1 Yield losses and control by sedaxane and fludioxonil of soil-borne Rhizoctonia , 2 Microdochium , and Fusarium species in winter wheat

21 Soil-borne Rhizoctonia , Microdochium , and Fusarium species are major causal agents of seedling 22 and stem-base diseases in wheat, and currently seed treatments are considered the most effective 23 solution for their control. Rhizoctonia solani anastomosis groups (AGs) 2-1 and 5, R. cerealis , 24 Microdochium , and Fusarium spp. were used in series of field experiments to determine their 25 capability to cause soil-borne and stem-base disease and to quantify their comparative losses in 26 establishment and yield of wheat. The effectiveness and the response to seed treatment formulated 27 of 10 g sedaxane and 5 g fludioxonil 100 kg -1 against these soil-borne pathogens were also 28 determined. Our results showed that damping off caused by soil-borne R. cerealis was associated 29 with significant reductions in emergence and establishment resulting in stunted growth and low 30 plant numbers. The pathogen also caused sharp eyespot associated with reductions in ear 31 partitioning index. R. solani AG 2-1 or AG 5 were weakly pathogenic and failed to cause 32 significant damping off, root rot, or stem-base disease in wheat. Fusarium graminearum and F. 33 culmorum applied as soil-borne inoculum failed to cause severe disease. Microdochium spp. 34 caused brown foot rot disease and soil-borne M. nivale reduced wheat emergence. Application of 35 sedaxane and fludioxonil increased plant emergence and reduced damping off, early stem-base 36 disease, and brown foot rot, thus providing protection against multiple soil-borne pathogens. R. 37 cerealis reduced thousand grain weight by 3.6% whilst seed treatment of fludioxonil and sedaxane 38 against soil-borne R. cerealis or M. nivale resulted in 4% yield increase.

In this study therefore, we aimed to determine the yield losses and effectiveness of seed 88 treatments containing sedaxane and fludioxonil against the main soil-borne pathogens found in 89 English wheat fields. The main objectives were to i) quantify the disease effects of soil-borne R. 90 solani AG5, AG2-1, and R. cerealis on the host from emergence through to harvest yield, ii) 91 compare yield loss due to the most pathogenic Rhizoctonia spp. with soil-borne Fusarium 92 graminearum, F. culmorum, or Microdochium nivale, and iii) determine the effectiveness of 93 fungicide seed treatments on disease severity and yield response.  Furthermore, since sedaxane is commercially available in a formulation with fludioxonil, only the 105 formulated seed treatment was included in the second series of experiments.

106
The first field series of experiments with winter wheat cv. Santiago were designed as 107 randomised block with two factors, pathogen inoculation (not-inoculated control, AG 2-1, AG 5, 108 or R. cerealis) and seed treatment (untreated, fludioxonil (5 g a.i 100 kg -1 ), or sedaxane (10 g a.i assessment. At each sampled growth stage plants were removed retaining all above ground 140 biomass and as much of the top 15 cm of the root system as feasible.  Real-time PCR assays were performed for AG 2-1, AG 5, and R. cerealis from DNA extracted 176 from plants grown in plots inoculated with the aforementioned pathogens. Primers and probes 177 used in this study are shown in Table S3. The qPCR conditions used are as described by Woodhall     solani AG 5 at 18 dpi (P = 0.019) (Fig. 1a). Reductions of 14% were observed in inoculated plots 218 of R. cerealis compared to the not-inoculated control by 26 dpi. Fludioxonil alone or applied with 219 sedaxane increased emergence (Fig. 1b) and plant numbers at GS39 (Fig. 1c) by 33% and 15%, 220 respectively.

221
Rhizoctonia diseases and effects of seed treatments. Fludioxonil and fludioxonil + sedaxane 222 reduced root rot symptoms by 29.1% and 35.1%, respectively, compared to the untreated (Fig. 2a).  (Table 1). The interaction between inoculation and season was significant (P = 0.002) 234 showing that AG 5 caused greater stem browning compared to the not-inoculated control in In contrast, sedaxane + fludioxonil significantly reduced stem browning compared to the untreated 241 by 47% and 38% in 2012/13 and 2013/14, respectively (Table 1).

252
Effect of inoculation and seed treatment on Rhizoctonia spp. DNA in soil and in planta. 253 Pathogen DNA in soil samples was quantified at GS 15 ( Table 2). The highest DNA 254 concentrations were quantified in the inoculated untreated plots. DNA of R. cerealis was found at 255 > 4000 pg g -1 of soil, followed by AG 2-1 DNA > 220 pg ng -1 of soil and then by DNA of AG 5, 256 which differed significantly in 2012/13 and 2013/14 at 141 and 0.10 pg g -1 of soil, respectively 257 ( Table 2). DNA of AG 2-1, AG 5, and R. cerealis was detected in the roots at GS 31 and GS 75.

258
There was a significant interaction between inoculation and season for the amount of Rhizoctonia 259 spp. in the roots of the wheat host at GS 31 (P = 0.049) and at GS 75 (P = 0.001), possibly due to 260 inconsistency in DNA amounts of AG 5 quantified in the two seasons (Table 2). Treatment had 261 no effect on pathogen DNA in soil and in roots at GS 31. At GS 75, DNA in roots of AG 2-1 and R. cerealis was found at lower concentrations than at the previous growth stage (Table 2). There 263 was a significant interaction between inoculation and treatment (P = 0.025) associated with 264 inconsistency in the effect of treatments in inoculated plots with AG 5. Overall, DNA of AG 2-1 265 and R. cerealis in roots in both seasons was less in plots treated with sedaxane + fludioxonil (Table   266 2).

267
At GS 15, there was a significant interaction between inoculation, season, and treatment with 268 higher DNA concentrations of R. cerealis and AG 5 in stems in the first season, in contrast to AG 269 2-1 DNA which accumulated more in the second season (Table 3). Furthermore, the effectiveness  (Table   273 3). AG 2-1 and R. cerealis DNA in stems was 4 and 12.5-fold higher than AG 5, respectively (P 274 = 0.002) (Table 3). However, there was no effect of seed treatment, and there were no interactions  1 compared to the not-inoculated plots in both seasons (Table 5). This effect was negated by 286 fludioxonil and sedaxane + fludioxonil treatments in AG 2-1 and R. cerealis, however the effect 287 of seed treatments on plants in AG 5 inoculated plots was less consistent (Table 5).

288
In contrast to plant height, inoculation had no effect on GAI at GS 15 but fludioxonil increased 289 GAI of inoculated plots. Sedaxane + fludioxonil treatment showed the same effect in the first 290 season of experimentation, but in the second experiment this effect was not consistent in AG 5 and 291 AG 2-1-inoculated plots.

292
Ear partitioning index (EPI) is the fraction of above-ground DM partitioned in the ear.

297
Yield is presented for 2012/13 since in 2013/14 plots were too small (1 x 1 m) to accurately 298 assess field harvest yield on per ha basis. Differences for inoculation were not significant at 299 P<0.05, however yields of infected plots with R. cerealis, AG2-1, and AG5 were 0.83, 0.44, and 300 0.22 t ha-1 , respectively lower than that of the control (Fig. 4a). R. cerealis reduced TGW 301 significantly by 3.6% in both seasons compared to the not-inoculated control (Fig. 4b). There was 302 no significant effect of seed treatment on yield or TGW.

303
Relationships between disease assessments and pathogen DNA. Regression analyses (R 2 ) 304 using disease indexes and pathogen DNA revealed that there were no significant correlations 305 between Rhizoctonia DNA in roots and root rot (data not shown). There were significant but generally weak to moderate (R 2 ≤ 0.40) relationships between Rhizoctonia spp. DNA in stems and 307 symptoms on the stems (Table S5). The strongest relationship (R 2 = 0.60) was between  (Table S5). inoculated plots at GS 15 by 43% overall (Fig. 5c). There were no interactions between factors for 325 yield, and yield response to treatment was 0.27 t ha -1 (P = 0.02) (Fig. 5d).

432
Artificially inoculated experiments provide useful information on worst case scenarios for 433 losses due to pathogens applied at high inoculum density, and this approach is appropriate to 434 establish comparative differences and effectiveness of control methods to individual pathogens.

435
Under natural infection, the inoculum density of these pathogens is likely to be lower and for some