Morphological and Molecular Characterization and Life Cycle of Meloidogyne graminicola Infecting Allium cepa

Abstract

To identify the root-knot nematodes (RKNs) infecting onions in Yuanmou County, Yunnan Province, morphological and molecular biological techniques were used. Observation of their life cycle and pathogenicity was conducted through artificial inoculation experiments in a greenhouse. The results show that the morphological characteristics and measurement data of the second-stage juveniles (J2s) and females of RKNs infecting onion roots are highly consistent with those of Meloidogyne graminicola (M. graminicola). Sequence alignment of the mitochondrial DNA (mtDNA) COXI region and 28S rDNA D2-D3 region revealed sequence similarities of 99.51–100.00% and 99.48–99.61%, respectively, compared with M. graminicola sequences from GenBank. The specific primers Mg-F3/Mg-R2 reliably amplified a characteristic 369 bp band. Inoculation experiments confirmed that the pathogen can complete its entire life cycle (approximately 26 days (ds)) on the roots of healthy onion seedlings, inducing typical root-knot symptoms and females. In conclusion, the pathogen was identified as M. graminicola, which is the first report of M. graminicola infecting onions in China. This study provides important theoretical support for the molecular diagnosis of onion root-knot nematode disease and the green control of Allium L. vegetables in China.

1. Introduction

Onion (Allium cepa L.) is an important economic vegetable crop cultivated worldwide [1]. According to FAO data, the global onion planting area is approximately 4.5 million hectares, with a production of 92.1 million tons and an average yield of 19.3 tons per hectare. India, China, the United States, Egypt, and Turkey are the top five countries in onion production [2]. During the production process, onions are susceptible to various diseases, leading to a decline in both yield and quality [3]. Onion rust [4], onion downy mildew [5], onion violet leaf spot [6], and onion gray mold disease [7] are major diseases affecting onions. Root-knot nematode (RKN) disease, as a soil-borne disease, is characterized by severe damage, widespread distribution, and difficulty to control, and has been reported on various crops [8]. Meloidogyne graminicola is one of the major RKNs that damage crops. M. graminicola was first discovered in 1965 on barnyard grass (Poaceae) in Louisiana, USA [9]. Due to its potential harm to crops (Oryza sativa L., Glycine max L., Allium L., etc.), this nematode has been listed as a quarantine pest in several countries [10]. Meloidogyne graminicola infestation was also observed in a rice–onion rotation system in the Philippines [11]. Anamika et al. [12] reported that onion crops in fields near the Jamuna and Ganga river belt in India suffered significant losses due to RKNs. Meloidogyne graminicola infection has also been detected on Allium fistulosum L. in Hainan Province, China [13].

After infection by M. graminicola, pathological changes in the roots include colonization at the root tip by J2s, followed by the formation of giant cells and root galls. Mature females then lay eggs, from which new J2s hatch and reinfect roots, completing the life cycle [14].This nematode has a short life cycle, completing its life history in just 15 days at 27–37 °C [15]. Consequently, even a small initial population of M. graminicola can rapidly increase to high densities within a single crop cycle [16]. Although M. graminicola has been reported on Allium L. species, systematic identification of the nematode species causing onion root-knot disease and studies on its life cycle remain lacking. This study discovered a severe RKN disease affecting onions at a site in Yuanmou County, Chuxiong Yi Autonomous Prefecture, Yunnan Province. Given the expanding distribution of M. graminicola in China and its potential threat to the onion industry, this research analyzes the characteristics of this pathogenic nematode from multiple perspectives to provide key theoretical support for scientific pest control. The main objectives of the study are as follows: (1) Identify the pathogen species to provide a basis for the selection of resistant varieties and targeted control strategies; (2) analyze the morphological characteristics of J2s and females to fill the taxonomic data gap of this nematode on onion hosts; (3) construct a phylogenetic tree based on coxI and 28S rDNA to reveal its genetic diversity and potential invasion sources; and (4) study the life cycle using acid fuchsin staining to gain an initial understanding of the infection dynamics and developmental cycle of plant-parasitic nematodes on Allium cepa, which is of significant practical importance for determining key control windows. This study is the first to systematically integrate the damage symptoms, biological characteristics, morphological features, and life history data of this pathogenic nematode. It also marks the first discovery of M. graminicola infecting Allium cepa in China, providing scientific support for the development of comprehensive management strategies based on species-specific characteristics.

2. Materials and Methods

2.1. Sample Collection and Field Symptom Observation

Samples were collected from an onion planting area in Yuanmou County, Chuxiong City, Yunnan Province (25°43′11″ N, 101°51′23″ E), where the white onion variety “Puxuenong” (from Beijing Tiandi Garden Seedling Co., Ltd., Beijing, China) was grown, and the previous crop was rice. Fresh onion samples with visible root galls were collected and brought back to the laboratory for pathogen isolation and identification. After cleaning and air-drying the collected onion RKN samples, they were observed and photographed under a stereomicroscope (Discovery. V12).

2.2. Population Purification and Breeding Preservation

Fresh onion root nodules were placed under a dissecting microscope, and a single egg mass was directly selected and inoculated onto the roots of large-leaf water spinach (Ipomoea aquatica F.) seedlings previously cultured in sterilized soil (loam:humus:perlite = 2:1:1). Breeding was conducted at room temperature.

2.3. Morphological Identification

The egg masses on the root surface were directly picked and placed in a 26–28 °C incubator to culture and collect J2s. The J2s were then placed in a 65–70 °C water bath for 2–3 min to kill them, followed by fixation in a 4% formaldehyde solution for later use. Subsequently, females were picked from the onion roots under a stereomicroscope using forceps and placed in physiological saline for later use. Morphological observation and photography of J2s and females were conducted using an Axio Vert. A1 inverted microscope, and morphological measurements were calculated using the De Man formula. A total of 25 samples of J2s and females were selected for analysis [17].

A single female was placed on a transparent acrylic plate with a drop of 45% lactic acid. The perineal pattern area was cut using a dissecting knife, and the tissue attached to the cuticle was cleaned using a fine soft brush. The perineal pattern was then transferred to a glass slide with a drop of pure glycerol, with the outer side facing up. A cover slip was placed, and the perineal pattern was observed and photographed under a microscope [18].

2.4. Molecular Biology Identification

2.4.1. DNA Extraction

DNA extraction from single female nematodes was performed following the method of Yang et al. [19]. A worm lysis buffer (100 μL) was prepared as follows: 20 μL of 10× PCR buffer (Mg²⁺-free) (Manufacturer: Beijing Biomed Gene Technology Co., Ltd., Beijing, China), 16 μL of MgCl₂ (25 mmol), 1 μL of proteinase K (20 mg/mL), and 63 μL of sterile ddH₂O. Individual female nematodes were transferred into 0.2 mL PCR tubes, pierced with a sterile bamboo stick, and mixed with 5 μL of the lysis buffer. After homogenization, the samples were frozen in liquid nitrogen for 20 min. Subsequently, 10 μL of mineral oil was added. After centrifugation using a handheld centrifuge, the sample were incubated in a PCR machine at 56 °C for 80 min, followed by 95 °C for 15 min, to obtain nematode DNA.

2.4.2. Phylogenetic Tree Construction

Mitochondrial DNA (mtDNA) COXI universal primers COXIF/COXIR (5′-TGGTCATCCTGAAGTTTATG-3′/5′-CTACAACATAATAAGTATCATG-3′) and 28S rDNA D2-D3 region universal primers D2A/D3B (5′-ACAAGTACCGTGAG51GGAAAGTTG-3′/5′-TCGGAAGGAACCAGCTACTA-3′) were used to amplify the mtDNA-COXI and 28S rDNA D2-D3 region sequences, respectively [19]. The primers were synthesized by Qingke Biotechnology Co., Ltd., Beijing, China. The PCR reaction mixture (25 μL) included 2.5 μL 10 × PCR buffer, 2 μL dNTPs (2.5 mM), 1 μL upstream and downstream primers (10 μmol/L), 2.5 μL template DNA, and 0.25 μL Taq polymerase (5 U/μL), and ddH₂O was added to reach a final volume of 25 μL. The reagents mentioned above were provided by Beijing Biomed Gene Technology Co., Ltd. The PCR program consisted of an initial denaturation at 94 °C for 4 min; 94 °C for 30 s; 54 °C or 60 °C for 30 s (COXIF/COXIR primers annealed at 54 °C, D2A/D3B primers annealed at 60 °C); and 72 °C for 1 min, for 35 cycles. This was followed by a final extension at 72 °C for 10 min.

The PCR products of the mtDNA-COXI region and the 28S rDNA D2-D3 region of the purified and recovered pathogenic nematodes were heat-shock-transformed and cloned into the pMD™18-T vector (Manufacturer: Takara Bio Inc., Shiga, Japan). After transformation, 50 μL of SOD medium was added to the cell suspension and incubated at 37 °C with shaking at 200 r/min for 1 h. Then, 150 μL of the bacterial suspension was spread onto LB (ampicillin) solid medium and incubated overnight. Single colonies were picked for PCR identification, and the positive clones were sent to Tsingke Biotechnology Co., Ltd. (Beijing, China) for sequencing. The sequencing results were analyzed using BLAST in GenBank [19]. A phylogenetic tree was constructed using MEGA12.0.11 software based on the maximum composite likelihood model, with Pratylenchus hippeastri (accession number: KY424098.1, Guangdong) and Pratylenchus hippeastri (accession number: MH324473.1, South Africa) used as the outgroups. The bootstrap values for each branch of the tree were tested with 1000 iterations [20].

2.4.3. Specific Primer Validation

DNA was extracted from 14 individual females and amplified using the nematode-specific primers Mg-F3 (5′-TTATCGCATCATTTTATTTG-3′) and Mg-R2 (5′-CGCTTTGTTAGAAAATGACCCT-3′) for M. graminicola [21]. The PCR protocol was as follows: 95 °C for 4 min; 94 °C for 30 s; 52 °C for 30 s; 72 °C for 35 s, for 35 cycles; 72 °C for 10 min; and storage at 4 °C. The PCR reaction mixture was prepared using the same system as that used for universal primers (COXIF/COXIR, D2A/D3B). PCR products from 14 individual female DNAs were analyzed using 1% agarose gel electrophoresis, with negative controls included after the PCR products.

2.5. Life Cycle and Pathogenicity Observation

Onion seedlings were cultivated by disinfecting “Pusuinong” onion seeds, which were then sown in sterilized soil. Seedlings were transplanted into small plastic pots (12 cm in diameter, 13 cm in height) 30 days after emergence, with one plant per pot. A total of 50 pots were planted and placed in a greenhouse with an ambient temperature of approximately 14–27 °C for natural growth. Once the plants developed 3–5 scale leaves, they were inoculated with nematodes, with approximately 1500 J2s per pot. Samples were collected every 2 days after inoculation, with three plants taken each time. The onion root systems were stained using an acidic fuchsine method [22], and the numbers of nematodes at different developmental stages (second-stage juveniles (J2s), third-stage juveniles (J3s), fourth-stage juveniles (J4s), and females) were observed and counted.

2.6. Data Statistics and Analysis

Data were statistically analyzed using Microsoft Excel 2016 and SPSS 18.0 software, with standard errors calculated. Figures were generated using Origin 2024, MEGA12.0.11, and Adobe Illustrator 2024.

3. Results

3.1. Symptoms of Onion Root-Knot Nematode Disease

After infection with RKNs, the aboveground parts of the onion exhibited poor growth, stunted development, yellowing and wilting of leaf tips, and inhibited bulb enlargement (Figure 1A). Upon uprooting the plant, numerous root galls were observed, with the roots showing typical hook-shaped swelling, mainly at the root tips (Figure 1B,C). The root-knot epidermis contained milky-white or yellowish egg masses, and severely affected the roots, which displayed symptoms such as shrinkage, deformation, and rotting (Figure 1D,E). Based on the above symptoms, the disease affecting the onion was initially identified as RKN disease, with further pathogen identification to follow.

3.2. Morphological Characteristics

J2s and females were isolated from diseased onion roots in Yuanmou County, Chuxiong Prefecture, Yunnan Province, with no males observed. The morphological characteristics of J2s and females are shown in Figure 2. J2s are slender with slightly pointed ends, exhibiting a slight ventral curve after heat treatment (Figure 2A). The basal part of the stylet is small, with a distinct esophageal bulb and valve structures (Figure 2B), and the tail is elongated (Figure 2C). Females are pear-shaped and milky white, with a slender neck (Figure 2E), a well-developed stylet, a straight stylet cone, a cylindrical stylet shaft, and a flattened bulb at the base of the stylet (Figure 2D). The eggs are oval (Figure 2F). The perineal pattern morphology of females is oval, with a round dorsal arch that is moderately low and smooth in texture, with indistinct lateral lines; a few striae may converge at both ends of the vulva, and the lateral region is either blurred or absent (Figure 2G,H). Based on the perineal pattern morphology of females, the pathogen was tentatively identified as M. graminicola.

The morphometric data of J2s and females are detailed in

Table 1. Morphometric values of J2s of Allium cepa RKN populations and other RKN populations.

Origin	Yunnan, China	M. graminicola (Zhejiang Jinhua, China) [23]	M. graminicola (USA) [9]	M. graminicola (Hainan, China) [13]	M. graminicola (Zhejiang Hangzhou, China) [24]	M. hapla (Yunnan, China) [25]	M. incognita (Yunnan, China) [26]
Host	Allium cepa L.	Oryza sativa L.	Echinochloa crusgalli (L.) Beauv.	Allium fistulosum L.	Oryza sativa L.	Gentiana crassicaulis	Achyranthes bidentata
Number of nematodes measured	25	20	20	25	11	20	-
Body length	477.46 ± 41.51 (447.88–492.07)	456.7 (402.7–509.0)	441 (415–484)	456.4 (410.0–510.0)	449.5 (437.0–461.0)	370.07 ± 13.73 (338.87–398.45)	434.97 ± 28.5 (384.65–468.15)
Body width	19.46 ± 3.73 (14.21–26.25)	16.1 (12.9–19.1)	-	-	15.7 (14.2–17.4)	11.88 ± 0.81 (10.48–13.28)	18.50 ± 2.66 (12.73 ~ 22.81)
Stylet length	12.16 ± 0.63 (10.96–13.14)	12.1 (10.6–13.1)	11.38 (11.20–12.32)	-	10.8 (10.0–11.6)	13.05 ± 0.50 (12.12–14.35)	13.74 ± 1.11 (11.38~16.05)
Distance from base of stylet to dorsal esophageal gland opening	3.74 ± 0.48 (2.66–4.76)	2.6 (2.1–2.8)	2.83 (2.80–3.36)	-	3.0 (2.1–4.3)	5.88 ± 7.908 (61.92–40.95)	2.81 ± 0.19 (2.56–3.46)
Stylet knob width	1.89 ± 0.4 (1.15–2.6)	-	-	-	2.3 (2.2–2.6)	1.23 ± 0.20 (0.79–1.57)	1.73 ± 0.35 (1.06–2.21)
Stylet knob height	1.35 ± 0.18 (1.07–1.69)	-	-	-	1.4 (1.1–1.6)	1.19 ± 0.17 (0.80–1.48)	1.27 ± 0.24 (0.92–1.97)
Metacorpus length	11.29 ± 1.67 (7.89–15.13)	-	-	-	-	10.32 ± 1.14 (8.74–12.89)	9.07 ± 0.96 (8.06–11.89)
Metacorpus width	8.03 ± 1.36 (6.16–10.61)	-	-	-	-	6.68 ± 0.49 (5.94–7.70)	7.17 ± 1.14 (6.45–10.67)
Anterior end to center of metacorpus	49.9 ± 4.69 (41.41–57.29)	-	-	-	-	53.60 ± 2.19 (7.72–57.49)	55.83 ± 7.46 (34.61–66.29)
Tail length	76.18 ± 6.05 (62.06–85.16)	70.2 (61.2–79.8)	70.9 (67.0–76.0)	72.9 (60.0–85.0)	74.3–2.5 (70.3–78.6)	43.64 ± 4.21 (29.88–49.95)	43.64 ± 12.67 (35.11–55.81)
Hyaline tail length	19.47 ± 1.72 (14.54–22.55)	19.5 (16.5–22.6)	17.9 (14.0–21.2)	22.1 (12.5–27.5)	21.6 (19.4–23.7)	11.53 ± 1.30 (8.40–13.41)	10.78 ± 2.36 (8.98–12.58)
Anal body diameter	12.03 ± 1.13 (10.05–15.23)	-	-	-	11.4 (10.5–12.1)	9.62 ± 0.692 (8.30–10.71)	-

Note: All measurements are in μm and number in brackets means range. - means no data. The value after “±” represents the standard error. The same below.

and

Table 2. Morphometric values of females of Allium cepa RKN populations and other RKN populations.

Origin	M. graminicola (Yunnan, China)	M. graminicola (Zhejiang, China) [23]	M. graminicola (USA) [9]	M. graminicola (Hainan, China) [13]	M. hapla (Yunnan, China) [25]	M. incognita (Yunnan, China) [26]
Host	Allium cepa L.	Oryza sativa L.	Echinochloa crusgalli (L.) Beauv.	Allium fistulosum L.	Gentiana crassicaulis	Achyranthes bidentata
Number of nematodes measured	25	7	20	-	20	-
Body length	633.72 ± 90.60 (504.42–806.78)	598.9 (499.1–818.7)	573 (445–765)	-	723.70 ± 66.77 (612.55–851.46)	736.18 ± 132.32 (555.39–1046.53)
Body width	432.35 ± 67.14 (309.05–587.46)	354.5 (277.3–455.5)	419 (275–520)	-	417.51 ± 39.04 (341.31–480.91)	544.49 ± 73.23 (321.27–724.13)
Stylet length	12.43 ± 1.06 (10.62–14.18)	10.2 (8.1–12.6)	11.08 (10.6–11.2)	12.1 (10.8–14.0)	10.35 ± 1.24 (8.11–12.86)	16.31 ± 1.16 (13.86–17.75)
Distance from base of stylet to dorsal esophageal gland opening	3.99 ± 0.38 (3.33–4.71)	3.7 (2.9–4.9)	3.2 (2.8–3.9)	4.3 (3.7–4.7)	4.95 ± 0.77 (3.70–7.02)	2.68 ± 0.59 (2.11–3.78)
Stylet knob width	3.27 ± 0.48 (2.26–4.24)	-	-	-	2.79 ± 0.35 (2.03–3.59)	3.75 ± 0.60 (2.78–4.95)
Stylet knob height	1.66 ± 0.28 (1.12–2.14)	-	-	-	1.97 ± 0.49 (1.32–3.55)	2.75 ± 0.92 (2.15–5.17)
Metacorpus length	29.2 ± 8.65 (11.19–49.27)	-	-	-	38.09 ± 3.94 (31.00–47.74)	40.36 ± 8.33 (37.36–59.18)
Metacorpus width	25.62 ± 8.3 (10.3–47.02)	-	-	-	37.11 ± 4.45 (28.29–46.88)	35.12 ± 4.76 (30.13–50.18)
Anterior end to center of metacorpus	69.35 ± 7.08 (55.69–82.89)	-	-	-	73.51 ± 7.31 (59.25–89.11)	81.69 ± 12.56 (56.55–91.48)

(Figures S1 and S2). Compared with RKN populations from other regions, the J2s in this study exhibited greater body length and width. The stylet length was similar to that in the Jinhua, Zhejiang population but longer than that found in other M. graminicola populations, while being shorter than that in M. hapla and M. incognita populations. The distance from the base of the stylet to the dorsal esophageal gland opening (DGO) was shorter than that in the M. hapla population but longer than in other populations. The height and width of the stylet knobs were both smaller than those in the Hangzhou, Zhejiang population but larger than in M. hapla and M. incognita populations. The length and width of the metacorpus exceeded those of M. hapla and M. incognita, although the anterior end to the center of the metacorpus was shorter than in these two populations. The tail length was comparable to the Hangzhou population but significantly longer than in other populations; the hyaline tail length was also similar to that in the Hangzhou population. Females’ body length and width were greater than those in the Jinhua and U.S. populations but smaller than those in M. hapla and M. incognita populations. Stylet length, the height and width of stylet knobs, and DGO distance were close to those in the Hangzhou population and larger than those in M. hapla but smaller than those in M. incognita. Overall, the morphometric values of J2s and females were generally larger than other populations, which may be attributed to individual variation and geographic factors. Most remaining morphological traits were consistent with the typical characteristics of M. graminicola, and all measurements fell within acceptable ranges.

3.3. Molecular Identification

Molecular identification was performed via the amplification and sequencing of a 413 bp fragment from the mtDNA COXI region (GenBank accession number: OR889158). The sequence exhibited 99.51–100% similarity with the reported reference sequences of M. graminicola (including MN017128 and OL587534 (India), OL587542 (India), OL587557 (India), LR215856 (France), LR215858 (France), MZ522756 (Guangdong, China), and MN017128 (Fujian, China)). Phylogenetic analysis based on 24 sequences showed that the RKNs isolated from the onion samples formed an evolutionary branch with 100% support that was closely related to the aforementioned M. graminicola reference sequences and significantly diverged from other groups (Figure 3A). A 767 bp fragment from the 28S rDNA D2-D3 region (GenBank accession numbers: OR897813-OR897815) was also obtained, and its sequence exhibited 99.48–99.61% similarity with known M. graminicola reference sequences (ON032628 (Fujian, China), ON032631 (Fujian, China), ON032633 (Fujian, China), MZ656128 (Hunan, China), MT159673 (Fujian, China), MT159675 (Fujian, China), MN513029 (Guangdong, China), MN647592 (Guangdong, China), etc.). Phylogenetic analysis based on 28 sequences revealed that the RKN sequences from the onion samples formed an independent evolutionary branch with high support (100%) that was closely related to the aforementioned M. graminicola reference sequences (Figure 3B). Molecular identification results indicate that the pathogen causing onion RKN disease is M. graminicola.

3.4. Specific Primer Diagnosis

The use of the M. graminicola-specific primers Mg-F/Mg-R successfully amplified a specific band of 369 bp in all 14 onion RKN disease samples. This band size was consistent with the M. graminicola PCR amplification band reported in the existing literature, further confirming that the RKN pathogen infecting the 14 onion samples was M. graminicola (Figure 4).

3.5. Life Cycle and Pathogenicity Observation Results

After acid fuchsin staining, it was observed that after inoculation with M. graminicola 2d, J2s invaded the root system and accumulated in the meristematic zone inside the roots (Figure 5A). After invading the vascular bundles, J2s established colonies and fed, extending from the meristematic zone near the root cap to the elongation zone. The larva swells during development, forming a carafe-like shape (Figure 5B). Under indoor inoculation conditions, J3s were observed after 4 days (Figure 5C). After a developmental period of 4–18 days, J4s (Figure 5D) and early-stage females (Figure 5E) were found. The immature females (Figure 5E), after feeding and developing, eventually transformed into pear-shaped females (Figure 5F). During the 4–8 days observation period, the females began to lay eggs, which adhered to a thin gelatinous substance, with the eggs connected by a uniform gelatinous material (Figure 5G). Subsequently, the eggs hatched into J2s, which again invaded the root system (Figure 5H).

At 26 days, pathogenicity observations of the root systems of both the control healthy plants and inoculated plants revealed that the nematodes had successfully infected the roots (Figure 5I). The roots of the infected plants appeared light yellow, with prominent galls primarily concentrated at the root tips. Some of the roots had already decayed and died (Figure 5J,K). Under greenhouse conditions (with an ambient temperature of approximately 14–27 °C), after inoculating with J2s, J3s were observed on the roots on day 4; J4s appeared on day 10; early females were visible on day 18; mature females and their egg sacs were observed after day 22; and newly hatched J2s were seen after day 26 (Figure 6). These results indicate that M. graminicola completes one developmental generation in approximately 26 d after infecting the Allium cepa roots.

4. Discussion

This study reports, for the first time, the discovery of RKN infestations on onions in the Yuanmou area of Chuxiong, Yunnan Province, China. Through a combination of morphological identification, molecular biological analysis, and pathogenicity tests, the pathogen was identified as M. graminicola. According to statistical data from 2022 to 2024, the onion planting area in this region reached 2687.01 ha, with an RKN infection rate of approximately 10%, resulting in significant economic losses. This is the first systematic report in China regarding the damage caused by this nematode to onion crops. Notably, multiple researchers have identified M. graminicola on shallots (Allium cepa var. aggregatum) in Thailand [27], onions (Allium cepa) in Karnataka, India [11], and scallions (Allium fistulosum) in Hainan, China [13]. This indicates that the nematode threatens a wide range of Allium L. crops, causing severe economic losses, and further investigation of the disease and the implementation of control measures are urgently needed.

4.1. Pathogen Identification Methods and Taxonomic Significance

Accurate identification of nematode species is a crucial foundation for studying their biological characteristics, pathogenic mechanisms, and developing effective control strategies, which is of significant importance for ensuring the safety of agricultural production systems. Currently, RKN identification has evolved into a system combining both morphology and molecular biology, effectively overcoming the limitations of traditional methods that relied solely on the characteristics of the female perineal pattern, significantly improving the accuracy of identification. This study found that the vulval patterns of onion RKN populations exhibited characteristics such as unclear lateral lines in the lateral region and blurred or absent lateral regions, which closely matched the morphological traits of M. graminicola reported by Tian et al. [23]. Further measurements showed that the morphological parameters of their J2s and females (including body length, stylet length, and DGO) were consistent with the data described for M. graminicola by Liu et al. [24] with measurement errors within the acceptable range. However, traditional morphological identification requires a high level of expertise from the operator, and the vulval pattern exhibits intraspecific variation, which may affect the accuracy of identification results. Given that some rare RKN species may also exhibit similar traits, molecular biology methods should be used for verification.

This study used universal primers for mtDNA COXI and 28S rDNA D2-D3 sequences to perform PCR amplification, resulting in specific bands of 413 bp and 767 bp, respectively. Sequence alignment revealed that the amplified sequences were identical in length to those obtained by Tian et al. [23], using the same primers for M. graminicola root-knot nematodes, with a sequence similarity exceeding 99% [23]. The phylogenetic tree based on molecular data further confirmed that the causal agent of onion RKN disease is M. graminicola, providing scientifically reliable identification. Additionally, the specific primers Mg-F3/Mg-R2 for M. graminicola were used in this study to successfully amplify a 369 bp fragment, which was consistent with the findings of Wang et al. [28], further validating that the nematode infecting onions is M. graminicola.

4.2. Life History Characteristics and Host Adaptability

In this study, the periodic staining method was used to systematically observe the infection, migration, colonization, and feeding behavior of M. graminicola J2s in onion roots. The results showed that the nematode J2s began to develop after migrating to the inner meristematic region of the root crown, eventually maturing into egg-laying females. The newly hatched J2s from these eggs were capable of reinfecting onion roots. The life cycle of M. graminicola is significantly influenced by environmental conditions: it takes only 19 d to complete its life cycle in Bangladesh (22–29 °C), 26 d under early summer conditions in India, and only 18 d in Fujian Province, China (26–37 °C) [14]. In this experiment, the nematode required approximately 26 d to complete a generation in onion roots. Notably, the life cycle of M. graminicola in onion-growing regions of Yunnan Province is relatively prolonged, which may be attributed to developmental variation caused by differences in host plants and cultivation environments.

4.3. Disease Epidemics and Control Strategies

Meloidogyne graminicola is widely distributed in tropical and subtropical regions, with a broad host range that includes over 100 plant species from families such as Poaceae and Brassicaceae [29]. It is one of the most important nematode pathogens of rice and is therefore commonly referred to as the rice RKN. This nematode was first identified in China on Welsh onion (Allium fistulosum) in Hainan Province [13], and has since been reported to cause damage in rice-growing areas of the Fujian, Hubei, Hunan, Jiangsu, Zhejiang, Jiangxi, and Sichuan provinces [23]. Under loose, well-aerated sandy loam and suitable temperature conditions, M. graminicola can efficiently infect and proliferate [30]. In an onion cultivation area in Yuanmou, Chuxiong, where the soil is sandy, the conditions favor nematode infection and reproduction, leading to a severe incidence of RKN disease. This study also found that when irrigated rice was grown in the preceding season, its roots may have been infected and colonized by M. graminicola during the seedling nursery or early submerged stages, serving as an initial source of infection for the subsequent onion crop. Although continuous or intermittent irrigation during the rice growing season mitigated visible yield losses or symptoms, the nematodes nonetheless became a primary inoculum for the next crop. Onion is widely cultivated in Yuanmou, Chuxiong, and to enhance land use efficiency and economic returns while reducing continuous cropping obstacles and soilborne diseases, rotational cropping with other species has become an important practice. As onion–rice or onion–vegetable rotation systems become more prevalent, the risk of the widespread occurrence of rice RKN disease increases. Integrated management strategies, including the use of resistant cultivars, agronomic practices, and chemical control, are recommended. Selecting resistant onion varieties is the most economical and effective approach, though systematic resistance evaluations are currently lacking [31]. Where necessary, nematicides such as abamectin and fosthiazate may be applied during soil preparation or the seedling stage [32].

5. Conclusions

This study provides the first confirmation of Meloidogyne graminicola infecting onions in Yunnan, China, with its taxonomic status clarified through morphological and molecular identification. The research reveals a characteristic life cycle of 26 days on onion hosts, which is significantly shorter than that on rice, suggesting host-adaptive evolution. The findings offer a theoretical foundation for developing early molecular detection techniques for onion RKN disease and formulating a “rice-avoidance rotation” control strategy. Additionally, future research directions are proposed, including screening resistant germplasm, developing biocontrol agents, and tracing pathogen populations. This work holds significant implications for ensuring the safe production of allium vegetables in China.