Polymerase chain reaction: a molecular diagnostic method for detection of Fusarium oxysporum f.sp.niveum

Abstract: 【Objective】Fusarium wilt of watermelon is a worldwide fungal soil-borne disease caused by Fusarium oxysporum f. sp. niveum(Fon), which is a yield-limiting factor in watermelon production and occurs at all growing stages of watermelon. Like all form special of Fusarium oxysporum, this fungus is a soil inhabitant and extremely difficult to control. Although the use of resistant rootstocks and cultivars provides some degree of protection away from Fusarium wilt, the development of new pathogenic races is a constant problem, and there are currently no commercially acceptable cultivars with adequate resistance to Fon. However, the prevention and control of the disease mainly include crop rotation, grafting and insecticide control, but there are some drawbacks, and the control of watermelon wilt has always been one of the main problems in watermelon production. Therefore, it is very important to establish a simple and rapid early detection method for controlling the disease in watermelon plants and soils.【Methods】Based on the existing genome database of Fusarium oxysporum, the specific primer for qualitative detection of Fon was designed and screened. The PCR detection technology system of Fon was established and optimized. The system was used to detect and verify the inoculated watermelon plants and soils.【Results】The DNA of Fon(race 0, 1, 2, watermelon), Fusarium oxysporum f. sp. melonis(melon), Fusarium oxysporum f. sp. cucumerinum(cucumber), Fusarium oxysporum f. sp. lycopersici(tomato), Fusarium oxysporum f. sp. conglutinans(cabbage), Fusarium oxysporum f. sp. vesinfectum(cotton) Rhizoctonia Solani(cotton) and Verticillium dahliae(tomato) were used as test materials. Fons(Rcae 0, Rcae 1 and Rcae 2) were all able to amplify the target band with a length of 556 bp using the specific primer FONDX-15, while those of other plants were negative. The sensitivity of PCR qualitative detection system was 365 pg · μL-1 by using the genomic DNA of Fon as a template. The final reaction system: 2 × Es TaqMaster Mix 12.5 μL, 1 μL(5 μmol · L-1) of upstream and downstream primers, 2μL of DNA and 8.5 μL of RNase-free ddH2O with total volume 25 μL. Amplification procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1 min, annealing at 56 ℃ for 1 min and extension at 74 ℃ for 30 s for 30 cycles; lasting 72 ℃ extension for 10 min. Detection of Fon was carried out in the watermelon and soil by PCR qualitative detection system. The results of the watermelon test showed that: On the first day after inoculation, the qualitative system can detect Fon in the resistant and susceptible watermelon. According to the strip brightness analysis, as for the'Zaojia 8424', the amount of fungus increased gradually from 1 to 7 days after inoculation, there was no significant change in the amount of fungus from 7 to 17 dpi, and the amount of fungus decreased slightly from 17 to 19 dpi. After the dipping root treatment of'Zaojia 8424', the plants of 1-4 dpi showed normal performance and the disease index was 0; the 5 dpi watermelon plants began to show mild wilting symptoms, the disease index was 1.7; the disease index continued to increase at 5-13 dpi, reaching 96.67 at 15 dpi; At 17-19 dpi, almost all of the watermelon plants died with wilting. For'Xinong 8', the amount of fungus increased gradually from 1 to 7 dpi, and the amount of fungus decreased gradually from 7 to 19 dpi.'Xinong 8'showed no symptoms at 0-13 dpi, and a slight wilting disease began at 15 dpi. The disease index was 2.67, and subsquently reached 12.72. It can be seen that the pathogen can be detected 1-5 days after the inoculation with both resistant and susceptible watermelon varieties before the plants showed symptoms, and the amount of the detected fungus was consistent with the disease index. The soil test results showed that the strips gradually weakened with the decrease of the inoculated spores, and the sensitivity was 5×102 cfu · g-1 soil. The host performance of 5×105 cfu · g-1 and 5×104 cfu · g-1 soil treated watermelon was similar. Resistant and susceptible hosts developed symptoms on the 8 th and 5 th day after inoculation, respectively, reaching the maximum on the 20 th day, and the incidences of plant disease was 8% and 100%. With watermelon plants treated in 5×103 cfu · g-1 and 5×102 cfu · g-1 soil, the disease was less than 5×104 cfu · g-1-5×105 cfu · g-1 treatments, and the incidences of both resistant and susceptible watermelon plants were low; the disease appeared only 12 days after inoculation for 50 cfu · g-1, and the disease progressed the slowest. The final incidences of resistant and susceptible watermelon plants were2% and 32%, respectively.【Conclusion】The specific primer FONDX-15 of Fon was obtained. The molecular diagnosis method was established and can be used for rapidly and accurately detecting Fon in watermelon plants and soils before thevisible Fusarium wilt symptom appeared.