No Pairwise Interactions of GmSNAP18, GmSHMT08 and AtPR1 with Suppressed AtPR1 Expression Enhance the Susceptibility of Arabidopsis to Beet Cyst Nematode.

GmSNAP18 and GmSHMT08 are two major genes conferring soybean cyst nematode (SCN) resistance in soybean. Overexpression of either of these two soybean genes would enhance the susceptibility of Arabidopsis to beet cyst nematode (BCN), while overexpression of either of their corresponding orthologs in Arabidopsis, AtSNAP2 and AtSHMT4, would suppress it. However, the mechanism by which these two pairs of orthologous genes boost or inhibit BCN susceptibility of Arabidopsis still remains elusive. In this study, Arabidopsis with simultaneously overexpressed GmSNAP18 and GmSHMT0 suppressed the growth of underground as well as above-ground parts of plants. Furthermore, Arabidopsis that simultaneously overexpressed GmSNAP18 and GmSHMT08 substantially stimulated BCN susceptibility and remarkably suppressed expression of AtPR1 in the salicylic acid signaling pathway. However, simultaneous overexpression of GmSNAP18 and GmSHMT08 did not impact the expression of AtJAR1 and AtHEL1 in the jasmonic acid and ethylene signaling pathways. GmSNAP18, GmSHMT08, and a pathogenesis-related (PR) protein, GmPR08-Bet VI, in soybean, and AtSNAP2, AtSHMT4, and AtPR1 in Arabidopsis could interact pair-wisely for mediating SCN and BCN resistance in soybean and Arabidopsis, respectively. Both AtSNAP2 and AtPR1 were localized on the plasma membrane, and AtSHMT4 was localized both on the plasma membrane and in the nucleus of cells. Nevertheless, after interactions, AtSNAP2 and AtPR1 could partially translocate into the cell nucleus. GmSNAP18 interacted with AtSHMT4, and GmSHMT4 interacted with AtSNAP2. However, neither GmSNAP18 nor GmSHMT08 interacted with AtPR1. Thus, no pairwise interactions among α-SNAPs, SHMTs, and AtPR1 occurred in Arabidopsis overexpressing either GmSNAP18 or GmSHMT08, or both of them. Transgenic Arabidopsis overexpressing either GmSNAP18 or GmSHMT08 substantially suppressed AtPR1 expression, while transgenic Arabidopsis overexpressing either AtSNAP2 or AtSHMT4 remarkably enhanced it. Taken together, no pairwise interactions of GmSNAP18, GmSHMT08, and AtPR1 with suppressed expression of AtPR1 enhanced BCN susceptibility in Arabidopsis. This study may provide a clue that nematode-resistant or -susceptible functions of plant genes likely depend on both hosts and nematode species.

Bacterial and Fungal Biocontrol Agents for Plant Disease Protection: Journey from Lab to Field, Current Status, Challenges, and Global Perspectives.

Plants are constantly exposed to various phytopathogens such as fungi, Oomycetes, nematodes, bacteria, and viruses. These pathogens can significantly reduce the productivity of important crops worldwide, with annual crop yield losses ranging from 20% to 40% caused by various pathogenic diseases. While the use of chemical pesticides has been effective at controlling multiple diseases in major crops, excessive use of synthetic chemicals has detrimental effects on the environment and human health, which discourages pesticide application in the agriculture sector. As a result, researchers worldwide have shifted their focus towards alternative eco-friendly strategies to prevent plant diseases. Biocontrol of phytopathogens is a less toxic and safer method that reduces the severity of various crop diseases. A variety of biological control agents (BCAs) are available for use, but further research is needed to identify potential microbes and their natural products with a broad-spectrum antagonistic activity to control crop diseases. This review aims to highlight the importance of biocontrol strategies for managing crop diseases. Furthermore, the role of beneficial microbes in controlling plant diseases and the current status of their biocontrol mechanisms will be summarized. The review will also cover the challenges and the need for the future development of biocontrol methods to ensure efficient crop disease management for sustainable agriculture.

Self-Assembled Nanonematicide Induces Adverse Effects on Oxidative Stress, Succinate Dehydrogenase Activity, and ATP Generation.

Long-term overuse of chemical nematicides has resulted in low control efficacy toward destructive root-knot nematodes, and continuous development in nanotechnology is supposed to enhance the utilization efficiency of nematicides to meet practical needs. Herein, a cationic star polymer (SPc) was constructed to load fluopyram (flu) and prepare a flu nanoagent. Hydrogen bonding and van der Waals forces facilitated the self-assembly of the flu nanoagent, leading to the breakdown of self-aggregated flu and reducing its particle size to 60 nm. The bioactivity of flu was remarkably improved, with the half lethal concentration 50 from 8.63 to 5.70 mg/L due to the help of SPc. Transcriptome analysis found that a large number of transport-related genes were upregulated in flu nanoagent-exposed nematodes, while the expression of many energy-related genes was disturbed, suggesting that the enhanced uptake of flu nanoagents by nematodes might lead to the disturbance of energy synthesis and metabolism. Subsequent experiments confirmed that exposure to flu nanoagents markedly increased the reactive oxygen species (ROS) level of nematodes. Compared to flu treatment alone, succinate dehydrogenase (SDH) activity was inhibited in flu nanoagent-exposed nematodes with an increase in the pIC50 from 8.81 to 11.04, which further interfered with adenosine triphosphate (ATP) biosynthesis. Furthermore, the persistence of SPc-loaded flu in soil was prolonged by 2.33 times at 50 days after application. The protective effects of flu nanoagents on eggplant seedlings were significantly improved in both greenhouse and field trials, and the root-knot number was consistently smaller in roots treated with flu nanoagents than in those treated with flu alone. Overall, this study successfully constructed a self-assembled flu nanoagent with amplified effects on oxidative stress, SDH activity, and ATP generation, leading to highly effective control of root-knot nematodes in the field.

Current advances in the identification of plant nematode diseases: From lab assays to in-field diagnostics.

Plant parasitic nematodes (PPNs) cause an important class of diseases that occur in almost all types of crops, seriously affecting yield and quality and causing great economic losses. Accurate and rapid diagnosis of nematodes is the basis for their control. PPNs often have interspecific overlays and large intraspecific variations in morphology, therefore identification is difficult based on morphological characters alone. Instead, molecular approaches have been developed to complement morphology-based approaches and/or avoid these issues with various degrees of achievement. A large number of PPNs species have been successfully detected by biochemical and molecular techniques. Newly developed isothermal amplification technologies and remote sensing methods have been recently introduced to diagnose PPNs directly in the field. These methods have been useful because they are fast, accurate, and cost-effective, but the use of integrative diagnosis, which combines remote sensing and molecular methods, is more appropriate in the field. In this paper, we review the latest research advances and the status of diagnostic approaches and techniques for PPNs, with the goal of improving PPNs identification and detection.

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.

Association of Globodera rostochiensis (Nematoda) with Stunted and Chlorotic Potato Plants in Yunnan and Sichuan Provinces in China.

On a global basis, potato cyst nematodes (Globodera spp. Skarbilovich 1959 [Behrens 1975]) are one of the most serious soilborne pathogens in potato (Solanum tuberosum L.) production. In 2019 to 2020, 188 soil samples were taken from rhizosphere soil associated with the roots of stunted and chlorotic potato plants in the main potato-growing areas of Yunnan and Sichuan Provinces of China. Globodera rostochiensis Wollenweber 1923 (Skarbilovich 1959) was recovered from 112 of the samples. Nematode identification was as confirmed by morphometric, light microscopy, electron microscopy, and molecular methodologies. Population densities of G. rostochiensis ranged from 47.0 to 69.0 eggs/g of soil. A BLASTn homology search program was used to compare the sequences of populations of G. rostrochienses from Yunnan and Sichuan Provinces with populations of other Heteroderinae spp. and populations of G. rostochiensis from other nations. Although potato has been grown in China for at least 400 years and the nation produces more potato than any other country, potato cyst nematodes were not reported in China until 2022.

Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a Technology for Rapid and Highly Sensitive Detection of Heterodera avenae and Heterodera filipjevi.

The cereal cyst nematodes Heterodera avenae and Heterodera filipjevi are recognized as cyst nematodes that infect cereal crops and cause severe economic losses worldwide. Rapid, visual detection of cyst nematodes is essential for more effective control of this pest. In this study, recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (formerly known as cpf1) was developed for the rapid detection of H. avenae and H. filipjevi from infested field samples. The RPA reaction was performed at a wide range of temperatures from 35 to 42°C within 15 min. There was no cross-reactivity between H. avenae, H. filipjevi, and the common closely related plant-parasitic nematodes, indicating the high specificity of this assay. The detection limit of RPA-Cas12a was as low as 10-4 single second-stage juvenile (J2), 10-5 single cyst, and 0.001 ng of genomic DNA, which is 10 times greater than that of RPA-lateral flow dipstick (LFD) detection. The RPA-Cas12a assay was able to detect 10-1 single J2 of H. avenae and H. filipjevi in 10 g of soil. In addition, the RPA-LFD assay and RPA-Cas12a assays could both quickly detect H. avenae and H. filipjevi from naturally infested soil, and the entire detection process could be completed within 1 h. These results indicated that the RPA-Cas12a assay developed herein is a simple, rapid, specific, sensitive, and visual method that can be easily adapted for the quick detection of H. avenae and H. filipjevi in infested fields.

Silicon Mediated Plant Immunity against Nematodes: Summarizing the Underline Defence Mechanisms in Plant Nematodes Interaction.

Silicon (Si) is known to stimulate plant resistance against different phytopathogens, i.e., bacteria, fungi, and nematodes. It is an efficient plant growth regulator under various biotic and abiotic stresses. Silicon-containing compounds, including silicon dioxide, SiO2 nanoparticles (NPs), nano-chelated silicon fertilizer (NCSF), sodium siliconate, and sodium metasilicate, are effective in damaging various nematodes that reduce their reproduction, galling, and disease severity. The defence mechanisms in plant-nematodes interaction may involve a physical barrier, plant defence-associated enzyme activity, synthesis of antimicrobial compounds, and transcriptional regulation of defence-related genes. In the current review, we focused on silicon and its compounds in controlling plant nematodes and regulating different defence mechanisms involved in plant-nematodes interaction. Furthermore, the review aims to evaluate the potential role of Si application in improving plant resistance against nematodes and highlight its need for efficient plant-nematodes disease management.

Origin and Phylogeography of Chinese Cereal Cyst Nematode Heterodera avenae Revealed by Mitochondrial COI Sequences.

Heterodera avenae, a globally distributed plant-parasitic nematode, is one of the most significant pests on cereal crops. In China, it is widely distributed in cereal-growing areas of 16 provinces and causes serious yield losses. In the present study, a total of 98 populations of H. avenae were collected from major wheat-growing regions in China and six other countries. The mitochondrial COI genes were amplified and analyzed. Forty-one mitochondrial COI haplotypes were identified, suggesting a high genetic diversity and endemism level of H. avenae in China. Phylogenetic analysis showed that H. avenae populations in China were divided into four clades. Significant evolutionary and genetic differences were found between Chinese (except Hubei) and foreign populations. Hap1, the most widely distributed haplotype, was considered to be a separate evolutionary origin in China. The gene flow of H. avenae from the northwestern region to the north China region and Huang-Huai-Hai region was significant, so as the direction between north China and Huang-Huai-Hai region. We speculate that water flowing from the Yellow River and mechanical harvesters promoted gene exchange among these groups. A distance-based redundancy analysis showed that genetic distances observed among H. avenae populations were explained foremost not only by geographic distance but also by temperature and precipitation. This study provides theoretical support for the origin and spread of H. avenae populations in China and elsewhere in the world.

Opposite Beet Cyst Nematode Infection Phenotypes of Transgenic Arabidopsis Between Overexpressing GmSNAP18 and AtSNAP2 and Between Overexpressing GmSHMT08 and AtSHMT4.

The rhg1-a GmSNAP18 (an α-SNAP) and Rhg4 GmSHMT08 are two major cloned genes conferring soybean cyst nematode resistance in Peking-type soybeans, but the application of α-SNAPs and SHMTs in cyst nematode management remains elusive. In this study, GmSNAP18 and GmSHMT08, together with their orthologs in Arabidopsis, AtSNAP2 (an α-SNAP) and AtSHMT4, were individually transformed into Arabidopsis Col-0 to generate the transgenic lines, and the growth of transgenic plants, beet cyst nematode (BCN) infection phenotypes, and AtSNAP2, AtSHMT4, and AtPR1 expression patterns were analyzed using Arabidopsis-BCN compatible interaction system, in addition with protein-protein interaction assay. Pulldown and BiFC assays revealed that GmSNAP18 and GmSHMT08 interacted with AtSHMT4 and AtSNAP2, respectively. Plant root growth was not impacted by overexpression of GmSNAP18 and AtSNAP2. However, overexpression of GmSHMT08 and AtSHMT4 both increased plant height, additionally, overexpression of GmSHMT08 decreased rosette leaf size. Overexpression of GmSNAP18 and GmSHMT08 both suppressed AtPR1 expression and significantly enhanced BCN susceptibility, while overexpression of AtSNAP2 and AtSHMT4 both substantially boosted AtPR1 expression and remarkably enhanced BCN resistance, in transgenic Arabidopsis. Overexpression of GmSNAP18 reduced, while overexpression of AtSNAP2 unaltered AtSHMT4 expression. Overexpression of GmSHMT08 and AtSHMT4 both suppressed AtSNAP2 expression in transgenic Arabidopsis. Thus, different expression patterns of AtPR1 and AtSHMT4 are likely associated with opposite BCN infection phenotypes of Arabidopsis between overexpressing GmSNAP18 and AtSNAP2, and between overexpressing GmSHMT08 and AtSHMT4; and boosted AtPR1 expression are required for enhanced BCN resistance in Arabidopsis. All these results establish a basis for extension of α-SNAPs and SHMTs in cyst nematode management.

Morphological characterization reveals new insights into giant cell development of Meloidogyne graminicola on rice.

MAIN CONCLUSION: Three types of nematode-feeding sites (NFSs) caused by M. graminicola on rice were suggested, and the NFS polarized expansion stops before the full NFS maturation that occurs at adult female stage. Root-knot nematodes, Meloidogyne spp., secrete effectors and recruit host genes to establish their feeding sites giant cells, ensuring their nutrient acquisition. There is still a limited understanding of the mechanism underlying giant cell development. Here, the three-dimensional structures of M. graminicola-caused nematode-feeding sites (NFSs) on rice as well as changes in morphological features and cytoplasm density of the giant cells (GCs) during nematode parasitism were reconstructed and characterized by confocal microscopy and the Fiji software. Characterization of morphological features showed that three types of M. graminicola-caused NFSs, type I-III, were detected during parasitism at the second juvenile (J2), the third juvenile (J3), the fourth juvenile (J4) and adult female stages. Type I is the majority at all stages and type II develops into type I at J3 stage marked by its longitudinal growth. Meanwhile, NFSs underwent polarized expansion, where the lateral and longitudinal expansion ceased at later parasitic J2 stage and the non-feeding J4 stage, respectively. The investigation of giant cell cytoplasm density indicates that it reaches a peak at the midpoint of early parasitic J2 and adult female stages. Our data suggest the formation of three types of NFSs caused by M. graminicola on rice and the NFS polarized expansion stopping before full NFS maturation, which provides unprecedented spatio-temporal characterization of development of giant cells caused by a root-knot nematode.

Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples.

Sugar beet cyst nematode (SBCN, Heterodera schachtii) is an important nematode that causes significant yield losses of 25-50% or more in most areas of sugar beet production worldwide. Rapid and accurate identification of this species is essential to support decisions on pest management. However, the difference between H. schachtii and other Heterodera spp. based on morphology is a challenging task. In the present study, a SCAR-PCR assay was developed to identify and differentiate H. schachtii in infected root and soil samples. H. schachtii-species-specific SCAR-PCR primers OPA06-HsF and OPA06-HsR were designed from the randomly amplified polymorphic DNA (RAPD) marker amplified with random primer OPA06. The developed primers specifically amplify a 922-bp fragment from the target populations but did not amplify DNA from non-target cyst nematodes including Heterodera, Globodera, Cactodera, and other related species tested in this study. The sensitivity detection indicated that 5 × 10-4 of a single cyst, 1/320 of a single second-stage juvenile (J2), or 10 pg of genomic DNA could be detected. The assay accurately identifies the different stages of H. schachtii in sugar beet and oilseed rape roots as well as a single J2 in 10 g of soil. Finally, the SCAR-PCR assay detected H. schachtii in seven samples out of the fifteen field samples. The assay will not only be useful for differentiating H. schachtii from mixed populations of Heterodera spp. but also for effective detection of the species directly from infested samples. The assay also requires no expertise in the taxonomy and morphology of the species but serves to improve the diagnosis of H. schachtii in infested fields.

Prevalence and Molecular Diversity of Plant-Parasitic Nematodes of Yam (Dioscorea spp.) in China, with Focus on Merlinius spp.

There is little information about nematode pests associated with yam in China. Between 2020 and 2021, surveys of yam fields were conducted to investigate the abundance and prevalence of plant-parasitic nematodes in major yam growing areas. A total of 110 bulk soil samples from the yam rhizosphere and 48 yam tubers were collected from seven counties in Jiangxi and Shandong provinces. Standard protocols were used to extract nematodes from soil and tubers and identified at the genus level. In this study, 16 species and 13 nematode genera were recorded. The five most prominent species on the yam rhizosphere according to mean population densities were Pratylenchus coffeae (291/individuals), Meloidogyne (262/individuals), Rotylenchulus reniformis (225/individuals), Merlinius (224/individuals), and Helicotylenchus dihystera (171/individuals). In the tubers, the three most prominent species were Pratylenchus coffeae (415/individuals), Meloidogyne (331/individuals), and Rotylenchulus reniformis (115/individuals). These species were verified with appropriate molecular analysis. The high prevalence of the ectoparasite (Merlinius spp.) on the rhizosphere of yam also revealed that Merlinius spp. May be more important to yam than previously thought. Morphological and molecular analyses further confirmed the identity of the species as Merlinius brevidens and were characterized for the first time on yam in China. Minor morphometrical differences (slightly longer body and stylet) were observed in Chinese populations of M. brevidens compared to the original description. Additionally, this study reveals that M. brevidens isolated from China showed a higher nucleotide sequence in the ITS region compared to M. brevidens populations from India. This finding provides baseline information on the nematode pest occurrence on yam in China and calls for effective management.

Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology.

Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10-4 single cysts and single females, 4-3 single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.

Correction: Dihydroxyacetone of wheat root exudates serves as an attractant for Heterodera avenae.

[This corrects the article DOI: 10.1371/journal.pone.0236317.].

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.

Plasmodesmata play pivotal role in sucrose supply to Meloidogyne graminicola-caused giant cells in rice.

On infection, plant-parasitic nematodes establish feeding sites in roots from which they take up carbohydrates among other nutrients. Knowledge on how carbohydrates are supplied to the nematodes' feeding sites is limited. Here, gene expression analyses showed that RNA levels of OsSWEET11 to OsSWEET15 were extremely low in both Meloidogyne graminicola (Mg)-caused galls and noninoculated roots. All the rice sucrose transporter genes, OsSUT1 to OsSUT5, were either down-regulated in Mg-caused galls compared with noninoculated rice roots or had very low transcript abundance. OsSUT1 was the only gene up-regulated in galls, at 14 days postinoculation (dpi), after being highly down-regulated at 3 and 7 dpi. OsSUT4 was down-regulated at 3 dpi. No noticeable OsSUTs promoter activities were detected in Mg-caused galls of pOsSUT1 to -5::GUS rice lines. Loading experiments with carboxyfluorescein diacetate (CFDA) demonstrated that symplastic connections exist between phloem and Mg-caused giant cells (GCs). According to data from OsGNS5- and OsGSL2-overexpressing rice plants that had decreased and increased callose deposition, respectively, callose negatively affected Mg parasitism and sucrose supply to Mg-caused GCs. Our results suggest that plasmodesmata-mediated sucrose transport plays a pivotal role in sucrose supply from rice root phloem to Mg-caused GCs, and OsSWEET11 to -15 and OsSUTs are not major players in it, although further functional analysis is needed for OsSUT1 and OsSUT4.

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.

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.