Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a destructive wheat disease in China. The Gansu–Ningxia region (GN) is a key area for pathogen over-summering in China, and northwestern Hubei (HB) is an important region for pathogen over-wintering, serving as a source of inoculum in spring epidemic regions. The spatiotemporal population genetic structure of Pst in HB and the pathogen population exchanges between GN and HB are important for estimating the risk of interregional epidemics. Here, 567 isolates from GN and HB were sampled from fall 2016 to spring 2018 and were genotyped using simple sequence repeat markers. The genotypic and genetic diversity of Pst subpopulations in HB varied among seasons and locations. Greater genetic diversification levels were found in the spring compared with fall populations using principal coordinate analysis and Bayesian assignments. In total, there were 17 common genotypes among the 208 determined, as shown by a small overlap of genotypes in the principal coordinate analysis and dissimilar Bayesian assignments in both regions, which revealed the limited genotype exchange between the populations of GN and HB.

Bletilla striata is a valuable medicine in China, belonging to the Orchidaceae family, and is used for treating various ailments such as hemoptysis, pyocutaneous disease, and anal fissure by preventing blood flow, reducing swelling, and promoting granulation. In June 2022, a disease with symptoms similar to root rot was observed on B. striata in the pineland (the area was 0.4 hectare) of Lancang County (22°48'17" N, 99°46'58"22 E), Yunnan Province, China. The root rot incidence rate reached 16% (Table S1). The root rot incidence was calculated as follows: root rot incidence (%) = (number of root rot seedlings/total number of seedlings investigated) × 100. In May 2023, the similar symptoms were observed in the field, and the disease incidence was 17% (Table S1). Initially, there were no obvious symptoms on the leaves. Subsequently, the leaves wilted and brown spots appeared. Later, the entire leaf browned, withered and eventually died (Fig. S1A, B). The roots were brown and the browning spread from the root edge to the center, causing vascular bundle browning and dead lignified fibers in the cortex (Fig. S1C, D). To isolate the causal pathogen, 20 symptomatic root tissues were collected from 20 plants. Cutting the diseased tissues into small pieces (0.5 × 0.5 cm). After surface sterilization (30s with 75% ethanol and 3 min with 2% sodium hypochlorite, rinsed three times with sterile water), the disinfected root tissues were plated onto potato dextrose agar (PDA) and incubated at 25℃ for 4 to 6 days with 12 h light/dark photoperiod. A total of 10 single-spore isolates with similar morphology and conidial characteristics were obtained. one representative isolate BJG6 was selected for identification and further study. The fungal colony was reddish-brown or orange-white on PDA after 8 days of incubation at 25℃. The mycelium was like carpet or cotton, and the edge of colony was uniform (Fig. S1E). Large conidia were formed on simple conidial peduncles (Fig. S1F, G). The conidia with 1~3 septates and 1 mostly, with cylindrical shapes and narrow tops but sharp bases (Fig. S1H-J). Conidia with 1 septate measured as 5.5 (4.3-6.7) × 20.7 (16.0-25.4) μm (n=30), while those with 2 septates measured as 6.6 (5.8-7.4) × 26.5 (21.7-31.3) μm (n=30), and those with 3 septates was 6.9 (6.2-7.8) × 31.8 (29.3-34.3) μm (n=30). Ellipsoidal microconidia could be formed on conidiophore and measured as 2.4 (1.9-2.9) × 4.9 (5.9-3.9) μm to 2.7 (2.2-3.2) × 5.4 (4.3-6.5) μm (n=30). Spherical or subspherical chlamydospores were produced on low-nutrient agar, with an average size of 5.8(5.0-6.6) μm×5.3 (4.4-6.2) μm (n=30) (Fig. S1K, L). According to the morphology and conidial features, the pathogen was consistent with the description of Ilyonectria coprosmae (Cabral et al. 2012). The total genomic DNA was extracted, and primer pairs ITS4/ITS5 were used to amplify and sequence the rDNA-ITS region (ITS1-5.8 S rRNA-ITS2 gene regions) (White et al. 1990). The sequences were deposited in GenBank (SUB13905750 for ITS). BLAST searches revealed BJG6 showed 98% homology with corresponding sequences of Ilyonectria coprosmae in GenBank (JF735260). A phylogenetic tree (MEGA 7.0) was constructed using maximum-likelihood methods (Fig. S2). To identify pathogenicity, a cultured medium in a size of 6mm containing isolate BJG6 was inoculated onto ten healthy roots of B. striata, PDA plugs alone were used as the uninoculated controls. All samples were placed in a dark inoculation chamber at 25℃. The pathogenicity test was replicated three times. After two weeks, all inoculated roots appeared similar symptoms identical to those observed on field plants (Fig. S1M, N-P), while control plants remained healthy (Fig. S1Q, R). The same pathogenic fungus was reisolated from the symptomatic root rot, and the characteristics of colony and conidia were the same as the original isolates (Fig. S1S, T). These results confirmed I. coprosmae as the causal pathogen of root rot disease on B. striata in China by Koch's postulates tests for the first time. Further exploration should be conducted to understand the occurrence and migration of this disease, so as to develop specific and efficient disease management strategies in the future.

Puccinia striiformis f. sp. tritici, causing stripe rust, is one of the most prominent pathogens of wheat worldwide. The biotrophic and obligate fungus is capable of rapidly developing new virulent races that can overcome race-specific resistance in host plants. The traditional virulence characterization of the pathogen requires strict conditions for testing isolates on wheat differentials with specific resistance genes, which is time-consuming. Developing molecular markers for avirulence genes could provide an efficient method for monitoring virulence changes in the pathogen population. In this study, secreted protein gene-based single-nucleotide polymorphism markers previously identified to be associated with avirulence genes of the pathogen were converted to kompetitive allele-specific PCR (KASP) markers. The KASP markers were screened with a diverse panel of 192 isolates selected from various countries based on their virulent races and molecular genotypes. The markers significantly correlated with the avirulence/virulence phenotypic data of the 192 isolates were further validated with 845 isolates collected from the United States in 2019 to 2021. Based on the results of both the screening and validation data, 21 KASP markers significantly associated with different avirulence genes were developed. Seventeen of the 21 markers were significantly associated with two or more avirulence genes, and except AvrYr10, and the remaining 15 avirulence genes had two or more markers. Different combinations of up to three markers could be used for specific detection of 16 avirulence genes in monitoring the pathogen population.