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Morphological and molecular analyses of a Meloidogyne mali population with high intragenomic rRNA polymorphism.

ABSTRACT: Root-knot nematode, Meloidogyne mali can cause damage in trees, shrubs, and herbaceous plants, and was placed on the EPPO Alert List in 2014. In the present study, we report a population isolated from Japanese maple. The recovered population is described by detailed morphological and molecular approaches. The molecular phylogentic analysis based on 28S rRNA, ITS, and mitochondrial COI genes places the population in the clade together with other M. mali sequences available in GenBank. The cloned sequences of the 28S rRNA gene revealed a high intragenomic rRNA polymorphism where the polymorphic copies are spread across M. mali clade. Similarly, we also found high variation in the mitochondrial COI gene. Among four haplotypes in M. mali, three occur in the newly found population. Our study provides the first report of intragenomic polymorphism in M. mali, and the results suggest that intragenomic polymorphism maybe widespread in Meloidogyne.

SUBMITTER: Gu J

PROVIDER: S-EPMC8015333 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Morphological and molecular analyses of a Meloidogyne mali population with high intragenomic rRNA polymorphism.

Gu Jianfeng J Fang Yiwu Y Liu Lele L

Journal of nematology 20201106

Root-knot nematode, Meloidogyne mali can cause damage in trees, shrubs, and herbaceous plants, and was placed on the EPPO Alert List in 2014. In the present study, we report a population isolated from Japanese maple. The recovered population is described by detailed morphological and molecular approaches. The molecular phylogentic analysis based on 28S rRNA, ITS, and mitochondrial COI genes places the population in the clade together with other M. mali sequences available in GenBan ...[more]

PMID: 33829187

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Project description:The discovery of Meloidogyne mayaguensis is confirmed in Florida; this is the first report for the continental United States. Meloidogyne mayaguensis is a virulent species that can reproduce on host cultivars bred for nematode resistance. The perineal patterns of M. mayaguensis isolates from Florida show morphological variability and often are similar to M. incognita. Useful morphological characters for the separation of M. mayaguensis from M. incognita from Florida are the male stylet length values (smaller for M. mayaguensis than M. incognita) and J2 tail length values (greater for M. mayaguensis than M. incognita). Meloidogyne mayaguensis values for these characters overlap with those of M. arenaria and M. javanica from Florida. Enzyme analyses of Florida M. mayaguensis isolates show two major bands (VS1-S1 phenotype) of esterase activity, and one strong malate dehydrogenase band (Rm 1.4) plus two additional weak bands that migrated close together. Their detection requires larger amounts of homogenates from several females. Amplification of two separate regions of mitochondrial DNA resulted in products of a unique size. PCR primers embedded in the COII and 16S genes produced a product size of 705 bp, and amplification of the 63-bp repeat region resulted in a single product of 322 bp. Nucleotide sequence comparison of these mitochondrial products together with sequence from 18S rDNA and ITS1 from the nuclear genome were nearly identical with the corresponding regions from a M. mayaguensis isolate from Mayaguez, Puerto Rico, the type locality of the species. Meloidogyne mayaguensis reproduced on cotton, pepper, tobacco, and watermelon but not on peanut. Preliminary results indicate the M. mayaguensis isolates from Florida can reproduce on tomato containing the Mi gene. Molecular techniques for the identification of M. mayaguensis will be particularly useful in cases of M. mayaguensis populations mixed with M. arenaria, M. incognita, and M. javanica, which are the most economically important root-knot nematode species in Florida, and especially when low (<25) numbers of specimens of these species are recovered from the soil.

Project description:BackgroundIntragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner.MethodsTotal genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5-V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the 'number of AluI bands' and 'type of eletropherogram'.ResultsTwo types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity.ConclusionThe approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

Dataset Information

Morphological and molecular analyses of a Meloidogyne mali population with high intragenomic rRNA polymorphism.

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Morphological and molecular analyses of a <i>Meloidogyne mali</i> population with high intragenomic rRNA polymorphism.

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