Development of rice conidiation media for Ustilaginoidea virens

Rice false smut, caused by the ascomycete Ustilaginoidea virens, is a serious disease of rice worldwide. Conidia are very important infectious propagules of U. virens, but the ability of pathogenic isolates to produce conidia frequently decreases in culture, which influences pathogenicity testing. Here, we developed tissue media with rice leaves or panicles that stimulate conidiation of U. virens. Generally, rice leaf media more effectively increased conidiation than panicle media, and certain non-filtered tissue media were better than their filtered counterparts. Among the tested media, the Indica rice leaf medium with 0.06 g/ml of Wanxian 98 leaf was most efficient for inducing conidiation, and it was also usable for conidiation-defective isolates. Although the conidia induced in rice tissue media were smaller, they were able to germinate on potato sucrose agar medium and infect rice normally. This method provides a foundation for the production of conidia in U. virens that will be widely applied in the pathogenicity testing as well as in genetic analyses for false smut resistance in rice cultivars.

Introduction depends on the time that the isolate has been in cultured and temperature, medium and 84 isolates, etc. Up to 10 8 conidia/ml can be produced after 6 days in potato dextrose 85 broth (PDB) and potato sucrose broth (PSB) (Wang et al. 1998). Previous research 86 has indicated that 2% sucrose-amended PDB containing barley seed produces more 87 conidia in a shorter period of time (Ashizawa et al. 2011) compared with other media. 88 The largest amount of conidia were detected after 6 or 7 days at 26-28℃ with shaking 89 at 140 to 210 rpm (He et al. 2011;Shi et al. 2017). In addition, some studies have 90 found that conidia production was related to individual isolate characteristics (Li et al. 91 2012a; Li et al. 2012b). We also found that the conidiation capacity varies in different 92 isolates and degradation with increasing numbers of transfers or, for certain isolates, 93 length of time kept in the laboratory. 94 In this study, we developed tissue media with rice leaves or panicles that could 95 promote the conidiation of U. virens. The optimal medium identified was especially 96 useful for stimulating conidiation in isolates with defective conidial production. 97 Although the conidia produced in this medium were small in size, they germinated 98 and infected rice similarly with these produced in normal PSB medium. 99

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U. virens isolates and rice cultivar panicles of the Indica and Japonica rice cultivars were collected for media preparation. 110 Subsequently, 0.5, 1.5, 3.0 and 5.0 g of leaves or panicles of Wanxian 98 or Huajing 111 952 were crushed with a 22, 000 rpm mixer (Midea, model number: MJ-250BP01B) 112 for 1 min in 40 ml distilled water respectively. Then they were used directly 113 (unfiltered) or filtered with three layers of gauze to prepare media with the final 114 concentrations of 0.01, 0.03, 0.06 and 0.10 g/ml respectively, and the media were 115 autoclaved at 121℃ for 30 min. 116 For conidiation tests, the strain G2 was incubated on PSA (potato sucrose agar: 200 117 ml of juice from 200 g of potato, 20 g of sucrose, and 15 g of agar per liter) at 27℃ 118 for 10 days, two 5-mm mycelial plugs were taken from the periphery of colony using 119 a cork borer and incubated in 50 ml of PSB (PSA without agar) or liquid rice tissue 120 media at 27℃ with shaking at 160 rpm. The production of conidia was investigated 121 from 3 rd to 7 th day using a hemacytometer. The conidiation from the filtered rice 122 tissue media were compared with unfiltered ones at the 7 th day post-incubation (DPI). 123 The conidiation in 0.06 g/ml leaf media were compared with 0.06 g/ml panicle media 124 at 7 DPI. The conidiation in 0.06 g/ml of Indica rice leaves (IRLs) were compared 125 with 0.06 g/ml of Japonica rice leaves (JRLs) at 7 DPI. Three replicates were

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Rice tissue media promoted the conidiation of U. virens 172 To develop a medium that promotes U. virens conidiation, different rice tissue 173 media were produced to investigate whether rice tissues can promote the conidiation of U. virens. Conidiation of strain G2 was evaluated at 3, 4, 5, 6 and 7 DPI. As shown 175 in Table 1, compared with PSB, Indica rice leaves or panicles media produced more 176 conidia at individual time points. These results indicated that Indica rice tissues could 177 promote conidia production. Among the four concentrations, in general, higher rice 178 tissue concentrations induced more conidia except for the medium with 0.10 g/ml 179 IRLs, which showed fewer conidia compared with other concentrations of IRLs 180 (Table 1). It is possible that too much fibrous tissue in the 0.10 g/ml IRL medium 181 hindered the smooth shaking that is important for conidia production.  For the different tissues, compared with IRP, the IRL media produced more conidia 208 at 0.01 (P = 3.218×10 -5 ) and 0.03 g/ml (P = 7.818×10 -5 ) at 7 DPI, but no significant 209 difference was observed at 0.06 g/ml (P = 0.086). However, at 0.10 g/ml, the IRLs 210 media produced fewer conidia compared with the IRP media (P = 2.867×10 -5 ). (P = 6.709×10 -7 ), and no significant difference was observed at 0.10 g/ml (P = 0.340). 219 JRP media produced more conidia at all four concentrations than JRPF media (Table   220 2). 221  collected, and the length and width were measured. The results showed that the conidia produced in PSB were larger than these produced in IRL, IRP, JRL and JRP 243 media, with these produced in the IRP media being the smallest (Table 3). These 244 results indicated that even though the rice tissue media could stimulate the conidiation 245 of U. virens, the conidia produced were smaller than these produced in PSB.
246 respectively, which were significantly lower than that from PSB (Fig 1). These results 267 showed that the germination of conidia from rice tissue medium was slower than that 268 from PSB, and generally the germination rate was higher on PSA than on WA. that mycelial growth was stimulated on some rice tissue media, the hyphae were loose 282 and mycelia were thin (Fig 2). These results showed that U. virens can grow on both 283 rice tissue media and PSA, but the overall biomass is decreased on rice tissue media, 284 suggesting that rice tissue media could not provide enough nutrients for mycelial 285 growth. inoculation results showed that more smut balls were observed for treatment III, 299 whereas few or no smut balls were observed for treatments I and II (Table 4). These 300 results showed that although the conidia produced in rice tissue media can germinate 301 and infect rice successfully, they should be incubated for 8-12 h in PSB before they 302 are used to inoculate rice.
303  313 As the best medium to induce conidiation among examined different media, the 314 0.06 g/ml IRL medium was used to investigate whether conidiation could be induced 315 in conidiation-defective isolates. The isolates D32-1, HWD2-2, UV-8, 09-11-1-1 and 316 09-14-21 were selected because they all fail to produce conidia or only produce few 317 conidia. Interestingly, for all of these isolates, conidia were produced at 3 or 4 DPI in 0.06 g/ml IRL medium, with substantial amounts of conidia produced at 6 or 7 DPI; 319 conversely, little or no conidia were observed in PSB. HWD2-2 and UV-8 produced 320 the most conidia (up to 2.3×10 7 conidia/ml) in the IRL medium, an amount equal to 321 that produced by non-defective isolates in PSB. The other isolates, D32-1, 09-11-1-1 322 and 09-14-21, produced fewer conidia but were still within the same order of 323 magnitude as that produced by HWD2-2 and UV-8. These results indicated that the 324 IRL medium could be used to induce conidiation in defective isolates (Table 5).