Morphological and Molecular Characterization of Prevalent Plant-Parasitic Nematodes from Turfgrasses in Guangdong, China

: The turfgrass industry has undergone a rapid development in Guangdong province, China, which has the largest number of golf courses in the country. Recent surveys of turfgrasses in the province revealed five plant-parasitic nematodes that are prevalent: Helicotylenchus dihystera , Mesocriconema xenoplax , Meloidogyne graminis , Hemicriconemoides rosae and Tylenchorhynchus leviterminalis . The most prevalent species are M. xenoplax and M. graminis , found in 60.6% and 27.3% of locations, respectively. These ﬁve species are morphologically and morphometrically described. Molecular characterization and phylogenetic analyses using 18S rRNA and the D2-D3 expansion segments of 28S rRNA sequences are provided. This is the ﬁrst report on molecular characterization and phylogenetic relationships of plant-parasitic nematodes associated with turfgrasses in Guangdong, China. This work was a ﬁrst step for future study including pathogenicity assay, relationship examination with other pathogens and development of control measures of these turf nematodes to provide more precise and effective management options to turf superintendents.


Introduction
With rapid urbanization and growing demand for high-quality life, the turfgrass industry has undergone a rapid expansion in Guangdong province, China. In 2019, Guangdong province ranked first in the country for green space, with an estimated 502,400 hectares [1]. Guangdong has always been at the forefront of golf course development in China. It currently has 97 golf courses [2]. These golf courses are mainly distributed throughout the economically developed Pearl River Delta region, including Guangzhou, Zhongshan, Foshan, Dongguan, Huizhou, Zhuhai and Shenzhen. To maintain a high-quality turfgrass, especially putting greens, turfgrass needs to be intensely managed with pesticides, fertilizer and irrigation to prevent diseases and pest insects, and to minimize deleterious effects due to extremes in environmental conditions [3]. However, due to economic pressures, restrictions on the application of pesticides and damage by pests, it is often difficult for management inputs to maintain a desirable turfgrass surface.
Of the pests that impact turfgrass, plant-parasitic nematodes are an important pathogen; however, they are often overlooked because their microscopic size makes it difficult to see them with the naked eye and because the symptoms they cause are similar to those caused by drought stress, nutrient deficiency and fungal root diseases, making it hard to diagnose them. Furthermore, there are no turfgrass cultivars resistant to nematodes at the present time, and few effective measures can be taken to manage the nematodes in turfgrass once their infestation is established. Soil fumigation is effective for controlling nematodes before planting, but it can't be used easily once turf is established, and some effective fumigants, for example, Dichloropropene, were restricted due to expensive cost (about $16,000 for 35 acres of fairway) and their being potentially phytotoxic to turfgrass [4]. Therefore, nematodes have become a serious problem in turfgrass worldwide, especially in subtropical and tropical regions like Guangdong province. Nematode problems highlight the need for a greater understanding of nematodes infecting turfgrasses; this includes species identification so that targeted and sustainable management strategies can be developed.
Several surveys of nematodes associated with turfgrasses have been conducted in the USA and other countries, and have shown a broad diversity with different countries and regions [5][6][7][8][9][10][11]. In Florida, USA, common genera of nematodes that damage turf include ectoparasitic Belonolaimus, Trichodorus, Nanidorus, Helicotylenchus and Mesocriconema, and endoparasitic Hoplolaimus and Meloidogyne [5]. A total of 29 species belonging to 22 genera in 15 families in North Carolina (NC) and South Carolina (SC), USA [10]; 28 species/taxa belonging to 16 genera and 12 families in Korea [6]; 52 different species/taxa belonging to 23 genera and 9 families in Belgium [8]; and 9 genera of plant-parasitic nematodes associated with turfgrass in southern Ontario, Canada [9] were reported. The nematodes associated with turfgrasses in NC and SC were molecularly characterized using 18S rDNA sequences [12]. Meloidogyne graminis (Sledge & Golden) Whitehead was reported to be occurring on golf greens of Yangjing and Zhuhai, Guangdong province [13]. However, an extensive survey of plant-parasitic nematodes associated with turfgrasses in Guangdong, China is needed. The objectives of this work were to: (i) identify the nematode species associated with turfgrasses in Guangdong province; (ii) characterize the most common species using morphological, morphometric and molecular methods; and (iii) analyze phylogenetic relationships among these nematode species using sequences of the 18S nuclear ribosomal RNA and the D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene.

Soil Sampling
During 2016-2020, samples for extracting nematodes were collected four times a year at a total of 33 locations of planted turfgrasses in Guangdong province (23.1317 • N, 113.2663 • E), China. Soil samples were collected from the root zone of bermudagrass (Cynodon dactylon (L.) Pers.) at 19 golf courses and 14 other locations in Guangdong province, China (sampling information shown in Table 1). Each sample consisted of 12 soil cores (1.5 cm diam. × 20 cm deep) sampled at roughly equal intervals in a zig-zag pattern across an area of 500 m 2 or less. Soil samples were combined for bulk sample and placed in sealed plastic bags. The sealed plastic bags were placed in sample boxes and stored at 4 • C before analysis to minimize changes in nematode populations.

Morphological Characterization
Nematodes were extracted from soil samples using the rapid centrifugal-flotation method [14]. Specimens were heat-killed, fixed in 3% formaldehyde and processed to glycerin using the formalin-glycerin method [15]. Measurements were performed with the aid of a camera lucida and a stage micrometer. The morphometric data were processed using Excel software [16]. Photomicrographs were taken with a Leica video camera (DFC490) attached via a C-mount Adapter fitted on a Leica microscope (DM4000B) and edited using Adobe Photoshop CS6.
The abbreviations and their definitions for the de Man's ratios and other indices used in tables are as follows: n = number of specimens on which measurements are based L = overall body length V = % distance of vulva from anterior relative to body length a = body length/greatest body diameter b = body length/distance from anterior to esophago-intestinal valve c = body length/tail length c = tail length/tail diameter at anus or cloaca VA = distance from vulva to anus VBD = diameter of body at vulva PUS = postuterine sac MB = % distance of center of the middle esophageal bulb from anterior relative to esophageal length T = % distance of testis relative to body length R = ring number of body cuticle Rs = ring number from anterior to base of stylet base Rex = ring number from anterior to excretory pore Roes = ring number from anterior to base of esophageal glands Rv = ring number from vulva to tail tip Ran = ring number from anus to tail tip Rvan = ring number between vulva and anus

Molecular Characterization
One male or female was hand-picked and placed into 50 µL of worm lysis buffer (WLB) containing Proteinase K for DNA extraction [17]. DNA samples were stored at −20 • C until used as a PCR template.
PCR reactions (25 µL) were performed using Dream Taq Green PCR Master Mix DNA polymerase (Thermo Fisher Scientific [China] Co. Ltd., Shanghai, China) according to the manufacturer's protocol. The thermal cycler program for PCR was as follows: denaturation at 95 • C for 5 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 55 • C for 45 s, and extension at 72 • C for 2 min. A final extension was performed at 72 • C for 10 min [20]. PCR products were cleaned using ExoSap-IT (Affymetrix Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. PCR products were sequenced by Guangzhou Tianyihuiyuan Gene Science & Technology Co., Ltd., Guangzhou, China, using an ABI PRISM 3730 sequencing system.
The rDNA SSU and LSU sequences from this project were deposited in GenBank under the accession numbers presented in Table 2 and compared with other nematode species in GenBank using the BLAST homology search program. The most similar sequences were downloaded for phylogenetic analysis. DNA sequences were aligned using Mega5.05 [21]. The model of base substitution in the SSU and LSU sets were evaluated using MODELTEST version 3.06 [22]. The Akaike-supported model, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 [23] running the chain for 1 × 10 6 generations and setting the 'burn-in' at 1000. The MCMC (Markov chain Monte Carlo) methods within a Bayesian framework were employed to estimate the posterior probabilities of the phylogenetic trees [24] using the 50% majority-rule.  (Table 3). Mesocriconema xenoplax was the most prevalent, with a detection rate (percentage of locations) of 60.6%, followed by Meloidogyne graminis with 27.3%, and other species with around 20% each.

Morphological Description
Because the SSU and LSU sequences aligned by ClustalW from the populations from different locations for each species in the present study were identical, they are considered as one species in the following description.

Description of Helicotylenchus dihystera
Female: Body spiral-shaped when heat-killed and distinctly annulated. Four incisures visible in lateral field with light microscopy. Stylet well-developed with rounded stylet knobs. Orifice of dorsal esophageal gland at about half stylet length behind stylet base. Lip hemispherical-shaped with lip rings. Median esophageal bulb oval with a well-developed valve. Excretory pore located at the anterior level of the esophageal gland. Hemizonion indistinct. The posterior esophageal glands overlapping intestines. Ovaries paired, outstretched, with oocytes in single row. Spermatheca without sperms. Vulva transverse without vaginal membrane. Tail ventrally curved, dorsally convex-conoid to a narrow terminus which may form a slight projection ( Figure 1).
Male: Not observed.

Description of Hemicriconemoides rosae
Female: Body slightly ventrally curved when heat-killed. Cuticular sheath and rounded annuli distinct. Lateral field absent and without anastomoses. Lip region not set off, rounded with a prominent labial disc and two annuli. Cephalic framework well-developed. Stylet strong, long, with well-developed, anchor-shaped knobs. Procorpus and metacorpus amalgamated. Median esophageal bulb oblong, with well-developed valve. Isthmus narrow followed by distinct basal bulb, becoming vase-shaped. Excretory pore posterior to basal bulb end, 28-31 annuli from anterior end. Vulva with a membranous sheath, without vulval flap. Vagina distinct, sigmoid. Ovary monodelphic, prodelphic, outstretched with oocytes in a single row. Spermatheca well-developed, oblong to ovoid-shaped, without sperms. Anus visible. Tail dorsally convex-conoid, with a bluntly rounded or pointed tip ( Figure 2).
Remarks: Hemicriconemoides rosae was first collected from the rhizosphere of rose (Rosa indica L.) in the Bareilly district, Uttar Pradesh, India, and was originally described by Rathour et al. [30]. It was collected from sugarcane and redescribed by Khan et al. [31]. Hemicriconemoides rosae is known to occur only in rose and sugarcane in India and Pilea cadierei Gagnep. & Guill. in China. In this study, it was found in Guangzhou Lihu Golf, Culture Park, People Park, Sand River Golf, Shenzhen Juhao Golf and Dongguan Zhongxin Golf. Both morphology and morphometrics match the original description [30]. This is the first report of H. rosae on turfgrasses.

Description of Meloidogyne graminis
Second-stage juvenile: Body cylindrical, tapering to the posterior end. Head without distinct cephalic framework and annuli. No obvious contraction between the head and the body. Four incisures visible in lateral field. Stylet small with rounded knobs. Median esophageal bulb elongated with well-developed valves. Tail with a distinct, relatively long hyaline terminus and rounded tip (Figure 3).    Male: Body long and vermiform-shaped. Cuticle striated. Four incisures visible in lateral field. Head with well-developed cephalic framework, without annuli. No obvious contraction exists between the head and the body. Stylet robust with distinct rounded knobs. Median esophageal bulb oval with a well-developed valve. Spicules paired, separated, slightly curved ventrally, with a slender gubernaculum, near tail terminus. Tail short, with rounded tip (Figure 3).
Female: Not observed.

Molecular Characterization and Phylogenetic Relationships
A 602-bp 18S rDNA and a 574-bp 28S D2-D3 expansion segment of H. dihystera in this study were amplified and sequenced. A BLASTN search of this species matches well with its corresponding species. From 18S sequence, the study H. dihystera and a population of H. dihystera (JX069950) in GenBank yielded 602 total characters with 99.83% identity; intraspecific sequence variation for H. dihystera was 0. 17% (1 nucleotide, nt)   Phylogenetic analyses of the partial 18S and 28S D2-D3 were performed to examine the relationships among the most common species from this study and related species from Genbank. The dendrogram inferred from 18S rDNA sequences ( Figure 6
The genus Hemicriconemoides Chitwood & Birchfield, 1957 morphologically differ from other genera within Criconematidae by possessing a loosened outer cuticular sheath cov-reported that M. graminis was associated with bermudagrass. Zeng et al. [10,11] showed that M. graminis was found in two grass species (bermudagrass and zoysiagrass). In the present study, M. graminis was also detected in bermudagrass from 9 golf courses. Our report, along with previous publications, shows a strong association of M. graminis and bermudagrass, but more extensive sampling is needed. Meloidogyne minor was previously associated with yellow patch disease on Agrostis stolonifera var. stolonifera L. on golf greens [63]. Meloidogyne spp. have variations in pathogenicity, but a recent survey in SC indicated severe damage caused by M. graminis on golf course turfgrasses, particularly bermudagrass [81]. Although the pathogenicity of some Meloidogyne species to turfgrasses still needs to be further examined, root-knot nematodes are undoubtedly worthy of attention in the future.

Conclusions
Morphologically, this study clarified the identity of the most common plant-parasitic nematodes on golf course turf in Guangdong. Molecularly, it characterized these species using sequences of the 18S nuclear ribosomal RNA (rRNA) and the D2-D3 expansion segments of the 28S rRNA gene, showing little intraspecific variation of the sequences with respective corresponding species from GenBank. Phylogenetically, it investigated the phylogenetic relationships among these plant-parasitic nematode species based on sequences of the 18S rRNA and the D2-D3 expansion segments of the 28S rRNA gene, revealing correct phylogenetic placements of the five species in this study. Both sequences of the 18S rRNA and the D2-D3 expansion segments of the 28S rRNA gene verified morphological-based identification of these species in the present study. Five nematode species are described, including H. dihystera, H. rosae, M. graminis, M. xenoplax and T. leviterminalis, with new records of H. rosae, and T. leviterminalis associated with turfgrass. This work was a first step for future study including pathogenicity assay, relationship examination with other pathogens and development of control measures of these turf nematodes to provide more precise and effective management options to turf superintendents. This is the first report on molecular characterization and phylogenetic relationships of plant-parasitic nematodes associated with turfgrasses in Guangdong, China.