{"paper_id":"03fe22e1-cf9a-4855-a8b0-be998a5a3da9","body_text":"1\n1 Quantitative Analysis of Meloidogyne incognita Population Density Using Real-Time PCR \n2 and Its Correlation with Root-Knot Disease Index in Tabacco (Nicotiana tabacum)\n3\n4 Zejun Cheng1,2*, Jingwen Chai1, Haoshuai Pu1, Zhe Zhao4, Xiaoxin Duan1,2, Wei Zheng1,2, \n5 Jianqiang Xu1,2, Pu Miao3, Wenbang Hou4*\n6\n7 1College of Horticulture and plant protection, Henan University of Science and Technology, \n8 Luoyang, Henan Province, China. \n9 2Henan Province Engineering Technology Research Center of Green Plant Protection, Luoyang \n10 471023, Henan Province, China.\n11 3China Tobacco Corporation of Luoyang, Henan Province, China.\n12 4College of Agricultural / Tree Peony, Henan University of Science and Technology, Luoyang, \n13 Henan Province, China.  \n14\n15 *Corresponding author: chengzj@haust.edu.cn (Z. Cheng)\n16 *Corresponding author: Houwenbang@haust.edu.cn (W. Hou)\n17\n18 Abstract: Tobacco root-knot disease represents a significant threat to tobacco production, \n19 particularly in the western Henan region, where Meloidogyne incognita is the predominant \n20 species. This study collected samples of M. incognita and soil from Luoyang, Henan, and \n21 designed specific primers MiF and MiR based on the amplified 735 bp sequence of the \n22 ITS1-5.8S-ITS2 region. These primers exhibit mismatches with related species, including M. \n23 javanica, M. minor, M. hapla, and M. arenaria, showing 1, 6, 10, and 10 base differences in the \n24 forward direction, respectively, 3, 10, and 9 base mismatches in the reverse direction. Although \n25 the primers were used to detect corn-wheat soil samples, no amplification was observed. \n26 Additionally a real-time quantitative PCR curve for M. incognita in soil was constructed, \n27 revealing a negative correlation between the Ct value (Y) and the log-transformed number of \n28 nematodes (x) per 20 g of dry soil, represented by the equation y = -0.9757x + 35.565; (R² = \n29 0.9999, P < 0.01). According to the decomposition efficiency of nematode DNA in soil, the \n30 results showed that nematode DNA degrades rapidly in soil, with a degradation rate of \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n2\n31 approximately 87.3% at 3 days and 99.97% at 14 days. Furthermore, significant differences were \n32 observed in the real-time PCR detection efficiency among various nematode forms: the Ct value \n33 of J1 was significantly higher than that of J2, while abnormal eggs (empty eggs or internal bubble \n34 eggs) exhibited the highest Ct value. The proportion of abnormal eggs in the soil before planting \n35 was significantly higher at 64.08% compared to only 15.3% at harvest, indicating that the activity \n36 of nematodes in the soil is significantly reduced after harvesting in October and planting in March \n37 of the following year. A survey of 126 tobacco plants indicated a significant positive correlation \n38 between the root-knot index (RKI) and root-knot nematode density (r = 0.80, p < 0.01). The study \n39 identified a minimum disease threshold of 234 individuals / 20 g soil at harvest and revealed a \n40 nonlinear relationship between disease severity and nematode density. Specifically, a weak \n41 correlation was observed at low density (Low RKI: 0-1; nematode density<2000 individuals/20 g \n42 soil, r = 0.49, P<0.05), while a significant correlation was noted at moderate density (High RKI: \n43 nematode density>2000 individuals/20 g soil, r = 0.69, P<0.05). This study showed that as the \n44 RKI increases, the rate of increase in nematode density in the soil diminishes. These findings \n45 provide valuable insights for the development of effective scientific strategies for nematode \n46 control.\n47\n48 Keywords: tobacco root-knot nematode; specific primers; tobacco root disease index (RKI);  \n49 nematode population density (NPD) \n50\n51 1. Introduction\n52 Tobacco (Nicotiana tabacum L.) is the primary raw material for cigarettes and constitutes a \n53 significant economic crop in China [1]. As the largest producer and consumer of tobacco globally, \n54 China accounts for over one-third of the world’s tobacco production [2]. Major pests and diseases \n55 affecting tobacco include the tobacco mosaic virus (TMV), black shank (Phytophthora \n56 nicotianae), bacterial wilt (Ralstonia solanacearum), and root-knot nematode [3]. Notably, \n57 root-knot nematodes cause wounds on the epidermal cells of tobacco roots, which increase the \n58 susceptibility of tobacco plants to soil-borne pathogens. Therefore, controlling tobacco root-knot \n59 nematodes is essential, as it may reduce the incidence of other soil-borne diseases[4]. \n60 Meloidogyne spp. are widely distributed plant pathogenic nematodes that significantly affect the \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n3\n61 growth and yield of various economically important crops, including tobacco, tomato, and \n62 cucumber [10]. By invading the root system of plants, these nematodes induce the formation of \n63 root galls, disrupting the normal structure and function of the roots [11]. This disruption leads to a \n64 decline in water and nutrient absorption, resulting in stunted growth, yellowing of leaves, reduced \n65 yields, and in severe cases, plant death [12]. Statistics indicate that agricultural losses caused by \n66 root-knot nematodes worldwide amount to billions of dollars [13]. Tobacco root-knot diseases \n67 typically reduce yields by 10% to 20%, however, in severe cases, losses can exceed 75%, with a \n68 higher incidence of disease reported in developing countries compared to developed ones [10]. In \n69 provinces such as Henan, Anhui, Sichuan, Guizhou, Zhejiang, and Yunnan in China, tobacco \n70 root-knot disease affects approximately 52,000 hm2 annually, accounting for about one-tenth of \n71 the total production area, leading to losses of around 9 million dollars [14]. Therefore, early \n72 detection and effective prevention and control of tobacco root-knot nematodes are critical issues \n73 that must be addressed in agricultural production [15].\n74 Currently, there are no tobacco varieties exhibiting high resistance to root-knot nematodes, and \n75 the interactions between these pests and host plants are complex, posing significant challenges for \n76 resistance breeding [5]. Consequently, root-knot nematode disease has emerged as a significant \n77 constraint on tobacco production in various countries and regions [6]. Presently, the most effective \n78 strategy for managing tobacco root-knot nematode disease focuses on suppressing the \n79 reproduction and population density of the pathogenic nematodes [7]. For instance, Zhang et al., \n80 cloned the tobacco RKN resistance gene NtRk1, which is induced upon nematode infection and \n81 enhances resistance by regulating salicylic acid and jasmonic acid pathways. Overexpression of \n82 this gene confers protection in susceptible cultivars while RNA interference (RNAi) silencing \n83 increases susceptibility to Meloidogyne incognita [8]. Additionally, Li et al. identified 5,206 \n84 high-confidence long non-coding RNAs (lncRNAs) in tobacco, with 565 being differentially \n85 expressed during nematode infection [9]. \n86   Traditional nematode detection methods primarily rely on morphological identification and \n87 microscopic observation [16]. Morphological identification necessitates a comprehensive \n88 understanding of the morphological characteristics of nematodes, as even minor differences \n89 among species can lead to misidentification [17]. Moreover, microscopic observation is \n90 time-consuming and often does not meet the rapid detection requirements for large-scale samples. \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n4\n91 Conventional quantitative methods are inadequate for detecting nematode eggs, J1 and inactive \n92 nematodes in soil, resulting in unreliable disease forecasts [18]. With the rapid advancements in \n93 molecular biology technology, real-time fluorescent quantitative PCR (Real-Time PCR) has \n94 become widely utilized in plant disease detection due to its high sensitivity, specificity, and \n95 quantitative capabilities [19]. This technology allows for the rapid and precise detection of \n96 nematode DNA through the design of specific primers and fluorescent probes, facilitating accurate \n97 quantification of nematode density [21]. Compared to traditional morphological methods, \n98 real-time PCR technology significantly reduces detection time and effectively distinguishes \n99 between different types of nematodes, thereby providing robust technical support for early \n100 warning and precision prevention and control of diseases [21-24].\n101   While real-time PCR technology demonstrates a significant advantage in detecting nematodes, \n102 current research remains limited [25]. The root-knot disease index (RKI) serves as a crucial \n103 indicator of disease severity, typically assessed through parameters such as the number of root \n104 knots and the extent of root damage [26]. Clarifying the quantitative relationship between \n105 root-knot nematode density and the disease index not only enhances our understanding of the \n106 infection mechanism of root-knot nematodes but also provides a scientific foundation for early \n107 diagnosis and comprehensive disease management. For instance, developing a mathematical \n108 model that correlates nematode density with disease severity enables the prediction of disease \n109 progression and the formulation of targeted prevention strategies [27]. This approach can \n110 minimize pesticide use, reduce environmental pollution, and enhance the sustainability of \n111 agricultural production [28]. \n112 The purpose of this study is to use real-time PCR technology for the quantitative detection of \n113 tobacco root-knot nematode density and to analyze the correlation between nematode density and \n114 the root disease index. This research will explore the manifestations of disease in the tobacco root \n115 system under varying nematode densities, thereby establishing a quantitative relationship between \n116 nematode density and the disease index. The findings will provide a theoretical foundation and \n117 technical support for early diagnosis, as well as comprehensive prevention and control of tobacco \n118 root diseases. Furthermore, the results of this research object not only to enhance the detection \n119 efficiency of tobacco root-knot nematodes but also to serve as a reference for the prevention and \n120 control of similar issues in other crops.\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n5\n121\n122 2. Materials and methods\n123 2.1 Soil and nematode collection\n124 The soil and tobacco samples were collected from four tobacco planting field in Luoyang: \n125 Luoning Xiaojie (111.59°E, 34.45°N), Yiyang Gaocun (111.88°E, 34.55°N), Ruyang Baipo \n126 (111.70°E, 34.50°N), and Kaiyuan Farm (112.42°E, 34.60°N). Prior to tobacco cultivation \n127 in March 2022, soil samples were collected from fields affected by root-knot nematodes at a depth \n128 of 0-30 cm. Egg masses of root-knot nematode in the soil were extracted and counted before \n129 transplantation. The Cobb's sieving and decanting method, along with the flotation method, were \n130 employed for the collection of egg masses from the soil [29]. A total of 100 g of soil samples were \n131 taken, to which an appropriate amount of distilled water was added. This mixture was then filtered \n132 through 850 µm and 250 µm sieves to remove larger particle impurities and collect the residues. \n133 The residue was re-suspended in a sucrose solution (454 g/L, with a density of approximately 1.18 \n134 g/cm³). After mixing, the solution was subjected to centrifugation at 1207 x g for 5 min. Due to \n135 the lower density of the egg masses, they floated to the surface of the sucrose solution, while \n136 heavier impurities settled at the bottom of the tube. A straw was used to carefully extract the \n137 liquid, which was then transferred to a new centrifuge tube. The mixture was centrifuged again to \n138 remove the sucrose, resulting in the isolation of the egg mass sample. On October 7, 2022, and \n139 October 15, 2023, tobacco root samples were collected following the tobacco harvest. Using a \n140 five-point sampling method, samples were taken from the 0-30 cm soil layer within a 50 cm radius \n141 centered on the tobacco plants. The tobacco root egg masses were separated from the tobacco \n142 roots, the fibrous roots were cut, and the egg within the root egg masses were extracted under a \n143 microscope.\n144\n145 2.2 Extraction of M. incognita DNA and Design of Specific Primers\n146 The nematodes used for the identification of the DNA sequence of root-knot nematodes were \n147 collected from infected roots of tobacco plants in four different tobacco planting fields in October \n148 2021. The egg masses were extracted from the root knots, and a single egg was used as a sample \n149 for DNA extraction. Using tweezers, the egg mass was broken to release the eggs and juvenile \n150 second-stage nematodes (J2). The eggs were then incubated at room temperature (25℃) for 3 h. A \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n6\n151 pipette was used to extract a single egg or J2, following the single nematode DNA extraction \n152 method described by Wang et al. (2011). A single J2 sample was placed on a sterilized glass slide \n153 alongside a drop of water. A No. 3 insect needle was then used to make a vertical incision in the \n154 J2 sample. Subsequently, 10 µL of the nematode solution was aspirated into a 200 µL centrifuge \n155 tube, and 1.5 µL of 10 x PCR Buffer (Mg2+-free) was added. The mixture was subjected to liquid \n156 nitrogen for 1 min, followed by heating at 85℃ for 2 min. Then, 1 µL of 1 mg/mL protease K was \n157 incorporated, and the solution was heated at 56℃ for 15 min followed by an additional 10 min at \n158 95 ℃. This DNA extraction solution was subsequently diluted 10 times with sterile water to serve \n159 as a template for PCR amplification.\n160   The amplification of the ITS1-5.8S-ITS2 region was conducted using the forward primer ITS-F \n161 (5'-ACA AGT ACC GTG GAA AGT TG-3') and the reverse primer ITS-R (5'-TCG GAA GGA \n162 ACC TAC TA-3'). PCR amplification was performed with Hieff Canace Plus High-Fidelity DNA \n163 Polymerase (Yeasen, Shanghai) under the following conditions: 94℃ for 2 min, (94℃ for 1 min, \n164 48℃ for 1 min, 72℃ for 1 min) x 35 cycles, 72℃ for 5 min. The PCR products were purified \n165 using the DiaSpin column PCR product purification kit (Sangon Biotech, Shanghai) and \n166 subsequently sent to Sangon Biotech for sequencing. The ITS region sequence of M. incognita \n167 obtained in this study was compared with sequence in the NCBI database for identification. The \n168 target sequence was analyzed using Mega 11.0 software to identify variant regions, and NCBI \n169 Primer-BLAST was employed to design primers specific to the nematode (MiF and MiR). \n170\n171 2.3 Establishing a real-time PCR method for the quantification of tobacco root-knot nematode\n172 The DNA solution extracted from a single J2 was diluted ten fold with sterile distilled water to \n173 serve as a template for Real-Time PCR. Real-Time PCR was conducted using the CFX96 \n174 (Bio-Rad, USA) for detection. The reaction mixture consisted of 10 µL, which included 5 µL of \n175 Hieff qPCR SYBR GREEN Master Mix (Yeasen, Shanghai), 0.4 µL of specific primers, and 2 µL \n176 of template DNA. The reaction conditions were as follows: 95℃ for 30 s; (95℃ for 5 s and 58℃ \n177 for 30 s) x 40 cycles. Distilled water was used as a negative control. Additionally, the DNA \n178 solution from a single M. incognita was diluted to 10^-1, 10^-2, 10^-3, 10^-4, and 10^-5 for use as \n179 templates, and specific primers MiF and MiR were employed across three replicates for treatment.\n180  Three soil samples were collected from corn-wheat crop rotation fields in Yiyang Gaocun, \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n7\n181 following the method described by Cheng et al. (2018) for extracting total biological DNA from \n182 the soil. 20 g of soil samples were dried at 60℃ for 24 h and processed using a planetary ball mill \n183 (LANENDE, Shandong) at a speed of 450 rpm for 2 min [30]. Subsequently, 5 g of the ball milled \n184 soil samples were added to 50 mL centrifugal tubes, along with 10 mL of phosphate buffer \n185 (Na2HPO4 and NaH2PO4, 0.12 M, pH 8.0), with three replicates for each sample. The soil-buffer \n186 mixture was agitated using a shaker (Hongke Technology HQY-C, Jiangsu, China) at 200 rpm for \n187 30 m. After an additional 10 min, the solution was purified using the DiaSpin column PCR \n188 product purification kit (Sangon Biotech, Shanghai). The DNA eluate was then diluted ten-fold to \n189 serve as a template for real-time PCR. All samples were prepared in triplicate.\n190 The DNA solution extracted from the soil sample of the corn-wheat crop rotation was \n191 detected using the MiF and MiR primers; however, no specific amplification was observed (data \n192 not shown). Consequently, a quantitative curve for the root-knot nematode of tobacco was created \n193 using soil collected from the corn-wheat rotation field. The tobacco roots containing egg masses \n194 were cut into 1 cm segments with scissors, and subsequently homogenized in a blender for 30 \n195 seconds (Midea WBL2521H, Guangdong). The fragmented roots were placed in a gauze bag, and \n196 both J2 and adults root-knot nematodes were extracted using the Baermann funnel method for \n197 inoculation. The soil from the corn-wheat rotation field was dried at 60℃ and then weighed to a \n198 mass of 20 g. Different quantities of M. incognita (J2 and adults) were artificially inoculated: 0, \n199 30, 150, and 750 individuals. Following inoculation, the soil was dried again at 60℃ for 6 h. Each \n200 inoculation amount was prepared in triplicate. \n201\n202 2.4 Rate of DNA degradation of M. incognita in soil\n203 In October 2023, tobacco roots infected with root-knot nematode were collected post-harvest. \n204 The infected roots were homogenized using a blender (Midea WBL2521H, Guangdong) for 30 s, \n205 with this process repeated twice. The nematode suspension, comprising both J2 and adult stages \n206 was subsequently extracted using the Baermann funnel method. Following treatment of the M. \n207 incognita suspension in a water bath at 75℃ for 10 minutes, approximately 2000 individuals of M. \n208 incognita were inoculated into 20 g of raw soil sourced from a corn-wheat rotation field using a \n209 pipette for precision. DNA extraction from the soil was performed at intervals of 0, 3, 7 and 14 \n210 days, employing the DNA extraction method outlined by Cheng et al. (2018)[]. The extracted \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n8\n211 DNA was then analyzed through real-time PCR detection, yielding Ct values with the MiF and \n212 MiR primers. The equivalent number of nematodes was calculated based on the quantitative curve \n213 of M. incognita.\n214\n215 2.5 Effect of the tobacco root-knot disease index (RKI) on nematode population density (NPD). \n216 On October 7, 2022, and October 15, 2023, samples were collected from four tobacco-growing \n217 fields in Luoyang City, Henan Province, specifically at Luoning, Yiyang, Ruyang Bai, and \n218 Kaiyuan Farm, following the tobacco harvest. At each location, 38, 32, 30, and 26 tobacco plants \n219 were sampled for roots, resulting in a total of 126 plants. Soil samples were also collected from \n220 the rhizosphere area (15 cm in diameter with the root; 0-30 cm in depth) of each tobacco plant and \n221 stored in sterile sealed bags. The established real-time PCR method for detecting M. incognita was \n222 used to employed to assess the nematode population density (NPD) in the soil samples. The \n223 diseased tobacco roots were washed with water to eliminate any adhering soil, and 30 g of root \n224 samples were cut and placed on a white tray for root-knot quantification. The number of galls was \n225 observed and counted either with the naked eye or using a magnifying glass. Concurrently, the \n226 Root-Knot Disease Index (RKI) of each tobacco plant was evaluated. Based on the number of root \n227 egg masses and the degree of root damage, a 0-5 scale grading standard from Taylor (1978) was \n228 used to evaluate the RKI of each tobacco root, with specific grading criteria as follows [31]:\n229 0 level: No root-knots present; \n230 1 level: 0% ≤ number of swollen roots or root-knots due to nematode damage < 10%; \n231 2 level: 10% ≤ number of swollen roots or root-knots due to nematode damage < 25%; \n232 3 level: 25% ≤ number of swollen roots or root-knots due to nematode damage < 50%; \n233 4 level: 50% ≤ number of swollen roots or root-knots due to nematode damage < 75%; \n234 5 level: 75% ≤ number of swollen roots or root-knots due to nematode damage ≤ 100%.\n235 The infected young roots develop spherical or irregular galls of varying sizes, which appear \n236 white or pale in color. In severe cases, the diseased roots turn brown and decay. Under conditions \n237 of high RKI level, the root system becomes severely deformed, exhibiting a beaded or claw-like \n238 morphology.\n239 2.6 Statistical analysis\n240 Statistical analyses were performed using SPSS version 26.0. A linear regression analysis was \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n9\n241 conducted to explore the relationship between the number of inoculated nematodes and the Ct \n242 values obtained from real-time PCR, with a correlation coefficient calculated (p < 0.01). \n243 Furthermore, the Pearson correlation coefficient was employed to assess the correlation between \n244 the root-knot disease index (RKI) and the nematode population density (NPD).\n245\n246 3. Result\n247 3.1 Identification of M. incognita and Design of Specific Primers\n248 The findings of this study indicate that the ITS region sequences of root-knot nematodes from \n249 tobacco in four planting areas of Luoyang, Henan Province, exhibit a high degree of consistency. \n250 Analysis using NCBI BLAST revealed a similarity of 99.57% with M. incognita from Fujian \n251 Province, China (accession number: OQ632600), with only three base pair mismatches in a 735 bp \n252 sequence. Additionally, a similarity of 99.28% was observed with the sequence designated as \n253 accession number MT209949, which contained five base pair mismatches. For further analysis, \n254 the sequence was compared with those of both closely related and distantly related species, as \n255 provided by the National Center for Biotechnology Information (NCBI) (Figure 1).\n256 Based on the ITS sequence of M. incognita, specific primers MiF and MiR were designed \n257 using the NCBI Primer-BLAST software. The forward primer exhibits a GC content of 38.1%, \n258 while the reverse primer has a GC content of 50%. A comparison of the primer sequences with the \n259 ITS sequences of other closely related species revealed that the forward primer had mismatches of \n260 1, 6, 6, 10, 10, and 10 base pairs with M. javanica (MW672262), M. minor (KX671108), M. \n261 artiellia (JX393300), M. hapla (OM864510), M. arenaria (EU364878), and M. enterolobii \n262 (KZ411228), respectively. The reverse primer exhibited mismatches of 1, 3, 6, 10, 9, and 6 base \n263 pairs with the same species, respectively (Table 1).\n264\n265 3.2 Ct value of different morphological nematodes\n266 The egg masses extracted from the tobacco roots affected by nematodes at harvest time \n267 exhibited a milky white color and a smooth surface. Upon breaking open the egg masses for \n268 observation, three distinct morphological types of nematodes were identified: J1, which comprised \n269 eggs with clearly formed nematode bodies; J2, which were larvae that had hatched; and abnormal \n270 eggs, which were either hollow or contained bubbles (Figure 2). We extracted DNA from these \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n10\n271 different morphological types of nematodes and used them as templates for Ct value detection. \n272 The results indicated significant differences in Ct values among the various morphological types \n273 of nematodes. The Ct values for J1 extracted from the pre-planting soil and the affected roots at \n274 harvest were 19.6 to 19.9 (n = 5), respectively, showing no significant difference between the two. \n275 In contrast, the Ct values for J2 were lower than those for J1, recorded at 18.4 to 17.3 (n = 5), \n276 respectively. Notably, the Ct values detected from the abnormal egg DNA were substantially \n277 lower than those for J1 and J2, measuring 28.8 and 27.8, respectively, with a higher variance than \n278 that of J1 and J2. Furthermore, the egg masses extracted from the pre-planting soil and the \n279 affected roots at harvest were opened, and the eggs of each morphological type were counted. The \n280 results revealed that the proportion of abnormal eggs in the pre-planting soil was as high as \n281 64.08%, whereas the proportion of abnormal eggs in the egg masses at harvest was 15.3% (Table \n282 2).\n283\n284 3.3 Quantitative Curve of M. incognita in Soil\n285 We diluted the DNA solution of individual M. incognita as a template and detected the \n286 amplification of the designed specific primers MiF and MiR. The study revealed that the Ct values \n287 (y) increased logarithmically with the increase in the DNA dilution rate (x), with the regression \n288 equation defined as y = -0.9841x + 33.9857 and R2 = 0.9964. This indicates a highly significant \n289 correlation between Ct values and DNA dilution rate (P < 0.01). Furthermore, when the DNA was \n290 diluted to 105 times, a linear relationship between the DNA dilution rate and Ct value was \n291 observed, with Ct values ranging from 19.8 to 32.9. However, when the dilution reached 107 \n292 times, the Ct value was compromised, and no Ct values were detected in the negative control \n293 samples (Figure 3 A).\n294 In this study, based on the number of inoculated M. incognita in the soil and the Ct values \n295 detected by real-time PCR, a soil quantitative curve was constructed. A correlation analysis was \n296 performed between the Ct values (y) and the logarithmically transformed number of inoculated \n297 nematodes (x). The results revealed a significant negative correlation between the two, with the \n298 regression equation defined as  y = -1.0859x + 32.025, R 2 = 0.9866, P < 0.01 (Figure 3 B). \n299\n300 3.4 Degradation rate of DNA from M. incognita in soil\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n11\n301 The initial inoculation density of dead M. incognita in the soil ranged from 2090 to 2275 \n302 individuals/20 g soil. By the third day, the DNA-based equivalent of nematode density decreased \n303 to 374±16 individuals/20 g soil, reflecting a degradation rate of 87.3%. On the seventh day,the \n304 DNA-based equivalent of nematode density further declined to 14±2 individuals / 20 g soil, \n305 indicating a degradation rate of 99.34% compared to the initial inoculation. By the fourteenth day, \n306 the DNA-based equivalent of nematode density in the soil had diminished to only 1.0±0.5 \n307 individuals/20 g soil, achieving a degradation rate of 99.97% relative to the initial inoculation \n308 (Figure 4).\n309\n310 3.5 Effect of M. incognita density in the rhizosphere on the root-knot index\n311  Following the tobacco harvest in October 2022 and October 2023, we conducted an \n312 investigation into the incidence of root diseases in 126 tobacco plants and assessed the density of \n313 M. incognita in the rhizosphere soil (Figure 5). The results indicated that at a Root-knot Index \n314 (RKI) level of 0, no galls or egg masses were detected in the tobacco root system; however, the \n315 density of M. incognita in the rhizosphere soil varied from 0 to 68 individuals/20 g soil. When the \n316 RKI reached level 1, the number of galls ranged from 2 to 9 galls/30 g root, with nematode \n317 densities in the soil ranging from 234 to 2321 individuals/20 g soil. At RKI level 2, the number of \n318 galls increased between 13and 23 galls/30 g root, with nematode density recorded at 2345-6764 \n319 individuals / 20 g soil. At RKI level 3, the count rose to 27 to 49 galls/30 g root, and the nematode \n320 density in the soil ranged from 4656 to 23434 individuals/20 g soil. At RKI level 4, the number of \n321 galls ranged from 53 to 73 galls/30g root, with a corresponding nematode density of 8233 to \n322 35291 individuals / 20 g soil. Finally, at RKI level 5, the number of galls or swellings ranged form \n323 71 to 86 galls/30 g root, with nematode density in the soil falling between 18345 and 49234 \n324 individuals/20 g soil. There were extremely significant differences between RKI level 0 and levels \n325 1-5 (P < 0.01). Significant or extremely significant differences were found between RKI level 1 \n326 and levels 2-5 as well as between RKI levels 2 and 3-5, RKI levels 3 and 4-5, and RKI levels 4 \n327 and 5 (P < 0.05). Figure 5 showed that at RKI levels 1-3, as the number of galls increased, the \n328 nematode density in the soil also significantly increased, indicating a notable enhancement in \n329 significance. However, at RKI levels 4-5, the significance of this growth decreased, indicating that \n330 the variation in nematode density in the soil was reduced when the root-knot disease was severe.\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n12\n331\n332 4. Discussion\n333 This study successfully established a real-time PCR analysis method for tobacco root-knot \n334 nematodes (M. incognita) in the western region of Henan Province, demonstrating that the \n335 population density of M. incognita in the soil significantly impacts the occurrence of root-knot \n336 disease. Furthermore, the nematode samples collected from four tobacco-growing areas in this \n337 region exhibited complete consistency in their ITS region sequences, all identified as M. \n338 incognita. However, when comparing the sequences of different Meloidogyne species retrieved \n339 from the NCBI database with our designed specific primers MiF and MiR, 1-10 base mismatches \n340 were identified. According to Ri, M.U. et al. (2023), a forward primer with 10 base mismatches at \n341 the 3' end significantly reduces amplification efficiency [32]. Moreover, if there are three or more \n342 base mismatches at the 5' end, amplification will not occur. The M. incognita specific primers \n343 designed by Toyota et al. (2010) share identical sequences with M. arenaria and M. javanica, \n344 preventing differentiation between these species [21]. Similarly, the primers designed by Zhao et \n345 al. (2010) share the same sequence with M. javanica but differ by a single base from M. arenaria, \n346 resulting in an absence of observable PCR amplification products [33]. Additionally, Wu et al. \n347 (2024) reported that M. arenaria is the dominant population among Guizhou M. incognita [34]. \n348 Xu et al. (2023) indicated that in the primary tobacco-growing area of Kunming, Yunnan, M. \n349 incognita comprises 89.0% of the tobacco root-knot nematode population, while M. arenaria \n350 constitutes 41.3%, M. javanica 53.2%, and other Meloidogyne sp. 12.8% [35]. Jiao et al. (2014) \n351 reported the population distribution ratio of tobacco root-knot nematodes in Henan Province, \n352 revealing that M. incognita constituted at 55.83%, M. arenaria 23.33%, M. hapla 17.50%, and M. \n353 javanica 3.33% [36]. This finding further confirms that M. incognita is the dominant species in re \n354 region [37]. In this study, the M. incognita identified from tobacco samples collected in the \n355 Luoyang area of Henan Province was confirmed through ITS region sequence analysis, aligning \n356 with Jiao et al. (2014), who also identified M. incognita as the predominant tobacco root-knot \n357 nematode in Henan [36]. Concerning the closely related species M. javanica, our primers MiF and \n358 MiR exhibited one base mismatch each in their forward and reverse sequences. When we used the \n359 nematode DNA for detection using the MiF and MiR primers, the Ct value increased by 6, and the \n360 amplification efficiency decreased by 64% (data not shown), indicating a high specificity of these \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n13\n361 primers for M. incognita.\n362 This study is the first to detect DNA in nematodes of varying morphologies within the egg \n363 mass, using the MiF and MiR primers for real-time PCR analysis of Ct values. The results \n364 indicated that the Ct values of abnormal eggs were significantly lower than those of normal eggs \n365 (J1), suggesting that these abnormal eggs are inactive and that their DNA has begun to degrade, \n366 rendering them incapable of hatching. This finding suggests that the hatching rate of M. incognita \n367 eggs in the soil during fallow periods, in the absence of a host plant, will significantly decrease. \n368 Additionally, this study examined the decomposition rate of DNA from deceased nematodes and \n369 found that seven days post-mortem, the amplification signal of their DNA was extremely low, \n370 nearly negligible. Consequently, the number of nematodes detected in the soil by real-time PCR \n371 could be  interpreted as representing active nematodes or viable eggs. Furthermore, this indicates \n372 that even if M. incognita eggs hatch into J2, the rate of DNA abnormalities remains relatively high \n373 in the absence of a host plant. In conclusion, implementing fallow or crop rotation measures can \n374 effectively reduce the population density of M. incognita in the soil and significantly impact \n375 disease control. These results provide crucial scientific evidence for managing M. incognita and \n376 advocate for the adoption of fallow or crop rotation strategies in practical applications to mitigate \n377 nematode damage.\n378 The results of this study on the relationship between RKI and NPD indicate a positive \n379 correlation between the number of M. incognita in the tobacco rhizosphere soil and the RKI levels \n380 of 1 to 3. This finding aligns with previous research reports, suggesting that during the early to \n381 mid-stages of the disease, the growth of the nematode population is significantly related to the \n382 severity of root-knot disease. In the initial stages of the disease (RKI 1-3), an increase in RKI \n383 levels corresponds with a significant rise in the number of nematodes within the rhizosphere soil. \n384 This indicates that following infection of the tobacco root system, nematodes could reproduce \n385 rapidly and establish a stable population. At this stage, the growth of the nematode population is \n386 primarily driven by the nutritional supply from the host root system, and the formation of root \n387 knots provides additional feeding sites for nematodes, thereby further promoting their \n388 reproduction [38]. However, as the disease progresses to higher levels (RKI 4-5), the nutritional \n389 capacity of the tobacco root system begins to decline, which restricts further nematode \n390 reproduction and results in a significant slowdown in the growth rate of the nematode population. \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n14\n391 Furthermore, the number of nematodes in the rhizosphere soil may have already reached the \n392 environmental carrying capacity, thus limiting the contribution of further increases in disease \n393 levels to the growth of the nematode population.\n394 The findings of this study provide scientific evidence for the integrated management of tobacco \n395 root-knot nematode disease. In the early stages of the disease (RKI 1-3), the nematode population \n396 has not yet reached the environmental carrying capacity. At this stage, implementing control \n397 measures, such as the application of nematicides or the cultivation of resistant varieties, could \n398 effectively curb the increase in nematode numbers and mitigate the disease’s negative impact on \n399 tobacco yield []. Once the nematode population reaches a specific threshold, its growth rate \n400 significantly declines. Therefore, monitoring the nematode population in the rhizosphere soil \n401 during agricultural production can help determine the economic threshold for disease \n402 management, allowing for the rational use of pesticides, thus reducing production costs and \n403 minimizing environmental pollution. The integration of agricultural management practices, such \n404 as crop rotation and soil disinfection, along with biological control methods including the \n405 application of antagonistic microorganisms or entomopathogenic nematodes, can effectively \n406 control nematode population growth, reduce reliance on chemical pesticides, and enhance the \n407 sustainability of tobacco production.\n408 Despite revealing the association between RKI and NPD, this study has several limitations. It \n409 did not account for the influence of environmental factors such as soil type and climatic \n410 conditions, on the dynamics of the nematode population. Future research should further \n411 investigate the specific roles of these factors. Additionally, genetic variations among M. incognita \n412 populations across different regions may impact their host infection capabilities and reproductive \n413 efficiency.\n414\n415 5. Acknowlegements\n416 Funding for this study was generously provided by the Science and Technology Project Fund \n417 of the China Tobacco Corporation of Luoyang #2022410300270069, the Science and Technology \n418 Project Fund of the China Tobacco Corporation of Xuchang #2024411000240026, the Key \n419 Scientific and Technological Projects of the China Tobacco Corporation #110202201026 (LS-10), \n420 and the Henan Province Science and Technology Research Project #22010174. 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PLoS Pathog. 2018;14 (3):e1006947.\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n19\n541\n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 31, 2025. ; https://doi.org/10.1101/2025.08.27.672540doi: bioRxiv preprint \n\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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