinernema carpocapsae responded only toB. germanica feces, whereas5. scapterisci did not significantly respond to any of the insect species. During contact with cockroach feces, all nematodes, exceptS. scapterisci, showed avoidance behavior. We suggest that ammonia present in cockroach feces is inhibitory to nematodes. Specific host recognition by entomopathogenic nematodes may be an important mechanism to maintain host affinities.     

Subterranean Herbivore-induced Volatiles Released by Citrus Roots upon Feeding by <Emphasis Type="Italic">Diaprepes abbreviatus</Emphasis> Recruit Entomopathogenic Nematodes

Herbivore-induced volatile emissions benefit plant hosts by recruiting natural enemies of herbivorous insects. Such tritrophic interactions have been examined thoroughly in the above-ground terrestrial environment. Recently, similar signals have also been described in the subterranean environment, which may be of equal importance for indirect plant defense. The larvae of the root weevil, Diaprepes abbreviates, are a serious pest of citrus. Infestations can be controlled by the use of entomopathogenic nematodes, yet the interactions between the plant, insect and nematode are poorly understood and remain unpredictable. In bioassays that used a root zone six-arm olfactometer, citrus roots (‘Swingle citrumelo’ rootstock) recruited significantly more entomopathogenic nematodes (Steinernema diaprepesi) when infested with root weevil larvae than non-infested roots. Infested plants were more attractive to nematodes than larvae alone. Roots damaged by weevil larvae attracted more nematodes than mechanically damaged roots and sand controls. By dynamic in situ collection and GC-MS analysis of volatiles from soil, we determined that four major terpene compounds were produced by infested plant roots that were not found in samples from non-infested roots or soil that contained only larvae. Solvent extracts of weevil-infested roots attracted more nematodes than extracts of non-infested roots in a two choice sand-column bioassay. These findings suggest that Swingle citrus roots release induced volatiles as an indirect defense in response to herbivore feeding, and that some of these induced volatiles function as attractants for entomopathogenic nematodes.     

Control of plant-parasitic nematodes by a nematicidal strain ofAspergillus niger

An isolate ofAspergillus niger (designated PD-42) was evaluated in laboratory, greenhouse, and field trials for efficacy in controlling plant-parasitic nematodes. In greenhouse experiments, PD-42 drenches containing spores of PD-42 on oatmeal significantly reduced galling on tomato due to root-knot nematode as compared to untreated controls. In a one-half acre field experiment, PD-42 incorporated in seed coats was associated with significantly increased yield and decreased root-knot galling on pepper. In a second one-half acre field experiment, PD-42 drenches significantly reduced tomato and pepper root galling due toMeloidogyne incognita, and nonsignificant yield increases occurred. In each field experiment, treatment with PD-42 reducedRotylenchulus reniformis populations. The nematicidal components of theA. niger culture filtrates include citric acid, oxalic acid, and undetermined molecules larger than 8000 MW.     

Glucosinolates and Differential Herbivory in Wild Populations of Brassica oleracea

Glucosinolates are known to elicit responses from Brassica herbivores in laboratory studies. To study their importance in interactions with herbivores in the field, glucosinolate profiles and levels of herbivory were ascertained for wild cabbage plants growing in four neighboring populations in the UK. Glucosinolate profiles differed between plant populations, but not between different habitats within populations. Within habitats, there was no link between individual plant glucosinolate profiles and herbivory by Pieris spp., slugs and snails, flea beetles or aphids. Plants attacked by the micromoth, Selania leplastriana, contained higher levels of 2–hydroxy-3–butenylglucosinolate and 3–indolylmethylglucosinolate than plants within the same population that were not attacked. It is concluded that the differences in glucosinolate profiles between the plant populations are unlikely to be due to differential selection pressures from herbivores feeding on the mature plants over the two years studies.     

Aphelenchoides besseyi Ab-FAR-1 Interacts with Arabidopsis thaliana AtADF3 to Interfere with Actin Cytoskeleton,and Promotes Nematode Parasitism and Pathogenicity

Fatty acid and retinol binding proteins (FAR) are unique proteins found in nematodes and are considered potential targets for controlling these parasites. However, their functions in nematode parasitism and pathogenicity and interaction with hosts are still unclear. In this study, we investigated the specific roles of rice white tip nematodes (RWTNs), Aphelenchoides besseyi, and a protein, Ab-FAR-1, to elucidate the parasitic and pathogenic processes of nematodes. The results showed that the expression level of Ab-far-1 was significantly up-regulated after A. besseyi infection of the plant. The immunofluorescence and subcellular localisation showed that Ab-FAR-1 was secreted into plant tissues mainly through the body wall of nematodes and might act in the nucleus and cytoplasm of plant cells. The pathogenicity of RWTNs was enhanced in Arabidopsis thaliana overexpressing Ab-FAR-1 and inhibited in Ab-far-1 RNAi A. thaliana. Yeast two-hybrid, Co-IP, BiFC, and nematode inoculation experiments showed that Ab-FAR-1 could interact with the A. thaliana actin-depolymerizing factor protein AtADF3, and the A. thaliana adf3 mutant was more susceptible to nematodes. An in vitro actin filament depolymerisation assay demonstrated that Ab-FAR-1 could inhibit AtADF3-mediated depolymerisation of actin filaments, and the turnover process of cellular actin filaments was also affected in A. thaliana overexpressing Ab-FAR-1. In addition, flg22-mediated host defence responses were suppressed in A. thaliana overexpressing Ab-FAR-1 and adf3 mutants. Therefore, this study confirmed that RWTNs can affect the turnover of actin filament remodelling mediated by AtADF3 through Ab-FAR-1 secretion and thus inhibit plant PAMP-triggered immunity (PTI), promoting the parasitism and pathogenicity of nematodes.     

Herbivore-induced Plant Volatiles Trigger Sporulation in Entomopathogenic Fungi: The Case of <Emphasis Type="Italic">Neozygites tanajoae</Emphasis> Infecting the Cassava Green Mite

A large body of evidence shows that plants release volatile chemicals upon attack by herbivores. These volatiles influence the performance of natural enemies. Nearly all the evidence on the effect of plant volatiles on natural enemies of herbivores concerns predators, parasitoids, and entomophagous nematodes. However, other entomopathogens, such as fungi, have not been studied yet for the way they exploit the chemical information that the plant conveys on the presence of herbivores. We tested the hypothesis that volatiles emanating from cassava plants infested by green mites (Mononychellus tanajoa) trigger sporulation in three isolates of the acaropathogenic fungus Neozygites tanajoae. Tests were conducted under climatic conditions optimal to fungal conidiation, such that the influence of the plant volatiles could only alter the quantity of conidia produced. For two isolates (Altal.brz and Colal.brz), it was found that, compared with clean air, the presence of volatiles from clean, excised leaf discs suppressed conidia production. This suppressive effect disappeared in the presence of herbivore-damaged leaves for the isolate Colal.brz. For the third isolate, no significant effects were observed. Another experiment differing mainly in the amount of volatiles showed that two isolates produced more conidia when exposed to herbivore-damaged leaves compared with clean air. Taken together, the results show that volatiles from clean plants suppress conidiation, whereas herbivore-induced plant volatiles promote conidiation of N. tanajoae. These opposing effects suggest that the entomopathogenic fungus tunes the release of spores to herbivore-induced plant signals indicating the presence of hosts.     

Host plant utilization and iridoid glycoside sequestration byEuphydryas anicia (Lepidoptera: Nymphalidae)

The iridoid glycoside content of individual adultEuphydryas anicia butterflies from two Colorado populations was quantitatively determined. At one site (Red Hill), larval host plants wereCastilleja integra andBesseya plantaginea, while at the other site (Cumberland Pass) a single host plant,B. alpina, was used. At Red Hill, macfadienoside and catalpol were sequestered, while at Cumberland Pass, catalpol and aucubin were sequestered. Artificial diet studies showed that larvae hydrolyzed a major iridoid ofB. plantaginea, 6-isovanilIylcatalpol, to catalpol (which was sequestered) and isovanillic acid (which was excreted). Large year-to-year and individual variation in butterfly iridoid content was established as was a female-male difference in macfadienoside vs. catalpol content. Larval host plant distributions and numbers were determined at Red Hill for two years and compared with changes in butterfly populations and sequestered iridoids.     

Glucosinolate Content and Susceptibility for Insect Attack of Three Populations of Sinapis alba

Sinapis alba is less susceptible to damage by insect pests than Brassica napus. We investigated the composition and distribution of glucosinolates in different plant parts in three populations of S. alba; two populations selected for low-seed-glucosinolate content and one commercial cultivar. We have assessed the susceptibility of low-seed-glucosinolate content populations of S. alba to four insect pests, a flea beetle, a pollen beetle, and two species of aphids. Over 90% of the total glucosinolates in the cotyledons of the three populations of S. alba consisted of sinalbin. There was no difference in feeding damage by flea beetles on different S. alba populations at the cotyledon stage, nor was there a difference in sinalbin concentration of cotyledons. Total glucosinolate levels were highest in younger plant tissues. Sinalbin declined as a proportion of total glucosinolate content in later growth stages, especially in the low breeding lines. Reproduction by aphids was the same on all three populations despite differences in sinalbin content of the S. alba leaves at the growth stage tested. The specialist aphid, Brevicoryne brassicae, was found mainly on young tissues, while the generalist aphid, Myzus persicae, was found predominantly on older plant parts. There was no difference in oviposition by pollen beetles between the S. alba populations, despite the fact that on one population total glucosinolate concentration and the proportion of sinalbin found in the buds were much lower than on the other two populations.     

Antifungal leaf-surface metabolites correlate with fungal abundance in sagebrush populations

A central component in understanding plant–enemy interactions is to determine whether plant enemies, such as herbivores and pathogens, mediate the evolution of plant secondary metabolites. Using 26 populations of a broadly distributed plant species, sagebrush (Artemisia tridentata), we examined whether sagebrush populations in habitats with a greater prevalence of fungi contained antifungal secondary metabolites on leaf surfaces that were more active and diverse than sagebrush populations in habitats less favorable to fungi. Because moisture and temperature play a key role in the epidemiology of most plant–pathogen interactions, we also examined the relationship between the antifungal activity of secondary metabolites and the climate of a site. We evaluated the antifungal activity of sagebrush secondary metabolites against two fungi, a wild Penicillium sp. and a laboratory yeast, Saccharomyces cerevisiae, using a filter-paper disk assay and bioautography. Comparing the 26 sagebrush populations, we found that fungal abundance was a good predictor of both the activity (r ² = 0.36 for Saccharomyces, r ² = 0.37 for Penicillium) and number (r ² = 0.34 for Saccharomyces) of antifungal secondary metabolites. This suggests that selection imposed by fungal pathogens has led to more effective antifungal secondary metabolites. We found that the antifungal activity of sagebrush secondary metabolites was negatively related to average vapor pressure deficit of the habitat (r ² = 0.60 for Saccharomyces, r ² = 0.61 for Penicillium). Differences in antifungal activity among populations were not due to the amount of secondary metabolites, but rather to qualitative differences in the composition of antifungal compounds. Although all populations in habitats with high fungal prevalence had secondary metabolites with high antifungal activity, different suites of compounds were responsible for this activity, suggesting independent outcomes of selection on plants by fungal pathogens. The location of antifungal secondary metabolites on the leaf surface is consistent with their putative defense role, and we found no evidence supporting other functions, such as protection from ultraviolet light or oxidation. That the antifungal activity of sagebrush secondary metabolites was similar for two different fungi provides support for broad antifungal defenses. The incidence and severity of fungal disease in the field (caused by Puccinia tanaceti) were similar in moist and dry habitats, possibly reflecting an equilibrium between plant defense and fungal attack, as sites with greater fungal abundance compensated with more effective secondary metabolites. The geographic correlation between fungal abundance and antifungal secondary metabolites of sagebrush, coupled with our other data showing heritable variation in these metabolites, suggests that pathogenic fungi have selected for antifungal secondary metabolites in sagebrush.     

Nematicidal Resorcylides from the Aquatic Fungus Caryospora callicarpa YMF1.01026

This study investigated metabolites with activities against plant parasite nematodes from the fresh-water fungus Caryospora callicarpa YMF1.01026. We obtained three novel tetradecalactone metabolites, caryospomycins A–C, with such activities. The chemical structures of these were determined through NMR spectroscopic analysis and were found to belong to the 14-membered macrolides with a fused 1,2-dimethoxy-4-hydroxybenzene ring, a rare structure among the resorcylides. In the in vitro tests, all three compounds exhibited moderate killing activity against the nematode Bursaphelenchus xylophilus. To our knowledge, this is the first report of secondary metabolites in the aquatic fungal genus Caryospora.     

The Role of Fresh <Emphasis Type="Italic">versus</Emphasis> Old Leaf Damage in the Attraction of Parasitic Wasps to Herbivore-Induced Maize Volatiles

The odor produced by a plant under herbivore attack is often used by parasitic wasps to locate hosts. Any type of surface damage commonly causes plant leaves to release so-called green leaf volatiles, whereas blends of inducible compounds are more specific for herbivore attack and can vary considerably among plant genotypes. We compared the responses of naïve and experienced parasitoids of the species Cotesia marginiventris and Microplitis rufiventris to volatiles from maize leaves with fresh damage (mainly green leaf volatiles) vs. old damage (mainly terpenoids) in a six-arm olfactometer. These braconid wasps are both solitary endoparasitoids of lepidopteran larvae, but differ in geographical origin and host range. In choice experiments with odor blends from maize plants with fresh damage vs. blends from plants with old damage, inexperienced C. marginiventris showed a preference for the volatiles from freshly damaged leaves. No such preference was observed for inexperienced M. rufiventris. After an oviposition experience in hosts feeding on maize plants, C. marginiventris females were more attracted by a mixture of volatiles from fresh and old damage. Apparently, C. marginiventris has an innate preference for the odor of freshly damaged leaves, and this preference shifts in favor of a blend containing a mixture of green leaf volatiles plus terpenoids, after experiencing the latter blend in association with hosts. M. rufiventris responded poorly after experience and preferred fresh damage odors. Possibly, after associative learning, this species uses cues that are more directly related with the host presence, such as volatiles from host feces, which were not present in the odor sources offered in the olfactometer. The results demonstrate the complexity of the use of plant volatiles by parasitoids and show that different parasitoid species have evolved different strategies to exploit these signals.     

A novelstreptomyces species for controlling plant-parasitic nematodes

A novel species ofSteptromyces isolated from nematode suppressive soils in Costa Rica was evaluated for efficacy in controlling plant-parasitic nematodes. This isolate, designated CR-43, was shown to inhibit reproduction ofCaenorhabditis elegans in a laboratory assay. Greenhouse trials utilizing three different methods of treatment with CR-43 gave significant reductions of tomato root galling due toMeloidogyne incognita. In a field experiment in Puerto Rico, in soil naturally infested withM. incognita, CR-43-treated pepper showed significant reductions in root galling and significant increases in yield as compared to untreated controls. In a second experiment in Puerto Rico, a significant reduction in tomato root galling and a slight reduction in root galling on pepper occurred. In this trial, yields on both tomato and pepper were higher in CR-43 treatments, but these differences were not statistically significant. In both experiments populations ofRotylenchulus reniformis were reduced by CR-43 treatment. In a field trial on strawberry in Massachusetts, CR-43-treated plants had lower numbers ofPratylenchus penetrons within roots and showed a significant decrease in black root rot disease. Studies on sterile filtrates from CR-43 cultures indicated that a major determinant of CR-43 antinematodal activity was mostly thermostable macromolecules of molecular weight higher than 6000. Culture filtrates of CR-43 exhibited antifungal activity in vitro.