Establishment of a rapid detection method for wheat crown rot

. We have established a real-time fluorescent quantitative PCR system that can detect wheat crown rot rapidly and accurately quantify fungi, Fusarium species and Fusarium pseudograminearum in the rhizosphere soil of infected wheat through the standard curve produced, with a view to the early stage of wheat Provide help in predicting the occurrence of wheat crown rot.


Introduction
Fusarium spp. is a pathogen of a variety of soil-borne diseases of wheat [1], which can infect the vascular system of plants and cause the roots, stems and leaves of plants to rot. For example, Fusarium pseudograminearum is the main pathogens of wheat crown rot [2]. The infection of fusarium on wheat tissues will greatly affect the yield of wheat and the quality of wheat ears [3].
Real-time fluorescent quantitative PCR (QPCR) technology is to use fluorescence accumulation to monitor the entire process in real time by adding fluorescent groups to the DNA amplification reaction, thereby quantitatively analyzing unknown templates [4,5]. This technology has been widely used in the detection of various plant pathogens after its emergence. QPCR generally has two types: SYBR Green and TaqMan probe method [4]. Both methods are involved in this experiment.
In this study, the established a rapid QPCR detection system for wheat crown rot, aiming to provide detection for the number of fungi Fusarium in rhizosphere soil, and also for the establishment of a technique for predicting the occurrence of early wheat stem rot.

Collection and processing of soil samples
Soil sample collection: Three plots with significant differences in the incidence of wheat crown rot (H), light (L) and non-infected plots (CK) were selected. Harvest 5 randomly selected wheat seedlings (including root system and attached rhizosphere soil) from each plot, transfer them to sterile sample bags and bring them back. Use sterile water to rinse the soil attached to the roots and then centrifuge to produce rhizosphere soil samples. Shake and combine the rhizosphere soil at each sampling point and store it at -80°C before analysis.
Preparation of standard products: Extract the positive clone plasmid as the QPCR reaction standard product. The concentration of the dilution standard was (1×10 -6 ng～ 1ng) seven different gradients, and ddH2O was used as the negative control.

Preparation of fungal standard plasmid
Amplification of the target fragment .Primer sequence: ITS-F(5'-3'): CTTGGTCATTTAGAGGAAGTAA; ITS-R(5'-3): GCTGCGTTCTTCATCGATGC. Use this primer to amplify the plasmid, and the reaction system and sequence determination are the same as those of Fusarium.
Preparation of standard plasmid: the same as the construction method of Fusarium
Preparation of standard plasmid: the same as the construction method of Fusarium.

Establishment of real-time QPCR detection system for F.pseudograminearum
The reaction system is the same as that of Fusarium. PCR conditions were 95 ℃ 10 min, 95 ℃ 30 s, 51 ℃ 30 s, 72 ℃ extension 1 min, 72 ℃ final extension 7 min, 39 cycles.

Application of fluorescence quantitative PCR in wheat rhizosphere soil
Extraction of soil genome: Take 0.3g fresh weight of each soil, and extract the genome with the DNeasy®PowerSoil®Kit.
Gene copy number calculation formula: (copies/g)=SQ×6.02×1023×10-9×100/(DNA length×660×0. 3) (1) where SQ is the result of fluorescence quantitative PCR and DNA length is the standard the size of the plasmid(fungal plasmid is 3171bp,Fusarium spp. 3135 bp and F.pseudograminearum 2776bp), 100 is the volume of DNA extracted from the soil sample (uL), and 0.3g is the mass of the soil sample. The result is shown in Figure 1. The fragment size is 479bp, and the PCR product size is correct. The amplified band is clearer, and there is no primer dimer influence. The result is shown in Figure 2. The fragment size is 443bp, the PCR product size is correct Note: Marker DL2000, lanes 1, 2, 3 ,4 are the fragments amplified for F. pseudograminearum. The result is shown in Figure 3. The fragment size is 84bp, the PCR product size is correct. The results of the fungal standard curve are shown in Figure 4. The amplification curves of the 7 plasmid standards are relatively smooth, and each cycle threshold is evenly spaced. The linear relationship is good, and the sensitivity can reach 1fg. Comprehensive primer PCR gel electrophoresis detection and QPCR system results can show that the established fungal QPCR system and reaction conditions meet our needs for accurate quantification of fungi.curve and melting curve are automatically drawn by Bio-rad fluorescent quantitative PCR instrument. The results of the standard curve of Fusarium are shown in Figure 5. The 7 gradients of standard products have a good linear relationship, and the sensitivity can reach 1fg. The amplification curve is smooth, typical s-shape. Combined with the above results, it can be shown that the established QPCR detection system for Fusarium in wheat rhizosphere soil is reasonable and sensitive, and can meet our requirements for accurate quantification of Fusarium. The results of the standard curve of F. pseudograminearum are shown in Figure 6. The 7 gradients of standard products have a good linear relationship, and the sensitivity can reach 1fg. Combined with the above results, it can be shown that the established QPCR detection system for F. pseudograminearum in wheat rhizosphere soil is reasonable and sensitive, and can meet our requirements for accurate quantification of Fusarium. The results are shown in Figure 7. The more severe the disease, the more fungi, Fusarium and Fusarium pseudograminearum, and the degree of disease is positively correlated with the amount of Fusarium. The result is consistent with the condition of wheat.

Conclusions
In this study, the primers used in the fluorescence quantitative PCR reaction, the amplification curve obtained from the reaction, the melting curve and the standard curve all reached the ideal conditions for the fluorescence quantitative reaction. The QPCR detection system for fungi, Fusarium and F.pseudograminearum established in the experiment can accurately quantify the content of fungi and Fusarium in wheat rhizosphere soil and can predict the occurrence of early wheat crown rot . The fluorescence quantitative detection method established in this experiment is more accurate than the traditional plate dilution method for the quantification of fungi and Fusarium in the soil. The copy number of fungi and Fusarium in wheat rhizosphere soil can be obtained through the established QPCR reaction system. Through the establishment of F.pseudograminearum quantitative PCR, we can predict the early incidence of wheat stem rot. It can be seen from the results that the amount of fungi and Fusarium in the rhizosphere soil in areas with severe disease is much higher than that in areas with mild or no disease, indicating that the degree of disease is positively correlated with the amount of fungi and Fusarium in the soil . There are also significant differences in the content of Fusarium pseudograminearum. The establishment of this method can provide help for predicting the occurrence of early wheat crown rot.