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

Abstract

Tobacco root-knot disease represents a significant threat to tobacco production, particularly in the western Henan region, where Meloidogyne incognita is the predominant species. This study collected samples of M. incognita and soil from Luoyang, Henan, and designed specific primers MiF and MiR based on the amplified 735 bp sequence of the ITS1-5.8S-ITS2 region. These primers exhibit mismatches with related species, including M. javanica, M. minor, M. hapla, and M. arenaria, showing 1, 6, 10, and 10 base differences in the forward direction, respectively, 3, 10, and 9 base mismatches in the reverse direction. Although the primers were used to detect corn-wheat soil samples, no amplification was observed. Additionally a real-time quantitative PCR curve for M. incognita in soil was constructed, revealing a negative correlation between the Ct value (Y) and the log-transformed number of nematodes (x) per 20 g of dry soil, represented by the equation y = -0.9757x + 35.565; (R2 = 0.9999, P < 0.01). According to the decomposition efficiency of nematode DNA in soil, the results showed that nematode DNA degrades rapidly in soil, with a degradation rate of approximately 87.3% at 3 days and 99.97% at 14 days. Furthermore, significant differences were observed in the real-time PCR detection efficiency among various nematode forms: the Ct value of J1 was significantly higher than that of J2, while abnormal eggs (empty eggs or internal bubble eggs) exhibited the highest Ct value. The proportion of abnormal eggs in the soil before planting was significantly higher at 64.08% compared to only 15.3% at harvest, indicating that the activity of nematodes in the soil is significantly reduced after harvesting in October and planting in March of the following year. A survey of 126 tobacco plants indicated a significant positive correlation between the root-knot index (RKI) and root-knot nematode density (r = 0.80, p < 0.01). The study identified a minimum disease threshold of 234 individuals / 20 g soil at harvest and revealed a nonlinear relationship between disease severity and nematode density. Specifically, a weak correlation was observed at low density (Low RKI: 0-1; nematode density<2000 individuals/20 g soil, r = 0.49, P<0.05), while a significant correlation was noted at moderate density (High RKI: nematode density>2000 individuals/20 g soil, r = 0.69, P<0.05). This study showed that as the RKI increases, the rate of increase in nematode density in the soil diminishes. These findings provide valuable insights for the development of effective scientific strategies for nematode control.