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Concept: Root-knot nematode

172

Taxonomically restricted genes (TRGs), i.e., genes that are restricted to a limited subset of phylogenetically related organisms, may be important in adaptation. In parasitic organisms, TRG-encoded proteins are possible determinants of the specificity of host-parasite interactions. In the root-knot nematode (RKN) Meloidogyne incognita, the map-1 gene family encodes expansin-like proteins that are secreted into plant tissues during parasitism, thought to act as effectors to promote successful root infection. MAP-1 proteins exhibit a modular architecture, with variable number and arrangement of 58 and 13-aa domains in their central part. Here, we address the evolutionary origins of this gene family using a combination of bioinformatics and molecular biology approaches. Map-1 genes were solely identified in one single member of the phylum Nematoda, i.e., the genus Meloidogyne, and not detected in any other nematode, thus indicating that the map-1 gene family is indeed a TRG family. A phylogenetic analysis of the distribution of map-1 genes in RKNs further showed that these genes are specifically present in species that reproduce by mitotic parthenogenesis, with the exception of M. floridensis, and could not be detected in RKNs reproducing by either meiotic parthenogenesis or amphimixis. These results highlight the divergence between mitotic and meiotic RKN species as a critical transition in the evolutionary history of these parasites. Analysis of the sequence conservation and organization of repeated domains in map-1 genes suggests that gene duplication(s) together with domain loss/duplication have contributed to the evolution of the map-1 family, and that some strong selection mechanism may be acting upon these genes to maintain their functional role(s) in the specificity of the plant-RKN interactions.

Concepts: Gene, Evolution, Biology, Organism, Species, Nematode, Nematodes, Root-knot nematode

170

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Concepts: Parasites, Nematode, Model organism, Nematodes, Plant pathogens and diseases, Pseudomonas syringae, Root-knot nematode, Heterodera

167

Strain HA10002 was isolated from mangrove sediment collected from Dongzhaigang Mangrove Reserve in Hainan, China. It was selected with potent nematicidal activity and was identified as Streptomyces albogriseolus. By bioassay-guided fractionation, a new active component A22-1(S1) against root-knot nematodes was separated from its fermentation broth. On the basis of spectroscopic analyses and comparison with the data from correlative literature, the structure of S1 was established to be 6'-methyl-fungichromin, named as fungichromin B in this paper. The LD50 values of fungichromin B to the 2-stage juveniles of Meloidogyne incognita and Meloidogyne javanica were 7.64 and 7.83 μg/ml, respectively. Further examination demonstrated fungichromin B still showed a wide antifungal spectrum, as with fungichromin.

Concepts: Nematodes, Plant pathogens and diseases, Root-knot nematode, Meloidogyne incognita, Meloidogyne

163

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.

Concepts: Parasites, Nematode, Nematodes, Solanum, Plant pathogens and diseases, Root-knot nematode, Meloidogyne incognita, Meloidogyne

142

Cuticle collagens form a major part of the nematode cuticle and are responsible for maintaining the overall shape of the animal and its protection from the external environment. Although substantial research on cuticle collagen genes has been carried out in Caenorhabditis elegans, their isolation and characterization in plant parasitic nematodes have been limited to a few genes only. In this study, a cuticle collagen gene, Mi-col-5, was isolated from root-knot nematode, Meloidogyne incognita. A partial segment of 402 bp was first cloned and analyzed on Gbrowse followed by subsequent cloning of the 1047 bp long full cDNA specifying the open reading frame. The deduced amino acid sequence showed 92% sequence identity with that of Mj-col-5. However, a transmembrane helix was predicted in Mi-col-5 which was not present in Mj-col-5. The conserved pattern of cysteine residues in Mi-col-5 suggested that it belonged to group 2 of nematode cuticle collagens but with a longer carboxy terminal region as was the case with Mj-col-5. Domain prediction revealed the presence of a nematode cuticle collagen N terminal domain and a pfam collagen domain along with collagen triple helix repeats. A phylogenetic tree based on the amino acid sequences showed evolutionary relationship of Mi-col-5 with cuticle collagens genes of other nematodes. 3D models for Mi-col-5 were predicted with the best confidence score of -2.78. Expression of Mi-col-5 transcript was found to be maximum in egg masses followed by adult females and J2s suggesting its role in the early stages of the development of the nematode during its life cycle.

Concepts: DNA, Protein, Amino acid, Collagen, Caenorhabditis elegans, Nematode, Nematodes, Root-knot nematode

28

Plant-parasitic nematodes secrete so-called effectors into their host plant which are able to suppress the plant’s defence responses, alter plant signalling pathways and, in the case of root knot nematodes, induce the formation of giant cells. Putative effectors have been successfully identified by genomics, transcriptomics and proteomics approaches. In this study, we investigated the transcriptome of the rice root knot nematode Meloidogyne graminicola by 454 sequencing of second-stage juveniles as well as mRNA-seq of rice infected tissue. Over 350 000 reads derived from M. graminicola preparasitic juveniles were assembled, annotated and checked for homologues in different databases. From infected rice tissue, 1.4% of all reads generated were identified as being derived from the nematode. Using multiple strategies, several putative effector genes were identified, both pioneer genes and genes corresponding to already known effectors. To check whether these genes could be involved in the interaction with the plant, in situ hybridization was performed on a selection of genes to localize their expression in the nematode. Most were expressed in the gland cells or amphids of the nematode, confirming possible secretion of the proteins and hence a role in infection. Other putative effectors showed a different expression pattern, potentially linked with the excretory/secretory system. This transcriptome study is a good starting point to functionally investigate novel effectors derived from M. graminicola. This will lead to better insights into the interaction between these nematodes and the model plant rice. Moreover, the transcriptome can be used to identify possible target genes for RNA interference (RNAi)-based control strategies. Four genes proved to be interesting targets by showing up to 40% higher mortality relative to the control treatment when soaked in gene-specific small interfering RNAs (siRNAs).

Concepts: Protein, Gene, Gene expression, Bacteria, RNA, Secretion, Model organism, Root-knot nematode

28

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 (J2). Both filtrates and ChA demonstrated strong adverse effects on J2 mortality with 99.8% at 300 μg ChA /mL (LC50=77.0 μg/mL) at 72 h. ChA and filtrates did not affect egg hatch until 72 h exposure. All filtrate treatment 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 egg per plant was significantly reduced in the treatment of 30 mg ChA per kg soil by 63% relative to control plants, indicating the apparent negative effect on reproduction of M. incognita. The report demonstrated the nematicidal activity of ChA and suggested that it could be a potential biocontrol agent for integrated management of M. incognita.

Concepts: Plant, Effect, Affect, Adverse drug reaction, Negative feedback, Root-knot nematode, Meloidogyne incognita, Meloidogyne

28

In the present study it was evaluated the paralysis activity, of the whole Italian and Algerian Melia azedarach fruits and parts (seeds, wood, kernel), against Meloidogyne incognita second stage juveniles (J2). Paralysis activity was evaluated in vitro after one hour and one day immersion periods of nematodes in test solutions. Phenolic composition of the extracts was elucidated by means of HPLC-DAD and quantification by HPLC-MS/MS. The water extract of the Italian M. azedarach fruit pulp (IPWE) showed significant nematicidal activity (EC50/48h=955 μg/mL) and among its active ingredient components were p-coumaric acid and p-hydroxybenzoic acid (EC50/48h = 840 and 871 μg/mL, respectively). This is the first report of the nematicidal activity of M. azedarach pulp water extract and phenolic acids against the root knot nematode M. incognita.

Concepts: Plant, Nematode, Nematodes, Fruit, Phenols, Root-knot nematode, Meloidogyne incognita, Melia azedarach

27

The nematicidal activity of selected aromatic aldehydes was tested against the root knot nematode Meloidogyne incognita. The most active aldehyde was phthalaldehyde with an EC50 value of 10.59 ± 5.95 mg/L followed by salicylaldehyde and cinnamic aldehyde with an EC50 of 10.80 ± 0.99 and 12.12 ± 5.28 mg/L respectively. On the other hand, structurally related aldehydes such us 2-methoxybenzaldehyde, 3,4-dimethoxybenzaldehyde and vanillin 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. We report that at the test concentration of 1 mM, the main adduct formation was observed for 3,4-diihydroxybenzaldehyde, 2-methoxybenzaldehyde, 3,4-dimethoxybenzaldehyde Considering that 2-methoxybenzaldehyde and 3,4-dimethoxybenzaldehyde were not active against M. incognita in in vitro experiments lead 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 was tested against M. incognita J2 nematodes an EC50 at 24 h of 72.1 ± 25.3 mg/L was found. This toxicity was comparable with those redox-active compounds such us salicylaldehyde and phthalaldehyde suggesting 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.

Concepts: Protein, Nematode, Nematodes, Benzaldehyde, Aldehyde, Root-knot nematode, Meloidogyne incognita, Aldehydes

26

Lysobacter capsici YS1215 isolated from soil previously showed nematicidal potential for biological control of root-knot nematode. In this study, lactic acid, nematicidal compound, was isolated from culture filtrate of the YS1215, and its ovicidal activity was investigated. Purification and identification of lactic acid was performed by series of column chromatographies and identified by (1)H and (13)C-NMR spectrum and GC-MS analysis. Our result showed that bacterial culture filtrate containing lactic acid significantly inhibited egg hatch. The lowest egg hatch rate (5.9%) was found in high concentration (25 μl/ml) of lactic acid at 5 days after incubation, followed by 20 (15.2%), 15 (23.7%), 10 (29.8%) and 5 (36.4%) μl/ml while egg hatch in control (sterile distilled water) was 44.5%. This is the first report of lactic acid as an ovicidal compound and it may be considered as alternative of chemical pesticide against root-knot nematodes.

Concepts: Bacteria, Parasites, Nematode, Nematodes, Lactic acid, Root-knot nematode, Meloidogyne incognita, Meloidogyne