Identification and pathogenicity of Pratylenchus scribneri on tomato in Sichuan Province of People's Republic of China

Root-lesion nematodes (Pratylenchus spp.) are a group of economically important pathogens that have caused serious economic losses in many crops. In 2019, root-lesion nematodes were recovered from tomato (Solanum lycopersicum) root samples collected from Sichuan Province, People's Republic of China (PRC). Extracted nematodes were disinfected, and one individual female was cultured on a carrot disc for propagation at 25 °C by parthenogenesis and designated the SC isolate. Afterwards, the isolate was identified on the basis of morphometric and molecular markers. Both morphometric characters and molecular analysis of the internal transcribed spacer region gene (ITS) of ribosomal DNA, the D2-D3 expansion region of the 28S rDNA gene and the mitochondrial cytochrome oxidase I (mtDNA-COI) gene revealed that the species of root-lesion nematode was Pratylenchus scribneri. The Bayesian tree inferred from the ITS rDNA, 28S rDNA and mtDNA-COI gene sequences also showed that this isolate formed a highly supported clade with other P. scribneri isolates. The pathogenicity of the root-lesion nematode SC isolate on tomato was assessed, showing that tomato was a suitable host for P. scribneri. To the best of our knowledge, this is the first report of P. scribneri on tomato in Sichuan Province, PRC. These are also the first molecular data obtained from P. scribneri on tomato in the PRC, and the pathogenicity of P. scribneri to tomato was studied for the first time. This study provides scientific data for the detection, identification and control of tomato root-lesion nematode disease.

Discovery of Root-Lesion Nematode (Pratylenchus scribneri) on Corn in Hainan Province of China

Corn (Zea mays L.) is a very important cereal crop and serves as food, feed, and industrial material (Liu et al. 2016). The root-lesion nematode (RLN) is considered one of the most important plant-parasitic nematodes and can cause economic losses in agriculture worldwide (Jones et al. 2013). In January 2020, five samples were collected from a corn field in Lingshui Lizu Autonomous County, Hainan Province, China. The collected corn plants (cv. Denghai 685) were growing poorly and roots showed distinct lesions and rot. Corn fields with symptoms of stunted plants, and brown lesions on roots were widespread. This corn disease was severe in Lingshui Lizu Autonomous County. RLN were extracted from soil samples by the modified Baermann funnel (Hooper et al. 2005). All the samples contained RLN ranging from 9 to 82 (average 39) RLN per 100 cm3 of soil and 113 to 257 (average 194) RLN per 5 g roots. The extracted RLN were sterilized and cultured on carrot disks at 25°C for 90 days. Afterwards, seeds of corn (cv. Denghai 685) were sown in pots containing 1.8 liters of sterilized soil. Eight plants, one per pot, were infested with 1,000 RLN, eight pots of noninfested corn plants were used as controls, and plants were kept in a greenhouse at 25°C. At 75 days after inoculation, symptoms were like those initially observed in corn fields, whereas no symptoms were observed in the control plants. Nematodes in the soil and roots were extracted using the same method as previously described (Hooper et al. 2005). The average number of RLN per pot was approximately 4,250 in soil and 820 in roots, the reproduction factor (final number of nematodes/initial number of nematodes) was 5.07, no RLN were found in the control. The experiment was conducted twice. The morphological and molecular studies of RLN were examined to confirm species identification. The main morphological measurements of adult females (n = 15) included body length = 526.0 μm ± 17.1 (standard error) (range = 498.0 to 560.5 μm), stylet = 16.0 μm ± 0.3 (15.5 to 16.5 μm), tail length = 29.0 μm ± 1.5 (26.5 to 31.0 μm), a = 23.6 ± 0.6 (22.6 to 24.4), b = 5.6 ± 0.3 (5.2 to 6.0), c = 18.3 ± 0.9 (16.4 to 19.7), V = 78.2% ± 0.6 (77.4 to 79.2%), lip region with two annules. No males were found in the samples. This population was identified as Pratylenchus scribneri, based on the morphological characters (Castillo and Vovlas, 2007). DNA was isolated from individual nematodes followed by proteinase K-based lysis (Wang et al. 2011). The D2/D3 expansion region of the 28S rRNA gene, rDNA-internal transcribed spacer (ITS) region, and mitochondrial cytochrome oxidase I (mtDNA-COI) gene were amplified with primers D2A/-D3B (De Ley et al. 1999), TW81/ AB28 (Vovlas et al. 2011) and JB3/ JB5 (Liu et al. 2018), respectively. The PCR products were purified and ligated into pJET 1.2/blunt cloning vectors and transformed to Escherichia coli strain DH5α, and then sequenced. The obtained 28S rRNA gene D2/D3 region sequences (785bp), ITS sequences (886 bp) and mtDNA-COI (447bp) in this study were submitted to GenBank. The D2/D3 region of the 28S rRNA sequences of the RLN collected in Lingshui (GenBank accession no. MZ701843) showed 99.75% identity with P. scribneri sequences available in the GenBank (KX842628 and KX842625). The ITS sequences of the RLN collected in this study (MZ701842) showed the highest identity of 97.06% with P. scribneri sequences available in the GenBank (KX842626). The mtDNA-COI sequences of the RLN collected in this study (OK040228) showed 100% identity with P. scribneri (MN366409). Both morphological and molecular data confirmed the identity of P. scribneri. P. scribneri has been reported on corn in Inner Mongolia, Hebei, Shanxi, Shandong, Henan, Jiangsu, and Liaoning provinces of China (Li et al. 2019). As far as we know, this is the first report of P. scribneri on corn in Hainan Province, China. Since the RLN can cause considerable damage to corn, strategic measures should be taken to prevent the spread of P. scribneri to other regions in China.

Developing a real-time PCR assay for direct detection and quantification of Pratylenchus scribneri in field soil

Summary The endomigratory root-lesion nematode, Pratylenchus scribneri, is one of the major plant-parasitic nematodes infecting potato. Accurate identification and quantification of this nematode are essential to develop management strategies but microscopic observations are particularly challenging and time consuming. In this study, a SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. scribneri from field soil DNA extracts. A primer set was designed from the internal transcribed spacer (ITS) region of the P. scribneri rDNA gene. Primer specificity to the target nematode was evaluated by both in silico analysis and qPCR and no detection or non-specific amplification was observed for other non-target nematode species/communities tested in this study. Standard curves were generated using DNA extracts from autoclaved soil infested with varying nematode numbers for calibration. The curves were supported by a high correlation between the P. scribneri numbers artificially added to soil or estimated from naturally infested field soils by traditional methods, and the numbers quantified using the qPCR assay. The assay was able to detect 1 out of 128 (0.0078) equivalents of the DNA of a single nematode in 0.5 g of soil. The qPCR assay developed in this study provides a specific and sensitive detection and quantification of P. scribneri from field soils and a rapid alternative to time-consuming traditional nematode identification and enumeration.

Specific Detection of the Root-Lesion Nematode Pratylenchus scribneri Using Conventional and Real-Time PCR

Pratylenchus scribneri is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological methods is an arduous task requiring extensive training on nematode taxonomy and years of experience. Thus, molecular methods for P. scribneri detection and identification were developed. Conventional and real-time polymerase chain reaction (PCR) assays with new species-specific primers were used in this study, which exclusively amplified DNA of P. scribneri but not DNA from other Pratylenchus spp. or non-Pratylenchus spp. tested. Compared with conventional PCR that was able to detect an equivalent to 1/4 of the DNA of a single nematode, real-time PCR was more sensitive and could amplify an equivalent to 1/128 of the DNA of one nematode. Both conventional and real-time PCR assays successfully identified P. scribneri and distinguished it from P. penetrans and P. neglectus isolated from field samples collected from various locations in North Dakota and Minnesota. The Blast-search based on the sequence information confirmed the reliability of the PCR assays for species identification. This is the first report of P. scribneri identification using a real-time PCR assay. The developed PCR assays are suitable for use in diagnostic laboratories and detection of field infestations with this nematode species.

Toxicity of Endophyte-Infected Tall Fescue Alkaloids and Grass Metabolites on Pratylenchus scribneri

Neotyphodium coenophialum, an endophytic fungus associated with tall fescue grass, enhances host fitness and imparts pest resistance. This symbiotum is implicated in the reduction of stresses, including plant-parasitic nematodes. To substantiate this implication, toxicological effects of root extracts, polyphenolic fraction, ergot, and loline alkaloids from endophyte-infected tall fescue were investigated using Pratylenchus scribneri, a nematode pest of tall fescue. In vitro bioassays and greenhouse studies were used as tests for effects of root fractions and compounds on motility and mortality of this lesion nematode. Greenhouse studies revealed that endophyte-infected tall fescue grasses are essentially nonhosts to P. scribneri, with root populations averaging 3 to 17 nematodes/pot, compared with 4,866 and 8,450 nematodes/pot for noninfected grasses. The in vitro assay indicated that root extracts from infected tall fescues were nematistatic. Polyphenols identified in extracts included chlorogenic acid, 3,5-dicaffeoylquinic acids, caffeic acid, and two unidentified compounds, but these were not correlated with endophyte status, qualitatively or quantitatively. Tests of several ergot alkaloids revealed that ergovaline and α-ergocryptine were nematicidal at 5 and 50 μg/ml, respectively, while ergocornine and ergonovine were nematistatic at most concentrations. Loline (N-formylloline), the pyrrolizidine alkaloid tested, was nematicidal (50 to 200 μg/ml). The ecological benefits of the metabolites tested here should assist in defining their role in deterring this nematode species while offering some probable mechanisms of action against plant-parasitic nematodes in general.

Bacillus thuringiensiscrystal protein toxicity against plant-parasitic nematodes

A bioassay method was developed to use the parasporal crystal protein ofBacillus thuringiensisagainst plant-parasitic nematodes. Using this method, the parasporal crystal proteins of tenBtstrains showed activity against plant-parasitic nematodes. The toxicity of YBT-021 againstMeloidogyne hapla,Pratylenchus scribneri,Tylenchorhynchussp.,Ditylenchus destructorandAphelenchoidessp. was also assayed. The resulting LC50values were 35.62 μg/ml, 75.65 μg/ml, 94.31 μg/ml, 215.21 μg/ml and 128.76 μg/ml, respectively.