Quantitative Analysis of Meloidogyne incognita Population Density Using Real-Time PCR and Its Correlation with Root-Knot Disease Index in Tabacco (Nicotiana tabacum)

1 1 Quantitative Analysis of Meloidogyne incognita Population Density Using Real-Time PCR 2 and Its Correlation with Root-Knot Disease Index in Tabacco (Nicotiana tabacum) 3 4 Zejun Cheng1,2*, Jingwen Chai1, Haoshuai Pu1, Zhe Zhao4, Xiaoxin Duan1,2, Wei Zheng1,2, 5 Jianqiang Xu1,2, Pu Miao3, Wenbang Hou4* 6 7 1College of Horticulture and plant protection, Henan University of Science and Technology, 8 Luoyang, Henan Province, China. 9 2Henan Province Engineering Technology Research Center of Green Plant Protection, Luoyang 10 471023, Henan Province, China. 11 3China Tobacco Corporation of Luoyang, Henan Province, China. 12 4College of Agricultural / Tree Peony, Henan University of Science and Technology, Luoyang, 13 Henan Province, China. 14 15 *Corresponding author: [email protected] (Z. Cheng) 16 *Corresponding author: [email protected] (W. Hou) 17 18 Abstract: Tobacco root-knot disease represents a significant threat to tobacco production, 19 particularly in the western Henan region, where Meloidogyne incognita is the predominant 20 species. This study collected samples of M. incognita and soil from Luoyang, Henan, and 21 designed specific primers MiF and MiR based on the amplified 735 bp sequence of the 22 ITS1-5.8S-ITS2 region. These primers exhibit mismatches with related species, including M. 23 javanica, M. minor, M. hapla, and M. arenaria, showing 1, 6, 10, and 10 base differences in the 24 forward direction, respectively, 3, 10, and 9 base mismatches in the reverse direction. Although 25 the primers were used to detect corn-wheat soil samples, no amplification was observed. 26 Additionally a real-time quantitative PCR curve for M. incognita in soil was constructed, 27 revealing a negative correlation between the Ct value (Y) and the log-transformed number of 28 nematodes (x) per 20 g of dry soil, represented by the equation y = -0.9757x + 35.565; (R² = 29 0.9999, P < 0.01). According to the decomposition efficiency of nematode DNA in soil, the 30 results showed that nematode DNA degrades rapidly in soil, with a degradation rate of .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 2 31 approximately 87.3% at 3 days and 99.97% at 14 days. Furthermore, significant differences were 32 observed in the real-time PCR detection efficiency among various nematode forms: the Ct value 33 of J1 was significantly higher than that of J2, while abnormal eggs (empty eggs or internal bubble 34 eggs) exhibited the highest Ct value. The proportion of abnormal eggs in the soil before planting 35 was significantly higher at 64.08% compared to only 15.3% at harvest, indicating that the activity 36 of nematodes in the soil is significantly reduced after harvesting in October and planting in March 37 of the following year. A survey of 126 tobacco plants indicated a significant positive correlation 38 between the root-knot index (RKI) and root-knot nematode density (r = 0.80, p < 0.01). The study 39 identified a minimum disease threshold of 234 individuals / 20 g soil at harvest and revealed a 40 nonlinear relationship between disease severity and nematode density. Specifically, a weak 41 correlation was observed at low density (Low RKI: 0-1; nematode density<2000 individuals/20 g 42 soil, r = 0.49, P2000 individuals/20 g soil, r = 0.69, P<0.05). This study showed that as the 44 RKI increases, the rate of increase in nematode density in the soil diminishes. These findings 45 provide valuable insights for the development of effective scientific strategies for nematode 46 control. 47 48 Keywords: tobacco root-knot nematode; specific primers; tobacco root disease index (RKI); 49 nematode population density (NPD) 50 51 1. Introduction 52 Tobacco (Nicotiana tabacum L.) is the primary raw material for cigarettes and constitutes a 53 significant economic crop in China [1]. As the largest producer and consumer of tobacco globally, 54 China accounts for over one-third of the world’s tobacco production [2]. Major pests and diseases 55 affecting tobacco include the tobacco mosaic virus (TMV), black shank (Phytophthora 56 nicotianae), bacterial wilt (Ralstonia solanacearum), and root-knot nematode [3]. Notably, 57 root-knot nematodes cause wounds on the epidermal cells of tobacco roots, which increase the 58 susceptibility of tobacco plants to soil-borne pathogens. Therefore, controlling tobacco root-knot 59 nematodes is essential, as it may reduce the incidence of other soil-borne diseases[4]. 60 Meloidogyne spp. are widely distributed plant pathogenic nematodes that significantly affect the .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 3 61 growth and yield of various economically important crops, including tobacco, tomato, and 62 cucumber [10]. By invading the root system of plants, these nematodes induce the formation of 63 root galls, disrupting the normal structure and function of the roots [11]. This disruption leads to a 64 decline in water and nutrient absorption, resulting in stunted growth, yellowing of leaves, reduced 65 yields, and in severe cases, plant death [12]. Statistics indicate that agricultural losses caused by 66 root-knot nematodes worldwide amount to billions of dollars [13]. Tobacco root-knot diseases 67 typically reduce yields by 10% to 20%, however, in severe cases, losses can exceed 75%, with a 68 higher incidence of disease reported in developing countries compared to developed ones [10]. In 69 provinces such as Henan, Anhui, Sichuan, Guizhou, Zhejiang, and Yunnan in China, tobacco 70 root-knot disease affects approximately 52,000 hm2 annually, accounting for about one-tenth of 71 the total production area, leading to losses of around 9 million dollars [14]. Therefore, early 72 detection and effective prevention and control of tobacco root-knot nematodes are critical issues 73 that must be addressed in agricultural production [15]. 74 Currently, there are no tobacco varieties exhibiting high resistance to root-knot nematodes, and 75 the interactions between these pests and host plants are complex, posing significant challenges for 76 resistance breeding [5]. Consequently, root-knot nematode disease has emerged as a significant 77 constraint on tobacco production in various countries and regions [6]. Presently, the most effective 78 strategy for managing tobacco root-knot nematode disease focuses on suppressing the 79 reproduction and population density of the pathogenic nematodes [7]. For instance, Zhang et al., 80 cloned the tobacco RKN resistance gene NtRk1, which is induced upon nematode infection and 81 enhances resistance by regulating salicylic acid and jasmonic acid pathways. Overexpression of 82 this gene confers protection in susceptible cultivars while RNA interference (RNAi) silencing 83 increases susceptibility to Meloidogyne incognita [8]. Additionally, Li et al. identified 5,206 84 high-confidence long non-coding RNAs (lncRNAs) in tobacco, with 565 being differentially 85 expressed during nematode infection [9]. 86 Traditional nematode detection methods primarily rely on morphological identification and 87 microscopic observation [16]. Morphological identification necessitates a comprehensive 88 understanding of the morphological characteristics of nematodes, as even minor differences 89 among species can lead to misidentification [17]. Moreover, microscopic observation is 90 time-consuming and often does not meet the rapid detection requirements for large-scale samples. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 4 91 Conventional quantitative methods are inadequate for detecting nematode eggs, J1 and inactive 92 nematodes in soil, resulting in unreliable disease forecasts [18]. With the rapid advancements in 93 molecular biology technology, real-time fluorescent quantitative PCR (Real-Time PCR) has 94 become widely utilized in plant disease detection due to its high sensitivity, specificity, and 95 quantitative capabilities [19]. This technology allows for the rapid and precise detection of 96 nematode DNA through the design of specific primers and fluorescent probes, facilitating accurate 97 quantification of nematode density [21]. Compared to traditional morphological methods, 98 real-time PCR technology significantly reduces detection time and effectively distinguishes 99 between different types of nematodes, thereby providing robust technical support for early 100 warning and precision prevention and control of diseases [21-24]. 101 While real-time PCR technology demonstrates a significant advantage in detecting nematodes, 102 current research remains limited [25]. The root-knot disease index (RKI) serves as a crucial 103 indicator of disease severity, typically assessed through parameters such as the number of root 104 knots and the extent of root damage [26]. Clarifying the quantitative relationship between 105 root-knot nematode density and the disease index not only enhances our understanding of the 106 infection mechanism of root-knot nematodes but also provides a scientific foundation for early 107 diagnosis and comprehensive disease management. For instance, developing a mathematical 108 model that correlates nematode density with disease severity enables the prediction of disease 109 progression and the formulation of targeted prevention strategies [27]. This approach can 110 minimize pesticide use, reduce environmental pollution, and enhance the sustainability of 111 agricultural production [28]. 112 The purpose of this study is to use real-time PCR technology for the quantitative detection of 113 tobacco root-knot nematode density and to analyze the correlation between nematode density and 114 the root disease index. This research will explore the manifestations of disease in the tobacco root 115 system under varying nematode densities, thereby establishing a quantitative relationship between 116 nematode density and the disease index. The findings will provide a theoretical foundation and 117 technical support for early diagnosis, as well as comprehensive prevention and control of tobacco 118 root diseases. Furthermore, the results of this research object not only to enhance the detection 119 efficiency of tobacco root-knot nematodes but also to serve as a reference for the prevention and 120 control of similar issues in other crops. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 5 121 122 2. Materials and methods 123 2.1 Soil and nematode collection 124 The soil and tobacco samples were collected from four tobacco planting field in Luoyang: 125 Luoning Xiaojie (111.59°E, 34.45°N), Yiyang Gaocun (111.88°E, 34.55°N), Ruyang Baipo 126 (111.70°E, 34.50°N), and Kaiyuan Farm (112.42°E, 34.60°N). Prior to tobacco cultivation 127 in March 2022, soil samples were collected from fields affected by root-knot nematodes at a depth 128 of 0-30 cm. Egg masses of root-knot nematode in the soil were extracted and counted before 129 transplantation. The Cobb's sieving and decanting method, along with the flotation method, were 130 employed for the collection of egg masses from the soil [29]. A total of 100 g of soil samples were 131 taken, to which an appropriate amount of distilled water was added. This mixture was then filtered 132 through 850 µm and 250 µm sieves to remove larger particle impurities and collect the residues. 133 The residue was re-suspended in a sucrose solution (454 g/L, with a density of approximately 1.18 134 g/cm³). After mixing, the solution was subjected to centrifugation at 1207 x g for 5 min. Due to 135 the lower density of the egg masses, they floated to the surface of the sucrose solution, while 136 heavier impurities settled at the bottom of the tube. A straw was used to carefully extract the 137 liquid, which was then transferred to a new centrifuge tube. The mixture was centrifuged again to 138 remove the sucrose, resulting in the isolation of the egg mass sample. On October 7, 2022, and 139 October 15, 2023, tobacco root samples were collected following the tobacco harvest. Using a 140 five-point sampling method, samples were taken from the 0-30 cm soil layer within a 50 cm radius 141 centered on the tobacco plants. The tobacco root egg masses were separated from the tobacco 142 roots, the fibrous roots were cut, and the egg within the root egg masses were extracted under a 143 microscope. 144 145 2.2 Extraction of M. incognita DNA and Design of Specific Primers 146 The nematodes used for the identification of the DNA sequence of root-knot nematodes were 147 collected from infected roots of tobacco plants in four different tobacco planting fields in October 148 2021. The egg masses were extracted from the root knots, and a single egg was used as a sample 149 for DNA extraction. Using tweezers, the egg mass was broken to release the eggs and juvenile 150 second-stage nematodes (J2). The eggs were then incubated at room temperature (25℃) for 3 h. A .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 6 151 pipette was used to extract a single egg or J2, following the single nematode DNA extraction 152 method described by Wang et al. (2011). A single J2 sample was placed on a sterilized glass slide 153 alongside a drop of water. A No. 3 insect needle was then used to make a vertical incision in the 154 J2 sample. Subsequently, 10 µL of the nematode solution was aspirated into a 200 µL centrifuge 155 tube, and 1.5 µL of 10 x PCR Buffer (Mg2+-free) was added. The mixture was subjected to liquid 156 nitrogen for 1 min, followed by heating at 85℃ for 2 min. Then, 1 µL of 1 mg/mL protease K was 157 incorporated, and the solution was heated at 56℃ for 15 min followed by an additional 10 min at 158 95 ℃. This DNA extraction solution was subsequently diluted 10 times with sterile water to serve 159 as a template for PCR amplification. 160 The amplification of the ITS1-5.8S-ITS2 region was conducted using the forward primer ITS-F 161 (5'-ACA AGT ACC GTG GAA AGT TG-3') and the reverse primer ITS-R (5'-TCG GAA GGA 162 ACC TAC TA-3'). PCR amplification was performed with Hieff Canace Plus High-Fidelity DNA 163 Polymerase (Yeasen, Shanghai) under the following conditions: 94℃ for 2 min, (94℃ for 1 min, 164 48℃ for 1 min, 72℃ for 1 min) x 35 cycles, 72℃ for 5 min. The PCR products were purified 165 using the DiaSpin column PCR product purification kit (Sangon Biotech, Shanghai) and 166 subsequently sent to Sangon Biotech for sequencing. The ITS region sequence of M. incognita 167 obtained in this study was compared with sequence in the NCBI database for identification. The 168 target sequence was analyzed using Mega 11.0 software to identify variant regions, and NCBI 169 Primer-BLAST was employed to design primers specific to the nematode (MiF and MiR). 170 171 2.3 Establishing a real-time PCR method for the quantification of tobacco root-knot nematode 172 The DNA solution extracted from a single J2 was diluted ten fold with sterile distilled water to 173 serve as a template for Real-Time PCR. Real-Time PCR was conducted using the CFX96 174 (Bio-Rad, USA) for detection. The reaction mixture consisted of 10 µL, which included 5 µL of 175 Hieff qPCR SYBR GREEN Master Mix (Yeasen, Shanghai), 0.4 µL of specific primers, and 2 µL 176 of template DNA. The reaction conditions were as follows: 95℃ for 30 s; (95℃ for 5 s and 58℃ 177 for 30 s) x 40 cycles. Distilled water was used as a negative control. Additionally, the DNA 178 solution from a single M. incognita was diluted to 10^-1, 10^-2, 10^-3, 10^-4, and 10^-5 for use as 179 templates, and specific primers MiF and MiR were employed across three replicates for treatment. 180 Three soil samples were collected from corn-wheat crop rotation fields in Yiyang Gaocun, .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 7 181 following the method described by Cheng et al. (2018) for extracting total biological DNA from 182 the soil. 20 g of soil samples were dried at 60℃ for 24 h and processed using a planetary ball mill 183 (LANENDE, Shandong) at a speed of 450 rpm for 2 min [30]. Subsequently, 5 g of the ball milled 184 soil samples were added to 50 mL centrifugal tubes, along with 10 mL of phosphate buffer 185 (Na2HPO4 and NaH2PO4, 0.12 M, pH 8.0), with three replicates for each sample. The soil-buffer 186 mixture was agitated using a shaker (Hongke Technology HQY-C, Jiangsu, China) at 200 rpm for 187 30 m. After an additional 10 min, the solution was purified using the DiaSpin column PCR 188 product purification kit (Sangon Biotech, Shanghai). The DNA eluate was then diluted ten-fold to 189 serve as a template for real-time PCR. All samples were prepared in triplicate. 190 The DNA solution extracted from the soil sample of the corn-wheat crop rotation was 191 detected using the MiF and MiR primers; however, no specific amplification was observed (data 192 not shown). Consequently, a quantitative curve for the root-knot nematode of tobacco was created 193 using soil collected from the corn-wheat rotation field. The tobacco roots containing egg masses 194 were cut into 1 cm segments with scissors, and subsequently homogenized in a blender for 30 195 seconds (Midea WBL2521H, Guangdong). The fragmented roots were placed in a gauze bag, and 196 both J2 and adults root-knot nematodes were extracted using the Baermann funnel method for 197 inoculation. The soil from the corn-wheat rotation field was dried at 60℃ and then weighed to a 198 mass of 20 g. Different quantities of M. incognita (J2 and adults) were artificially inoculated: 0, 199 30, 150, and 750 individuals. Following inoculation, the soil was dried again at 60℃ for 6 h. Each 200 inoculation amount was prepared in triplicate. 201 202 2.4 Rate of DNA degradation of M. incognita in soil 203 In October 2023, tobacco roots infected with root-knot nematode were collected post-harvest. 204 The infected roots were homogenized using a blender (Midea WBL2521H, Guangdong) for 30 s, 205 with this process repeated twice. The nematode suspension, comprising both J2 and adult stages 206 was subsequently extracted using the Baermann funnel method. Following treatment of the M. 207 incognita suspension in a water bath at 75℃ for 10 minutes, approximately 2000 individuals of M. 208 incognita were inoculated into 20 g of raw soil sourced from a corn-wheat rotation field using a 209 pipette for precision. DNA extraction from the soil was performed at intervals of 0, 3, 7 and 14 210 days, employing the DNA extraction method outlined by Cheng et al. (2018)[]. The extracted .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 8 211 DNA was then analyzed through real-time PCR detection, yielding Ct values with the MiF and 212 MiR primers. The equivalent number of nematodes was calculated based on the quantitative curve 213 of M. incognita. 214 215 2.5 Effect of the tobacco root-knot disease index (RKI) on nematode population density (NPD). 216 On October 7, 2022, and October 15, 2023, samples were collected from four tobacco-growing 217 fields in Luoyang City, Henan Province, specifically at Luoning, Yiyang, Ruyang Bai, and 218 Kaiyuan Farm, following the tobacco harvest. At each location, 38, 32, 30, and 26 tobacco plants 219 were sampled for roots, resulting in a total of 126 plants. Soil samples were also collected from 220 the rhizosphere area (15 cm in diameter with the root; 0-30 cm in depth) of each tobacco plant and 221 stored in sterile sealed bags. The established real-time PCR method for detecting M. incognita was 222 used to employed to assess the nematode population density (NPD) in the soil samples. The 223 diseased tobacco roots were washed with water to eliminate any adhering soil, and 30 g of root 224 samples were cut and placed on a white tray for root-knot quantification. The number of galls was 225 observed and counted either with the naked eye or using a magnifying glass. Concurrently, the 226 Root-Knot Disease Index (RKI) of each tobacco plant was evaluated. Based on the number of root 227 egg masses and the degree of root damage, a 0-5 scale grading standard from Taylor (1978) was 228 used to evaluate the RKI of each tobacco root, with specific grading criteria as follows [31]: 229 0 level: No root-knots present; 230 1 level: 0% ≤ number of swollen roots or root-knots due to nematode damage < 10%; 231 2 level: 10% ≤ number of swollen roots or root-knots due to nematode damage < 25%; 232 3 level: 25% ≤ number of swollen roots or root-knots due to nematode damage < 50%; 233 4 level: 50% ≤ number of swollen roots or root-knots due to nematode damage < 75%; 234 5 level: 75% ≤ number of swollen roots or root-knots due to nematode damage ≤ 100%. 235 The infected young roots develop spherical or irregular galls of varying sizes, which appear 236 white or pale in color. In severe cases, the diseased roots turn brown and decay. Under conditions 237 of high RKI level, the root system becomes severely deformed, exhibiting a beaded or claw-like 238 morphology. 239 2.6 Statistical analysis 240 Statistical analyses were performed using SPSS version 26.0. A linear regression analysis was .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 9 241 conducted to explore the relationship between the number of inoculated nematodes and the Ct 242 values obtained from real-time PCR, with a correlation coefficient calculated (p < 0.01). 243 Furthermore, the Pearson correlation coefficient was employed to assess the correlation between 244 the root-knot disease index (RKI) and the nematode population density (NPD). 245 246 3. Result 247 3.1 Identification of M. incognita and Design of Specific Primers 248 The findings of this study indicate that the ITS region sequences of root-knot nematodes from 249 tobacco in four planting areas of Luoyang, Henan Province, exhibit a high degree of consistency. 250 Analysis using NCBI BLAST revealed a similarity of 99.57% with M. incognita from Fujian 251 Province, China (accession number: OQ632600), with only three base pair mismatches in a 735 bp 252 sequence. Additionally, a similarity of 99.28% was observed with the sequence designated as 253 accession number MT209949, which contained five base pair mismatches. For further analysis, 254 the sequence was compared with those of both closely related and distantly related species, as 255 provided by the National Center for Biotechnology Information (NCBI) (Figure 1). 256 Based on the ITS sequence of M. incognita, specific primers MiF and MiR were designed 257 using the NCBI Primer-BLAST software. The forward primer exhibits a GC content of 38.1%, 258 while the reverse primer has a GC content of 50%. A comparison of the primer sequences with the 259 ITS sequences of other closely related species revealed that the forward primer had mismatches of 260 1, 6, 6, 10, 10, and 10 base pairs with M. javanica (MW672262), M. minor (KX671108), M. 261 artiellia (JX393300), M. hapla (OM864510), M. arenaria (EU364878), and M. enterolobii 262 (KZ411228), respectively. The reverse primer exhibited mismatches of 1, 3, 6, 10, 9, and 6 base 263 pairs with the same species, respectively (Table 1). 264 265 3.2 Ct value of different morphological nematodes 266 The egg masses extracted from the tobacco roots affected by nematodes at harvest time 267 exhibited a milky white color and a smooth surface. Upon breaking open the egg masses for 268 observation, three distinct morphological types of nematodes were identified: J1, which comprised 269 eggs with clearly formed nematode bodies; J2, which were larvae that had hatched; and abnormal 270 eggs, which were either hollow or contained bubbles (Figure 2). We extracted DNA from these .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 10 271 different morphological types of nematodes and used them as templates for Ct value detection. 272 The results indicated significant differences in Ct values among the various morphological types 273 of nematodes. The Ct values for J1 extracted from the pre-planting soil and the affected roots at 274 harvest were 19.6 to 19.9 (n = 5), respectively, showing no significant difference between the two. 275 In contrast, the Ct values for J2 were lower than those for J1, recorded at 18.4 to 17.3 (n = 5), 276 respectively. Notably, the Ct values detected from the abnormal egg DNA were substantially 277 lower than those for J1 and J2, measuring 28.8 and 27.8, respectively, with a higher variance than 278 that of J1 and J2. Furthermore, the egg masses extracted from the pre-planting soil and the 279 affected roots at harvest were opened, and the eggs of each morphological type were counted. The 280 results revealed that the proportion of abnormal eggs in the pre-planting soil was as high as 281 64.08%, whereas the proportion of abnormal eggs in the egg masses at harvest was 15.3% (Table 282 2). 283 284 3.3 Quantitative Curve of M. incognita in Soil 285 We diluted the DNA solution of individual M. incognita as a template and detected the 286 amplification of the designed specific primers MiF and MiR. The study revealed that the Ct values 287 (y) increased logarithmically with the increase in the DNA dilution rate (x), with the regression 288 equation defined as y = -0.9841x + 33.9857 and R2 = 0.9964. This indicates a highly significant 289 correlation between Ct values and DNA dilution rate (P < 0.01). Furthermore, when the DNA was 290 diluted to 105 times, a linear relationship between the DNA dilution rate and Ct value was 291 observed, with Ct values ranging from 19.8 to 32.9. However, when the dilution reached 107 292 times, the Ct value was compromised, and no Ct values were detected in the negative control 293 samples (Figure 3 A). 294 In this study, based on the number of inoculated M. incognita in the soil and the Ct values 295 detected by real-time PCR, a soil quantitative curve was constructed. A correlation analysis was 296 performed between the Ct values (y) and the logarithmically transformed number of inoculated 297 nematodes (x). The results revealed a significant negative correlation between the two, with the 298 regression equation defined as y = -1.0859x + 32.025, R 2 = 0.9866, P < 0.01 (Figure 3 B). 299 300 3.4 Degradation rate of DNA from M. incognita in soil .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 11 301 The initial inoculation density of dead M. incognita in the soil ranged from 2090 to 2275 302 individuals/20 g soil. By the third day, the DNA-based equivalent of nematode density decreased 303 to 374±16 individuals/20 g soil, reflecting a degradation rate of 87.3%. On the seventh day,the 304 DNA-based equivalent of nematode density further declined to 14±2 individuals / 20 g soil, 305 indicating a degradation rate of 99.34% compared to the initial inoculation. By the fourteenth day, 306 the DNA-based equivalent of nematode density in the soil had diminished to only 1.0±0.5 307 individuals/20 g soil, achieving a degradation rate of 99.97% relative to the initial inoculation 308 (Figure 4). 309 310 3.5 Effect of M. incognita density in the rhizosphere on the root-knot index 311 Following the tobacco harvest in October 2022 and October 2023, we conducted an 312 investigation into the incidence of root diseases in 126 tobacco plants and assessed the density of 313 M. incognita in the rhizosphere soil (Figure 5). The results indicated that at a Root-knot Index 314 (RKI) level of 0, no galls or egg masses were detected in the tobacco root system; however, the 315 density of M. incognita in the rhizosphere soil varied from 0 to 68 individuals/20 g soil. When the 316 RKI reached level 1, the number of galls ranged from 2 to 9 galls/30 g root, with nematode 317 densities in the soil ranging from 234 to 2321 individuals/20 g soil. At RKI level 2, the number of 318 galls increased between 13and 23 galls/30 g root, with nematode density recorded at 2345-6764 319 individuals / 20 g soil. At RKI level 3, the count rose to 27 to 49 galls/30 g root, and the nematode 320 density in the soil ranged from 4656 to 23434 individuals/20 g soil. At RKI level 4, the number of 321 galls ranged from 53 to 73 galls/30g root, with a corresponding nematode density of 8233 to 322 35291 individuals / 20 g soil. Finally, at RKI level 5, the number of galls or swellings ranged form 323 71 to 86 galls/30 g root, with nematode density in the soil falling between 18345 and 49234 324 individuals/20 g soil. There were extremely significant differences between RKI level 0 and levels 325 1-5 (P < 0.01). Significant or extremely significant differences were found between RKI level 1 326 and levels 2-5 as well as between RKI levels 2 and 3-5, RKI levels 3 and 4-5, and RKI levels 4 327 and 5 (P < 0.05). Figure 5 showed that at RKI levels 1-3, as the number of galls increased, the 328 nematode density in the soil also significantly increased, indicating a notable enhancement in 329 significance. However, at RKI levels 4-5, the significance of this growth decreased, indicating that 330 the variation in nematode density in the soil was reduced when the root-knot disease was severe. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 12 331 332 4. Discussion 333 This study successfully established a real-time PCR analysis method for tobacco root-knot 334 nematodes (M. incognita) in the western region of Henan Province, demonstrating that the 335 population density of M. incognita in the soil significantly impacts the occurrence of root-knot 336 disease. Furthermore, the nematode samples collected from four tobacco-growing areas in this 337 region exhibited complete consistency in their ITS region sequences, all identified as M. 338 incognita. However, when comparing the sequences of different Meloidogyne species retrieved 339 from the NCBI database with our designed specific primers MiF and MiR, 1-10 base mismatches 340 were identified. According to Ri, M.U. et al. (2023), a forward primer with 10 base mismatches at 341 the 3' end significantly reduces amplification efficiency [32]. Moreover, if there are three or more 342 base mismatches at the 5' end, amplification will not occur. The M. incognita specific primers 343 designed by Toyota et al. (2010) share identical sequences with M. arenaria and M. javanica, 344 preventing differentiation between these species [21]. Similarly, the primers designed by Zhao et 345 al. (2010) share the same sequence with M. javanica but differ by a single base from M. arenaria, 346 resulting in an absence of observable PCR amplification products [33]. Additionally, Wu et al. 347 (2024) reported that M. arenaria is the dominant population among Guizhou M. incognita [34]. 348 Xu et al. (2023) indicated that in the primary tobacco-growing area of Kunming, Yunnan, M. 349 incognita comprises 89.0% of the tobacco root-knot nematode population, while M. arenaria 350 constitutes 41.3%, M. javanica 53.2%, and other Meloidogyne sp. 12.8% [35]. Jiao et al. (2014) 351 reported the population distribution ratio of tobacco root-knot nematodes in Henan Province, 352 revealing that M. incognita constituted at 55.83%, M. arenaria 23.33%, M. hapla 17.50%, and M. 353 javanica 3.33% [36]. This finding further confirms that M. incognita is the dominant species in re 354 region [37]. In this study, the M. incognita identified from tobacco samples collected in the 355 Luoyang area of Henan Province was confirmed through ITS region sequence analysis, aligning 356 with Jiao et al. (2014), who also identified M. incognita as the predominant tobacco root-knot 357 nematode in Henan [36]. Concerning the closely related species M. javanica, our primers MiF and 358 MiR exhibited one base mismatch each in their forward and reverse sequences. When we used the 359 nematode DNA for detection using the MiF and MiR primers, the Ct value increased by 6, and the 360 amplification efficiency decreased by 64% (data not shown), indicating a high specificity of these .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 13 361 primers for M. incognita. 362 This study is the first to detect DNA in nematodes of varying morphologies within the egg 363 mass, using the MiF and MiR primers for real-time PCR analysis of Ct values. The results 364 indicated that the Ct values of abnormal eggs were significantly lower than those of normal eggs 365 (J1), suggesting that these abnormal eggs are inactive and that their DNA has begun to degrade, 366 rendering them incapable of hatching. This finding suggests that the hatching rate of M. incognita 367 eggs in the soil during fallow periods, in the absence of a host plant, will significantly decrease. 368 Additionally, this study examined the decomposition rate of DNA from deceased nematodes and 369 found that seven days post-mortem, the amplification signal of their DNA was extremely low, 370 nearly negligible. Consequently, the number of nematodes detected in the soil by real-time PCR 371 could be interpreted as representing active nematodes or viable eggs. Furthermore, this indicates 372 that even if M. incognita eggs hatch into J2, the rate of DNA abnormalities remains relatively high 373 in the absence of a host plant. In conclusion, implementing fallow or crop rotation measures can 374 effectively reduce the population density of M. incognita in the soil and significantly impact 375 disease control. These results provide crucial scientific evidence for managing M. incognita and 376 advocate for the adoption of fallow or crop rotation strategies in practical applications to mitigate 377 nematode damage. 378 The results of this study on the relationship between RKI and NPD indicate a positive 379 correlation between the number of M. incognita in the tobacco rhizosphere soil and the RKI levels 380 of 1 to 3. This finding aligns with previous research reports, suggesting that during the early to 381 mid-stages of the disease, the growth of the nematode population is significantly related to the 382 severity of root-knot disease. In the initial stages of the disease (RKI 1-3), an increase in RKI 383 levels corresponds with a significant rise in the number of nematodes within the rhizosphere soil. 384 This indicates that following infection of the tobacco root system, nematodes could reproduce 385 rapidly and establish a stable population. At this stage, the growth of the nematode population is 386 primarily driven by the nutritional supply from the host root system, and the formation of root 387 knots provides additional feeding sites for nematodes, thereby further promoting their 388 reproduction [38]. However, as the disease progresses to higher levels (RKI 4-5), the nutritional 389 capacity of the tobacco root system begins to decline, which restricts further nematode 390 reproduction and results in a significant slowdown in the growth rate of the nematode population. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 14 391 Furthermore, the number of nematodes in the rhizosphere soil may have already reached the 392 environmental carrying capacity, thus limiting the contribution of further increases in disease 393 levels to the growth of the nematode population. 394 The findings of this study provide scientific evidence for the integrated management of tobacco 395 root-knot nematode disease. In the early stages of the disease (RKI 1-3), the nematode population 396 has not yet reached the environmental carrying capacity. At this stage, implementing control 397 measures, such as the application of nematicides or the cultivation of resistant varieties, could 398 effectively curb the increase in nematode numbers and mitigate the disease’s negative impact on 399 tobacco yield []. Once the nematode population reaches a specific threshold, its growth rate 400 significantly declines. Therefore, monitoring the nematode population in the rhizosphere soil 401 during agricultural production can help determine the economic threshold for disease 402 management, allowing for the rational use of pesticides, thus reducing production costs and 403 minimizing environmental pollution. The integration of agricultural management practices, such 404 as crop rotation and soil disinfection, along with biological control methods including the 405 application of antagonistic microorganisms or entomopathogenic nematodes, can effectively 406 control nematode population growth, reduce reliance on chemical pesticides, and enhance the 407 sustainability of tobacco production. 408 Despite revealing the association between RKI and NPD, this study has several limitations. It 409 did not account for the influence of environmental factors such as soil type and climatic 410 conditions, on the dynamics of the nematode population. Future research should further 411 investigate the specific roles of these factors. Additionally, genetic variations among M. incognita 412 populations across different regions may impact their host infection capabilities and reproductive 413 efficiency. 414 415 5. Acknowlegements 416 Funding for this study was generously provided by the Science and Technology Project Fund 417 of the China Tobacco Corporation of Luoyang #2022410300270069, the Science and Technology 418 Project Fund of the China Tobacco Corporation of Xuchang #2024411000240026, the Key 419 Scientific and Technological Projects of the China Tobacco Corporation #110202201026 (LS-10), 420 and the Henan Province Science and Technology Research Project #22010174. The isolates of the .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint 15 421 tobacco root-knot nematode and tobacco plant samples were collected in 2023 by Mr. Pu Miao 422 from the China Tobacco Corporation of Luoyang. 423 424 References 425 1. 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