Integrating spore trapping technology with loop-mediated isothermal amplification assay for surveillance and sustainable management of rice false smut disease

Abstract

Rice (Oryza sativa L.) is a vital crop feeding more than half of the world's population, with production occurring predominantly in Asian countries. However, rice cultivation faces challenges from various fronts, including biotic stresses intensified by climate change. False smut, caused by Ustilaginoidea virens, has emerged as a significant threat to rice production globally. The application of curative fungicides after symptom appearance has limited scope in managing this disease since the infection process usually starts during the early flowering stage of rice crops. This study investigates the utilization of spore-trapping technology coupled with Loop-Mediated Isothermal Amplification (LAMP) assay for monitoring airborne U. virens inocula in rice fields. For early detection and quantification of U. virens, sampling rods coated with silicone grease were deployed to collect airborne spores, and DNA extraction was performed using a modified method. Both PCR and LAMP assays were employed for detection, with LAMP offering advantages of rapidity, sensitivity, and simplicity. The study demonstrated the superior sensitivity of LAMP compared to PCR, detecting U. virens DNA at concentrations as low as 100 femtograms. Continuous monitoring of U. virens inoculum using spore trapping revealed the spatio-temporal dynamics of U. virens dispersal, providing valuable insights for disease management. Implementing a fungicidal application schedule based on airborne inoculum detection led to significant reductions in both false smut incidence and severity and improved crop yield. The meteorological parameters including minimum temperature, relative humidity in the morning and evening, sunshine, and solar radiation were found to be correlated with disease incidence. Multi-operator validation confirmed the robustness and specificity of the LAMP assay. Overall, this integrated approach offers a proactive strategy for monitoring and managing false smut disease, enhancing sustainable rice production and food security.

Keywords: LAMP; PCR; Ustilaginoidea virens; false smut; rice; spore trap.

Figure 1

Solar power operated impaction spore trap. (A) Layout of impaction spore trap. a: Solar panel (18V); b: Tilt angular cum panel holder; c: Battery (6V); d: On/Off switch; e: G.I pipe; f: Coupling; g: Height adjuster screw; h: Wire (connecting solar panel—battery-motor); i: L rod (motor holder); j: iron plate attached to L rod; k: Plastic causing; l: DC motor (12 V); m: Sampling arm; n: Sampling rods. (B) A solar operated impaction spore trap deployed at Paddy Breeding Station, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu.

Figure 2

The graphical representation of three different treatments associated with weather parameters. The different colour bars represent each weather parameter (Maximum Temperatre, Minimum Temperature, Relative Humidity morning, Relative Humidity evening, Rain fall, Wind speed, Sunshine, Solar Radiation). The line graph represents treatment 1, 2, and 3.

Figure 3

Overall flow on detection of Ustilaginoidea virens. (a) Ustilaginoidea virens culture grown on PSA media; (b) microscopic observation of U. virens spores; (c) sampling rods; (d) silicone grease coated over sampling rod; (e) the spore inocula added to rods in different dilution and DNA extracted; (f) as well as the DNA was extracted from the mycelial mat and diluted serially (100 ng, 10 ng, 1 ng, 100 pg,10 pg, 1 pg, 100 fg, 10 fg, 1 fg); (g) the samples were then subjected to PCR and LAMP assay.

Figure 4

Detection of Ustilaginoidea virens using PCR assay. (A) Agarose gel electrophoresis analysis of the PCR products, each Lane represents L:1Kb Ladder; 1-100 ng; 2-10 ng; 3-1 ng; 4-100 pg; 5-10 pg; 6-1 pg; 7-100 fg; 8-10 fg; 9-1 fg; C-NFW (Negative control). (B) Agarose gel electrophoresis analysis of the PCR products, each Lane represents L:1Kb Ladder; 1–1 × 10⁴ conidia; 2-1 × 10³ conidia; 3-1 × 10² conidia; 4-1 × 10 conidia; 5-1 conidia; C-NFW (Negative control). ng, nano gram; pg, pico gram; fg, femtogram; NFW, nuclease free water.

Figure 5

Detection of U. virens using LAMP assay. (A) Agarose gel electrophoresis analysis of the LAMP products, each Lane represents L:1Kb Ladder; 1-100 ng; 2-10 ng; 3-1 ng; 4-100 pg; 5-10 pg; 6-1 pg; 7-100 fg; 8-10 fg; 9-1 fg; C-NFW (Negative control). (B) Agarose gel electrophoresis analysis of the LAMP products, each Lane represents L:1Kb Ladder; 1-1 × 104 conidia; 2-1 × 103 conidia; 3-1 × 102 conidia; 4-1 × 10 conidia; 5-1 conidia; C-NFW (Negative control). ng, nano gram; pg, pico gram; fg, femtogram; NFW, nuclease free water.