Virulence characteristics of Blumeria graminis f. sp. tritici and its genetic diversity by EST-SSR analyses

Collection of Bgt isolates

From May to July 2015, diseased leaves with fresh powdery mildew spores were collected from Liaoning and Heilongjiang and kept in falcon tubes containing water agar medium with 1% of 6-benzylaminopurine preservation solution (40 mg∙L⁻¹). Diseased leaves carrying the cleistothecium of Bgt were also collected from Sichuan, brought back to the laboratory, dried in a cool place, and stored at 4 °C. Xianxin Wu and Wanlin Wang identified all isolates used in the study. All isolates were deposited (no deposition number) in the College of Plant Protection, Shenyang Agricultural University (our lab).

Isolation, purification, and propagation of Bgt

The highly susceptible Little Club (provided by the College of Plant Protection of Shenyang Agricultural University) was sowed in tile pots. When the seedlings had grown to the one-leaf stage, 5–6 cm leaf segments were cut and placed in a Petri dish lined with double filter paper. Five to six-leaf segments were placed face up in each Petri dish and held in place with glass strips on both ends. The filter paper was moistened with 40 mg∙L⁻¹ of 6-benzylaminopurine (6-BA) preservation solution. The collected powdery mildew was first attached to the front of the leaf with a toothpick sharpened into a flattened tip. Three spots were attached to each leaf, lightly and evenly applied taking care not to scratch the leaf. After inoculation, the leaves were incubated for 5–7 days in a growth chamber at 18–22 °C with a 14-h/10-h light/dark cycle. After white spore mounds appeared on the leaves, the leaves were inoculated by shaking. The spores on one leaf segment were gently shaken off on the freshly isolated leaf segment. After incubation at 18–22 °C for 5–7 days under light, single colonies were inoculated at three points on the isolated leaf segments with a flat toothpick, and this process was repeated several times for the isolation and purification of monospores. The isolated and purified single pustules were numbered and multiplied for preservation (Zhu et al., 2015).

Release of Bgt cleistothecium ascospores

A cleistothecium was picked from diseased leaves using an inoculating needle and incubated on moistened filter paper soaked with distilled water. After 5 days, the cleistothecium was picked and observed under a microscope. When ascospores were formed, a cleistothecium was randomly picked from diseased leaves and transferred to moistened filter paper sheets, with one cleistothecium per sheet. The filter paper sheet was then placed upside down in the center of a Petri dish lid, the Petri dish lid was placed on the petri dish with the isolated wheat leaves, and the Petri dish was sealed with parafilm and placed in a light incubator at 17 °C (Chi, 2009).

Analysis of virulence frequency of Bgt

A total of 31 lines with known Pm genes (provided by the College of Plant Protection of Shenyang Agricultural University) were sown in 10 cm diameter tile pots according to their numbers and marked. The highly susceptible variety Chancellor was used as the susceptible control. The first leaf was cut when the seedlings reached the one-leaf stage and the cut leaf segments were placed in order in the petri dishes, and the propagated single pustules were gently shaken off the leaves. The culture was incubated for 5–7 days. When the susceptible control was fully developed, the infection types (ITs) were assessed and recorded following the method described by the previous study (Si et al., 1987): ITs 0–2 were marked as resistant (R), and the corresponding isolates as avirulent; ITs 3–4 were marked as susceptible (S) and the corresponding isolates as virulent. The test was repeated three times.

Genomic DNA extraction and polymerase chain reaction (PCR) analysis

Genomic DNA was extracted from conidia using the Omega Bio-Tek fungal DNA kit (Norcross, GA, USA) following the manufacturer’s protocol. The total PCR reaction volume was 20 μL, consisting of 1 μL DNA template (30 ng·μL⁻¹), 1 μL forward primer, 1 μL reverse primer, 10 μL 2 × Power Taq PCR Master Mix, and 7 μL ddH₂O. The PCR procedure was as follows: initial denaturation at 94 °C for 5 min; then 35 cycles of a denaturation step at 94 °C for 30 s and an extension step at 72 °C for 1.5 min, followed by a final extension at 72 °C for 10 min.

Selection of the EST-SSR primers

Seven pairs of EST-SSR primers (Table 1) were designed according to Xu (2012) and were screened for the occurrence of clear and stable polymorphisms. Two pairs were chosen for the genetic polymorphism analysis. The primers were synthesized by Sangon Biotech Inc. (Shanghai, China; http://www.sangon.com/).

Polyacrylamide gel electrophoresis and genetic diversity analysis

The procedure for polyacrylamide gel electrophoresis (PAGE) was as previously published (Chen et al., 2015). The silver staining method was used to visualize the PCR products as described by Bassam, Caetano-Anollés & Gresshoff (1991). Based on the PAGE results, ‘1’ or ‘0’ were assigned to the presence or absence of bands, respectively, and the same method was used for frequency of virulence analysis, ‘1’ or ‘0’ were assigned to resistance (ITs: 0–2) or susceptibility (ITs: 3–4), respectively, in the host. According to the ‘1, 0’ data matrix, the genetic similarity was calculated using NTSYSpc 2.10_e. The unweighted pair group arithmetic method was used for gene diversity cluster analysis of the expression sequences and then the classification trees were constructed. In addition, the genetic and virulence correlations of different isolates were described by Principal Component Analysis (PCA) analysis. The isolates were divided into three groups by ‘K-mean’ method. The optimal number of groups was determined by the ‘gap_stat’ method in ‘cluster’ package of R software 4.0.0.

Virulence frequencies of 80 isolates to 36 single gene lines

A total of 80 Bgt isolates, collected from Liaoning (29), Heilongjiang (26), and Sichuan (25), were isolated (Table S1). Virulence frequencies of these isolates were assessed on 36 differential lines individually containing single powdery mildew (Pm) resistance gene. The results show that the virulence frequency of isolates from Northeast China against resistance genes including Pm1, Pm3c, Pm3d, Pm3e, Pm3f, Pm4a, Pm4b, Pm5, Pm6 (Coker747), Pm6 (Timgalen), Pm7, Pm8, Pm16, Pm17, Pm19, Pm23, Pm30, Pm4 + 8, Pm4b + mli, Pm4 + 2X, and Era was above 60%, indicating that the effectiveness of these resistance genes had been partially or completely lost. Virulence to Pm 3a, Pm3b, Pm13, Pm18 (1c), Pm20, Pm22 (1e), Pm5 + 6, and PmV2 + 6 was 40–60%. Virulence to PmVXBD and Pm21 were 27.5% and 0 (Table 2).

EST-SSR analysis of Bgt isolates

Two pairs of EST-SSRs, Blu ssr3-1-blu ssr3-2 and Blu ssr2-1-blu ssr32-2, were selected from seven reported EST-SSR primers, 9 and 20 bands of clear and stable polymorphisms were amplified respectively. These two pairs of primers were specific to Bgt isolated from Northeastern China and Gansu Province. Figures 1 and 2 show the PAGE results of EST-SSR polymorphism using primer pairs Blu SSR3-1- plus Blu SSR3-2 and Blu SSR32-1- plus Blu SSR32-2, respectively. Genetic similarity analysis of EST-SSR PAGE was assessed on 80 Bgt isolates collected from Liaoning, Heilongjiang, and Sichuan using NTSYSpc 2.10_e and PCA. NTSYSpc 2.10_e dates analysis showed when the genetic similarity coefficient was 0.52, the 80 isolates were divided into three groups: Group I consisted of 36 isolates, including 23 isolates from Sichuan, nine isolates from Liaoning, and four isolates from Heilongjiang (Fig. 3); Group II consisted of 38 isolates, including 20 isolates from Heilongjiang, 16 isolates from Liaoning, and two isolates from Sichuan; Group III consisted of six isolates, including four isolates from Liaoning, and two isolates from Heilongjiang. PCA dates analysis is similar to NTSYSpc 2.10_e, and all isolates were also divided into three groups (Fig. 4). The PCA model described 26.5% variance. 15.1% variance was described by Dim 1, and 11.4% was by Dim 2. Overall, there was a certain degree of transmission among Bgt isolates in different regions. However, when the genetic similarity coefficient was high, some isolates from Heilongjiang and Liaoning were clustered into subcategories. In contrast, isolates from Sichuan were clustered into subcategories separately, indicating that the genetic exchange between isolates from Heilongjiang and Liaoning was extensive. At the same time, it showed that there were genetic differences among these Bgt groups from different regions.

Virulence diversity and genetic diversity of Bgt

The virulence diversity “0, 1” matrix of these isolates was constructed based on the infection type of 80 Bgt isolates on 34 identified hosts. The phylogenetic tree clustered according to the similarity of infecting hosts is shown in Fig. 5. When the genetic similarity coefficient is 0.31, except for isolates L30, L31, L35, L42, and L45 from Liaoning; isolates H19, H21, and H29 from Heilongjiang, and C2-4 from Sichuan, 71 out of 80 isolates were divided into the same group. The PCA model described 34.0% variance. A total of 23.8% variance was described by Dim 1, and 11.2% was by Dim 2. All isolates were divided into three groups (Fig. 6).

The EST-SSR polymorphism and virulence diversity dendrograms of these isolated genes revealed that when the coefficient was <0.21, there were 10 clusters of genetic and virulence diversity. The positions of other isolates in the genetic polymorphism and virulence diversity dendrograms were different and did not correspond to each other. Thus, there was a correlation between genetic polymorphism and virulence diversity of 36 isolates (45%) (Table S2). However, the EST-SSR polymorphism of isolated genes did not correspond to the virulence diversity of isolates in the single-gene lineage identification of hosts.