Incognita Juveniles Research Articles

RNA Interference of Dual Oxidase in the Plant Nematode Meloidogyne incognita

RNA interference (RNAi) is a powerful tool for the analysis of gene function in model organisms such as the nematode Caenorhabditis elegans. Recent demonstrations of RNAi in plant parasitic nematodes provide a stimulus to explore the potential of using RNAi to investigate disruption of gene function in Meloidogyne incognita, one of the most important nematode pests of global agriculture. We have used RNAi to examine the importance of dual oxidases (peroxidase and NADPH oxidase), a class of enzyme associated with extracellular matrix cross-linking in C. elegans. RNAi uptake by M. incognita juveniles is highly efficient. In planta infection data show that a single 4-h preinfection treatment with double-stranded RNA derived from the peroxidase region of a dual oxidase gene has effects on gene expression that are phenotypically observable 35 days postinfection. This RNAi effect results in a reduction in egg numbers at 35 days of up to 70%. The in vitro feeding strategy provides a powerful tool for identifying functionally important genes, including those that are potential targets for the development of new agrochemicals or transgenic resistance strategies.

Occurrence of Pasteuria spp. Associated with Plant-parasitic Nematodes in Korea

A survey was conducted on the occurrence of Pasteuria spp. associated with plant-parasitic nematodes in greenhouse and open field soils in Korea from 1996 to 2000. Among the 372 soil samples assayed, 20 samples had plant-parasitic nematodes attached with Pasteuria spp. endospores. The nematodes found with Pasteuria endospores and their collection sites were Meloidogyne spp. in Sungju, Cheju, and Yeoju and Helicotylenchus sp., Pratylenchus sp., Heterodera sp., and Aphelenchus sp. in Cheju. Morphological characteristics of the endospores on Meloidogyne incognita juveniles observed under light, scanning and transmission electron microscopes were same as the previous description of Pasteuria penetrans Sayre & Starr 1988.

Effects of DL‐methionine on hatching and activity of Meloidogyne incognita eggs and juveniles

Several concentrations of DL-methionine were tested in the laboratory for their effects on Meloidogyne incognita (Kofoid & White) Chitwood egg hatching and juvenile activity in aqueous suspensions and infested soil. After 7 days in methionine solutions, the proportions of hatched eggs were reduced by 23.3% at 0.25 mg litre(-1) methionine and by 76.4% at 25 g litre(-1) when compared with controls in tap water. An effect of methionine solutions on juvenile activity was also apparent after 24 h in the 25 g litre(-1) treatment, where the percentage of active M incognita juveniles was reduced by 16.3%. Further reductions in nematode mobility were observed as the time of exposure increased, and at lower methionine concentrations over longer exposure times. When methionine solutions were applied to soil infested with M incognita, reductions in egg hatching and juvenile activity were observed at 0.1 and 1 mg methionine g(-1) soil in both sand and clay-loam soils. The percentage of hatched eggs one week after methionine application at rates of 1 mg g(-1) was lower in sand (5.6%) and clay-loam soil (20.8%) than in controls where egg hatch was 52.0% and 48.0% in sand and soil, respectively. Non-active juveniles were found at 1 mg methionine g(-1) soil one week after its application to soil.

Phenylacetic acid-producing Rhizoctonia solani represses the biosynthesis of nematicidal compounds in vitro and influences biocontrol of Meloidogyne incognita in tomato by Pseudomonas fluorescens strain CHA0 and its GM derivatives

The aim of the present investigation was to determine the influence of Rhizoctonia solani and its pathogenicity factor on the production of nematicidal agent(s) by Pseudomonas fluorescens strain CHA0 and its GM derivatives in vitro and nematode biocontrol potential by bacterial inoculants in tomato. One (Rs7) of the nine R. solani isolates from infected tomato roots inhibited seedling emergence and caused root rot in tomato. Thin layer chromatography revealed that culture filtrates of two isolates (Rs3 and Rs7) produced brown spots at Rf-values closely similar to synthetic phenylacetic acid (PAA), a phytotoxic factor. Filtrates from isolate Rs7, amended with the growth medium of P. fluorescens, markedly repressed nematicidal activity and PhlA'-'LacZ reporter gene expression of the bacteria in vitro. On the contrary, isolate Rs4 enhanced nematicidal potential of a 2,4-diacetylphloroglucinol overproducing mutant, CHA0/pME3424, of P. fluorescens strain CHA0 in vitro. Therefore, R. solani isolates Rs4 and Rs7 were tested more rigorously for their potential to influence biocontrol effectiveness of the bacterial agents. Methanol extract of the culture filtrates of PAA-producing isolate Rs7 resulting from medium amended with phenylalanine enhanced fungal repression of the production of nematicidal agents by bacteria, while amendments with zinc or molybdenum eliminated such fungal repression, thereby restoring bacterial potential to cause nematode mortality in vitro. A pot experiment was carried out, 3-week-old tomato seedlings were infested with R. solani isolates Rs4 or Rs7 and/or inoculated with Meloidogyne incognita, the root-knot nematode. The infested soil was treated with aqueous cell suspensions (10(8) CFU) of P. fluorescens strain CHA0 or its GM derivatives or left untreated (as a control). Observations taken 45 days after nematode inoculation revealed that, irrespective of the bacterial treatments, galling intensity per gram of fresh tomato roots was markedly higher in soil amended with isolate Rs4 than in Rs7-amended soils. Soil amendments with R. solani and the bacterial antagonists resulted in substantial reductions of the number of galls per gram of root. These results are contradictory to those obtained under in vitro conditions where culture filtrates of PAA-positive Rs7 repressed the production of nematicidal compounds. Plants grown in Rs7-amended soils, with or without bacterial inoculants, had lesser shoot and root weights than plants grown in nonamended or Rs4-amended soils. Moreover, amendments with Rs7 substantially retarded root growth and produced necrotic lesions that reduced the number of entry sites for invasion and subsequent infection by nematodes. Populations of P. fluorescens in the tomato rhizosphere were markedly higher in Rs7-amended soils. PAA-producing virulent R. solani drastically affects the potential of P. fluorescens to cause death of M. incognita juveniles in vitro and influences bacterial effectiveness to suppress nematodes in tomato roots. As most agricultural soils are infested with root-infecting fungi, including R. solani, it is likely that some PAA-producing isolates of the fungus may also be isolated from such soils. The inhibitory effect of PAA-producing R. solani on the biosynthesis of nematicidal agent(s) critical in biocontrol may reduce or even eliminate the effectiveness of fluorescent pseudomonads against root-knot nematodes, both in nursery beds and in field conditions. Introduction of bacterial inoculants, for the control of any plant pathogen, should be avoided in soils infested with PAA-producing R. solani. Alternatively, the agents could be applied together with an appropriate quantity of fungicide or chemicals such as zinc to create an environment more favourable for bacterial biocontrol action.

Micro-moulded substrates for the analysis of structure-dependent behaviour of nematodes

AbstractMicrostructures, consisting of networks of channels of rectangular cross sections (50 μm high, 40-200 μm wide), were formed in 5 mm square areas on transparent substrates made of silicon rubber. An experimental set-up using the network sealed with a flat glass plate has the potential to function in a way similar to the pore space in soil, and is therefore useful for studies of nematode migration. The set-up allowed the migratory activity of nematodes in water-filled, porous and transparent microstructures to be observed with a microscope. By means of substrates with two different channel dimensions, the structure-dependent behaviour of second-stage juveniles of Meloidogyne incognita was visually demonstrated. Their behaviour was examined on the basis of the migration patterns obtained by superimposing recorded serial images of individual juveniles. In a micro-channel network with 40 μm high channels of 200 μm wide elements, juveniles showed marked activity in migration, forming consistent zigzag patterns spread over the network area. In contrast, in a micro-channel network with 80 μm high channels of 400 μm wide elements, migration showed thick, sparse patterns, restricted around the area where the juveniles were initially deposited. This comparison showed that M. incognita juveniles in a narrow, fine network tended to migrate actively and, in contrast, those in a wide, coarse network were prevented from migrating by the network configuration.

Influence of soil conditions, spore densities and nematode age of Pasteuria penetrans attachment to Meloidogyne incognita

Several soil variables as soil texture, moisture and temperature, Pasteuria penetrans spore densities and nematode age were tested in the laboratory for their effects on spore attachment to Meloidogyne incognita juveniles. Percentage of juveniles with endospores attached and number of spores per juvenile increased with concentration of spores applied to soil. In a clay-loam soil, hundred-percent attachments were reached at densities of 5 × 105 spores per g of soil and above. Attachment was greater in loamy-sand than in sandy-loam and clay-loam soils, but lower when soil moisture was under 10% than when it was over 25%. Numbers of juveniles with spores attached were greater when soil temperatures were 25 deg C and 35 deg C than at 15 deg C and lower in 7-30 days old juveniles than in 0-6 days old juveniles. All factors that favored nematode mobility in soil increased Pasteuria spore attachment to M. incognita juveniles.

Salicylic acid-induced suppression of Meloidogyne incognita infestation of okra and cowpea

AbstractSalicylic acid (SA) applied as 10 mM foliar spray to okra (Abelmoschus esculentus) cv. Purbani Kranti and cowpea (Vigna unguiculata) cv. Pusa Ruby plants 24 h before inoculation of roots with Meloidogyne incognita juveniles, reduced infestation. Salicylic acid had no direct influence on plant growth and did not kill nematodes in an in vitro test. It induced increased accumulation of a 14 kDa pathogenesis-related protein (PR-1) in roots of both non-inoculated and inoculated salicylic acid-sprayed okra but not in the treated leaves. Salicylic acid sprays enhanced phenylalanine ammonia lyase (PAL) activity in roots of both non-inoculated and inoculated cowpea, estimated at 15 days after inoculation. Infected roots had higher PAL activity than roots of the corresponding non-inoculated treatments. Both PAL activity and SA-induced resistance gradually declined with plant age after treatment.

Purificação de metabólitos fúngicos com efeitos tóxicos sobre Meloidogyne incognita

Objetivando o desenvolvimento de novas metodologias de controle de fitonematóides, este trabalho buscou purificar as substâncias nematicidas produzidas por Cunninghamella elegans, Fusarium sp., Paecilomyces lilacinus eP. variotii. Esses fungos foram cultivados em meio líquido Czapek-Dox durante 15 dias, a 25 ºC, em agitador orbital. Em seguida, filtraram-se as misturas, o que permitiu a obtenção de soluções que foram concentradas sob vácuo e submetidas à purificação direcionada por testes in vitro com Meloidogyne incognita. Observou-se que os filtrados de P. lilacinus e P. variotii perdiam suas atividades nematicidas após a concentração sob vácuo, sugerindo que as substâncias ativas produzidas por esses fungos são consideravelmente voláteis. Para o filtrado de Fusarium sp., observou-se perda total da atividade contra M. incognita após fracionamento em coluna de sílica gel, indicando instabilidade da substância nematicida frente às condições empregadas.Do filtrado de C. elegans isolou-se uma substância que, em solução aquosa na concentração de 250 ppm, imobilizou 94% dos juvenis do segundo estádio de M. incognita expostos a tal solução durante 48 h.

Combined application of Glomus sp. and Pasteuria penetrans for reducing Meloidogyne incognita(Tylenchida: Meloidogynidae) populations and improving tomato growth.

The beneficial effects of application of spores of the mycorrhizal fungus Glomus sp. and the nematode parasitic bacterium Pasteuria penetrans to Meloidogyne incognita-infested soil on tomato growth and nematode reproduction were assessed in pot and microplot experiments. Tomato shoot and fruit weights in M. incognita-infected plants receiving a combined application of Glomus sp. and P. penetrans were greater than in untreated nematode-infected plants. In addition, the use of the mycorrhizal fungus and the nematode parasitic bacterium together conferred greater beneficial effects than their use alone. Final densities of M. incognita juveniles in soil were lower than the control by 40% and 18% in mycorrhizal plants (cvs. Kyoryoku-beiju and Pritz, respectively), by 38% and 53% in P. penetrans application, and by 61% and 57%, when both treatments were applied together. M. incognita did not affect root colonization by Glomus sp. when mycorrhizal inoculum was added two or three weeks before nematode inoculum. No significant effects of the mycorrhizal treatment on P. penetrans attachment to M. incognita or number of females infected by P. penetrans were observed.

A Technique for Screening Grape Germplasm for Resistance to Meloidogyne incognita.

A technique to evaluate the root-knot nematode resistance of grape seedlings was developed. Seedlings of rootstock crosses and nematode-susceptible Vitis vinifera cvs. Colombard and Carignane were inoculated with Meloidogyne incognita juveniles. Reproduction of nematodes on individual plants was measured by counting the number of egg masses stained with eosin and the number of eggs present. Egg mass counts were highly correlated with egg counts. Resistant and susceptible cultivars could be clearly distinguished by the number of egg masses produced on vegetatively propagated cuttings. It is concluded that egg mass counting can substitute for the more laborious and time-consuming methods of counting nematode eggs or juveniles in the evaluation of root-knot nematode resistance in Vitis.

Interactions between Meloidogyne incognita and endophytic bacteria in cotton and cucumber

Greenhouse studies with cotton and cucumber were made to determine the effects of inoculation of the parasitic nematode Meloidogyne incognita on population dynamics of indigenous bacterial endophytes and introduced endophytic bacterial strains JM22 ( Enterobacter asburiae) and 89B-61 ( Pseudomonas fluorescens) applied as seed treatments. Internal communities of endophytic bacteria in roots were generally largest in the presence of M. incognita. Recovery of JM22 from cucumber roots was positively, but not significantly, associated with soilborne nematode inoculum size, except at 2 weeks after inoculation. The internal populations of 89B-61 applied to seed also increased with nematode applications. The diversity of indigenous bacterial endophytes changed within 7 d after M. incognita inoculation. Species richness and diversity of endophytic bacteria were slightly, but not significantly, greater for nematode-infested plants than for non-infested plants. Alcaligenes piechaudii and Burkholderia pickettii occurred only in nematode-infested plants, whereas Brevundimonas vesicularis was mainly isolated from nematode-free plants. Agrobacterium radiobacter and Pseudomonas spp. were the most common taxa found in both treatments, accounting for a total of 41% and 37% of the community for non-inoculated and inoculated plants, respectively. JM22 colonized cotton roots internally and was also found in high numbers on the root surface around nematode penetration sites and on root galls where the root tissue had been disruptured due to gall enlargement. Single cells of JM22 were attached to the cuticle of M. incognita juveniles. Endophytic bacteria and M. incognita form complex associations, and an understanding of these associations will aid efforts to develop and manage microbial communities of endophytic bacteria for practical use as biocontrol agents against plant-parasitic nematodes and soil-borne pests and pathogens.

Differences in survival time of Meloidogyne incognita juveniles inside and outside of soil aggregates in an Andosol

Differences in survival time of Meloidogyne incognita juveniles inside and outside of soil aggregates in an Andosol

Influence of size of soil aggregates on the survival of Meloidogyne incognita juveniles

Influence of size of soil aggregates on the survival of Meloidogyne incognita juveniles

Toxicity of fungal endophyte secondary metabolites to plant parasitic nematodes and soil-borne plant pathogenic fungi

Fungi isolated from the cortical tissue of surface sterilized tomato roots collected from field plots produced secondary metabolites in nutrition broth that were highly toxic toMeloidogyne incognita. Especially strains ofFusarium oxysporum were highly active with 13 of 15 strains producing culture filtrates toxic to nematodes. The mechanism of action of the toxic metabolites produced by the non-pathogenicF. oxysporum strain 162 with proven biological control ofM. incognita in pot experiments was investigated. These metabolites reducedM. incognita mobility within 10 min of exposure. After 60 min, 98% of juveniles were inactivated. Juveniles were initially inactivated within a few minutes of exposure, but with exposure of 5 h 50% of the juveniles were dead and 24 h exposure resulted in 100% mortality. In a bioassay with lettuce seedlings metabolite concentrations > 100 mg/l reduced the number ofM. incognita juveniles on the roots comparing to the water control. TheF. oxysporum toxins were highly effective towards sedentary parasites and less effective towards migratory endoparasites. Nonparasitic nematodes were not influenced at all. Metabolites of strain 162 also reduced significantly the growth ofPhytophthora cactorum, Pythium ultimum andRhizoctonia solani in vitro. Secondary metabolites of endophytic fungi on plant-parasitic nematodes and soil-borne fungi should be considered for control of plant parasitic nematodes and plant pathogenic fungi. The results also show the need for proper selection of target nematodes inin vitro bioassays.

Alginate films for delivery of root-knot nematode inoculum and evaluation of microbial interactions

A method was developed for utilizing alginate films to deliver inoculum into soil and evaluate microbial antagonistic activity against nematode eggs. Eggs of Meloidogyne incognita were harvested from galled tomato roots (Lycopersicon esculentum), surface disinfested, suspended in 2% (w/v) aqueous sodium alginate, and applied to 2.5 × 5.0 cm polyvinyl chloride coated fiberglass screens (1.5 mm2 mesh size) at a uniform thickness of 0.5 mm. The alginate solution was gelled by dipping in 0.25 M CaCl2. Films containing eggs were observed in vitro and egg development was evaluated. The number of immature eggs and eggs with first stage juveniles declined linearly over time while the number of empty eggs shells, and hatched juveniles increased over time, indicating that the alginate gel did not inhibit development and motility of M. incognita juveniles. In a greenhouse experiment using cucumber (Cucumis sativus) the number of galls g-1 root was correlated with the number of eggs in alginate films placed in each pot at planting. Films containing M. incognita eggs were buried in field soil containing organic amendments, incubated, removed from soil, rinsed with water, and observed. The number of immature eggs in grids from soil amended with chitin or flax seed meal were lower than in untreated soil; percent parasitized eggs was also greater in films from amended soil than from untreated soil.

Observations on the pathogenicity of Pasteuria penetrans, a parasite of root‐knot nematodes

SUMMARYA simple test was used to determine whether or not Pasteuria penetrans spores would attach to 17 species of nematodes. All susceptible individuals had spores attached to their cuticles after 24 h of gentle agitation in suspensions containing 105spores/ml. Spores of P. penetrans from six populations of Meloidogyne only adhered to species of Meloidogyne and they adhered in greatest numbers to the species from which they had been originally isolated. Sonication of spores from infected females increased attachment but the effect was dependent on pH and whether the test was conducted in tap or distilled water. Invasion of tomato roots was reduced by up to 86% when, rather than using healthy juveniles, second‐stage juveniles bearing 15 or more spores were added to soil at high densities (1000 or 3000/plant); at low densities (500/plant) invasion was not significantly affected. The rate of development of M. incognita juveniles infected with P. penetrans was slower than that of healthy juveniles. The numbers of second‐generation of M. incognita were reduced by 82–93% when juveniles encumbered with 1–15 spores were added to soil instead of those bearing no spores. Pasteuria penetrans populations differed in their aggressiveness and when juveniles encumbered with the same number of spores from two populations were added to soil there were differences in the numbers of females that became infected. The implications of these results for the development of P. penetrans as a biological control agent are discussed.

Differences in Resistant Capsicum Annuum Attacked By Different Meloidogyne Species

A comparison of the responses of two resistant lines of Capsicum annuum with those of a cultivar Doux Long des Landes susceptible to infection by the root-knot nematodes Meloidogyne incognita, M. arenaria, M. javanica and M. sp. population, was carried out under greenhouse conditions (max. 30°C). Fewer M. incognita juveniles penetrated the roots of the resistant peppers, especially those of PM 687. In this line the necrotic reaction arose immediately after nematodes penetrated the roots. When PM 217 was infected with M. incognita or some M. arenaria, occasionally imperfect giant cells developed with which a few immature females were sometimes associated. PM 687 is susceptible to the Seville population while 217 is highly resistant. Close correlations were found between these observations and those obtained with five genes previously identified as conferring resistance to root-knot nematodes.

Improved Techniques for Measuring the Co2 Exchange Rate of Meloidogyne Infected Bean Plants

An improved open system that measures CO 2 exchange rate of up to 20 plants per day is described. The CO 2 exchange rate was determined in experiments using Phaseolus vulgaris inoculated with one of four inoculum levels of Meloidogyne incognita juveniles. Measurements were taken at intervals up to 27 d after inoculation using either a destructive or a non-destructive assay. The normal pattern of photosynthetic rate was decreased in nematode-infected plants relative to the controls from 3 d after inoculation. Dark respiration rates significantly increased over controls only at 3 d after nematode inoculation. The change in photosynthetic and dark respiration pattern of beans after nematode inoculation is discussed in relation to the ontogenetic development of the host and nematode. The advantages of this improved open system in facilitating an understanding of the mechanisms by which nematodes affect CO 2 exchange rate are identified.

Effect of Crotalaria Spectabilis and Two Nematicides, On Numbers of Meloidogyne Incognita and Helicotylenchus Dihystera

An experiment in an infested field indicated that Crotalaria spectabilis at 17 or 34 plants/m2, Nemagon 20G (granular formulation with 20% dibromochloropropane) at 3, 6, or 9 g/m2, or Basamid (powder formulation with 85 % dazomet at 25, 50 or 75 g/m2, decreased numbers of Meloidogyne incognita juveniles by 90% within 2 months. During the first 2 months after planting, C. spectabilis did not decrease numbers of Helicotylenchus dihystera, whereas Basamid at all three rates and Nemagon at 6 and 9 g/m2 decreased numbers by approximately 90%. Nemagon at 3 g/m2 gave less than 50% control of H. dihystera. Okra (Hibiscus esculentus) grown for 6 months on land previously treated with Basamid or Nemagon doubled numbers of M. incognita compared with untreated land planted with the same crop. No such increase occurred in land planted with C. spectabilis for 2 months followed by okra. Okra had no effect on H. dihystera whereas M. incognita was scarce in soil where C. spectabilis had been grown for 8 months.

Behaviour of the systemic nematicide oxamyl in plants in relation to control of invasion and development of Meloidogyne incognita

SUMMARYA single foliar application of oxamyl (12.5 μg) in acetone significantly reduced invasion of cucumber seedlings by Meloidogyne incognita juveniles for at least 21 days but did not affect the early stages of development of juveniles which had already invaded the roots. In contrast, application of oxamyl to the roots significantly reduced both invasion and development of juveniles. Concurrent studies using radiolabelled oxamyl showed that the amount of toxicant in the roots after 3 days was 13 times greater following root application than after foliar treatment.It is probable that oxamyl concentrates at the sites of nematode attack as an overall concentration of only 3 ng oxamyl g‐1 root was sufficient to prevent invasion. Much greater concentrations than this were required to affect the nematode in vitro. Oxamyl appeared to be lost from the roots into the soil principally in the form of its non‐toxic oxime and it is suggested that the site of action following foliar application is at the root surface or outer cortex. Studies on the invasion behaviour of M. incognita juveniles on agar showed that the action of oxamyl had a sensory component.