Quantitative Loop-Mediated Isothermal Amplification Detection of Ustilaginoidea virens Causing Rice False Smut

Abstract

Rice false smut caused by Ustilaginoidea virens is one of the most devastating diseases in rice worldwide, which results in serious reductions in rice quality and yield. As an airborne fungal disease, early diagnosis of rice false smut and monitoring its epidemics and distribution of its pathogens is particularly important to manage the infection. In this study, a quantitative loop-mediated isothermal amplification (q-LAMP) method for U. virens detection and quantification was developed. This method has higher sensitivity and efficiency compared to the quantitative real-time PCR (q-PCR) method. The species-specific primer that the UV-2 set used was designed based on the unique sequence of the U. virens ustiloxins biosynthetic gene (NCBI accession number: BR001221.1). The q-LAMP assay was able to detect a concentration of 6.4 spores/mL at an optimal reaction temperature of 63.4 °C within 60 min. Moreover, the q-LAMP assay could even achieve accurate quantitative detection when there were only nine spores on the tape. A linearized equation for the standard curve, y = -0.2866x + 13.829 (x is the amplification time, the spore number = 10^0.65y), was established for the detection and quantification of U. virens. In field detection applications, this q-LAMP method is more accurate and sensitive than traditional observation methods. Collectively, this study has established a powerful and simple monitoring tool for U. virens, which provides valuable technical support for the forecast and management of rice false smut, and a theoretical basis for precise fungicide application.

Keywords: detection; quantitative loop-mediated isothermal amplification (q-LAMP); rice false smut; ustiloxins biosynthetic gene.

Figure 1

The species-specific primers for detecting Ustilaginoidea virens in the quantitative loop-mediated isothermal amplification (q-LAMP) and quantitative real-time PCR (q-PCR). The species-specific primers designed based on the sequence of the ustiloxins biosynthetic gene segments for identification and quantification of U. virens in q-LAMP assay and q-PCR assay. The forward and reverse primer sequences were highlighted with shade and arrow for orientation.

Figure 2

Optimization of the q-LAMP assay via reaction temperature screening. The influence of temperature ranged from 61.8 °C to 66 °C in the q-LAMP detection system and showed that the strongest fluorescence intensity and the shortest reaction time were obtained at 63.4 °C (black line).

Figure 3

Specificity validation of the q-LAMP assay system. The q-LAMP assay system (using UV-2 primer sets and 63.4 °C as reaction temperature) was highly specific for the detection of U. virens. The q-LAMP assay system showed that fluorescence signals were only detected in the samples with DNA template of U. virens (black line), while the samples with DNA template of the other 9 fungi (including Fusarium fujikuroi, F. oxysporum, F. proliferatum, F. solani, F. graminearum, Penicillium sp., Pyricularia oryzae, Alternaria alternata and Rhizoctonia solani) or negative control (nucleic acid-free water) did not show any fluorescence signal.

Figure 4

Sensitivity validation of q-LAMP system. The fluorescence signals in q-LAMP assays were detected in the samples with DNA template of 2 × 10⁴ spores/mL, 4 × 10³ spores/mL, 8 × 10² spores/mL, 1.6 × 10² spores/mL, 32 spores/mL, and 6.4 spores/mL within 60 min, while no signals were detected in sample with DNA template of 1.28 spores/mL and CK. The bolded dark green line (horizontal) indicates fluorescence threshold. Fluorescence signals above this threshold marked as a successful detection of U. virens in q-LAMP assays.

Figure 5

Standard curve of q-LAMP detection system. A standard curve between logarithmic values of the spore number (y) and the amplification time quantitated using the cycle threshold (Ct) values (x): y = −0.2866x + 13.829. The correlation coefficient (R²) is 0.9942, showing a good linear relationship.

Figure 6

Quantitative detection of U. virens spores on Melinex tape using q-LAMP system. Serial numbers 1, 2, 3, and 4 represent 450, 116, 29, and 9 spores, respectively. The green line (horizontal) indicates fluorescence threshold. Fluorescence signals above this threshold marked as a successful detection of U. virens in q-LAMP assays.

Figure 7

Field application of U. virens spores using q-LAMP system. (A) Flow chart of field U. virens spore sample detection, q-LAMP assay system and microscope observation were used for the collected samples, respectively. (B) The results of U. virens spore concentration measured by different methods in rice fields in 2018 (q-LAMP assay system is the gray line; microscope observation method is the blue line). (C) The results of U. virens spore concentration measured by different methods in rice fields in 2019 (q-LAMP assay system is the gray line; microscope observation method is the blue line). The green arrow is the first detection of spores by q-LAMP assay system, the orange arrow is the first observation of spores by microscope observation, and the red arrow is the occurrence time of rice false smut in the field.