Mophological and Molecular Identification of Potato Cyst Nematode from Sichuan, China

Baolin SHAO Jianfeng GU Yiwu FANG Xinxin MA Xingyue LI Jingwu ZHENG

Abstract ［Objectives］The infection symptoms of cyst nematodes were found in Yuexi, Sichuan. In order to identify the pathogen, the isolated nematodes were identified by morphology and molecular biology.

［Methods］ The potato roots and soil around the roots were collected, the nematodes in the roots were stained and observed, and the cysts were separated by the simple floating method. The second-stage juveniles (J2s), females and cysts were found, and they were photographed and morphologically measured. The DNA of cysts and J2s were extracted and identified by species-specific PCR. The DNA sequences of 18S gene, 28S D2-D3 region and ITS region in ribosomal DNA were obtained. Sequences of some cyst nematode species were downloaded from GenBank for sequence alignment, and MrBayes 3.2.3 software was used to construct a Bayesian phylogenetic tree.

［Results］ The nematodes could invade hosts’ root system. The basal knobs of J2s was nearly round and inclined backward; the average length of the stylet was shorter than 23 μm; the Granek ratio of cysts was greater than or equal to 3, which is highly consistent with that of Globodera rostochiensis. The DNA templates of three cysts and four J2s were amplified by species-specific PCR, and a band of about 430 bp was obtained. After further sequencing, the length was 434 bp, which is consistent with G. rostochiensis. The evolutionary analysis of rDNA 18S and 28S showed that the cyst nematode population was a Globodera species, and further evolutionary analysis of ITS gene confirmed that the population was G. rostochiensis.

［Conclusions］ The nematodes are G. rostochiensis, which is a quarantine species of great concern to both domestic and import quarantine. Once introduced and colonized, it is hard to eradicate. It is necessary to establish a monitoring system as soon as possible, strengthen the quarantine, supervision and management, increase investment in G. rostochiensis research and development, and develop detection and control technologies, so as to escort the healthy and sustainable development of China’s potato industry.

Key words Potato cyst nematode; Morphology; Molecular identification; Quarantine; Identification; Monitoring

Received: June 5, 2022  Accepted: August 6, 2022

Supported by Sichuan Science and Technology Program (2021YFN0009); Science and Technology Project of General Administration of Customs (2020HK161); Key Project of Ningbo Public Welfare Science and Technology Program (2021S024); Technology Development Project of Ningbo Joysun Product Testing Service Company (2020ZS003).

Baolin SHAO (1980-), male, P. R. China, devoted to research about quarantine pests.

*Corresponding author. Jianfeng GU (1972-), male, P. R. China, devoted to research about nematodes, E-mail: jeffgu00@qq.com.

The potato is the third largest food crop in the world and the fourth largest in China. It is an advantageous crop integrating grain, vegetables, feed and industrial raw materials. China is the world’s largest potato production and planting country, and Sichuan ranks first in China’s potato planting, with an area of 730 000 hm2. Since 2012, Sichuan’s potato planting area and total yield have ranked first in the country, and the potato industry has gradually become a pillar industry for increasing agricultural production and farmers’ income in Sichuan Province. In recent years, Sichuan Province has listed the potato industry as a characteristic and advantageous agricultural industry in Sichuan Province, vigorously promoted the development strategy of potato as a staple food, and greatly improved the rapid development of the potato industry by relying on its unique geographical advantages and a sound breeding system for improved varieties. However, while strengthening the breeding and introduction of exotic varieties, the risk of introduction and spread of potato cyst nematodes is very high, which seriously threatens the sustainable development of the potato industry.

Potato cyst nematodes mainly include Globodera rostochiensis Skarbilovich, 1959［1］ and Globodera pallida Behrans, 1975［2］. They are important pathogenic nematodes that seriously threaten the potato industry, and they are very harmful and internationally recognized as the most important quarantine pests. They are also important imported phytosanitary pests in China. In 2020, potato cyst nematodes were included in National Agricultural Phytosanitary Pest List by the Ministry of Agriculture and Rural Affairs. According to foreign reports, G. rostochiensis generally causes a 20% reduction in potato yield. In tropical regions, when the damage is serious, it can cause a yield loss of 80 to 90%, or even no harvest［3］. In order to prevent its introduction, China has banned the commercial introduction of seed potatoes for a long time, and has not yet approved the import of any edible potatoes.

In June 2021, we found in Yuexi County, Sichuan Province that some plants in potato fields were short and showed yellow leaves and poor growth. The roots of the plants were dug up and observed with light yellow to golden spherical cysts thereon, and such plants were suspected to be infected by potato cyst nematodes. At present, no potato cyst nematodes have been reported in China, so the identification of pathogens is of great significance. The isolation and morphological and molecular biological identification of pathogenic nematodes were studied, aiming to identify pathogenic nematode species and provide reference for further monitoring, identification and prevention and control of potato cyst nematodes.

Materials and Methods

Sample collection

The samples were collected from potato planting fields in Guer Township, Zhu’ajue Town, Yuexi County (GPS: Longitude 102°41′37.3″E; Latitude: 28°25′46.8″N; Elevation: 2 315.1 m). The potato planted was Qingshu 9, and the collection time was the flowering period of potato. For the fields with poor potato growth and dwarfing, mixed samples of rhizosphere soil of about 500 g were collected from multiple points, and potato seedlings were uprooted to observe and collect a little root system.

Nematode isolation

The second-instar larvae in the soil were isolated by the modified funnel method (Ningbo Zhenhai Baichuan Biotechnology Co., Ltd.). A 100 g of soil sample was weighed, wrapped with double-layer gauze, put in a funnel with an appropriate amount of water (the amount of water was appropriate to just cover the soil), and incubated at 25 ℃ for 24 h. Then, 5-10 ml of the nematode isolation solution was collected with a watch glass, and whether there were nematodes was observed under a Zeiss Stemi 305 dissecting microscope.

Cysts in soil were separated by a simple flotation method. A 100 g of air-dried soil sample was weighed and put in a 2 000 ml triangular flask. The soil sample was added with water to a water depth of about 5 cm and shaken fully. Then, water was added while stirring until the water surface reached the mouth of the bottle, and the bottle was stood for a while. The floating materials at the bottle mouth were filtered through a set of sieves (including 20-mesh and 100-mesh sieves), and the collection on the 100-mesh sieve was poured gently into a funnel with filter paper for filtration. After filtration, the filter paper was taken to observe and count cysts under a dissecting microscope.

Fresh cysts were cut with a scalpel, and a large number of eggs were found in them. An appropriate amount of eggs were transferred with a pipette to a glass slide, which was then covered with a cover glass, and pressed gently with fingers to break some of the eggs, and the released nematodes were J2s.

Morphological identification

Cyst vulva cone preparation

Cysts were transferred to a drop of water on a plastic petri dish. The rear end of the cysts (that is, the side opposite to the neck) was cut with a scalpel under a dissecting microscope, and a bamboo needle or a No. 0 wolf brush was used to gently remove the adhesions and eggs in each vulva cone, the edge of which was properly trimmed with a scalpel. Next, a small drop of glycerol was dropped in the middle of a clean glass slide, and the trimmed vulva cone was moved to the center of the glycerol drop with a teasing needle under a dissecting microscope, and pressed down with the teasing needle to make the outer surface upward. Two small wax blocks were symmetrically placed on both sides of the glycerol drop, and a cover glass was added. The obtained slide was heated on a heating plate at 64 ℃. After the wax blocks were melted and cooled naturally, the slide was sealed with neutral balsam.

Microscope observation, photography and measurement

The overall morphology and internal characteristics of J2s, cysts, and vulva cones were observed, photographed, and measured with Zeiss Imager Z1 microscope and Axioscop MRm digital camera

Staining of nematodes inside roots

Potato plant roots were collected during flowering, and the seedling roots were stained with sodium hypochlorite-acid fuchsin as a whole［4-5］. The infection of cyst nematodes in the root system was observed.

Molecular biological identification

Extraction of Cyst and larval DNA

Three cysts and six second-instar larvae isolated were taken, and DNA was extracted according to the method of Wang et al.［6］, and stored at -20 ℃ until use.

Species-specific PCR identification

The DNA samples obtained were amplified by PCR using ITS5 and PITSr3 primers［7］, and a group of negative control and a group of blank control were also set. The DNA template of the negative control was soybean cyst nematodes. The reaction system (25 μl) contained 10×PCR buffer (Mg2+ free) 2.5 μl, 25 mmol/l MgCl2 2 μl, 2.5 mmol/L dNTP Mix 2 μl, 10 μmol/L primers 0.5 μl each, 5 U/μl Taq DNA polymerase 0.4 μl, template DNA2 μl, and ddH2O to 25 μl. The reaction program was started with 94 ℃ for 3 min, followed by 35 cycles of 94 ℃ for 30 s, 60 ℃ for 30 s and 72 ℃ for 45 s, and completed with 72 ℃ for 5 min. After PCR, the products were electrophoresed on a 1% agarose gel in 1×TAE solution with Gelred dye added, using DL100 bp (TaKaRa) as Marker, and a UV gel imager was used for observation and photographing to confirm that the amplification was effective. The remaining amplification products were sent to Hangzhou Tsingke Biotechnology Co., Ltd. for bidirectional sequencing.

Amplification and sequencing analysis of nematode ribosomal gene

The DNA templates of a single cyst and two J2s were taken for amplification, respectively. Partial 18S gene of ribosomal rDNA was amplified using primers: 988 (5’-CTCAAAGATTAAGCCATGC-3’) and 1912 (5’-TTTACGGTCAGAACTAGGG-3’), and 1813 (5’-CTGCGTGAGAGGTGAAAT-3’) and 2646 (5’-GCTACCTTGTTACGACTTTT-3’)［8］. The D2-D3 region of the ribosomal rDNA 28S gene used amplification primers 391A (5’-AGCGGAGGAAAAGAAACTAA -3’) and 501 (5’-TCGGAAGGAACCAGCTACTA-3’)［9］. For the ITS region of ribosomal rDNA, amplification primers TW81 (5’-GTTTCCGTAGGTGAACCTGC-3’) and AB28 (5’-ATATGCTTAAGTTCAGCGGGT-3’) were used［10］. The above primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd., and the PCR amplification procedure referred to the literature where each primer is involved. After PCR, the products were electrophoresed on a 1% agarose gel in 1×TAE solution with Gelred dye added, using DL100 bp (TaKaRa) as Marker, and a UV gel imager was used for observation and photographing to confirm that the amplification was effective. The amplified products of the 18S gene and the D2-D3 region of 28S gene sent to Hangzhou Tsingke Biotechnology Co., Ltd. for bidirectional sequencing. The direct sequencing of the ITS sequence could not obtain a valid sequence, and cloning and sequencing were performed by the above-mentioned company.

The chromas files obtained by sequencing were spliced using the ChormasPro software, and redundant bases at both ends of the sequencing results were removed to obtain sequences for subsequent alignment and phylogenetic analysis. Alignment was performed on the BLAST (basic local alignment search tool) page on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Partial sequences of various registered similar species (Globodera) in GenBank were downloaded, and sequence alignment was performed under the default settings of Clustal W［11］. Also, pairwise distance calculation was performed on the obtained sequences and their similar groups under the p-distance setting in the MEGA software.

With Heterodera schachtii MW834323 and Heterodera glycines EF611124 as outgroups, the evaluation of nucleotide substitution model was carried out on the obtained sequence alignment files under the Akaike information criterion (AIC) using the jModeltest software［12］, and corresponding supporting models were obtained. A Bayesian phylogenetic tree was constructed based on the obtained sequence alignment files using the MrBayes 3.2.3 software［13］. According to the obtained nucleotide substitution models, the program was set according to the instructions of Ronquist & Huelsenbeck［13］. Specifically, 5×107 runs of 3 hot chains and 1 cold chain were independently performed under 4 Markov Chains, with the chain sampled every 1 000 generations and the burn-in value was 25%. Adopting the principle of majority agreement (50%), the posterior probability value of the obtained Bayesian phylogenetic tree was evaluated by the MCMC (Markov Chain Monte Carlo) method［14］, and the phylogenetic tree was viewed and edited in the TreeGraph 2 software［15］.

Agricultural Biotechnology2022

Results and Analysis

Field symptoms

Potato plants in the sampling fields were short, and showed yellow leaves and poor growth. When pulling up the plants, there were light yellow to golden spherical particles on part of the root system, which were females or cysts of Globodera (Fig. 1). The particles appeared in a large number and were visible to the naked eye, and there were many hundreds of particles or more in severe cases.

Morphological characterization

More than 100 cysts were isolated from 100 g of air-dried soil by the flotation method, and a large number of J2s of cyst nematodes were isolated by the modified funnel method, but no males were found.

Females: The body is nearly spherical, and has a prominent neck. They are white to pale yellow. The head has fused lips and 1 or 2 distinct lip pieces. The neck rings are irregular. Most of the body walls become reticulated ridges, and the head skeleton is

weakly developed. The conus of the stylet accounts for about 50% of the length of the stylet and is obviously different from the shaft, and the basal knobs of the stylet are tilted backwards. The stylet extends from the head frame to about 75% of the length of the stylet. The middle esophagus bulb is large, almost spherical, and the valve door is crescent-shaped. The excretory hole is obvious, located at the base of the neck. The two ovaries fill the entire body cavity. The vulva is transversely fissured, and the surrounding stratum corneum is slightly depressed, forming a vulvar membrane hole. The vulva is located between two thin probola-like crescent-shaped regions. There are about 12 parallel ridges in the stratum corneum between the anus and the vulval fenestrae, a few of which are cross-connected.

Cysts: Cysts are spherical or nearly spherical, and have prominent neck and rounded tail, and no protrusions. They are golden or dark brown. The epidermis has "Z"-shaped ridges. The vulva cone is a single circumfenestrate, having no vulval bridge, inferior bridge and other internal gland processes, and no vesicular process. Eggs are present in cysts and do not form oocysts. The vulva area of fresh cysts is intact, but some or all of the vulval fenestrae are lost in older cyst specimens. The anus is obvious, and no fenestrae are formed, but a "V"-shaped structure is sometimes seen. The cuticle pattern is clearer than in females.

J2s: A mixture of various nematodes was isolated. Cyst nematodes are easily distinguished from various other nematodes based on their strong stylets, which are about 20 μm in length and have markedly enlarged knobs. A total of about 500 J2s of Globodera nematodes were isolated. They are worm-shaped, with regular rings on the cuticle and 4 lateral lines in the lateral area. After heat-killing, the worm body is usually slightly ventral, and the body length is 405-444 μm. The head is round, slightly constricted, with 4 to 6 rings. The stylet is strong, 19.4-21.9 μm in length. The basal knobs of the stylet are nearly round, slightly inclined backward, and the front is relatively flat. The conus of the stylet accounts for about 50% of the length of the stylet. The esophageal glands extend posteriorly to about 35% of the body length. The hemizonid is conspicuous, 2 body rings long, located at the body ring in front of the excretory pore. The tail is tapered, thin and round at the end, and the clear tail accounts for about 1/2 of the length of the tail.

J2s and cyst morphometric measurements

20 J2s and 15 cysts of the Yuexi population in Sichuan were measured and compared with the data of foreign reference information of G. rostochiensis. The results are shown in Table 1.

The cysts of the Yuexi population in Sichuan are spherical and golden yellow. The number of stratum corneum ridges between vulva membrane pores is 15 to 24, and the Granek ratio is 2.7 to 4.7. The length of the second-instar larvae is 19.4 to 21.9 μm. The stylet knobs are nearly round, slightly inclined backward, and the front is relatively flat. The DGO distance is 3.0-5.2 μm. The clear tail length is 17.7-25.0 μm. The above characteristics or measured values are consistent with G. rostochiensis.

Staining results of nematodes inside roots

After transparent staining of the root system, it could be observed that a large number of nematodes parasitized in the roots, which were pink (Fig. 4). The body length was about 400 μm and the body width was slightly shorter than 20 μm, which was consistent with the measured values in Table 2. Further observation of morphological characteristics showed that it was consistent with J2s of the above-mentioned cyst nematodes, so the nematodes could invade the interior of potato roots.

DNA sequence analysis of ribosomal genes

The nematode population isolated from Yuexi, Sichuan was subjected to gene amplification and sequencing of the 18S region of ribosomal rDNA. The fragments obtained from the three templates were all 1 740 bp in size, and the GenBank accession number is MZ613180. The similarity values of the 18S gene sequence from the Yuexi population with the sequences from G. rostochiensis reported in other countries and regions ranged from 99.41% (AY593877 1696/1701 bp) to 99.94% (KJ636271 1700/1701 bp); the values with the sequences from other G. pallida were in the range of 99.53% (G. pallida strain GlobPal10 KJ636269) to 99.77% (G. pallida isolate GlobPal3 AY284620); and the values with the sequences from other Globodera ranged from 99.12% (G. achilleae strain GlobAch2 KJ636274) to 100% (Globodera tabacum isolate 1094 FJ040401 1701/1701 bp). Therefore, the 18S region gene of ribosomal rDNA could not accurately distinguish the specific species of Globodera, but it could be clearly seen that the group belongs to Globodera.

The fragment size of the ribosomal 28S region gene of this population obtained by amplification and sequencing was all 953 bp, and the GenBank accession number is MZ613167. The similarity values of the 28S gene sequence from the population with the sequences from G. rostochiensis reported in other countries and regions ranged from 99.06% (AY592987 947/956 bp) to 100% (MK311333 953/953 bp); the values with the 28S gene sequences from G. pallida were in the range of 97.59% (EU855119 932/955 bp) to 98.89% (LT159821 711/719 bp); the values with the 28S gene sequences from G. artemisiae ranged from 98.01% (EU855121 937/956 bp) to 98.19% (MT233316 702/720 bp); and the value with the 28S gene sequences from G. tabacum was 99.40% (GQ294492). Similarly, the gene in the 28S region of ribosomal rDNA could not accurately distinguish the specific species of Globodera, but it could be clearly seen that the Yuexi group belongs to Globodera.

The direct sequencing on the gene amplification products of the ribosomal ITS region of this population failed. After cloning and sequencing, two sequences were obtained, with lengths of 1 040 and 1 041 bp, respectively. The GenBank accession numbers are MZ613152 and MZ613153, and the similarity between the two sequences was 99.33% (1035/1041 bp). The similarity of the ITS gene sequence of the Yuexi population with the sequences from G. rostochiensis reported from other countries and regions was the highest, ranging from 97.90% (JF907553 981/1002 bp) to 99.90% (GQ294513 1040/1041 bp). Meanwhile, the sequence similarity with the phylogenetically sister G. tabacum ranged from 97.26% (FJ667945 924/950 bp) to 97.90% (GQ294525 1024/1045 bp), and the highest sequence similarity with G. pallida was 96.5% (LT159833). It can be seen from the phylogenetic tree (Fig. 6) that the Yuexi population and G. rostochiensis populations reported in other countries and regions were clustered in the same branch with high confidence (post-validation probability=100), indicating that the Yuexi population in Sichuan is indeed G. rostochiensis.

Identification conclusions

Globodera belongs to the subfamily Heteroderinae, and common ones in this family are Globodera, Punctodera, Cactodera, and Heterodera［19］. Punctodera has oval cysts and forms membrane pores in the anal area; Cactodera has lemon-shaped cysts with simplified vulva cones; Heterodera has lemon-shaped cysts with prominent vulva cones; and Globodera has spherical cysts, without vulva cones, and no fenestrae are formed in the anal area, so it can be clearly distinguished from similar genus. The cysts of the Yuexi nematodes are spherical, and have no vulva cones, and no fenestrae are formed in the anus, so the population obviously belongs to Globodera.

According to Subbotin et al.［19］, Globodera includes 10 species of nematodes. Since then, G. ellingtonae was discovered in Idaho, USA in 2012［20］; G. capensi was found on weeds in South Africa in 2013［21］; and in 2017, G. agulhasensis was discovered in South Africa［22］, and G. sandveldensis was found on weeds in South Africa［23］. Therefore, a total of 14 species of Globodera have been reported so far［23-24］. According to the key list of Subbotin et al.［19］, the cuticle of G. mali cysts is thin and transparent; the average stylet length of the J2s of G. zelandica is equal to or greater than 27 μm; the average stylet length of the J2s of G. leptonepia is smaller than 19 μm; and the average hyaline tail region of the J2s of G. bravoae is greater than 31 μm. G. capensis, G. millefolii, G. artemisiae, G. agulhasensis and G. sandveldensis are mostly parasitic on Compositae plants, and the average Granek ratio was less than or equal to 2. Therefore, the samples to be tested can be easily distinguished from the above-mentioned 9 species of nematodes. The difference between the Yuexi population and G. tabacum and G. ellingtonae is the number of cuticular ridges between the anus and vulval fenestrae ［18 (15-24) vs 7 (5-15)］, 13 (10 to 18); and the difference from G. mexicana is the Granek ratio (mean 3.7 vs 2.8).

According to hosts and morphological characteristics, the Globodera population from Yuexi, Sichuan can be easily distinguished from other nematodes, and the most similar one is G. pallida. The differences from G. pallida lie in female and cyst color (females gradually turning from white to yellow, golden or brown cysts vs white females, brown cysts), slightly shorter body length of J2s ［(405.0-443.5) μm vs (440-525) μm］, slightly shorter stylet length of J2s ［20.2 (19.4-21.9) μm vs 23.6 (21-26) μm］, slightly shorter tail length of J2s ［46.8 (40.0-51.0) μm vs 51.9 (46-52) μm］, the shape of basal knobs of J2s (nearly round, slightly inclined backward, and relatively flat anterior surface vs. anterior surface protruding forward), the number of cuticular ridges between the anus and vulval fenestrae ［18 (15-24) vs 12.2 (8-24) 20)］, the distance from the anus to fenestrae ［(43-76) μm vs (88-102) μm］, and the Granek ratio ［3.7 (2.7-4.7) vs 2.2 (1.2-3.6)］［3,25］.

The Yuexi nematode population is basically the same as G. rostochiensis morphologically. The specific PCR method and ribosomal DNA sequence analysis showed that its ITS gene sequence was very similar to the already registered G. rostochiensis, with a similarity of 99.9%, and the highest similarity with G. pallida was only 96.5%. Therefore, both morphological and molecular biological methods support that the nematodes are G. rostochiensis.

Discussion and Conclusions

The damage caused by G. rostochiensis is concealed with a long incubation period, and there are no obvious symptoms in the early stage of infection. It is difficult to confirm whether nematodes exist by field observation only. Serious damage can lead to yellowing, necrosis, wilting, dwarfing and even death of hosts, but the above symptoms are difficult to distinguish from symptoms such as lack of water and fertilizers, and it is often necessary to collect soil and root samples for the isolation and identification of cysts or larvae in the laboratory. Microscopic examination showed that the cysts were spherical and had no vulva cones, and no fenestrae were formed in the anus, so it could be judged that the nematodes belong to Globodera. Then, for J2s, the basal knobs of the stylet were nearly round, and inclined backward; the average length of the stylet was smaller than 23 μm; and the Granek ratio was greater than or equal to 3. Consequently, the group could be judged as G. rostochiensis.

Nematodes are small in size, and their identification features are very subtle, so morphological identification requires extensive experience. Therefore, molecular biology methods are important auxiliary means for nematode identification. According to the method of EPPO［25］, in this study, the specific primers ITS5 and PITSr3 reported by Bulman & Marshall［7］ were used to amplify nematode cysts or J2s by PCR. The results of electrophoresis and sequencing showed that the amplified product was 434 bp, which is completely consistent with the reported characteristic fragment of G. rostochiensis. Meanwhile, we cloned, sequenced and analyzed the ribosomal rDNA 18S, rDNA 28S and ITS genes of the Yuexi population, and found that the 18S and 28S sequences of this population were highly similar to those of G. rostochiensis and G. pallida, but specific species could not be distinguished, and it could only be determined that the nematodes belong to the genus Globodera. Further, the ribosomal ITS gene sequence analysis showed that this species of nematode had the highest similarity with the ITS gene sequence of G. rostochiensis populations reported abroad. The phylogenetic tree results also showed that this group and G. rostochiensis populations reported in other countries and regions were clustered in the same branch with high confidence (post-validation probability = 100), which confirmed that the Yuexi group in Sichuan was indeed G. rostochiensis. Therefore, the ITS gene as a molecular target is more effective in the identification of Globodera species, and it is recommended to use specific PCR method or ITS gene for molecular identification of G. rostochiensis.

With the increasing frequency of economic globalization and international trade, G. rostochiensis has been introduced into China through personal carrying, exchange of potato germplasm resources, or the introduction of other root materials. Li et al.［27］ used niche models GARP and MaxEnt to analyze the suitable habitats of G. rostochiensis and G. pallida in China. The results showed that northeastern Yunnan, Guizhou, Chongqing, eastern Sichuan, Hunan, southern Hubei, southern Shandong, Henan, Anhui, and Jiangsu were the medium and high risk areas for G. rostochiensis. Because the nematodes have a wide range of suitable habitats in China, difficult prevention and control, high potential damage loss and huge social impact, once introduced and established, eradication is difficult. In the absence of a host crop, the cysts can survive in soil for more than 20 years［28］. Therefore, it is recommended to take strict emergency control measures in the area where the epidemic occurs, and strive to eliminate it. Meanwhile, monitoring points should be set up around the epidemic and high-risk areas to strictly control the cross-regional transportation of potatoes and other root and stem materials at epidemic points, in order to prevent the spread of the epidemic. In view of the economic importance of G. rostochiensis damage, it is necessary to improve the potato cyst nematode epidemic monitoring and control technology system as soon as possible, strengthen the quarantine supervision and management of seed potatoes, carry out national census of G. rostochiensis, and increase investment in the research and development of G. rostochiensis prevention and control technology, so as to escort the healthy and sustainable development of China’s potato staple food industry.

References

［1］ WOLLENWEBER H. Krankheiten und Besch dingung der Kartoffel［J］. Arbeiten Forschungs Institut für Kartoffel, Berlin, 1923(7): 1-56.

［2］ STONE AR. Heterodera pallida n. sp. (Nematoda: Heteroderidae), a second species of potato cyst nematode［J］. Nematologica, 1973, 18(1972): 591-606.

［3］ PENG H, LIU H, JIANG R, et al. Potential invasion of the potato cyst nematode Globodera rostochiensis and G. pallida into China［J］. Plant Protection, 2020, 46(6): 1-9. (in Chinese).

［4］ BYRD D W, KIRKPATRICK T, BARKER K R. An improved technique for clearing and staining plant tissues for detection of nematodes［J］. Nematology, 1983(15): 142-143.

［5］ FAN JW, HU CL, ZHANG LN. Jasmonic acid mediates tomato’s response to root-knot nematodes［J］. Journals of Plant Growth Regulation, 2015, 34(1): 196-205.

［6］ WANG JL, ZHANG JC, GU JF. Method of extract DNA from a single nematode［J］. Plant Quarantine, 2011, 25(2): 32-35. (in Chinese).

［7］ BULMAN SR, MARSHALL JW. Differentiation of Australasian potato cyst nematode (PCN) populations using the polymerase chain reaction (PCR)［J］. New Zealand Journal of Crop and Horticultural Science, 1997(25): 123-129.

［8］ HOLTERMAN M, VAN DER WURFF A, VAN DEN ELSEN S, et al. Phylum-wide analysis of SSU rRNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades［J］. Molecular Biology and Evolution, 2006(23): 1792-1800.

［9］ NADLER SA, CARRENO RA, MEJIA-MADRID H, et al. Molecular phylogeny of clade III nematodes reveals multiple origins of tissue parasitism［J］. Parasitology, 2007(134): 1421-1442.

［10］ CURRAN J, DRIVER F, BALLARD JWO, et al. Phylogeny of metarhizium-analysisi of ribosoma DNA-sequence data［J］. Mycological Research, 1994(98): 547-552.

［11］ THOMPSON JD, HIGGINS DG, GIBSON TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice［J］. Nucleic Acids Research, 1994(22): 4673-4680.

［12］ DARRIBA D, TABOADA GL, DOALLO R, et al. jModelTest 2: more models, new heuristics and parallel computing［J］. Nature Methods, 2012(9): 772.

［13］ RONQUIST F, HUELSENBECK JP. MrBayes 3: Bayesian phylogenetic inference under mixed models［J］. Bioinformatics, 2003(19): 1572-1574.

［14］ LARGET B, SIMON DL. Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees［J］. Molecular Biology and Evolution, 1999(16): 750-759.

［15］ ST VER BC, M LLER KF. TreeGraph 2: combining and visualizing evidence from different phylogenetic analyses［J］. BMC Bioinformatics, 2010(11): 7.

［16］ GOLDEN AM, ELLINGTON DMS. Redescription of Heterodera rostochiensis (Nematoda: Heteroderidae) with a key and notes on closely related species［J］. Proceedings of the Helminthological Society of Washington, 1972(39): 64-77.

［17］ KAZACHENKO IP. Cyst-forming nematodes of the Russian Far East and measures of their control［J］. Vladivostok, USSR, Dalnauka, 1993: 77.

［18］ SIRCA S, UREK G. Morphometrical and ribosomal DNA sequence analysis of Globodera rostochiensis and Globodera achilleae from Slovenia［J］. Russian Journal of Nematology, 2004(12): 161-168.

［19］ SUBBOTIN SA, HALFORD PD, WARRY A, et al. Variations in ribosomal DNA sequences and phylogeny of Globodera parasitising Solanaceae［J］. Nematology, 2000(2): 591-604.

［20］ HANDOO ZA, CARTA LK, SKANTAR AM, et al. Description of Globodera ellingtonae n. sp. (Nematoda: Heteroderidae) from oregon［J］. Journal of Nematology, 2012, 44(1): 40-57.

［21］ KNOETZE RAB, SWART AC, TIEDT L RD. Description of Globodera capensis n. sp. (Nematoda: Heteroderidae) from South Africa［J］. Nematology, 2013(2): 233-250.

［22］ KNOETZE RAB, SWART AC, WENTZEL RA, et al. Description of Globodera agulhasensis n. Sp. (Nematoda: Heteroderidae) from South Africa［J］. Nematology, 2017(3): 305-322.

［23］ KNOETZE R, SWART A, WENTZEL R, et al. Description of Globodera sandveldensis n. sp (Nematoda: Heteroderidae) from South Africa［J］. Nematology, 2017(7): 805-816.

［24］ Ministry of Agriculture of the People’s Republic of China, General Administration of Quality Supervision, Inspection and Quarantine of the people’s Republic of China. Imported plant quarantine pests of People’s Republic of China［S］. China, 2017, 47(2): 174-197.

［25］ EPPO. PM 7/40 (4) Globodera rostochiensis and Globodera pallida. Bulletin OEPP/EPPO Bulletin Bulletin OEPP/EPPO Bulletin. 2017, 47(2): 174-197.

［26］ LI JZ, PENG DL. Potential geographic distribution and management strategy of potato cyst nematode: (Globodera rostochiensis and G. pallida) in China［C］//Proceedings of the Annual Meeting of Chinese Society for Plant Pathology, 2009: 401.

［27］ WINSLOW RD, WILLIS RJ. Nematode diseases of potatoes. II. Potato cyst nematode, Heterodera rostochiensis. In Economic Nematology; Webster, J., Ed［J］. Academic Press: New York, NY, USA, 1972: 18-34.