Meloidogyne graminicola Population Structure in China Suggests a South-to-North Expansion.

The distribution range of root-knot nematode Meloidogyne graminicola is rapidly expanding, posing a severe threat to rice production. In this study, the sequences of cytochrome oxidase subunit I (COI) genes of rice M. graminicola populations from all reported provinces in China were amplified and sequenced by PCR. The distribution pattern and phylogenetic tree showed that all 54 M. graminicola populations in China have distinct geographical distribution characteristics; specifically, cluster 1 (southern China), cluster 2 (central south and southwest China), and cluster 3 (central and eastern China). The high haplotype diversity (Hd = 0.646) and low nucleotide diversity (π = 0.00682), combined with the negative value of Tajima's D (-1.252) and Fu's Fs (-3.06764), suggested that all nematode populations were expanding. The existence of high genetic differentiation (Fst = 0.5933) and low gene flow (Nm = 0.3333) indicated that there was a block of gene exchange between most populations. Mutation accumulation with population expansion might be directly responsible for the high genetic differentiation; therefore, the tested nematode population showed high within-group genetic variation (96.30%). The haplotype Hap8 was located at the bottom of the network topology, with the widest distribution and the highest frequency (59.26%), indicating that it was the ancestral haplotype. The populations in cluster 3 were newly invasive according to the lowest frequency of occurrence of Hap8, the highest number of endemic haplotypes, and the highest total haplotype frequency (60%). In contrast, cluster 1 having the highest genetic diversity (Hd = 0.772, π = 0.01127) indicated that it was the most primitive. Interestingly, the highest gene flow (Nm > 1), lowest genetic differentiation (Fst ≤ 0.33), and closest genetic distance (0.000) only occurred between the Guangdong/Hainan population and others, which suggested that there might be channels for gene exchange between them and that long-distance dispersal occurred. This suggestion is further confirmed by the weak correlation between genetic distance and geographical distance. Based on these data, a hypothesis can be drawn that M. graminicola populations in China were spreading from south to north, specifically from Guangdong and Hainan Provinces to other regions. Natural selection (including anthropogenic) and genetic drift were the main drivers of their evolution. Coincidentally, this hypothesis was consistent with the gradual warming trend and the chronological order of reporting these populations. The main factors influencing current M. graminicola population expansion and distribution patterns might be geography, climate, long-distance seedling transport, interregional operations of agricultural machinery, and rotation mode. It reminds human beings of the necessity to be vigilant about preventing nematode disease according to local conditions all year round.

First Report of Meloidogyne graminicola on Rice in Henan Province, China.

Rice (Oryza sativa) is an important food crop in China and root-knot nematode Meloidogyne graminicola has been one of the most important diseases on rice in recently five years (Ju et al. 2020). In August 2020, rice plants were found to be maldeveloped, yellow leaves and hooked root tips in an irrigated paddy field of Yuanyang County, Xinxiang City, Henan Province. Fifty rice plants were randomly collected and 84.0 percent plants were infected with root-knot nematodes, with root-gall index of 56.0. Then nematodes from rice roots were isolated with 100-µm and 25-µm sieves. A large number of females, some third-stage juveniles (J3s), and a small number of males of Meloidogyne spp. were found in root galls of all samples after dissected, and then were identified and measured under the microscope. In females (n = 20), the perineal pattern was dorsoventrally oval with low and round dorsal arch, and the lateral field was not obvious or absent, striae are usually smooth, with occasional short and irregular striatal fragmentation. The morphological data of females are as follows: body length (BL) = 516.9 ± 72.5 µm (424.2 to 611.6 µm), body width (BW)= 328.4 ± 80.7 µm (232.1 to 437.4 µm), stylet length = 11.2 ± 1.3 µm (7.7 to 13.9 µm), dorsal pharyngeal gland orifice to stylet base (DGO) = 3.9 ± 0.5 µm (3.2 to 4.5 µm), vulval slit length = 24.3 ± 4.6 µm (15.2 to 31.4 µm), vulval slit to anus distance = 16.2 ± 2.5 µm (10.1 to 20.2 µm). Males are long cylindrical, wormlike, with a short round tail. Morphological measurements of males (n = 20) were BL = 1,218.0 ± 150.7µm (1,085.7 to 1,692.2 µm), BW = 34.2 ± 4.6 µm (28.5 to 39.7 µm), stylet = 17.4 ± 0.7 µm (15.9 to 19.3 µm), DGO = 3.6 ± 0.7 µm (2.5 to 4.5 µm), tail = 10.8 ± 2.1 µm (8.0 to 14.8 µm), spicule = 30.3 ± 2.6 µm (24.7 to 36.3 µm). The egg masses from the females were incubated at 28℃ for 48 hours. Measurements of J2s (n = 20) were BL = 444.2 ± 37.8 µm (315.7 to 547.5 µm), BW = 21.2 ± 2.7 µm (16.7 to 26.4 µm), stylet = 14.2 ± 0.3 µm (13.6 to 14.8 µm), DGO = 3.5 ± 0.5 µm (2.7 to 4.5 µm), tail = 70.8 ± 5.1 µm (61.3 to 80.8 µm), hyaline tail length = 21.0 ± 2.5 µm (16.3 to 26.1 µm). These morphological features are consistent with the original description by Golden and Birchfield (1965). DNA of a single female from each sample was extracted for molecular identification. Primer pairs D2A/D3B (5´-ACAAGTACCGTGAGGGAAAGTTG-3´/ 5´-TCGGAAGGAACCAGCTACTA-3´) (De Ley et al. 1999) and the species-specific primers Mg-F3/Mg-R2 (5'-TTATCGCATCATTTTATTTG-3'/ 5'-CGCTTTGTTAGAAAATGACCCT-3') (Htay et al. 2016) were used to amplify D2/D3 region of 28S RNA and the internal transcribed spacer (ITS) region, respectively. The amplified sequences of D2/D3 region (GenBank MW490724, 766 bp) shared 99.9% and 99.7% homology with the sequences of M. graminicola (MN647592, MT576694) isolated from Guangxi and Anhui Province (Ju et al. 2020), respectively, while ITS region sequences (MW487239, 369 bp) shared 100% and 99.7% homology to M. graminicola isolate GXL3 (MN636702) and FQJJ01 (MT159690), respectively. In order to verify the pathogenicity of nematodes, about 300 J2s were inoculated on ten 14-week-old rice (Oryza sativa cv. Nipponbare) planted in pots with sterilized sandy soil, respcectively, and maintained in a greenhouse at 28°C/26°C with a 16h/8h light/dark photoperiod and 75% relative humidity. At 14 days post inoculation, obvious symptoms of hook galls were observed on roots in all inoculated rice plants, and females and males in the same shape as the collected samples were found in the root galls under the stereoscopic microscope. No symptoms were observed on non-inoculated rice plants. After 28 days, the growth of the inoculated rice plants was significantly worse than that of uninoculated ones, with yellow leaves and short plants. These results confirmed the pathogenicity of M. graminicola on rice and it indicated that M. graminicola was already spread from the main rice-producing areas to the wheat and rice rotation areas. To our knowledge, this is the first report of M. graminicola in the Henan Province of China.

αß-Dehydrocurvularin isolated from the fungus Aspergillus welwitschiae effectively inhibited the behaviour and development of the root-knot nematode Meloidogyne graminicola in rice roots.

BACKGROUND: The root-knot nematode Meloidogyne graminicola has become a serious threat to rice production as a result of the cultivation changes from transplanting to direct seeding. The nematicidal activity of Aspergillus welwitschiae have been investigated in vitro, and the disease control efficacy of the active compound has been evaluated under greenhouse and field conditions. RESULTS: The active compound αß-dehydrocurvularin (αß-DC), isolated by nematicidal assay-directed fractionation, showed significant nematicidal activity against M. graminicola, with a median lethal concentration (LC50) value of 122.2 µg mL- 1. αß-DC effectively decreased the attraction of rice roots to nematodes and the infection of nematodes and also suppressed the development of nematodes under greenhouse conditions. Moreover, αß-DC efficiently reduced the root gall index under field conditions. CONCLUSIONS: To our knowledge, this is the first report to describe the nematicidal activity of αß-DC against M. graminicola. The results obtained under greenhouse and field conditions provide a basis for developing commercial formulations from αß-DC to control M. graminicola in the future.

Proteome-Wide Analyses Provide New Insights into the Compatible Interaction of Rice with the Root-Knot Nematode Meloidogyne graminicola.

The root-knot nematode Meloidogyne graminicola is an important pathogen in rice, causing huge yield losses annually worldwide. Details of the interaction between rice and M. graminicola and the resistance genes in rice still remain unclear. In this study, proteome-wide analyses of the compatible interaction of the japonica rice cultivar "Nipponbare" (NPB) with M. graminicola were performed. In total, 6072 proteins were identified in NPB roots with and without infection of M. graminicola by label-free quantitative mass spectrometry. Of these, 513 specifically or significantly differentially expressed proteins were identified to be uniquely caused by nematode infection. Among these unique proteins, 99 proteins were enriched on seven Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparison of protein expression and gene transcription, LOC_Os01g06600 (ACX, a glutaryl-CoA dehydrogenase), LOC_Os09g23560 (CAD, a cinnamyl-alcohol dehydrogenase), LOC_Os03g39850 (GST, a glutathione S-transferase) and LOC_Os11g11960 (RPM1, a disease resistance protein) on the alpha-linolenic acid metabolism, phenylpropanoid biosynthesis, glutathione metabolism and plant-pathogen interaction pathways, respectively, were all associated with disease defense and identified to be significantly down-regulated in the compatible interaction of NPB with nematodes, while the corresponding genes were remarkably up-regulated in the roots of a resistant rice accession "Khao Pahk Maw" with infection of nematodes. These four genes likely played important roles in the compatible interaction of rice with M. graminicola. Conversely, these disease defense-related genes were hypothesized to be likely involved in the resistance of resistant rice lines to this nematode. The proteome-wide analyses provided many new insights into the interaction of rice with M. graminicola.

Complete genome sequence of a Chinese isolate of pepper vein yellows virus and evolutionary analysis based on the CP, MP and RdRp coding regions.

The genome sequence of pepper vein yellows virus (PeVYV) (PeVYV-HN, accession number KP326573), isolated from pepper plants (Capsicum annuum L.) grown at the Hunan Vegetables Institute (Changsha, Hunan, China), was determined by deep sequencing of small RNAs. The PeVYV-HN genome consists of 6244 nucleotides, contains six open reading frames (ORFs), and is similar to that of an isolate (AB594828) from Japan. Its genomic organization is similar to that of members of the genus Polerovirus. Sequence analysis revealed that PeVYV-HN shared 92% sequence identity with the Japanese PeVYV genome at both the nucleotide and amino acid levels. Evolutionary analysis based on the coat protein (CP), movement protein (MP), and RNA-dependent RNA polymerase (RdRP) showed that PeVYV could be divided into two major lineages corresponding to their geographical origins. The Asian isolates have a higher population expansion frequency than the African isolates. Negative selection and genetic drift (founder effect) were found to be the potential drivers of the molecular evolution of PeVYV. Moreover, recombination was not the distinct cause of PeVYV evolution. This is the first report of a complete genomic sequence of PeVYV in China.

HaCRT1 of Heterodera avenae Is Required for the Pathogenicity of the Cereal Cyst Nematode.

Cereal cyst nematodes are sedentary biotrophic endoparasites that secrete effector proteins into plant tissues to transit normal cells into specialized feeding sites and suppress plant defenses. To understand the function of nematode effectors in Heterodera avenae, here, we identified a calreticulin protein HaCRT1, which could suppress the cell death induced by Bax when expressed in Nicotiana benthamiana. HaCRT1 is synthetized in the subventral gland cells of pre-parasitic second-stage nematodes. Real-time PCR assays indicated that the expression of HaCRT1 was highest in parasitic second-stage juveniles. The expression of an HaCRT1-RFP fusion in N. benthamiana revealed that it was localized in the endoplasmic reticulum of the plant cell. The ability of H. avenae infecting plants was significantly reduced when HaCRT1 was knocked down by RNA interference in vitro. Arabidopsis thaliana plants expressing HaCRT1 were more susceptible than wild-type plants to Pseudomonas syringae. The induction of defense-related genes, PAD4, WRKY33, FRK1, and WRKY29, after treatment with flg22 was suppressed in HaCRT1-transgenic plants. Also, the ROS accumulation induced by flg22 was reduced in the HaCRT1-transgenic plants compared to wild-type plants. HaCRT1 overexpression increased the cytosolic Ca2+ concentration in A. thaliana. These data suggested that HaCRT1 may contribute to the pathogenicity of H. avenae by suppressing host basal defense.