Molecular And Morphological Characterization Of Rice Root Nematodes(M.graminicola,Hirschmanniella Spp.and H.elachista)

Rice(Oryza sativa L.)is one of world’s most widely consumed staple foods,and also plays a major part in the diet of more than half of the human’s population.Rice production is limited by several abiotic and biotic factors.Among biotic factors,plant-parasitic nematodes are considered as important harmful pathogens which can cause either direct or indirect damage to their host.The two foliar and six root parasitic nematodes are recorded as economically important pathogens on various rice crops.In this study,nematodes from three most important genera,Meloidogyne(root-knot nematodes),Hirschmanniella(rice root nematodes),and Heterodera(rice cyst nematodes)were focused by means of molecular and morphological characterization.Meloidogyne graminicola is a major constraint in rice production because its unique biology and life cycle allow it to adapt to flood conditions.Using rDNA-ITS(ribosomal DNA Internal Transcribed Spacers)sequence data alignments,the genetic variation among twenty-one populations of M.graminicola(sixteen from Myanmar and five from China)was investigated.The results showed that all the populations were clearly separated from other species and that there was a low level of genetic variation among the isolates.A set of species-specific primers was designed to develop a species-specific molecular tool for the precise identification of M.graminicola.The primer reliability,specificity and sensitivity tests showed that the primer set(Mg-F3 and Mg-R2)amplified the expected fragment size of 369 bp from the template DNA of target nematode populations but not from non-target organisms.A duplex PCR test allows for saving diagnostic time and costs by amplifying the species of interest from a nematode mixture.Therefore,this species-specific primer set may be a powerful tool to improve taxonomic identification by non-specialists and the design of successful management practices as well.Rice root nematodes(RRN)belonging to the genus Hirschmanniella are one of the most serious pests and commonly found in majority of rice-growing regions.In this research,a total of twenty-four populations of Hirschmanniella nematodes were examined by means of molecular and morphology.Two species,Hirschmanniella oryzae and Hirschmanniella mucronata,were observed from the collected samples based on the molecular diagnosis;H.oryzae in eighteen samples,H.mucronata in two samples,and a mixture of these two species in four specimens.Analysis of the ITS-rDNA Sequence showed that high intraspecific variation among the nucleotide sequence of ITS regions of tested Hirschmanniella isolates(sequence homology 88-99% to H.oryzae and 99% to H.mucronata).Twelve H.oryzae and two H.mucronata populations were selected out of the tested groups identified through molecular diagnosis and their morphological features were studied to look for morphological variations and confirm their species identity.The morphology and morphometric characterization of H.mucronata was largely similar to the original description of type specimens,but slightly differed by having wider maximum body diameter.In H.oryzae,comparison with the reported data indicated that the nematode in the present measurements have wider body width,higher ‘a’ value,longer pharyngeal glands and median bulb distance from anterior end,tail terminus with subterminal notch.These two species can be easily separated by their stylet length and body length.For detection and identification of H.oryzae,sixty random 10-mer primers were used in RAPD assay.OPB-04 10-mer primer gave reproducible amplification patterns and designed into sequence-characterized amplified region(SCAR)marker specific for target species.The expected fragment size of 475 bp was observed in all H.oryzae isolates but not in other species.Due to their variation in nucleotides and morphology,the combination of morphology and molecular identification of rice root nematodes will be more accurate way to avoid misinterpretation of the species and overestimation of genetic diversity of Hirschmanniella nematodes.The simple,rapid and sensitive nucleic acid amplification technique,called Loop-mediated isothermal amplification(LAMP),for detection of Heterodera elachista(rice cyst nematode)was developed in this study.The LAMP primers were designed targeting the internal transcribed spacer(ITS)sequences of ribosomal DNA of H.elachista.The optimum condition for the LAMP reaction was 64 oC for 1 h and 80 oC for 5 min.Under the optimized LAMP mixture and temperature conditions,typical ladder-like pattern in agarose gel electrophoresis was observed in positive reaction tubes containing genomic DNA from H.elachista.Moreover,validation of LAMP products was also achieved by visualization of color changes with the naked eye after adding SYBR Green I,and presence of both test and check lines in lateral flow stick(LFD)detection assay.The specificity is very high based on the negative results when the genomic DNA from other nematode species was used as templates for LAMP reaction.It is 10 times more sensitive than the traditional PCR as well.Due to its low cost,simplicity,specificity and sensitivity,LAMP assay for detection and diagnosis of H.elachista will be a future useful alternative tool for effective quarantine inspection and planning suitable management principles.The information from the present research will be useful in better understanding and accurate diagnosis of economically important plant-parasitic nematodes in rice,leading to help in selection of proper management,studying plant-nematode interactions and planning plant quarantine strategies at border.