Molecular characterization of internal transcribed spacer (ITS) of ribosomal RNA gene, haplotypes and pathogenicity of potato rot nematode Ditylenchus destructor in China

Potato rot nematode Ditylenchus destructor is one of the most damaging pests in potato-producing regions and causes severe yield losses worldwide. However, D. destructor has been rarely reported in potato crops in China. We collected 542 samples from 17 Chinese provinces during 2016–2020 for the detection of D. destructor using species-specific primers and universal primers targeting ribosomal internal transcribed spacer (ITS) or 28S ribosomal RNA (rRNA) sequences. D. destructor was detected in 14.94% of total samples, primarily in Inner Mongolia Autonomous Region (43 sites), Jilin Province (12 sites), and Shaanxi Province (9 sites). The nematode was for the first time detected in Guizhou Province and Ningxia Hui Autonomous Region. Phylogenetic analysis of 33 ITS sequences along with the prediction of the secondary structure of the helix H9 of ITS1 sequences revealed that haplotypes A, B, and C accounted for 6.06%, 9.09%, and 69.70% of our detections, respectively, while 15.15% were comprised of haplotypes H–L, and extensive genetic diversity of ITS sequences was detected in samples from Gansu Province. Using RNAfold software, we analyzed the haplotypes of 124 Chinese D. destructor populations based on their ITS sequences, and the results showed that haplotypes A, B, C, E, F, and new haplotypes M–P accounted for 49.19%, 12.10%, 15.32%, 3.23%, 1.61%, and 18.55% of the total sequences, respectively. Infection tests on the potato cultivar ‘Helan 15’ showed significant difference in infection capacity among different D. destructor populations. The information obtained in this study on spatial patterns of D. destructor haplotypes in China provides valuable insights into the development of an integrated approach for the management of this plant-parasitic nematode.


Background
Potato (Solanum tuberosum L.) is the third major food crop in the world after rice and wheat and plays a crucial role in global food security (Mackay 2009;Hancock et al. 2014). The total annual potato production in China will be increased to 220 million tons by 2025 (www. china bgao. com). During their growth and development, potato plants are often attacked by many plant-parasitic nematodes, which feed on tubers or stems to cause direct or indirect yield losses (Mugniéry and Phillips 2007). Potato rot nematode Ditylenchus destructor Thorne (Thorne 1945), commonly known as a stem nematode, is the second-ranking nematode pest of potato after the potato cyst nematode and usually causes significant yield losses,
In 1931, D. destructor was first found attacking potatoes in Prince Edward Island, Canada (Baker 1946). Then, the damage caused by D. destructor to potato plants was found in Europe, South Africa, Central Asia, and North America (EPPO, https:// gd. eppo. int/ taxon/ DITYDE/ distr ibuti on). In China, D. destructor was first detected in sweet potatoes in Hebei Province in 1937 (Fan et al. 1999) and was subsequently reported in other sweet potatoproducing areas of Beijing, Henan, and Anhui provinces (Yu et al. 2009). Infestation of potato plants by D. destructor was first found in Hebei Province in 2006 (Liu et al. 2006) and now has been reported in many regions in China. Potato yield losses caused by D. destructor generally reach 20% to 50%, and even 100% in endemic regions. In China, the planting area of potato has exceeded 4200 hectares only in Chongqing, Gansu, Guizhou, Shaanxi, Shanxi, Sichuan, and Yunnan provinces (National Bureau of Statistics of the People's Republic of China 2017). Despite the economic importance of potato in China, there has been no comprehensive survey of D. destructor on this crop.
Accurate identification of a pathogen is critical for understanding the occurrence of the disease caused by this pathogen and to develop effective control measures accordingly. Ditylenchus spp. are very similar in morphology but differ in molecular characteristics (Brodie et al. 1993;Wendt et al. 1993;Vovlas et al. 2011), thus it is essential to develop more molecular detection methods to differentiate between species of the genus. Diverse molecular tools provide better support for accurate and fast identification of the species under investigation. Ribosomal RNA (rRNA) genes have been widely used for species identification, including Ditylenchus spp.. D. destructor populations exhibit a high level of intraspecific variations in ITS regions and 28S rRNA gene sequences. In the previous studies, 22 D. destructor populations were divided into two different types, type A (940 bp) and type B (1100 bp), based on their ITS sequences (Wan et al. 2008); through phylogenetic analysis based on the sequences of D2/D3 regions of 28S rRNA, those 22 D. destructor populations were also classified into types A and B (Yu et al. 2009). Following the proposal by Wan et al. (2008), Subbotin et al. (2011) kept the haplotypes A and B and identified 5 additional haplotypes, i.e. haplotypes C, D, E, F, and G, based on the secondary structure of ITS1-H9. Meanwhile, they clustered 78 ITS rRNA gene sequences of D. destructor into two main clades based on the presence or absence of the repetitive elements in the ITS1. Additionally, the species-specific primer pair D1/ D2 was developed, which can detect all haplotypes of D. destructor (Liu et al. 2007); two primer pairs: DdS1/DdS2 and DdL1/DdL2 for the detection of haplotype A and all other haplotypes, respectively, were developed by Wan et al. (2008); another pair of primers (Des2-F/Des1-R) that can also detect all haplotypes of D. destructor except haplotype A was designed by Marek et al. (2010). In this study, we used the species-specific primer set D1/D2 (Liu et al. 2007) and ITS-rRNA sequences to identify and study the genetic diversity of D. destructor.
The objectives of this study were to survey the occurrence and distribution of D. destructor in potato-producing regions in China, to analyze the genetic diversity of the nematode populations in ITS sequences, and to evaluate population pathogenicity on potato plants. The data on the occurrence and genetic diversity of D. destructor in potato fields obtained in this study will help understand the evolutionary processes of the populations, including the development of haplotypes and adaptation to selection pressure. The results provide insights into the effective use of resistant potato cultivars as a strategy for management of this plant-parasitic nematode.

Geographic distribution of D. destructor in China
In 2016-2017, we collected a total of 158 soil samples from different potato-producing regions of 7 provinces/ autonomous regions in China (72 from Jilin, 2 from Liaoning, 55 from Inner Mongolia, 22 from Qinghai, 2 from Xinjiang, 4 from Yunnan, and 1 from Guizhou). D. destructor-positive samples were identified based on the morphological characteristics and molecular detection (specific amplicons) of the nematode. D. destructor was detected in a total of 45 samples (28%), all containing more than 30 individuals per 100 g soil (Additional file 1: Table S1). Of the 33 positive samples collected from Inner Mongolia Autonomous Region, three contained 30 to 100 individuals per 100 g soil, while the remainder contained more than 100 individuals per 100 g soil. Of the 9 positive samples collected from Jilin Province, three contained 30 to 100 individuals per 100 g soil, with more than 100 individuals per 100 g soil in the remainders. All three positive samples collected from Qinghai Province contained over 100 individuals per 100 g soil.
In 2018-2020, a total of 384 samples (one sample from each site) were collected from potato-producing regions, and it was shown that 36 sampling sites were infested by D. destructor through morphological identification and PCR detection. No nematodes were detected in Anhui, Beijing,Henan,Qinghai,Shanxi,and Yunnan,with 4,3,16,14,4, and 6 sampling sites, respectively. However, one potato tuber sample in 12 sampling sites of Gansu, 3 samples in 38 sites of Guizhou, one sample in 17 sites of Hebei, 2 samples (one was potato tuber) in 7 sites of Heilongjiang, 10 samples (4 were potato tubers) in 148 sites of Inner Mongolia, 3 samples in 39 sites of Jilin, 2 samples (one was potato tuber) in 13 sites of Liaoning, 4 samples in 47 sites of Ningxia Hui Autonomous Region, one potato tuber sample in Shandong, and 9 samples (2 were potato tubers) in 15 sites of Shaanxi, were infested by nematodes. Of 36 positive samples, on the average, 15 contained 2 to 6 individuals per 100 g soil, 11 had 20-70 individuals per 100 g soil, and 10 had over 100 individuals per 1 g potato (Additional file 1: Table S1). Morphological characteristics of D. destructor recovered from infected potato tubers are shown in Fig. 1.

Phylogenetic analysis of D. destructor
D. destructor-positive DNA samples, which had been successfully detected by species-specific primer pair, were amplified with universal primers targeting ITS region or 28S rRNA gene of the nematode. Thirty-three ITS sequences and one 28S-rRNA sequence (GenBank accession number OK330255) were obtained from 29 samples collected during 2018-2020. All amplicons were sequenced and blasted against the sequences of D. destructor that were already present in the GenBank database.
To distinguish different haplotypes of D. destructor, phylogeny analysis was performed based on the ITS rRNA gene sequences. All 33 ITS sequences obtained in this study were nested within the clade of the D. destructor complex, which was highly supported by the Bayesian posterior probability values (Fig. 2). In addition to the previously described three haplotypes (haplotypes A, B, and C), five new haplotypes (haplotypes H, I, G, K, and L) were also included in this clade. Among them, two were haplotype A (6.06%), three were haplotype B (9.09%), twenty-three were haplotype C (69.70%), and the five new haplotypes (haplotypes H-L) comprise 15.15% of the total. Thus, haplotype C is a dominate type among the D. destructor populations isolated from the potato. Representative D. destructor isolates with different haplotypes detected in this study were deposited in the National Center for Biotechnology Information (NCBI) GenBank database under the accession numbers shown in Table 1. Four samples (JL18-35, GS18-11, NM19-15, and ShaanX18-12) showed mixed infection by two or three haplotypes (Table 1).

Haplotypes of D. destructor in China
In this study, the populations of D. destructor in Anhui, Beijing, Henan, Guizhou, and Xinjiang Uygur Autonomous Region were identified as haplotype A, while those in Heilongjiang and Liaoning were identified as haplotype C. Four haplotypes A, B, C, and L were detected in Inner Mongolia, with haplotype C being the most frequent haplotypes. Haplotypes A and B were detected in Hebei. Gansu had nine haplotypes (A, B, C, F, H, I, M, N, and O), with haplotype B comprising 66.7% of the isolates. Jilin, Jiangsu, Shandong, Shaanxi, Qinghai, and Shanxi had four (A, B, C, and J), three (A, B, and C), five (A, B, C, E, and F), three (A, C, and K), three (N, O, and P), and two (A and N) haplotypes, respectively (Figs. 2, 4). The new haplotypes identified in Jilin, Inner Mongolia, Gansu, Qinghai, Shanxi, and Shaanxi differed from those reported previously (Subbotin et al. 2011). Gansu had a highly diverse collection of haplotypes including five new haplotypes, indicating that the D. destructor populations in this province were genetically diverse.

Infection test of D. destructor on potato
Not all the D. destructor populations we obtained in this study can infect the potato cultivar 'Helan 15' . Pathogenic D. destructor populations induced typical soft white spots, dry rot, and papery, cracked skin on potato tubers (Fig. 5), and ITS rRNA sequences of nematodes isolated from all the symptomatic potatoes were identical to those of the populations used as inoculum.
Significant difference (P < 0.05) in disease index was produced by the different D. destructor populations tested in this study. The results showed that 'Lulong' and 'BX' were the most virulent populations, with an average disease index of 75% and 68.75%, and RF of 23.52 and 5.61, respectively. By contrast, 'PH19-07' , 'CX' , and 'GS18-11' were significantly less virulent than other populations (Table 2).

Discussion
D. destructor, an economically important plant-parasitic nematode, affects potato production mostly in temperate climates. In China, the gradual increase in potato-producing areas has resulted in outbreaks of D. destructor in several provinces/autonomous regions such as Inner Mongolia and Liaoning in the past decade (Ou et al. 2017;Mao et al. 2020). In this study, during a survey of potato nematodes in 2016-2020, a total of 542 samples were collected from 17 main potato-producing provinces Fig. 4 Phylogenetic tree based on 101 ITS-rRNA sequences of Ditylenchus destructor from China. The sequence data were obtained from NCBI (https:// www. ncbi. nlm. nih. gov). The 50% majority-rule consensus tree from Bayesian analysis was generated from the ITS-rRNA gene sequence dataset for D. destructor using the HKY+I model. D. myceliophagus was used as an outgroup. The scale bar indicates the number of expected changes per site. The haplotypes are color-marked as given in the legend including Jilin and Shaanxi, and Inner Mongolia Autonomous Region, of which 81 samples (14.94%) were D. destructor-positive (Additional file 1: Table S2). Meanwhile, D. destructor was for the first time detected in Guizhou Province and Ningxia Hui Autonomous Region, China. Previously, the potential distribution areas of D. destructor in China were predicted using MAXENT model, and risk levels for occurrence of the nematode in these areas, including Inner Mongolia Autonomous Region, Jilin and Shaanxi provinces, were assessed (Li 2008). D. destructor can adapt to low temperature, northern China is therefore suitable for its propagation (Huang et al. 2012). Additionally, Yulin City of Shaanxi Province was also predicted to be a suitable habitat for D. destructor (Hong et al. 2017). Gansu Province is a special area with complicated geomorphology and diverse climate regimes where the genetic diversity of D. destructor was the highest among all the D. destructor populations investigated in this study, and we detected nine haplotypes of ITS sequences in the samples collected from Gansu (Figs. 2, 4 and Table 1). Serious damage would be caused by D. destructor when its population density reaches 20-50 individuals per 1000 g soil (Butorina et al. 2006). Our data showed that the density of D. destructor during 2016-2020 was no less than 20 individuals per 1000 g soil in China (Additional file 1: Table S1). Accordingly, D. destructor has posed a serious threat to potato production in China.
D. destructor produces conical pits along with skin splitting and rotting on potato tubers (Fig. 5, Jenkins and Taylor 1967). In this study, we evaluated the virulence of D. destructor isolates collected from 8 provinces  of China on potato and sweet potato and biologically characterized 12 geographic D. destructor populations, which exhibited significant difference in genotypes and pathogenicity (Table 2; Additional file 1: Table S3). Previously, it was revealed that the D. destructor populations from Hebei Province were more virulent than those from Henan and Jiangsu provinces (Ji et al. 2006), and the population 'DeHB' was more virulent than populations 'DeJJ' and 'DeLY' on sweet potato (Wang 2009). In addition, the same D. destructor population was found to produce multiple symptoms on potato plants depending on potato variety, initial population density of the nematode, temperature, and soil moisture, as such factors can influence the distribution and behavior of D. destructor and eventually the incidence and severity of potato rot caused by this nematode (Fiers et al. 2012;Ivanyuk and Ilyashenko 2008). Therefore, the reported results may not reflect the true potential virulence of these populations. Potato tubers infested by D. destructor are generally the primary source of potato rot, thus transportation of D. destructorinfested seed potatoes can promote the spread of the disease especially when light infestation occurred. In this case, the infested potatoes present healthy appearance and are easily neglected by a visual check (Kikas 1969), the nematode populations are thus transmitted to other potato-producing areas. D. destructor may be spread not only by planting materials but also by farm operations, such as ploughing and irrigating (PLH 2014). The results of our survey in this study clearly indicate a broad distribution of D. destructor in China (Additional file 1: Tables S1, S2). Therefore, as described by EFSA Panel on Plant Health (PLH) (2014), farmland management and plant quarantine practices such as the utilization of certified planting material and disinfested farm equipment and machinery, and avoidance of flood irrigation should be encouraged to prevent introduction and further spread of this nematode into new potato-producing regions. High genetic diversity of D. destructor populations has been detected in China according to the restriction map of ITS and D2/D3 regions or using an inter-simple sequence repeat (ISSR) molecular marker (Yang and Peng 2005;Huang et al. 2012). In this study, as it showed based on the haplotypes of ITS sequences, an abundant genetic diversity existed in D. destructor populations in China (Table 1). We identified several different haplotypes of ITS sequences of D. destructor including 5 new haplotypes (H-L) that can feed on potato, and some samples were even infected by a mix of two or three haplotypes (Table 1), which may be related to genetic exchange among different haplotypes of D. destructor populations and subsequent migrations of this nematode.

Conclusions
D. destructor is a migratory plant-parasitic nematode widely distributed in temperate regions. This nematode can parasitize more than 100 species of plants and weeds, such as potato, sweet potato, carrot, Codonopsis pilosula, and Angelica sinensis. This study is the first to uncover the occurrence and geographical distribution of D. destructor in China. The genetic diversity of D. destructor populations on different host plants was examined and the variation in pathogenicity was detected. To the best of our knowledge, this is the first report of D. destructor infesting potatoes in Guizhou Province and Ningxia Hui Autonomous Region in China. Surveys of the occurrence and distribution of this crop-parasitic nematode by precise detection and an understanding of its population diversity are crucial for making phytosanitary decisions as well as for breeding resistant cultivars.

Sample collection and nematode isolation
An approximately 0.5 kg of soil was obtained from each sampling site using the five-spot sampling method. The locations of the sampling sites were recorded using a Global Positioning System (GPS) receiver. Rhizosphere soil or potato tuber samples were collected during the entire growing season. In 2016-2020, a total of 542 soil samples and potato tuber samples were collected from different potato fields in China (Additional file 1: Table S1). We isolated nematodes from 100 g (dry weight) soil samples in an Oostenbrink dish (Oostenbrink 1960). The nematodes were settled in a glass beaker and the supernatant was removed. Nematodes was counted under an inverted microscope and individual specimens were transferred to a microscope slide with a needle for extraction of DNA. D. destructor was identified by general morphology and molecular identification (FAO 2015). Males and females are similar in general appearance: cuticular and head annulation fine, head often narrower than adjacent body; stylet length 10-12 µm, stylet cone round and sloping backwards, median bulb muscular; excretory pore opposite oesophageal glands; lips of vulva thick, elevated; tail of both sexes conical, usually ventrally curved, terminus rounded (FAO 2015). Additionally, the density of D. destructor was estimated by counting the number of individuals in 100 g soil (dry weight) or in 1 g potato tuber samples.

DNA extraction, PCR amplification and sequencing
Nematode DNA was extracted from single individuals as described by Subbotin et al. (2011). The number of individuals analyzed per population varied depending on the nematode populations found in each sample. If the number of the target nematode was over 5 per 100 g soil, five nematodes were used for DNA extraction. In brief, a single nematode was washed twice and transferred into a 0.2 mL Eppendorf tube containing 6 μL of double-distilled H 2 O, 2 μL of 10 × PCR buffer (TaKaRa, Japan), and 2 μL of proteinase K (600 μg/mL) (Promega, USA). The nematode was chopped into pieces under a stereomicroscope. The tubes were incubated at 65 °C for 90 min and then kept at 90 °C for 10 min.
Species-specific primer pair D1/D2 (Additional file 1: Table S4) was used to identify D. destructor following a preliminary morphological identification (Liu et al. 2007). The PCR reaction volume was 25 μL containing 4.0 μL of DNA, 2.5 μL of 10 × PCR buffer (Mg 2+ was added), 2 μL dNTP (10 mmol/L), 1 μL each of species-specific primers D1 (12.5 μmol/L) and D2 (12.5 μmol/L), 0.5 U Taq DNA polymerase (5 U/μL), and 14.4 μL of sterilized ddH 2 O. PCR amplification was performed at 94 °C for 4 min; 35 cycles of 1 min at 94 °C, 45 s at 55 °C and 45 s at 72 °C; and then a final extension step of 10 min at 72 °C. PCR products were separated by agarose gel electrophoresis, and the expected product size was 346 bp. The primer pair D3A/D3B (Additional file 1: Table S4) was used as an internal reference with an expected product size of 345 bp under the same amplification conditions.
In 2018-2020, D. destructor-positive DNA samples were further amplified with universal primers targeting ITS region or 28S rRNA gene of nematode. The D2A/ D3B primer pair (Yu et al. 2009) was used to amplify the D2-D3 sequence of 28s rRNA, and the rDNA1/rDNA2 and AB28/TW81 primer pairs (Liu et al. 2007;Wan et al. 2008) were used to amplify the ITS region (Additional file 1: Table S4). The PCR was performed in a 50 μL reaction volume containing 4.0 μL of DNA, 5 μL of 10 × PCR buffer (Mg 2+ was added), 4 μL dNTP (10 mmol/L), 1 μL of each forward (12.5 μmol/L) and reverse primer (12.5 μmol/L), 1 U Taq DNA polymerase (5 U/μL), and 34.8 μL of sterilized ddH 2 O. The PCR products were purified using a TIANgel Midi Purification Kit (TIAN-GEN, DP209) and cloned into a PMD19-T Vector (TAKARA, 6013), then transformed into E.coli DH5α (TIANGEN, CB101). Purified plasmid DNA was then sequenced at BGI (China, Beijing). DNA sequences were edited by Edit Seq software package (Lasergene, Madison, WI, USA) and blasted in NCBI. The newly obtained consensus sequences were deposited in the NCBI Gen-Bank database under the accession numbers indicated in Table 1.

Molecular characterization and phylogenetic analysis
All ITS sequences obtained in this study were used for haplotype analysis, and the secondary structures of helix H9 of ITS1 sequences were predicted by the energy minimization approach using the RNAfold web-server software (http:// rna. tbi. univie. ac. at/ cgi-bin/ RNAWe bSuite/ RNAfo ld. cgi). We extracted the sequences from the GenBank files using PhyloSuite v1.2.2 (Zhang et al. 2020), then aligned the sequences in batches using MAFFT v7.313 (Katoh and Standley 2013). ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the optimal evolutionary model. MrBayes 3.2 was used for the Bayesian inference analysis (Ronquist et al. 2012). We used the BI for phylogenetic reconstruction 1,000,000 generations, four Markov chain Monte Carlo chains, and the trees were sampled every 100 generations; an initial 25% of trees were discarded as burn-in. The topologies were used to generate a 50% majority-rule consensus tree. Trees from all analyses were visualized using iTOLv6 (http:// itol. embl. de).

Infection test of D. destructor on potato
In this study, D. destructor populations consisting of three ITS haplotypes were collected from different host plants in different provinces/autonomous regions (Additional file 1: Table S3). Sweet potato 'Yanshu 25' (susceptible cultivar) was used to maintain and multiply these populations, and Baermann-funnel was used to isolate nematodes. Nematode suspensions were adjusted to 500 nematodes/mL of water.
Infection tests were carried out on the potato cultivar 'Helan 15' (Solanum tuberosum L.), whose seeds were planted in 1 L pots filled with growth medium (slowrelease fertilizer was added to the growth medium at a ratio of 1.5 g/kg). Two weeks after planting, growing medium was infected with D. destructor suspension (2000 mixed nematodes of all stages). Each nematode population had four replicates, while plants treated with deionized water was used as a negative control. The potato plants were grown with sterile substrate in a greenhouse at 17-23 °C under a 13-h photoperiod and watered regularly. At 12-week post inoculation, the virulence of 12 nematode populations was assessed by measuring the lesion area on the potato and the RF of 12 nematode populations. The index rating and its corresponding lesion area in parentheses were: 0 (0%), 1 (0-25.0%), 2 (25-50.0%), 3 (50.1-75.0%), and 4 (75.1-100%). Differences in virulence caused by different D. destructor populations was determined by a one-way analysis of variance, and the least significant difference test at the 5% significance level was compared using IBM SPSS Statistics software.
Disease index = ∑(Number of diseased plants in that rating × Index rating)/(Total number of plants investigated × Highest rating) × 100.