Development and application of multiplex PCR for the rapid identification of four Fusarium spp. associated with Fusarium crown rot in wheat

Fungal strains, culture conditions, and DNA extraction

A total of 22 strains of F. graminearum, F. pseudograminearum, F. proliferatum and F. verticillioides were collected by Hubei Academy of Agricultural Sciences, and a total of 46 fungal strains, e.g., Fusarium solani, Fusarium incarnatum, Fusarium equiseti, Fusarium oxysporum, Fusarium humuli, Fusarium brachygibbosum, Fusarium fujikuroi were kindly provided by Nanjing Agricultural University, Jiangsu Academy of Agricultural Sciences, Northwest Agriculture and Forestry University, and Yulin Normal University of China. Fungal strains culture and DNA extraction method were as previously described in Liu et al. (2024). All strains were routinely cultured on potato dextrose agar (PDA) plates (200 gL⁻¹ of potato extracts, 1% glucose, and 2% agar), and incubated at 25 °C culture for 7–10 days. Mycelia of each isolate were collected with a sterile spatula for DNA extraction. Genomic DNA was extracted from mycelia using the Plant Genomic DNA Kit DP305 (TIANGEN, Beijing, China) according to the manufacturer’s instructions. DNA samples were measured with spectrophotometry to determine quality and concentration and stored at −20 °C until use.

Comparative genomics for identifying multiplex PCR primers

The genome sequences of F. pseudograminearum Class 2-1C (GenBank accession number CP064755.1), F. graminearum PH-1 (GenBank accession number HG970332.2), F. proliferatum ET1 (GenBank accession number NW_022194799.1), F. verticillioides 7600 (GenBank accession number CM000579.1), F. gerlachii CBS 119176 (GenBank accession number GCA_017656835.1), F. boothii CBS 316.73 (GenBank accession number GCA_017656985.1), F. culmorum Class2-1B (GenBank accession number CP064747.1), F. aethiopicum CBS 122858 (GenBank accession number GCA_017657045.1) and F. vorosii CBS 119178 (GenBank accession number GCA_017656575.1) were downloaded from the National Center for Biotechnology Information (NCBI) database. The primer sets design method were as previously described in Liu et al. (2024). We performed multiple alignments of the conserved sequences using Mauve software (version 2.3.1) to obtain homologous gene sequence fragments of these genomes. The same ≥20 bp sequences were selected from the homologous fragments of these genomes. Then specific downstream primers were designed from downstream non-homologous sequences. Therefore, a ≥20 bp genome sequence was selected from homologous fragments in F. pseudograminearum, F. graminearum, F. proliferatum, and F. verticillioides, and served as a universal upstream primer. A 1,000 bp downstream sequence was obtained in each genome for sequence alignment using BioEdit software (version 7.0.9.0). Then, nucleotide sequence of the designed specific downstream primers of each target strain was verified in the Basic Local Alignment Search Tool (BLAST) of the NCBI database. The primers are described in Table 1. The primer sets were synthesized by Sangon Biotech (Shanghai, China).

Optimization of multiplex PCR condition for detection of four Fusarium spp.

Multiplex PCR assay-related parameters were evaluated and optimized, including primer annealing temperatures, primer, dNTPs and Mg²⁺ concentrations. The test method were as previously described in Liu et al. (2024). Multiplex PCR was performed in 50 μl reaction volumes containing 0.25 μl TaKaRa Ex Taq polymerase (5 U/μl), 5 μl 10 × Ex Taq buffer (Mg²⁺-free), 1–8 μl (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mM) of MgCl₂ (25 mM), 2–16 μl (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mM) of dNTPs mixture (2.5 mM each), and 1μl for each of the four fungal DNA templates (each DNA concentration: 1 ng/μl). Primer length and G+C content were important factors that influence the amplification efficiency of multiplex PCR. Therefore, to determine primer sets, primers with different lengths and G+C contents were designed in previous assays, and then the optimal primer set was verified by an annealing temperature gradient experiment. To adjust optimal concentration of each primer in the multiplex PCR system, different primer concentration combinations were tested, including four groups of concentration ratios for the universal upstream primers (Fu-4F) and downstream primers (Fgram-R, Fpseu-R, Fprol-R, and Fvert-R) (group I: 1:1; group II: 2:1; group III: 3:1; and group IV: 4:1). The final concentrations of each specific downstream primer were set at 0.05 μmol/L, 0.1 μmol/L, 0.15 μmol/L, and 0.2 μmol/L, respectively (Table S1). Multiplex PCR amplification was performed with the following program: 95 °C for 5 min, 32 cycles of denaturation at 95 °C for 30 s, annealing at 45–65 °C for 30 s, extension at 72 °C for 1 min and final extension for 10 min at 72 °C. Twelve temperature gradients were set, including 45, 46.1, 47.7, 50.5, 53, 55, 57.2, 59.4, 61.6, 63.4, 64.6 and 65 °C to determine the optimal reaction conditions for annealing temperature. PCR products were visualized under UV light after being size-fractionated by electrophoresis through a 2% agarose gel made with TAE buffer and stained with ethidium bromide solution. These experiments were repeated three times.

Multiplex PCR specificity test

The specificity test method was done as previously described in Liu et al. (2024). To evaluate the specificity of the multiplex PCR primer set, 1 μl of 22 target pathogen DNA (six F. graminearum, eight F. pseudograminearum, five F. proliferatum and three F. verticillioides) from different hosts and other 46 fungal strains were used as templates for multiplex PCR amplification under the optimized multiplex PCR system and conditions. In addition, in order to demonstrate DNA was present for each sample, the internal transcriptional spacer (ITS) segments of 46 fungal pathogens were amplified with ITS4/ITS5 primers (White et al., 1990). All strains are listed in Table 2. PCR products were visualized under UV light after being size-fractionated by electrophoresis through a 2% agarose gel made with TAE buffer and stained with ethidium bromide solution. This experiment was repeated three times.

Multiplex PCR sensitivity test

The sensitivity test method was done as previously described in Liu et al. (2024). To determine the sensitivity of the multiplex PCR assay, genomic DNA from the four target pathogens was serially diluted to 10 ng/μl, 1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, and 10 fg/μl by a 10-fold gradient with sterile double distilled water 1 μl of each DNA dilution concentration was used as a single PCR template to test the detection limit of each target pathogen by single PCR. Subsequently, each DNA dilution concentration was mixed, respectively, as a multiplex PCR template to test the detection limit of multiplex PCR for each target pathogen. PCR was performed according to the optimized conditions. Finally, PCR products were visualized under UV light after being size-fractionated by electrophoresis through a 2% agarose gel made with TAE buffer and stained with ethidium bromide solution. This experiment was repeated three times.

Detection of target pathogen DNA from field wheat samples and artificially inoculated wheat samples

To evaluate the applicability of the multiplex PCR assay for four Fusarium pathogens of FCR, we collected 22 wheat samples in a wheat growing area of Xiangyang (32.2015913°N, 110.901005°E) and Suizhou (31.9938899°N, 113.0270585°E) in Hubei Province of China in June 2022. DNA extraction method was done as previously described in Liu et al. (2024). After a small piece of tissue was excised from the stem of the 22 wheat samples using a sterilized scalpel, genomic DNA was extracted from field wheat samples using the Plant Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions.

Artificial inoculation method was done as previously described in Liu et al. (2024). For the artificial inoculation test, fungal strains were cultured on PDA for three days at 25 °C, then mycelium plugs were transferred to mung bean medium and cultured at 25 °C for seven days with shaking at 200 rpm. Conidial suspensions were filtered through four layers of cheesecloth to separate conidia from mycelia. Concentration of the conidial spore suspensions was estimated using a hemocytometer and adjusted to 1 × 10⁷ spores/ml. Wheats were inoculated with conidia suspensions of each fungus (1 × 10⁷ spores/ml) in the stem of each wheat. Genome DNA was extracted after inoculation of 17 healthy wheats with four Fusarium strains in different combinations and seven wheats with sterile water for 3 days. Wheat samples inoculated with four Fusarium strains served as positive controls, while samples treated with sterile water were used as negative controls. Genomic DNA from all wheat samples was extracted using the Plant Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. All DNA extracted from the wheat sample used as a template for the multiplex PCR, which was performed using an optimized multiplex PCR system. PCR products were visualized under UV light after being size-fractionated by electrophoresis through a 2% agarose gel made with TAE buffer and stained with ethidium bromide solution. Amplified products of multiplex PCR were verified by sequencing of Sangon Biotech (Shanghai, China).

Specific primers for four Fusarium spp. were designed via whole genome sequence comparison

To detect DNA from F. graminearum, F. pseudograminearum, F. proliferatum and F. verticillioides simultaneously, we screened specific primer combinations and established a multiplex PCR system (Fig. 1A). First, whole genome sequence comparison analysis identified a 20 bp sequence located within a tRNA-lle gene in the genomes of four Fusarium strains. This 20 bp sequence is located at nucleotide positions 1,558,947 to 1,558,966, 1,532,724 to 1,532,743, 2,012,079 to 2,012,098, and 2,435,141 to 2,435,160 in F. pseudograminearum Class 2-1C (GenBank accession number CP064755.1), F. graminearum PH-1 (GenBank accession number HG970332.2), F. proliferatum ET1 (GenBank accession number NW_022194799.1), F. verticillioides 7,600 (GenBank accession number CM000579.1) genome respectively (Fig. 1B). This sequence was selected as an upstream universal primer (Fu-4F), and specific downstream primers (Fgram-R, Fpseu-R, Fprol-R, and Fvert-R) of four pathogens with different amplicon sizes were designed. The amplicon size of F. graminearum, F. pseudograminearum, F. proliferatum and F. verticillioides were 206, 482, 680 and 963 bp, respectively (Fig. 1 and Table 1). In addition, the downstream primers matched only the sequence of the target pathogens.

Standardization of for the multiplex PCR system

The length of the primers and the G+C content were closely related to the annealing temperature of PCR. The optimal primer set was selected by testing the annealing temperature of different primer sets (Fig. S1). Then, we tested the effects of different primer dNTPs, and Mg²⁺ concentration on the efficiency of multiplex PCR amplified DNA from the target pathogens. Our results showed that more PCR product was amplified under following primer concentrations (Fu-4F: 0.8 μmol/L, Fgram-R: 0.2 μmol/L, Fpseu-R: 0.2 μmol/L, Fprol-R: 0.2 μmol/L, Fvert-R: 0.2 μmol/L) when 2 mM MgCl₂ and 0.2 mM dNTPs were added with 53 °C annealing temperature (Fig. 2).

DNA from four target pathogens were specifically and sensitively detected by multiplex PCR

Using the optimal multiplex PCR system, an unambiguous detection result was obtained by multiplex PCR using mixed or individual genomic DNA of F. graminearum, F. pseudograminearum, F. proliferatum and F. verticillioides as templates. This result indicates that the established multiplex PCR method could specifically detect DNA of 22 target strains from different hosts (Fig. 3 and Table 2). As expected, DNA from other 46 fungal pathogens had no amplified product (Table 2 and Fig. S2). In addition, the fragments of the internal transcribed spacer (ITS) of 46 fungal pathogens could be amplified with the ITS4/ITS5 primer, demonstrating the presence of DNA in each sample (Fig. S3). Under the optimized PCR reaction conditions, no fragments were amplified without the addition of DNA templates, indicating that the fragments amplified by the primer set were due to the target DNA (Fig. S4).

In addition, the results of the sensitivity test in three replicate tests show that the PCR detection limit for individual DNA was 10 pg/μl for F. verticillioides, 1 pg/μl for F. proliferatum, 100 pg/μl for F. pseudograminearum, and 100 pg/μl for F. graminearum (Figs. 4B–4E). However, the detection limit for multiplex PCR was about 100 pg/μl for DNA mixture from F. verticillioides, F. proliferatum, F. pseudograminearum, and F. graminearum (Fig. 4A).

Multiplex PCR was successfully applied to detect pathogen DNA within wheat samples from the field and artificially inoculated samples

To determine the applicability of this multiplex PCR assay, we detected pathogen DNA in 22 wheat samples from the field and 24 artificially inoculated wheat samples. Our results showed that F. pseudograminearum, F. graminearum, and F. verticillioides were identified in 15, 10, and 3, respectively, of the 22 wheat samples from the field (Fig. 5A and Table S2). Among them, 10 wheat samples were co-infected with F. pseudograminearum and F. graminearum, two wheat samples were co-infected with F. pseudograminearum and F. verticillioides, and one wheat sample was co-infected with F. pseudograminearum, F. graminearum, and F. verticillioides (Fig. 5A). In addition, the results of the 24 artificially inoculated wheat samples were consistent as expected, F. pseudograminearum, F. graminearum, F. proliferatum, and F. verticillioides were identified in 12, eight, four, and four of the 24 artificially inoculated wheat samples, respectively (Fig. 5B and Table S2). Among, 10, 11, 14, 15, 16, 20, and 21 lanes were wheat samples inoculated with sterile water, and no amplification bands were found (Fig. 5B and Table S2). The amplified products were further verified by sequencing.