Development of Recombinase Polymerase Amplification Combined with Lateral Flow Detection Assay for Rapid and Visual Detection of Ralstonia solanacearum in Tobacco.

Bacterial wilt caused by Ralstonia solanacearum is a serious soilborne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 103 CFU/g artificially inoculated tobacco stems, and 104 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

First report of Meloidogyne graminicola on rice in Anhui province, China.

Meloidogyne graminicola, a devastating plant pathogen of rice (Oryza sativa), is considered to a severe constraint to productivity in rice-growing areas (Zhan et al., 2018). In April 2020, irrigated paddy rice field in Qianshan City, Anhui Province, China, showed symptoms with stunting, thinning, chlorosis, and typical hook-shaped root tips. Females and egg masses of Meloidogyne sp. were found inside the cortex of the root galls, males were found in soil and roots. The morphological characteristics of females, males and second-stage juveniles (J2) were identified as described previously (Tian et al., 2017; Wang et al., 2017). The perineal pattern of the females (n=12) was dorsoventrally oval with low and round dorsal arches, with lateral fields obscure or absent. Most of the striae were smooth, and occasionally broken by short and irregular striae. Morphological measurements (mean±sd, range) of female nematodes (n=20) were body length (543.0±66.0 µm, 448.0-629.0 µm), stylet (11.6±1.9 µm, 7.9-14.2 µm), dorsal pharyngeal gland orifice to stylet base (DGO) (4.0±0.4 µm, 3.4-4.7 µm), vulval slit length (24.1±4.9 µm, 14.8-32.8 µm), vulval slit to anus distance (16.1±3.0 µm, 8.4-20.2 µm). The male nematodes were cylindroid, vermiform, and rounded tail, with the measurements (n=20) body length (1673.0±125 µm, 1346.0-1822.0 µm), stylet (15.5±0.8 µm, 14.0-17.1 µm), DGO (3.7±0.5 µm, 2.9-5.5 µm), and spicule (30.7±2.5 µm, 23.4-34.6 µm). The J2 were vermiform with a gradually tapering hyalines, its measurements (n=20) were body length (452.0±33.0 µm, 391.0-511.0 µm), stylet (13.4±0.8 µm, 12.0-15.2 µm), DGO (3.6±0.6 µm, 2.5-4.7 µm), tail length (72.1±5.2 µm, 59.8-84.8 µm) and hyaline tail terminus (21.7±2.5 µm, 18.0-29.7 µm). DNA extracted from individual females (n=10) were used for molecular identification. The D2/D3 region of 28S RNA was amplified with D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') (De Ley et al. 1999). The ITS region was amplified with AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') and TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') (Curran et al. 1994). The fragments of D2/D3 region (GenBank accession No. MT576694) and ITS region (GenBank accession No. MT573412) were 766 bp and 579 bp respectively, they all exhibited 99%-100% similarity with those of M. graminicola isolates available in the GenBank. Therefore, both morphological and molecular characterization confirmed the status of this nematode as Meloidogyne graminicola. In green house test, twenty 2-week-old rice seedlings (cv. Longliangyou) were individually maintained in pots with sterilized sand and soil (3:1) and inoculated with 300 J2, other ten non-inoculated rice seedlings as negative control. Rice seedlings were grown in green house at 28℃/25℃ with a 16 h/8 h light/dark photoperiod. After 30 days, all inoculated rice seedling showed symptoms with stunting, chlorosis, and typical hook-shaped root tips, which were similar with that in fields. The nematode reproduction index (final population density/initial population density) were 7.86-10.32. No symptoms were observed on non-inoculated rice seedlings. These results confirmed the pathogenicity of M. graminicola on rice. To our knowledge, this is the first report of M. graminicola in Anhui Province, China. References Curran, J., et al. 1994. Mycol. Res. 98:547. https://doi.org/10.1016/S0953-7562(09)80478-4. De Ley, P., et al. 1999. Nematology. 1:591. https://doi.org/10.1163/156854199508559. Tian, Z., et al. 2017. Plant Disease. http://dx.doi.org/10.1094/PDIS-06-17-0832-PDN. Wang, G., et al. 2017. Plant Disease. http://dx.doi.org/10.1094/PDIS-12-16-1805-PDN. Zhan, L., et al. 2018. BMC Plant Biol. 18:50. https://doi.org/10.1186/s12870-018-1266-9.

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus.

The glycoside hydrolase family 18 (GH18) of chitinases is a gene family widely expressed in archaes, prokaryotes and eukaryotes, and hydrolyzes the ß-1,4-linkages in chitin. The pinewood nematode Bursaphelenchus xylophilus is one of the organisms that produces GH18 chitinases. Notably, B. xylophilus has a higher number of GH18 chitinases compared with the obligate plant-parasitic nematodes Meloidogyne incognita and Meloidogyne hapla. In this study, seven GH18 chitinases were identified and cloned from B. xylophilus based on genomic analyses. The deduced amino acid sequences of all these genes contained an N-terminal signal peptide and a GH18 catalytic domain. Phylogenetic analysis showed that the origin of B. xylophilus GH18 chitinases was independent of those from fungi and bacteria. Real-time quantitative reverse transcription PCR analysis indicated that GH18 chitinase genes had discrete expression patterns, representing almost all the life stages of B. xylophilus. In situ hybridisation showed that the mRNA of GH18 chitinase genes of B. xylophilus were detected mainly in the spermatheca, esophageal gland cells, seminal vesicle and eggs. RNA interference (RNAi) results revealed different roles of GH18 chitinase genes in B. xylophilus. Bx-chi-1, Bx-chi-2 and Bx-chi-7 were associated with reproduction, fungal cell-wall degradation and egg hatching, respectively. Bx-chi-5 and Bx-chi-6 may be involved in sperm metabolism. In conclusion, this study demonstrates that GH18 chitinases have multiple functions in the life cycle of B. xylophilus.

Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae.

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.

Exposure to double-stranded RNA mediated by tobacco rattle virus leads to transcription up-regulation of effector gene Mi-vap-2 from Meloidogyne incognita and promotion of pathogenicity in progeny.

Meloidogyne spp. are economically important plant parasites and cause enormous damage to agriculture world-wide. These nematodes use secreted effectors which modify host cells, allowing them to obtain the nutrients required for growth and development. A better understanding of the roles of effectors in nematode parasitism is critical for understanding the mechanisms of nematode-host interactions. In this study, Mi-vap-2 of Meloidogyne incognita, a gene encoding a venom allergen-like protein, was targeted by RNA interference mediated by the tobacco rattle virus. Unexpectedly, compared with a wild type line, a substantial up-regulation of Mi-vap-2 transcript was observed in juveniles collected at 7 days p.i. from Nicotiana benthamiana agroinfiltrated with TRV::vap-2. This up-regulation of the targeted transcript did not impact development of females or the production of galls, nor the number of females on the TRV::vap-2 line. In a positive control line, the transcript of Mi16D10 was knocked down in juveniles from the TRV::16D10 line at 7 days p.i., resulting in a significant inhibition of nematode development. The up-regulation of Mi-vap-2 triggered by TRV-RNAi was inherited by the progeny of the nematodes exposed to double-stranded RNA. Meanwhile, a substantial increase in Mi-VAP-2 expression in those juvenile progeny was revealed by ELISA. This caused an increase in the number of galls (71.2%) and females (84.6%) produced on seedlings of N. benthamiana compared with the numbers produced by control nematodes. Up-regulation of Mi-vap-2 and its encoded protein therefore enhanced pathogenicity of the nematodes, suggesting that Mi-vap-2 may be required for successful parasitism during the early parasitic stage of M. incognita.