ldentification of Heterodera schachtii on sugar beet in Xinjiang Uygur Autonomous Region of China

PENG Huan ,LlU Hui ,GAO Li ,JlANG Ru ,Ll Guang-kuo ,GAO Hai-feng,WU Wei,WANG Jun,ZHANG Yu,HUANG Wen-kunKONG Ling-anPENG De-liang

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

2 The National Agro-Tech Extension and Service Center,Beijing 100026,P.R.China

3 Key Laboratory of Integrated Pest Management on Crops in Northwestern Oasis of Ministry of Agriculture and Rural Affairs,Institute of Plant Protection,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,P.R.China

4 Agricultural Technology Extension Service Center of Xinyuan County,Yining 835800,P.R.China

5 Plant Protection Station of Xinjiang Uygur Autonomous Region,Urumqi 830006,P.R.China

Abstract The sugar beet cyst nematode,Heterodera schachtii,is a major parasite of sugar beet which has been recognized and listed as a quarantine nematode in China and more than 20 countries and regions worldwide. A survey for important nematodes was undertaken in the sugar beet planting area of China during 2015-2018,and numerous cysts were collected from sugar beet fields in Xinyuan County,Xinjiang Uygur Autonomous Region of China. The observations of morphological and morphometric characteristics revealed that cysts,vulval cones and second-stage juveniles of the Xinjiang population were in the same range of each other and within those of other reported H.schachtii populations.Molecular analysis of rDNA-ITS,28S-D2/D3 and mtDNA cytochrome c oxidase subunit 1 (COI) gene sequences suggested that the Xinjiang population clustered in a branch with those foreign populations,and the sequence similarity was as high as 99.81-100%. Moreover,this result was confirmed by PCR assay with species-specific primer SHF6 and rDNA2 of H.schachtii,and the pathogenicity test confirmed successful Xinjiang population reproduction in both plant hosts. In conclusion,based on morphological and molecular characterization,this study confirmed that the cyst nematode population collected from sugar beet fields in Xinjiang is H.schachtii. As far as we know,this is the first report of H.schachtii on sugar beets in Xinjiang,China.

Keywords:sugar beet cyst nematode,Heterodera schachtii,morphological,molecular identification

1.lntroduction

The sugar beet nematode,HeteroderashcachtiiSchmidt(1871),is one of the major nematodes on sugar beet worldwide (Müller 1999). It has a wide host range and can infect and complete its life cycle on more than 218 plants species in 95 genera and 23 families including agricultural fields,cultivated vegetable plants and weeds such as sugar beet,radish,rape,spinach,broccoli,cabbages,tomatoes and other closely related crops(Steele 1965),and yield loss can be as much as 50%when populations are high (Curto 2008). In Europe,the annual yield loss caused byH.schachtiiwas estimated at 90 million EUR (Müller 1999).

This species was first found on sugar beet in 1859 when it caused stunted growth and declining yields in Germany,one of the earliest plant nematode species described;and it was later found widely distributed throughout the world,especially in Europe,North America,Australia,the Middle East,Africa and South America (Evans and Rowe 1998). Recently,more than 50 countries and regions have reported it (Subbotinet al.2010). In Asia,this cyst nematode has been reported on Chinsese cabbage in South Korea (Kim 2016) and cauliflower in Jordan (Lafiet al.2016),Japan (IPPC website 2018) and other countries. There are records of beet cyst nematode in Guangdong Province of China in the 1990s (Zhang and Huang 1990);however,this record has not been confirmed until today. Recently,H.schachtiiwas listed as a quarantine pest in China.The potential distribution ofH.schachtiiin China using Maxent and GARP models predicted that this species could occur in the major sugar beet planting area of China including Xinjiang,Heilongjiang and Inner Mongolia (Liet al.2008).

A survey for important plant parasitic nematodes was undertaken in the sugar beet planting area of China,during 2015-2018. In a sugar beet field of Xinyuan County,Xinjiang Uygur Autonomous Region,the leaves of sugar beet were yellow,while dwarf plants,small patches of wilting and dead young plants were observed,and an excessive number of hair-like roots and white to brown,lemon-shaped cysts could be observed clinging to the roots. The morphological identification and molecular characterization of the cyst nematode fully indicated that it belongs to sugar beet nematodeH.schachtii. To our knowledge,this is the first record ofH.schachtiiin Xinjiang.

2.Materials and methods

2.1.Sample collection and nematode extraction

The soil samples were taken from a depth of 20 cm using a soil auger with a blade measuring 25 cm×3 cm. The root of sugar beet was also collected from the same locations as the soil at the same time and was generally combined in the same sample bag. Cysts were extracted from the soil according to the method of Trudgillet al.(1972). The females were extracted from the roots by direct dissecting,while the tray method was used to collected the second-stage juveniles (J2s) and males following Whitehead and Hemming (1965).

2.2.Morphological characterization

The juveniles and males were killed and fixed in thermal 4% formalin and kept warm for 20-40 min,processed in glycerin and mounted on microscope slides (Goldenet al.1988). The vulva cones of cysts prepared after dissection were cleaned and trimmed. All slides were observed and measured for morphological characteristics under a light microscope (BX53,Olympus,Japan) with a microscope digital camera (DP73,Olympus,Japan).

For scanning electron microscopy (SEM),the cysts and J2s were fixed with 10% formalin solution and 2.5%glutaraldehyde and then rinsed in 0.1 mol L-1phosphate buffer 2-3 times,followed by dehydration in a gradual transitional series of ethanol,CO2critical-point dried,mounted on stubs and sputter coated with gold-palladium(Lax and Doucet 2002). The SEM pictures were observed under a scanning electron microscope (Hitachi Regulus 8100,Hitachi,Japan) at 10 kV.

2.3.Molecular identification

Genomic DNA was extracted from single cysts as described by Penget al.(2013). The internal transcribed spacer (ITS) rRNA and D2-D3 of 28S rRNA were amplified with universal primer pairs(TW81,5′-GTTTCCGTAGGTGAACCTGC-3′,AB28,5′-ATATGCTTAAGTTCAGCGGGT-3′) (Curranet al.1994)and (D2A,5′-ACAAGTACCGTGAGGGAAAGTTG-3′;D3B,5′-TCGGAAGGAACCAGCTACTA-3′) (Leyet al.1999) respectively. The cytochrome c oxidase subunit 1(COI) mtDNA gene was amplified using the primers JB3 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′ and JB5 5′-AGCACCTAAACTTAAAACATAATGAAAATG-3′(Deryckeet al.2005). The PCR products were purified and sequenced. The obtained sequences were investigated by the BLASTn and BLASTx programs in GenBank at the NCBI website. The published sequences of ITS,D2-D3 region of 28S rRNA and COI genes fromH.schachiiand other cyst nematode species were selected and downloaded,combined with the sequences obtained in this study,and the phylogenetic tree was produced using MEGA6 with maxinum likehood model (Tamuraet al.2013). Finally,a previously described method for SBCN detection using a specific primer (SHF6) in combination with a universal primer (rDNA2) was used in our study to confirm the results (Amiriet al.2002).

2.4.Pathogenicity test

Sugar beetcv.SD21816 and oilseed rapecv.Deyou 6 were planted in 10 cm pots with autoclaved sand soil and incubated in a greenhouse maintained with a photoperiod of 16 h at 25°C. Cysts were hand-picked and cleaned with sterile water,eggs were collected from cysts and hatched pre-parasitic J2 were collected by placing the eggs on a sieve (25 μm pore size) in 3 mmol L-1ZnSO4at 25°C. Approximately 500 J2 nematodes were inoculated into the sugar beet andBrassicacampestrisroots per pot. Five pots of plants were collected at 5,10,15,and 30 days after infection (dai),the roots were cleaned gently with water and stained with acid fuchsin (Byrdet al.1983). The cysts in soil were isolated and counted under a Stereomicroscope (SZX16,Olympus,Japan). The cysts were detected by the molecular method described above.

3.Results

3.1.Morphological identification

The female:white,flask-shaped,with short neck embedded in host root and swollen body on the root surface. Yellow phase absent. A thin,white waxy material refered to as the ‘sub-crystalline layer’ present on female body surface,terminal vulva on vulva cone covered with a gelatinous matrix forming egg sac containing eggs (Fig.1-A-D).

Cyst:light to dark brown,with symmetry of neck and vulva cone. The terminal vulval slit is about as long as the vulval bridge which is flanked on either side by a thin,kidney-shaped area of cyst wall breaking down in older cyst. Terminal with strongly distinct vulval cone (Fig.1-E).Ambifenestrate,a regular zigzag pattern is formed on the surface (Fig.2-C). The underbridge well developed (Fig.1-F),a number of irregularly arranged,dark brown molarshaped bullae situated a short distance beneath vulval bridge. Cysts containing 500-600 eggs.

Fig.1 Cysts and females of Heterodera schachtii. A,females and cysts on the beet root. B,females and cysts on the beet. C,females with subcrystalline layer. D,females with egg sac. E,cysts. F,vulva cone.

J2s slightly curved ventrally (Fig.3-A). Labial region offset,hemispherical,with four indistinct annuli (Fig.2-BD). Stylet moderately heavy with prominent,forwardlydirected knobs (Fig.3-B-C). Median bulb is oval,the pharyngeal glands were well developed,and overlaps the intestine dorsoventrally,pharyngo-intestinal junction is clearly observed (Fig.3-B). Lateral field with four incisures with outer bands irregularly areolated (Figs.2-E and 3-D). Excretory pore distinct,genital primordium located slightly posterior to mid-body. Tail conical,gradually tapering to a rounded terminus,with hyaline portion (Figs.2-F and 3-E).

Fig.2 Scanning electron micrographs of Heterodera schachtii.A,cyst (arrow show the second-stage juveniles). B,vulva cone.C,cyst surface with ‘zigzag’ pattern. D,labia disc. E,lateral field with four incisures. F,tail region (arrow shows the annus).

Fig.3 The second-stage juveniles (J2s) of Heterodera schachtii.A,body. B,anterior region (arrow shows the excretory pore).C,head region. D,lateral field. E,tail (arrow shows the annus).F,genital primordium.

The key morphometrics of cysts (n=20) were:length excluding neck (range=(622.0-867.4) μm,mean=(752.0±87.2) μm);width ((391.1-594.1) μm,(485.2±55.3) μm);length of fenestra ((32.5-43.5) μm,(37.2±5.5) μm);width of fenestra ((22.4-37.9) μm,(27.9±4.5) μm);length of vulval slit ((37.2-48.9) μm,(40.2±4.4) μm);and length of underbridge ((88.1-116.7) μm,(105.4±9.9) μm).Measurements of J2s (n=26) included length of body(range=(429.2-512.3) μm,mean=(442.1±13.7) μm),stylet((22.3-26.5) μm,(24.2±0.5) μm),tail ((47.5-52.7) μm,(50.8±2.1) μm),hyaline tail terminus ((24.7-30.9) μm,(27.8±2.7) μm),tail/hyaline tail (1.8±0.1,1.7-1.9),and hyaline tail/stylet (1.1±0.07,1.0-1.2) (Table 1).Morphological and morphometric characteristics of the cyst and J2 were consistent with those ofH.schachtii.

Table 1 Morphometrical comparisons of cysts and second-stage juveniles (J2s) of Heterodera schachtii between Xinjiang population and other populations

3.2.Molecular characterization

The length of the obtained rDNA-ITS sequence was 1 028 bp (GenBank accession numbers MW856648-MW856650),Blast results confirmed that those sequences were 99.81-100% identical to those ofH.schachtiifrom the USA (EF611102.1),Belgium (EF611105.1 and EU616694.1),South Africa (MF754150.1),and Germany(EF611115.1). The phylogenetic tree based on the rDNA-ITS sequences was constructed,and the results indicated thatH.schachtiiof Xinjiang population was clustered with the USA population (EF611102.1),South Africa (MF754150.1),Belgium (EF611105.1),and France(EF611103.1) within a group at a value of 100% (Fig.4).The D2-D3 region of the 28S rDNA sequence ofH.schachtiiwas amplified using primers D2A and D3B,and yielded a single fragment of 737 bp (GenBank accession numbers MW534417-MW534418). Blast results showed that it hasa consistency of 100% withH.schachtiireported in Korea(MT644118.1 and MN720062.1),Japan (LC421660.1)and Netherlands (LC208672.1). In the phylogenetic tree obtained from the D2-D3 region of the 28S rRNA sequences,H.schachtiXinjiang population formed a clade with Korea (MT644118.1 and MN720062.1),Japan(LC421660.1) and Netherlands (LC208672.1) at a value of 99% (Fig.5).

Fig.4 Phylogenetic relationships of Heterodera schachtii populations and other Heterodera species based on the rDNA-ITS sequences by using the maxinum likehood model. Meloidogyne incognita was selected as outgroup. Numbers are bootstrap values.

Fig.5 Phylogenetic relationships of Heterodera schachtii populations and other Heterodera species based on the 28S D2-D3 sequences by using the Maxinum Likehood model. Meloidogyne incognita was selected as outgroup. Numbers are bootstrap values.

Amplification of the COI gene ofH.schachtiiusing the JB3 and JB5 primer set,yielded a single fragment of 446 bp in length (GenBank accession numbers MW856645-MW856647). Blast results showed that these sequences are highly identical withH.schachtiireported in the USA (EU855121.1 and KY884308.1),South Korea(MK621901.1 and KY775597.1),Poland (KC172918.1),and Netherlands (LC208708.1) with a consistency of 100%. In the phylogenetic tree based on the COI gene sequences,H.schachtifrom Xinjiang of China and other country’s populations were clustered in one branch at a value of 100% (Fig.6).

Fig.6 Phylogenetic relationships of Heterodera schachtii populations and other Heterodera species based on the cytochrome c oxidase subunit 1 (COI) sequences by using the maxinum likehood model. Meloidogyne incognita was selected as outgroup.Numbers are bootstrap values.

3.3.Specific primer for the detection of SBCN

For further confirmation,the previously reportedH.schachtiispecific primer SHF6 combined with universal primer rDNA2 were used in the present study,a single band at about 255 bp was observed inH.schachtiiXinjiang population. TheH.schachtiifrom Germany,Belgium,andTurkey were used in this study as positive controls,and the same bands were obtained from those populations (Fig.7).These results supported its identity asH.schachtii.

Fig.7 Identification of Heterodera schachtii (Hs) populations using the species-specific primers SHF6/rDNA2. M,D2000 DNA marker;Hg,Heterodera glycines;CK,negative control with no template DNA.

3.4.Pathogenicity test

The pathogenicity ofH.schachtiito sugar beet and oilseed rape was tested by inoculating 500 J2s onto healthy roots. At 5 dai,157±37 J2s per plant were observed on oilseed rape root,whereas 137±29 J2s and 35±7 J3s were found on sugar beet root. The females were found at 15 dai on both plant hosts,41±12 cysts and 32.6±7 cysts and females were isolated from sugar beet and oilseed at 30 dai,respectively. No females or cysts were observed on the control plants. The cysts obtained from the pathogenicity test were identified as the same species as those used in the inoculations based on morphology and molecular characteristics.

4.Discussion

The sugar beet nematode,H.schachtii,is one of the most important nematode pathogens which poses a significant economic threat to sugar beet production worldwide,and it has been recognized and listed as a quarantine nematode by China and more than 20 other countries and regions. This nematode has occurred and is distributed in more than 50 countries and regions and its occurrence area has been expanding.Unfortunately,the present study confirmed thatH.schachtiihas been found on sugar beet in Xinyuan County,Xinjiang according to the morphological identification and molecular analysis. Although this species was reported in Guangdong Province in 1990s,the symptom caused byH.schachtiiin beet was given in this record as “root knot” (Zhang and Huang 1990).According to the current reports,H.schachtiiinfection of sugar beet or other host crops will not cause the symptoms of root knot,moreover,that record lacks geographical location details,and it has not been confirmed so far. For this reason,this study is the first molecular and morphologcial confirmation of this species in China. During the filed investigation,we found that the sugar beet showed the following obvious symptoms:yellow leaves,dwarf plants,small patches of wilting and dead young plants. An excessive number of hair-like roots and white females and brown cysts can be observed on the roots (Fig.1). These results are consistent with the typical symptoms of beet cyst nematode infection (Subbotinet al.2010).

Morphology is the essential basis for identification of cyst nematodes (Handoo and Subbotin 2018).Heteroderaschachtiibelongs to the Schachtii group and is distinguished from closely related species by a combination of morphological and morphometric characteristics (Subbotinet al.2010). Yellow beet cyst nematodeH.betae,is another cyst nematode that can parasitize sugarbeet,however,this species can be easily distinguished morphologically fromH.schachtiiby longer body length,length of the tail and hyaline of the J2 (Maaset al.1982). In this paper,the morphological characteristics of the Xinjiang populations were observed and measured. Compared with theH.schachtiipopulations from South Korea (Mwamulaet al.2018),Turkey (Cuiet al.2016),Italy (Ambrogioni and Irdani 2001),USA (Graney and Miller 1982) and Germany (Subbotinet al.2010),the cyst and J2 characteristics of the Xinjiang population were almost consistent with each other. Moreover,molecular analyses of rDNA-ITS,28S-D2/D3 and mtDNA COI gene sequences revealed that the Xinjiang population clustered in a branch with the foreign populations ofH.schachtii,and the sequence similarity was as high as 98.47-100%. Finally,the identification of the cyst nematode was confirmed by the PCR method with species-specific primers SHF6/rDNA2.

Heteroderaschachtiiis one of the important quarantine nematodes in China (Penget al.2015). In our study,although we found beet cyst nematode in Xinyuan County of Xinjiang for the first time,fortunately,its distribution was only limited to individual counties of Xinjiang according to the later survey results. Furthermore,strict control measures should be taken to prevent this nematode from spreading or even eliminate it completely.

5.Conclusion

Based on morphological and molecular characterization,this study confirmed that the cyst nematode collected from sugar beet fields in Xinjiang Uygur Autonomous Region,China isH.schachtii. As far as we know,this is the first reliable report ofH.schachtiion the sugar beet in Xinjiang.

Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (31972247),the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (ASTIP-02-IPP-15),the Open Fund of Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis,Ministry of Agriculture and Rural Affairs (KFJJ202101),and the China Agriculture Research System of MOF and MARA(CARS170307).

Declaration of competing interest

The authors declare that they have no conflict of interest.