Zizania latifolia plants infected with MT- or T-type U. esculenta were acquired from the Aquatic Vegetable Research Base of Jinhua Institute of Agricultural Science (Zhejiang Province). The infected plants were transplanted on July 20, 2021, and sampled every 7 days starting from the transplanting date. Twenty plants were collected each time, including seven plants for analyzing the U. esculenta content in different plant issues, four plants for paraffin embedding, sectioning, and microscopic observation, and eight plants for studying the gene expression of U. esculenta. Zizania latifolia was sampled 11 times in total. Zizania latifolia plant samples after overwintering were collected on February 20, 2022.

Approximately, 0.1 g of plant tissue was collected, frozen in liquid nitrogen, and ground in a mortar. Total DNA was extracted from roots, stems, and stem tips following the user manual of the plant genomic DNA extraction kit [Tiangen Biotech (Beijing) Co., Ltd., DP 305]. The DNA content was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and diluted to approximately 100 ng/μL using sterile water. The diluted DNA samples were stored at −20°C until further use.

For the detection of U. esculenta in Z. latifolia, specific probes and primers (Table 1) were designed based on the SNP on the ITS sequences. The primers and probes were synthesized by Nanjing GenScript Biotech Corporation. The qPCR was performed using the following items: 10 μL PerfectStart II Probe qPCR SuperMix (2x; Beijing TransGen Biotech, AQ 711, AQ 401), 1.5 μL T fluorescent probe (10 μmol·L⁻¹), 1.5 μL MT fluorescent probe (10 μmol·L⁻¹), 1.5 μL forward primer ITS-F (10 μmol·L⁻¹), 1.5 μL reverse primer ITS-R (10 μmol·L⁻¹), 1 μL DNA template extracted from Z. latifolia tissues, and 3 μL sterile nuclease-free water. The amplification was carried out for 40 cycles under the following conditions: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 52°C for 15 s, and extension at 72°C for 30 s.

The collected plant tissues were washed with normal saline, and fixed in a Carnoy fixative (3:1 ethanol: acetic acid) for 48 h. After fixation, the samples were washed thrice with PBS, and dehydrated in a series of ethanol solutions (70, 80, 90, 95, and 100%). They were then transparentized using a balanced mixture of ethanol: xylene for 15 min, and then xylene for 15 min. The transparentized samples were successively immersed in a 1:1 mixture of paraffin: xylene for 15 min, and in paraffin twice for 60 min. The samples were placed into embedding boxes, and melted paraffin was added to generate embedding blocks.

The paraffin embedding block was sectioned at a thickness of 5 μm using a Leica rotating microtome (Leica RM2125 RTS). Sections were then dewaxed as instructed in the user manual of the environmentally friendly dewaxing solution (Wuhan Servicebio Co., Ltd., G1128). The dewaxed sections were stained as per the PAS staining kit (Wuhan Servicebio Co., Ltd., G1008). Upon staining, high-sugar U. esculenta in plant tissues got a dark red color while plant tissues had a light red color, thus distinguishing plant tissues from U. esculenta.

6 mL of 80% ethanol was added to 0.2 g of fresh plant tissue and incubated at 65°C for 20 min. The supernatant was collected, and the sediment was subjected to two additional extraction cycles following the same procedure. The collected supernatant was combined and concentrated. The soluble sugars were analyzed using an HPLC system comprising the Waters 600 separation module and the Waters 2414 RI detector (Waters Corp, Milford, MA, United States; Zhao et al., 2020). Sugar content was determined by the external standard method with 10 biological replicates per sample, and sugar concentration was measured in mg·g⁻¹ of fresh weight.

The paraffin embedding block was transversely sectioned at a thickness of 3 μm using a Leica rotating microtome (Leica RM2125 RTS), and sections were dewaxed as instructed in the user manual of the environmentally friendly dewaxing solution (Wuhan Servicebio Co., Ltd., G1128). Sections were stained using 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature, rinsed with PBS, and sealed using a water-soluble sealer.

Plant tissue sections were observed using a Leica Aperio VERSA 8 scanner. PAS-stained tissue sections were observed under bright field conditions; DAPI staining was excited with 405 nm light, and observed at 440–460 nm.

Image J software¹ was used to measure the average cell size and cell count in each cross-section.

Approximately, 0.1 g of plant tissue was collected, frozen in liquid nitrogen, and ground using a pestle and mortar. The total RNA of U. esculenta tissues was extracted according to the user manual of the FastPure Plant Total RNA Isolation kit (Polysaccharides & Polyphenolics–rich; Nanjing Vazyme Biotech Co., Ltd., RC 401) and stored at −80°C.

The integrity of RNA was verified by 1% agarose gel electrophoresis. High-quality RNA samples were selected for cDNA synthesis using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper; Nanjing Vazyme Biotech Co., Ltd., R223), following the manufacturer’s instructions.

β-actin was selected as the reference gene. Table 2 lists the primer sequences for the Ros1 gene and the reference gene. The relative expression levels were analyzed using the 2^-△△CT (Livak and Schmittgen, 2001) method. The qPCR reaction system contained the following items: 0.5 μL forward primer F (10 μmol·L⁻¹), 0.5 μL reverse primer R (10 μmol·L⁻¹), 1 μL cDNA template (100 mg·L⁻¹), 10 μL MonAmp™ SYBR® Green qPCR mix (Mona Biotechnology Co. Ltd., MQ00401), and 8 μL sterile water. The amplification was carried out for 40 cycles under the following conditions: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s.

Macroscopic phenotype observation revealed the following: (1) Zizania latifolia stems with MT-type U. esculenta significantly expanded on the 42nd day after transplanting, reaching a nearly edible size on the 120th day; (2) Zizania latifolia stems with T-type U. esculenta showed significant expansion on the 56th day after transplanting, and teliospores were observed on the 70th day. Smut galls induced by T-type U. esculenta were noticeably smaller compared to white and juicy galls induced by MT-type. The content of MT-type U. esculenta in plant stems was significantly higher than in roots and stem tips (value of p < 0.001) at the early stage (0–21 days after transplanting). Moreover, the content of MT-type U. esculenta in Z. latifolia stems peaked at least 0.1% on the 28 and 35th days after transplanting, whereas the content of T-type U. esculenta remained below 0.01%. U. esculenta exhibited extensive proliferation in stem tips and induced gall formation. The content profiles of T- and MT-type U. esculenta were similar in stem tips at the early stage. However, MT-type U. esculenta began to massively proliferate on the 28th day after transplanting, reaching peak content on the 35th day. Meanwhile, T-type massively proliferated since the 42nd day after transplanting, with peak content observed on the 56th day. The content of MT-type U. esculenta rapidly decreased after massive proliferation in stem tips, while T-type U. esculenta was kept at a high level. The gradual decline in the content of T-type U. esculenta at the later stage may be attributed to the low efficiency of DNA extraction from teliospores (Figure 1).

To investigate the rapid decrease of MT-type U. esculenta on the 42nd day after transplanting, the stem tip of white and juicy galls and smut galls were sectioned and observed on 0, 14, 28, 42, 56, and 70 days after transplanting. Ustilago esculenta was PAS-stained into a dark red color (Figure 2). Initially, a small quantity of U. esculenta was scattered in plant tissues. A few mycelia of MT-type U. esculenta started to aggregate in Z. latifolia on the 28th day after transplanting; this phenomenon was observed a little later in T-type. The mycelia aggregation coincided with the rapid increase in U. esculenta content observed in section 3.1. The aggregated mycelia of MT-type U. esculenta gradually disappeared and diffused outward by the 56th day after transplanting. MT-type U. esculenta failed to form teliospores. In contrast, T-type U. esculenta formed a larger mycelia aggregation and generated teliospores by the 70th day after transplanting. These results indicate a critical difference between T- and MT-type U. esculenta that occurred after the mycelia aggregation.

According to previous reports, massively proliferation and mycelia aggregation happened after karyogamy (Doehlemann et al., 2008; Tollot et al., 2016). The Ros1 gene, belonging to the WOPR family, is closely associated with these processes (Tollot et al., 2016). In our study, the expression of the Ros1 gene in T- and MT-type U. esculenta started to be upregulated on the 21st day after transplanting, and reached the maximum level on the 42nd day (Figure 3). The expression level in T-type U. esculenta was significantly higher on the 42nd and 49th days after transplanting compared to other time points, and it was significantly higher than that in MT-type. These results suggested that the karyogamy of U. esculenta might occur between the 21st and 42nd days after transplanting.

To further analyze the karyogamy of U. esculenta, stem tips of Z. latifolia on the 14th and 42nd days after transplanting were selected, and DAPI staining was performed to label cell nuclei (Figure 4). On the 14th day after transplanting, a few adjacent pairs of cell nuclei were observed in both MT- and T-type U. esculenta. Additionally, a few mononuclear cells were observed, possibly because the other cell nucleus was not in the current layer of sections. On the 42nd day after transplanting, the massive aggregation of mycelia made it difficult to distinguish the nuclei of different cells. Therefore, the size of cell nucleus was compared instead. The cell nucleus size ranged from 0.8 to 1.2 μm on the 14th day after transplanting, and significantly increased on the 42nd day (1.4–2.2 μm; p < 0.05). It suggested that karyogamy occurred in both MT- and T-type U. esculenta. The growth characteristics of U. esculenta undergo significant changes after karyogamy, including massively proliferation and mycelia aggregation.

The stems of infected and uninfected Z. latifolia plants were collected after 42 days of transplantation and analyzed. No matter what the type is (T or MT), the infected plants showed lower levels of fructose, glucose, maltose, and sucrose compared to the uninfected plants, indicating a high consumption rate of carbohydrates in the infected plant stems (Figure 5). On the other hand, the content of trehalose, which serves as an energy reserve of microbial carbohydrates, was significantly lower in MT-type U. esculenta than in T-type. Insufficient sugar supply in the stems appears to be the direct reason for the dramatic decrease in biomass of MT-type U. esculenta after 49 days of transplantation.

As diploid mycelia will largely consume the nutrition in the host plant, the longevity of it in Z. latifolia plants was investigated. After overwintering, the plant tissues were collected, DAPI-stained, and observed to visualize the cell nuclei of U. esculenta (Figure 6). In both T- and MT-type U. esculenta, the dikaryotic form with a single nucleus diameter ranging from 0.7 to 1.2 μm was observed. This diameter was not significantly different from the cell nucleus diameter on the 14th day after transplanting in the previous year (p = 0.902). These findings indicate the absence of diploid mycelia generated in plant tissues in the previous year. Diploid mycelia of U. esculenta could not sustain long-term survival in Z. latifolia plants.

The growth characteristics of U. esculenta undergo significant changes after karyogamy, which can potentially impact host plants. To assess these effects, plant tissue sections were analyzed to measure cell number and cell size in the cross-section of the first segment of stem tip (Figure 7). Zizania latifolia infected with U. esculenta exhibited a similar cell number but larger cell size, resulting in an enlarged stem tip compared to uninfected Z. latifolia plants. Between day 0 and day 35 after transplanting, the number of cells in infected Z. latifolia gradually increased, although their stem tip size did not significantly increase. This was mainly due to two reasons: (1) a gradual decrease in average cell size; (2) the progressive occupation of aerenchyma by plant cells. Notably, the number of cells in Z. latifolia infected by MT-type U. esculenta remained nearly unchanged between the 42nd and 70th day after transplanting, whereas when infected with T-type, the number of cells gradually decreased due to the substantial occupation of space by mycelia aggregations and teliospores. Meanwhile, cell size significantly increased in infected Z. latifolia (both T- and MT-type) between the 42nd and 70th day after transplanting. The massive proliferation of T-type U. esculenta led to cell death at the later stage, while MT-type U. esculenta did not cause any cell death. However, the effects on other aspects of plant cells were similar between MT- and T-type U. esculenta.