The chrysanthemum foliar nematode CFN , Aphelenchoides ritzemabosi , is a plant parasitic nematode that attacks many plants. The CFN with the most shared sequences was B. This research provides comprehensive gene expression information at the transcriptional level, which will facilitate the elucidation of the molecular mechanisms of CFN and the distribution of gene functions at the macro level, potentially revealing improved methods for controlling CFN. The chrysanthemum foliar nematode CFN , Aphelenchoides ritzemabosi Schwartz Steiner and Buhrer, , belongs to Nematoda, Secernentea, Aphelenchida, Aphelenchoididae and is an obligate ecto- and endoparasite of the above-ground parts of plants [ 1 ]. CFN is widely distributed globally and is economically important for chrysanthemums and many other ornamental plants. CFN can also severely damage strawberry [ 2 — 4 ].
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The chrysanthemum foliar nematode CFN , Aphelenchoides ritzemabosi , is a plant parasitic nematode that attacks many plants. The CFN with the most shared sequences was B. This research provides comprehensive gene expression information at the transcriptional level, which will facilitate the elucidation of the molecular mechanisms of CFN and the distribution of gene functions at the macro level, potentially revealing improved methods for controlling CFN.
The chrysanthemum foliar nematode CFN , Aphelenchoides ritzemabosi Schwartz Steiner and Buhrer, , belongs to Nematoda, Secernentea, Aphelenchida, Aphelenchoididae and is an obligate ecto- and endoparasite of the above-ground parts of plants [ 1 ]. CFN is widely distributed globally and is economically important for chrysanthemums and many other ornamental plants. CFN can also severely damage strawberry [ 2 — 4 ]. Next-generation sequencing NGS technology has enabled impressive scientific achievements and novel biological applications.
NGS has also contributed to a massive expansion of transcriptomics in many fields of biology, reducing the cost, time and performance barriers associated with conventional approaches [ 6 , 7 ]. NGS has been used to study several plant parasitic nematodes, including tylench nematodes, Globodera pallida , Heterodera avenae , H. These secreted proteins, which include cell-wall modifying enzymes, encompass a variety of functions and manipulate the interactions between nematodes and hosts [ 16 ].
Plant parasitic nematodes produce enzymes that break down the cell wall, the primary barrier faced by plant parasite nematodes to invade the plant cytoplasm. Since the first plant cell wall-degrading enzymes carbohydrate-active enzymes, CAZymes from nematodes were characterized in cyst nematodes [ 19 ], plant cell wall enzymes have been identified in many plant parasitic nematodes, including Aphelenchoides , Bursaphelenchus , Ditylenchus , Globodera , Heterodera , and Meloidogyne [ 16 , 17 , 20 ].
Some CAZymes that are secreted by parasitic nematodes of the below-ground parts of plants have been predicted to be involved in enabling nematode access to the roots and feeding on host cells [ 21 ]. A total of 26, transcripts were generated and analyzed from the CFN mixed-stage library.
We shed light on the basic biology and genetic background of CFN, and the transcriptome data generated in this study will facilitate the characterization of the mechanisms of CFN at the molecular level.
In addition, this research also provides significant new information on expressed genes that may aid the elucidation of the parasitic abilities of CFN and improved strategies for its control. We collected the CFN in areas where chrysanthemum foliar nematodes occurred and no specific permit was required. The field for nematodes collection was neither privately owned nor protected, and did not involve endangered or protected species.
SAMN used in this research were collected from the leaves of infected Dendranthema morifolium Ramat. The mRNA was then mixed with fragmentation buffer Ambion and fragmented into short fragments. Short fragments were purified and dissolved in ethidium bromide EB buffer for end repair and single nucleotide A adenine addition.
Then, the short fragments were connected with adapters, and suitable fragments were selected as templates for PCR amplification. Short reads were first assembled into contigs with no gap, then connected the contigs with Trinity, and obtained sequences that no longer could be extended, such sequences are defined as transcripts.
The best-aligned results were used to determine the sequence direction of the transcripts. To confirm validly assembled sequences and identify nematode orthologues with other nematodes, the annotated CFN transcripts were compared with the proteins of the completely sequenced genomes of B.
The CDS were translated into amino acid sequences using the standard codon table. The CAZy database describes families of structurally related catalytic and carbohydrate-binding modules or functional domains of enzymes that degrade, modify, or create glycosidic bonds. Expansin-like proteins were detected by BLAST search with the core modules of known expansin proteins as the query.
Based on the amino acid sequences of 30 GH5 proteins from 27 different species, 20 GH16 proteins from 19 different species, 10 GH43 proteins from 10 different species and 13 GH45 proteins from 9 different species, four phylogenetic trees were constructed using the neighbor-joining method in MEGA Molecular Evolutionary Genetics Analysis, USA 5. Bootstrap values were calculated from replicates. Thirty-seven C. The presence of these genes in A. In this study, we obtained an overview of the gene expression profile of a mixed-stage population of CFN.
The average length was 90 bp, and the proportion of high-quality clean reads was The proportions of nucleotides with a quality value greater than 20, unknown nucleotides, and guanidine and cytosine nucleotides among the total CFN nucleotides, were The clean reads were assembled to the longest assembled sequences also called contigs with Trinity, and resulted in 38, contigs with a mean length of bp and an N50 of 1, bp, respectively.
A total of 26, transcripts with a mean length of 1, bp and an N50 of 1, bp were obtained after further clustering and assembly. Of these transcripts, 15, Of the CFN transcriptome, 16, transcripts The CFN transcripts were annotated as the top organisms and plant parasitic nematodes based on the top BLASTX hit information in the species distribution statistics, the top 5 organisms were Loa loa 3, transcripts, A The E-value distribution of the results of the NR annotation.
B The similarity distribution of the NR annotations. C The species distribution of the NR annotations. Annotation of the CFN transcripts with the GO database classified the transcripts into 55 small classes in three ontologies: molecular function, cellular component and biological process.
The largest class of transcripts in the molecular function ontology was binding activity, which constituted In the cellular component ontology, the largest class of transcripts 4,, In the biological process ontology, the largest class was single-organism processes, which constituted The x-axis shows the COG function classes, and the y-axis shows the number of transcripts in one class.
The notation on the right shows the full names of the function classes. GO functions are shown on the x-axis. The right side of the y-axis shows the number of genes with the GO function, and the left side shows the percentage. The result of CFN transcripts orthologues present in four selected completely sequenced genomes of B. In the total of 26, CFN transcripts, 16, In addition, 10, A total of 11, transcripts were mapped to KEGG pathways, including metabolic pathways 1, transcripts, In addition, 25 signaling pathways and 5 secretion pathways were predicted in the CFN transcriptome analysis.
A group of important signaling pathways predicted in the CFN was orthologues with those of other nematodes; the largest group of these transcripts , 2. These signaling pathways play important roles in the growth and development of nematodes [ 34 , 35 ]. Approximately 1. Only a few tetranucleotide 1, 0. The x-axis shows the number of repeats of a repeat unit. The y-axis shows the number of SSRs. Some transcripts were assigned to more than one related protein type S7 File.
Fewer transcripts were predicted as homologues of plant cell wall-degrading enzymes than fungal cell wall-degrading enzymes. In CFN, 13 transcripts were predicted as homologues of 3 types with plant cell wall-degrading activities, including 4 transcripts predicted as homologues of GH5 proteins, 7 to GH45 proteins and 2 to GH43 proteins.
GH43 proteins had been predicted only in M. GH45 proteins had been predicted only in B. GH28 proteins, which are present in other nematodes, were not predicted in CFN. A total of 98 transcripts of the CFN transcriptome were predicted as homologues of 4 types with fungal cell wall-degrading activity, including 68, 11, 4 and 15 transcripts predicted as homologues of GH18, GH19 and GH20 proteins and GH16 proteins, respectively.
Thirteen GH45 proteins from 9 different species were divided into three groups. Sixteen types of genes in the CFN transcriptome were predicted as orthologues of 7 of 8 gene families compared with C.
RNAi suppressor genes were predicted in M. The proteins involved in the RNAi pathway of C. In this study, the transcriptome of mixed-stage population of CFN was investigated using the Illumina Hiseq system. A total of 26, transcripts with a mean length of 1, bp and an N50 of 1, bp were generated, and 16, transcripts The unannotated sequences might be sequences specific to this species and require further research.
In the CFN transcriptome, a total of 1, transcripts were assigned to six classes of CAZymes containing related protein types, and sixteen types of genes in the CFN transcriptome were predicted as orthologues of 7 of 8 gene families compared with C. Among foliar nematodes, only the transcriptome of A. Wang et al.
Kikuchi et al. Among nematodes that parasitize above-ground plant parts, Kikuchi et al. A systematic evolutionary study of plant cell wall-modifying genes in Tylenchoidea concluded that genes acquired by multiple horizontal gene transfer HGT events from bacteria or fungi were closely associated with the ancestors of these nematodes [ 39 , 40 ].
CFN can feed on a variety of fungi and is closely associated with fungi [ 41 , 42 ]. GH5 protein had been predicted in many plant parasitic tylench nematodes, including Meloidogyne , Globodera , Heterodera , Hirschmanniella and Pratylenchus [ 16 ]. The phylogenetic analyses [ 16 , 44 ] suggested that these proteins were not closely related to those from tylench nematodes and were likely acquired independently from different sources.
CAZymes that degrade plant cell walls and potentially degrade fungal cell walls are present in the CFN transcriptome, and more transcripts were predicted as homologues of fungal cell wall-degrading CAZymes than as homologues of plant cell wall-degrading CAZymes Table 3. In addition, more chitin-related CAZymes were predicted in the CFN transcriptome than in other plant parasitic nematodes.
Chitin is one of the main components of fungal cell walls, and the greater number of chitin-related CAZymes in CFN may be related to the ability of CFN to feed on a variety of plant tissues and fungi.
More orthologues of GH18 protein were predicted in the CFN transcriptome compared to other nematodes, and migratory plant-parasitic nematodes encode more GH18 protein than sedentary plant-parasitic nematodes, indicating that GH18 protein may have special biological significance in the nematode life cycle and in the evolution of nematodes from a free-living to parasitic lifestyle.
Fifteen GH16 proteins were predicted in the CFN transcriptome, and 1,3-glucan is another core component of the fungal cell wall. GH16 protein had also been predicted in A. GH16 protein is most homologues of genes from bacteria, possibly indicating that these genes were acquired from bacteria that were closely associated with the ancestors of these nematodes [ 16 , 45 ]. This research confirms that aphelench nematodes, CFN, A. CFN, A. Therefore, there is difference in the parasitic strategies, feeding behavior and niches occupied between aphelench and tylench nematodes.
But systematic and comprehensive studies are necessary to determine whether there is relationship between RNAi gene components and parasitic strategies, feeding behavior and niches occupied. In conclusion, we have performed the first de novo analysis of the CFN transcriptome, thus increasing the understanding of the molecular biology of foliar nematodes.
These results can be used to select interesting genes of possible importance in plant parasitism for new functional studies. Additionally, this transcriptomic information should be a valuable resource for future research on novel strategies for controlling CFN.
Aphelenchoides ritzemabosi is 0. Males are common. Reported median body size for this species Length mm; width micrometers; weight micrograms - Click:. Chrysanthemum, strawberry and many others.
EPPO Global Database
Aphelenchoides ritzemabosi Black currant nematode, Chrysanthemum foliar nematode, Chrysanthemum leaf nematode, Chrysanthemum nematode, Chrysanthemum Foliar eelworm is a plant pathogenic nematode. It was first scientifically described in in England. This nematode has a wide host range. Among the most important species affected are Chrysanthemums and strawberries.
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