Showing papers on "Meloidogyne incognita published in 2013"

Diversity and Evolution of Root-Knot Nematodes, Genus Meloidogyne: New Insights from the Genomic Era

Abstract: Root-knot nematodes (RKNs) (Meloidogyne spp.) are obligate endoparasites of major worldwide economic importance. They exhibit a wide continuum of variation in their reproductive strategies, ranging from amphimixis to obligatory mitotic parthenogenesis. Molecular phylogenetic studies have highlighted divergence between mitotic and meiotic parthenogenetic RKN species and probable interspecific hybridization as critical steps in their speciation and diversification process. The recent completion of the genomes of two RKNs, Meloidogyne hapla and Meloidogyne incognita, that exhibit striking differences in their mode of reproduction (with and without sex, respectively), their geographic distribution, and their host range has opened the way for deciphering the evolutionary significance of (a)sexual reproduction in these parasites. Accumulating evidence suggests that whole-genome duplication (in M. incognita) and horizontal gene transfers (HGTs) represent major forces that have shaped the genome of current RKN species and may account for the extreme adaptive capacities and parasitic success of these nematodes.

Mycorrhiza-induced resistance against the root–knot nematode Meloidogyne incognita involves priming of defense gene responses in tomato

Abstract: Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen); Short Term Scientific Mission (STSM) grant from EU-COST action 872 Exploiting Genomics to understand Plant-Nematode Interactions

Utility of Host Delivered RNAi of Two FMRF Amide Like Peptides, flp-14 and flp-18, for the Management of Root Knot Nematode, Meloidogyne incognita

Abstract: Root knot nematode, Meloidogyne incognita, is an obligate sedentary endoparasite that infects a large number of crop species and causes substantial yield losses. Non-chemical based control strategies for these nematodes are gaining importance. In the present study, we have demonstrated the significance of two FMRFamide like peptide genes (flp-14 and flp-18) for infection and development of resistance to M. incognita through host-derived RNAi. The study demonstrated both in vitro and in planta validation of RNAi-induced silencing of the two genes cloned from J2 stage of M. incognita. In vitro silencing of both the genes interfered with nematode migration towards the host roots and subsequent invasion into the roots. Transgenic tobacco lines were developed with RNAi constructs of flp-14 and flp-18 and evaluated against M. incognita. The transformed plants did not show any visible phenotypic variations suggesting the absence of any off-target effects. Bioefficacy studies with deliberate challenging of M. incognita resulted in 50-80% reduction in infection and multiplication confirming the silencing effect. We have provided evidence for in vitro and in planta silencing of the genes by expression analysis using qRT-PCR. Thus the identified genes and the strategy can be used as a potential tool for the control of M. incognita. This is the first ever report that has revealed the utility of host delivered RNAi of flps to control M. incognita. The strategy can also be extended to other crops and nematodes.

The 8D05 parasitism gene of Meloidogyne incognita is required for successful infection of host roots.

Abstract: Parasitism genes encode effector proteins that are secreted through the stylet of root-knot nematodes to dramatically modify selected plant cells into giant-cells for feeding. The Mi8D05 parasitism gene previously identified was confirmed to encode a novel protein of 382 amino acids that had only one database homolog identified on contig 2374 within the Meloidogyne hapla genome. Mi8D05 expression peaked in M. incognita parasitic second-stage juveniles within host roots and its encoded protein was limited to the subventral esophageal gland cells that produce proteins secreted from the stylet. Constitutive expression of Mi8D05 in transformed Arabidopsis thaliana plants induced accelerated shoot growth and early flowering but had no visible effects on root growth. Independent lines of transgenic Arabidopsis that expressed a double-stranded RNA complementary to Mi8D05 in host-derived RNA interference (RNAi) tests had up to 90% reduction in infection by M. incognita compared with wild-type control plants, suggesting that Mi8D05 plays a critical role in parasitism by the root-knot nematode. Yeast two-hybrid experiments confirmed the specific interaction of the Mi8D05 protein with plant aquaporin tonoplast intrinsic protein 2 (TIP2) and provided evidence that the Mi8D05 effector may help regulate solute and water transport within giant-cells to promote the parasitic interaction.

Nematicidal activity of chaetoglobosin A poduced by Chaetomium globosum NK102 against Meloidogyne incognita.

Abstract: The nematicidal activity of Chaetomium globosum NK102, culture filtrates, and chaetoglobosin A (ChA) purified by HPLC was evaluated on Meloidogyne incognita . The results showed that C. globosum NK102 significantly repelled second-stage juveniles (J2s). Both filtrates and ChA demonstrated strong adverse effects on J2 mortality with 99.8% at 300 μg ChA/mL (LC(50) = 77.0 μg/mL) at 72 h. ChA and filtrates did not affect egg hatch until 72 h of exposure. All filtrate treatments inhibited the J2 penetration even in 12.5% dilution treatment. Similarly, ChA (300 and 30 μg/mL) showed a significant inhibitory effect on J2 penetration. The number of eggs per plant was significantly reduced in the treatment of 30 mg ChA/kg soil by 63% relative to control plants, indicating the apparent negative effect on reproduction of M. incognita. The study demonstrated the nematicidal activity of ChA and suggested that it could be a potential biocontrol agent for integrated management of M. incognita.

Nematicidal activity of mint aqueous extracts against the root-knot nematode Meloidogyne incognita

Abstract: The nematicidal activity and chemical characterization of aqueous extracts and essential oils of three mint species, namely, Mentha × piperita , Mentha spicata , and Mentha pulegium , were investigated The phytochemical analysis of the essential oils was performed by means of GC-MS, whereas the aqueous extracts were analyzed by LC-MS The most abundant terpenes were isomenthone, menthone, menthol, pulegone, and carvone, and the water extracts yielded mainly chlorogenic acid, salvianolic acid B, luteolin-7-O-rutinoside, and rosmarinic acid The water extracts exhibited significant nematicidal activity against Meloidogyne incognita , and the EC50/72h values were calculated at 1005, 745, and 300 mg/L for M × piperita, M pulegium, and M spicata, respectively Only the essential oil from M spicata showed a nematicidal activity with an EC50/72h of 358 mg/L Interestingly, menthofuran and carvone showed EC50/48h values of 127 and 730 mg/L, respectively On the other hand, salicylic acid, isolated in the aqueous extracts, exhibited EC50 values at 24 and 48 h of 298 ± 92 and 288 ± 79 mg/L, respectively

Chemical Composition and Nematicidal Activity of Essential Oil of Agastache rugosa against Meloidogyne incognita

Abstract: The aim of this research was to determine the chemical composition and nematicidal activity of essential oil of Agastache rugosa flowering aerial parts against the root knot nematode, Meloidogyne incognita, and to isolate and identify any nematicidal constituents from the essential oil. The essential oil of A. rugosa aerial parts was obtained by hydrodistillation and analyzed by GC-FID and GC-MS. A total of 37 components of the essential oil were identified, with the principal compounds being methyleugenol (50.51%), estragole (8.55%), and eugenol (7.54%), followed by thymol (3.62%), pulegone (2.56%), limonene (2.49%) and caryophyllene (2.38%). Based on bioactivity-guided fractionation, the three active constituents were isolated from the essential oil and identified as methyleugenol, estragole and eugenol. The essential oil of A. rugosa exhibited strong nematicidal activity against M. incognita, with a LC50 value of 47.3 μg/mL. The components eugenol (LC50 = 66.6 μg/mL) and methyleugenol (LC50 = 89.4 μg/mL) exhibited stronger nematicidal activity against M. incognita (LC50 = 185.9 μg/mL). The results indicate that the essential oil of A. rugosa aerial parts and its constituent compounds have potential for development into natural nematicides for control of the root knot nematode.

Knocking-Down Meloidogyne incognita Proteases by Plant-Delivered dsRNA Has Negative Pleiotropic Effect on Nematode Vigor

Abstract: The root-knot nematode Meloidogyne incognita causes serious damage and yield losses in numerous important crops worldwide. Analysis of the M. incognita genome revealed a vast number of proteases belonging to five different catalytic classes. Several reports indicate that M. incognita proteases could play important roles in nematode parasitism, besides their function in ordinary digestion of giant cell contents for feeding. The precise roles of these proteins during parasitism however are still unknown, making them interesting targets for gene silencing to address protein function. In this study we have knocked-down an aspartic (Mi-asp-1), a serine (Mi-ser-1) and a cysteine protease (Mi-cpl-1) by RNAi interference to get an insight into the function of these enzymes during a host/nematode interaction. Tobacco lines expressing dsRNA for Mi-ser-1 (dsSER), Mi-cpl-1 (dsCPL) and for the three genes together (dsFusion) were generated. Histological analysis of galls did not show clear differences in giant cell morphology. Interestingly, nematodes that infected plants expressing dsRNA for proteases produced a reduced number of eggs. In addition, nematode progeny matured in dsSER plants had reduced success in egg hatching, while progeny resulting from dsCPL and dsFusion plants were less successful to infect wild-type host plants. Quantitative PCR analysis confirmed a reduction in transcripts for Mi-cpl-1 and Mi-ser-1 proteases. Our results indicate that these proteases are possibly involved in different processes throughout nematode development, like nutrition, reproduction and embryogenesis. A better understanding of nematode proteases and their possible role during a plant-nematode interaction might help to develop new tools for phytonematode control.

Potent Nematicidal Activity of Phthalaldehyde, Salicylaldehyde, and Cinnamic Aldehyde against Meloidogyne incognita

Abstract: The nematicidal activity of selected aromatic aldehydes was tested against the root knot nematode Meloidogyne incognita. The most active aldehyde was phthalaldehyde (1) with an EC(50) value of 11 ± 6 mg/L followed by salicylaldehyde (2) and cinnamic aldehyde (3) with EC(50) values of 11 ± 1 and 12 ± 5 mg/L, respectively. On the other hand, structurally related aldehydes such as 2-methoxybenzaldehyde (21), 3,4-dimethoxybenzaldehyde, and vanillin (23) were not active at the concentration of 1000 mg/L. By liquid chromatography-mass spectrometry the reactivity of tested aldehydes against a synthetic peptide resembling the nematode cuticle was characterized. At the test concentration of 1 mM, the main adduct formation was observed for 3,4-dihydroxybenzaldehyde (22), 2-methoxybenzaldehyde (21), and 3,4-dimethoxybenzaldehyde. Considering that 2-methoxybenzaldehyde (21) and 3,4-dimethoxybenzaldehyde were not active against M. incognita in in vitro experiments led us to hypothesize a different mechanism of action rather than an effect on the external cuticle modification of nematodes. When the toxicity of the V-ATPase inhibitor pyocyanin (10) was tested against M. incognita J2 nematodes, an EC(50) at 24 h of 72 ± 25 mg/L was found. The redox-active compounds such as phthalaldehyde (1) and salicylaldehyde (2) may share a common mode of action inhibiting nematode V-ATPase enzyme. The results of this investigation reveal that aromatic redox-active aldehydes can be considered as potent nematicides, and further investigation is needed to completely clarify their mode of action.

Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita

Abstract: The potential of 24 indigenous isolates of Purpureocillium lilacinum (Paecilomyces lilacinus) (Thom) Samson collected from different agro-climatic zones of India was investigated against the root-knot nematode, Meloidogyne incognita. The studies were conducted in vitro (larvicidal, ovicidal and egg-parasitising capacity) and under naturally infested field conditions with selected strains. Repeated field trials were conducted with talc-based preparations of fungal strains at 10 kg ha−1, which were applied mixed in farm yard manure (FYM) at 1.5 t ha−1. Results (in vitro) showed that all tested isolates were capable to parasitise eggs, inhibit egg hatching and cause juvenile mortality of M. incognita at various levels. Based on the performance under in vitro studies, eight isolates (NDPL-01, ANDPL-02, SHGPL-03, HYBPL-04, AHDPL-05, PTNPL-06, SNGPL-07 and VNSPL-08) were re-tested to confirm the results. HYBDPL-04 was found causing highest mortality (80%), inhibition of egg hatching (90%) as well as parasitisat...

Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes

Abstract: The gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling. We expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN. Our experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

The amino acid permeases AAP3 and AAP6 are involved in root-knot nematode parasitism of Arabidopsis

Abstract: The root-knot nematode, Meloidogyne incognita, is an obligate parasite which depends entirely on the host plant for its nutrition. Root-knot nematodes induce the formation of a highly specialized feeding site consisting of several giant cells surrounded by a network of vascular tissues. Nutrients, including amino acids and sugars, are transferred apoplastically from the vascular tissues to the feeding site. Using Arabidopsis thaliana lacking the vascular-expressed amino acid permeases (AAP) AAP3 or AAP6, we demonstrate that disruption of amino acid transport can affect nematode parasitism. Nematode infestation levels are significantly reduced on the aap3 and aap6 mutants. AAP3 and AAP6 act distinctly in the transport of amino acids to the feeding site, as demonstrated by differences in their carrying capacity profiles. Furthermore, analyses of promoter:β-glucuronidase lines show different expression patterns for AAP3 and AAP6 in infected roots. In the aap3-3 mutant, part of the decrease in infestation is connected to a defect in early infection, where juveniles enter but then leave the root. Both aap3-3 and aap6-1 produce fewer females and produce more adult male nematodes. Additionally, detrimental effects are observed in the nematodes harvested from aap3-3 and aap6-1 mutants, including decreased egg hatching and infectivity and lower levels of lipid reserves. The transport of amino acids by AAP3 and AAP6 is important for nematode infection and success of the progeny.

Solanum torvum responses to the root-knot nematode Meloidogyne incognita

Abstract: Solanum torvum Sw is worldwide employed as rootstock for eggplant cultivation because of its vigour and resistance/tolerance to the most serious soil-borne diseases as bacterial, fungal wilts and root-knot nematodes. The little information on Solanum torvum (hereafter Torvum) resistance mechanisms, is mostly attributable to the lack of genomic tools (e.g. dedicated microarray) as well as to the paucity of database information limiting high-throughput expression studies in Torvum. As a first step towards transcriptome profiling of Torvum inoculated with the nematode M. incognita, we built a Torvum 3’ transcript catalogue. One-quarter of a 454 full run resulted in 205,591 quality-filtered reads. De novo assembly yielded 24,922 contigs and 11,875 singletons. Similarity searches of the S. torvum transcript tags catalogue produced 12,344 annotations. A 30,0000 features custom combimatrix chip was then designed and microarray hybridizations were conducted for both control and 14 dpi (day post inoculation) with Meloidogyne incognita- infected roots samples resulting in 390 differentially expressed genes (DEG). We also tested the chip with samples from the phylogenetically-related nematode-susceptible eggplant species Solanum melongena. An in-silico validation strategy was developed based on assessment of sequence similarity among Torvum probes and eggplant expressed sequences available in public repositories. GO term enrichment analyses with the 390 Torvum DEG revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no GO term enrichment was found with eggplant DEG. The genes identified from S. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism. By combining 454 pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species. In addition, the development of an in silico validation strategy allowed to further extend the use of the custom chip to a related species and to assess by comparison the expression of selected genes without major concerns of artifacts. The expression profiling of S. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance. The availability of the long sequence tags in S. torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for exploitation of these resistance mechanisms in other species.

Toxicity of silver, titanium and silicon nanoparticles on the root-knot nematode, Meloidogyne incognita, and growth parameters of tomato

Abstract: The toxicity of three nanoparticles, silver (AgNP, 20 nm), silicon oxide (SiO2NP, 11-14 nm) and titanium oxide (TiO2NP, 20 nm), to the root-knot nematode, Meloidogyne incognita, was studied in laboratory and pot experiments. Application rates of all nanoparticles were 1.5, 3, 6, 12.5, 25, 50, 100, 200, 400 and 800 mg nanoparticles (ml water)−1 for laboratory experiment to determine their effects on motility and mortality of second-stage juveniles (J2). In pot experiment, 0.02, 0.01, 0.005, 0.0025, 0.00125 and 0.0007% (w/w) of nano-silver and nano-titanium were applied to examine effects on nematode activity and plant growth components of tomato (Solanum lycopersicum). Results showed 100% J2 immobility and mortality in treatments of 800, 400 and 200 mg ml−1 of AgNP; the LC50 for AgNP was 100 mg ml−1. J2 mortality was 48, 16 and 7.3% in 100, 50 and 25 mg ml−1 of AgNP, respectively. J2 mortality was 4.3 and 2% in 800 and 400 mg ml−1 of TiO2NP, respectively. No J2 mortality was seen in all other treatments after 24 h exposure. In a pot experiment, all treatments of AgNP and 0.02% TiO2NP completely controlled M. incognita. Treatments of 0.02, 0.01 and 0.005% of AgNP as well as 0.02% of TiO2NP were toxic to tomato plants and significantly reduced tomato root and stem length and fresh weights in comparison to control.

Validation of reference genes aiming accurate normalization of qPCR data in soybean upon nematode parasitism and insect attack

Abstract: Background: Soybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode (Meloidogyne incognita) parasitism and velvetbean caterpillar (Anticarsia gemmatalis) attack. Findings: The expression stability of nine classical reference genes (GmCYP2, GmELF1A, GmELF1B, GmACT11, GmTUB, GmTUA5, GmG6PD, GmUBC2 and GmUBC4) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with M. incognita and leaves attacked by A. gemmatalis. Two different algorithms (geNorm and NormFinder) were used to determine expression stability. GmCYP2 and GmUBC4 are the most stable in different organs. Considering the developmental stages, GmELF1A and GmELF1B genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using GmUBC4, GmUBC2, GmCYP2 and GmACT11 as reference genes. Our data indicate that both GmELF1A and GmTUA5 are the most stable reference genes for data normalization obtained from soybean roots infected with M. incognita, and GmCYP2 and GmELF1A are the most stable in soybean leaves infested with A. gemmatalis. Conclusions: Future expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

Biocontrol potential of Pasteuria penetrans, Pochonia chlamydosporia, Paecilomyces lilacinus and Trichoderma harzianum against Meloidogyne incognita in okra

Abstract: The root-knot nematode, Meloidogyne incognita , is a sedentary endoparasitic plant pathogen with a very wide host range, which causes annual crop losses amounting to millions of dollars. The small number of available nematicides and restrictions on the use of non-fumigant nematicides due to high toxicity to humans and non-target organisms hinder effective nematode control. A possible alternative to chemical nematicides is the use of biological control agents for the management of this nematode. In the present study, the efficacy of four biocontrol agents was tested against M. incognita at different doses. The biocontrol agents Pasteuria penetrans , Pochonia chlamydosporia , Paecilomyces lilacinus and Trichoderma harzianum were mass produced and mixed with the formalin sterilized soil at the rates of 2 × 103, 4 × 103, 6 × 103, 8 × 103, and 1 × 104 endospores/chlamydospores/cfu per g of soil. Okra seeds (cv. Sabz Pari) were sown in pots of soil amended with the different agents, and 10 d after emergence, the plants were inoculated with 2000 freshly hatched second stage juveniles of M. incognita. Data on plant growth parameters and nematode infestations were recorded 7 weeks after inoculation. The antagonists varied significantly in enhancing various growth parameters and reducing nematode infestations in a dose-responsive manner. Both P. penetrans and P. lilacinus were equally effective and caused maximum reductions in number of galls, egg masses, nematode fecundity and build up as compared with T. harzianum and P. chlamydosporia . Reductions in these parameters at the concentration of 8 × 103 were statistically similar with those caused at the concentration of 1 × 104 chlamydospores/ endospores/cfu. Our results indicate that application of antagonists can suppress galling and reproduction of M. incognita resulting in enhancement of plant growth.

Nematicidal activity of allylisothiocyanate from horseradish (Armoracia rusticana) roots against Meloidogyne incognita.

Abstract: In recent years, there has been a great development in the search for new natural pesticides for crop protection aiming a partial or total replacement of currently used chemical nematicides. Glucosinolate breakdown products are volatile and are therefore good candidates for nematodes fumigants. In this article, the methanol-aqueous extract (1:1, w/v) of horseradish (Armoracia rusticana) fresh roots (MAH) was in vitro tested for nematicidal activity against second stage (J2) Meloidogyne incognita. The EC50 of MAH after 3 days of J2 immersion in test solutions was 251 ± 46 mg/L. The chemical composition analysis of the extract carried out by the GC-MS technique showed that allylisothicyanate was the most abundant compound. This pure compound induced J2 paralysis with an EC50 of 52.6 ± 45.6 and 6.6 ± 3.4 mg/L after 1 h and 3 days of incubation. The use of LC-MS/MS showed for the first time that horseradish root is rich in polyphenols. The study of isothiocyanate degradation in soil showed that allylisothiocyanate was the most quickly degradable compound (half-life <10 min), whereas no significant differences in half-life time were noted between degradation in regular and autoclaved soil.

Post-infection development of Meloidogyne incognita on tomato treated with the endophytes Fusarium oxysporum strain Fo162 and Rhizobium etli strain G12

Abstract: The endophytic fungus Fusarium oxysporum strain Fo162 and the endophytic bacterium Rhizobium etli strain G12 have been shown to enhance plant resistance toward the root-knot nematode Meloidogyne incognita. The individual inoculation of tomato seedlings with these antagonists lead to significant reductions in the number of juveniles that penetrated the root and ultimately the number of galls and egg-masses produced. The present study determined the influence of Fo162 and G12 root colonization on juvenile development inside the root system over time after a synchronized nematode infection. The results showed that 14 and 21 days after nematode inoculation, the development into the third-stage juvenile as well as into the adult-stage was significantly lower in endophyte-treated plants when compared to the untreated control, respectively. In addition, Fo162 and G12 treatment led to a significant reduction in the number of eggs per female 35 days after nematode inoculation. The results demonstrated that both Fo162 and G12 not only reduce M. incognita root penetration, but also reduce their development and reproduction.

Ectopic expression of Kip-related proteins restrains root-knot nematode-feeding site expansion.

Abstract: The development of nematode feeding sites induced by root-knot nematodes involves the synchronized activation of cell cycle processes such as acytokinetic mitoses and DNA amplification. A number of key cell cycle genes are reported to be critical for nematode feeding site development. However, it remains unknown whether plant cyclin-dependent kinase (CDK) inhibitors such as the Arabidopsis interactor/inhibitor of CDK (ICK)/Kip-related protein (KRP) family are involved in nematode feeding site development. This study demonstrates the involvement of Arabidopsis ICK2/KRP2 and ICK1/KRP1 in the control of mitosis to endoreduplication in galls induced by the root-knot nematode Meloidogyne incognita. Using ICK/KRP promoter-GUS fusions and mRNA in situ hybridizations, we showed that ICK2/KRP2, ICK3/KRP5 and ICK4/KRP6 are expressed in galls after nematode infection. Loss-of-function mutants have minor effects on gall development and nematode reproduction. Conversely, overexpression of both ICK1/KRP1 and ICK2/KRP2 impaired mitosis in giant cells and blocked neighboring cell proliferation, resulting in a drastic reduction of gall size. Studying the dynamics of protein expression demonstrated that protein levels of ICK2/KRP2 are tightly regulated during giant cell development and reliant on the presence of the nematode. This work demonstrates that impeding cell cycle progression by means of ICK1/KRP1 and ICK2/KRP2 overexpression severely restricts gall development, leading to a marked limitation of root-knot nematode development and reduced numbers of offspring.

Transcriptional changes of the root-knot nematode Meloidogyne incognita in response to Arabidopsis thaliana root signals.

Abstract: Root-knot nematodes are obligate parasites that invade roots and induce the formation of specialized feeding structures. Although physiological and molecular changes inside the root leading to feeding site formation have been studied, very little is known about the molecular events preceding root penetration by nematodes. In order to investigate the influence of root exudates on nematode gene expression before plant invasion and to identify new genes potentially involved in parasitism, sterile root exudates from the model plant Arabidopsis thaliana were produced and used to treat Meloidogyne incognita pre-parasitic second-stage juveniles. After confirming the activity of A. thaliana root exudates (ARE) on M. incognita stylet thrusting, six new candidate genes identified by cDNA-AFLP were confirmed by qRT-PCR as being differentially expressed after incubation for one hour with ARE. Using an in vitro inoculation method that focuses on the events preceding the root penetration, we show that five of these genes are differentially expressed within hours of nematode exposure to A. thaliana roots. We also show that these genes are up-regulated post nematode penetration during migration and feeding site initiation. This study demonstrates that preceding root invasion plant-parasitic nematodes are able to perceive root signals and to respond by changing their behaviour and gene expression.

THE INFLUENCE OF ENDOPHYTIC BACTERIA ON Meloidogyne incognita INFECTION AND TOMATO PLANT GROWTH

Abstract: Plant parasitic nematodes cause significant damage and losses to vegetable crops. Control of plant parasitic nematodes with pesticides is often restricted due to their high toxicity and negative impact on the environment. The need for environmentally safe control strategies has increased interest in developing biological control measures. The objective of this research was to study the effect of selected endophytic bacteria, Pantoea agglomerans MK-29, Cedecea davisae MK-30, Enterobacter spp. MK-42 and Pseudomonas putida MT-19 on early root penetration and gall formation of Meloidogyne incognita on tomato. The results showed that application of bacterial endophytes significantly reduced M. incognita infestation on tomato either as a seed treatment, root dipping or as a soil drench application. The four selected endophytic bacteria also significantly reduced early root penetration of Meloidogyne juveniles into tomato roots up to 56 %, when applied as a root dipping and soil drench. Seed treatment of endophytic bacteria followed by soil drench application apparently gave a higher reduction in the number of galls than the single application.

New sources of resistance to Meloidogyne incognita race 3 in wild cotton accessions and histological characterization of the defence mechanisms

Abstract: Accessions of Gossypium spp., some of them never previously tested, were evaluated for resistance to a local isolate of Meloidogyne incognita race 3 in greenhouse experiments. Nematode infection was characterized based on the galling and egg mass indexes and the reproduction factors (RF). Root-knot nematode reproduction among the newly tested accessions varied from highly susceptible (AS0188, AS0189) to moderately resistant (MT123 no. 3), and some accessions showed highly reduced nematode reproduction (CIR1343, CIR1348, Fai Mui). Histological observations of two resistant accessions (G. barbadense CIR1348 and G. hirsutum TX-25, respectively) showed that resistance occurs through a two-stage mechanism in the first accession and through a single-stage mechanism in the second. Parasitism is blocked early after second-stage juvenile (J2) penetration or during its initial tissue migration (CIR1348) and the development of later-stage juveniles into female adults is suppressed at a later stage (TX-25 and CIR1348). Fluorescence and bright light microscopy showed that root cells surrounding nematodes exhibit a hypersensitivity-like reaction, with the accumulation of presumably phenolic compounds and the presence of necrotic cells that limit the development of nematodes and the formation of giant cells. Underdeveloped giant cells with degenerated cytoplasmic content were found in small numbers in CIR1348 and in large numbers in TX-25, along with deformed nematodes. The full characterization of the defence mechanisms of novel sources of resistance to the root-knot nematode in cotton constitutes a first step towards their use in crop improvement. (Resume d'auteur)

Nematicidal activity of nonacosane-10-ol and 23a-homostigmast-5-en-3β-ol isolated from the roots of Fumaria parviflora (Fumariaceae).

Abstract: Two bioactive nematicidal phytochemicals, viz., nonacosane-10-ol and 23a-homostigmast-5-en-3β-ol, were isolated from the n-hexane fraction of the roots of Fumaria parviflora through activity-guided isolation. The structures of the compounds were elucidated using ¹³C and ¹H nuclear magnetic resonance. Activity of the two compounds against eggs and juveniles (J2s) of Meloidogyne incognita was evaluated in vitro at the concentrations of 50, 100, 150, and 200 μg mL⁻¹. Over 120 h of incubation, the cumulative percent mortality and hatch inhibition of both of the compounds tested ranged from 20 to 100% and from 15 to 95.0%, respectively. In pot trials with tomato cultivar Riogrande, the two compounds, applied as soil drenches at the concentrations of 100, 200, and 300 mg/kg, significantly decreased the nematodes and plant growth parameters. Nonacosane-10-ol and 23a-homostigmast-5-en-3β-ol reduced the numbers of galls (42.6 and 60.3), galling index (1.6 and 2.8), females per gram of root (37.3 and 57.0), eggs per gram of root (991.3 and 1273.0), reproduction factor (Rf) (0.1 and 0.2), and fresh root weight (14.33 and 17.0 g) at 300 mg/kg concentration and increased fresh shoot weight (49.0 and 48.4 g), dry shoot weight (28.0 and 25.3 g), and plant height (53.5 and 49.6 cm), respectively. These compounds could provide new insight in the search for novel nematicides against M. incognita.

In vitro and in planta nematicidal activity of Fumaria parviflora (Fumariaceae) against the southern root-knot nematode Meloidogyne incognita

Abstract: Root and stem extracts of Fumaria parviflora showed strong nematicidal activity against Meloidogyne incognita in in vitro and in planta experiments. Phytochemical screening of F. parviflora revealed the presence of seven classes of bioactive compounds (alkaloids, flavonoids, glycosides, tannins, saponins, steroids and phenols). Quantitative determination of the plant extracts showed the highest percentages of alkaloids (0·9 ± 0·04) and saponins (1·3 ± 0·07) in the roots and total phenolic contents in the stem (16·75 ± 0·07 μg dry g ―1 ). The n-hexane, chloroform, ethyl acetate and methanol extracts of roots and stems at concentrations of 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL ―1 , significantly inhibited hatching and increased mortality of second-stage juveniles (J2s) compared with water controls. Percentage J2 mortality and hatch inhibition were directly related to exposure time. In pot trials with tomato cv. Rio Grande, root and stem extracts at concentrations of 1000, 2000 and 3000 ppm, applied as soil drenches, significantly reduced the number of galls, galling index, eggs masses, eggs and reproduction factor compared with the water control. Regardless of concentration, all the extracts significantly increased the host plant growth parameters studied. The n-hexane extracts from the roots and stem were the most active, followed by the methanol ones, at all concentrations. The in vitro and in planta results suggest that extracts from the roots and stem of F. parviflora may be potential novel nematicides.