Part I: identification and pathogenicity characterization

Figures and analyses

Figure 1. Map of sampled soybean fields in 2011 and 2012, and intensity of planted soybean acres demonstrated by color intensity at the county/parish level.

Figure 2. Frequency at which different Oomycete species were recovered from diseased soybean seedlings in 2011 and 2012. (*) Species previously reported as associated with soybean in the fungal-host database (http://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm).

Figure 3. Frequency of the oomycete species summarized by clade for 2011 and 2012. Oomycete genera outside of Pythium and Phytophthora were summarized by genus. Those species designated as spp. are not well resolved based only on the ITS sequence.

For seed rot analyses in detail, please check the seed_rot_data_analysis

Figure 4. Mean disease severity index of 84 oomycete species screened in a seed rot assay at 13ºC and 20ºC. Bars represent standard error and disease severity index values from 0 = non-pathogenic to 100 = highly pathogenic.

Figure 5. (A) Hierarchical analysis of the disease severity index of 84 oomycete species to establish three clusters related to pathogenicity on soybean and (B) boxplot of disease severity index by clusters at 13°C and 20°C as evaluated in a seed rot assay.

For seedling root rot analyses in detail, please check the seedling_data_analysis

Figure 6. Mean soybean root area (cm2), mean root length (cm), and mean weight per root (mg) after being challenged by 84 oomycete species using a soybean seedling root rot assay. Bars represent standard error and darker points represent species significantly different from the non-rice control (P < 0.05).

Figure 7. Phylogram for the ITS sequences of the rDNA for oomycete species found during the survey. Phylogenetic distribution of pathogenicity traits mapped to taxa represent in the tree. Tip colors indicate members of different clades. Parameters in light gray represent taxa not isolated in the study.

Figure 8. Prevalence of pathogenic oomycete species, designated based on seedling assay data, across the states sampled during the current study. Color gradient indicates number of isolates per species collected per state during 2011 and 2012.

Tables

Table 1. Mean disease severity index (DSI) of soybean cv. Sloan seeds in response to multiple oomycete species as compared to the non-inoculated control at 13ºC or 20ºC. Only species with significant differences from the control at either temperature are represented (Complete table, see Supplementary Table 2).
A seed rot assay was used to determine pathogenicity of oomycete species using a disease severity scale of 0-4 to rate individual seeds. Data were transformed to disease severity index (0=non-pathogenic; 100=highly aggressive).

Species N mean_DI13 std_DI13 Pval_13C mean_DI20 std_DI20 Pval_20C
Phytophthora drechsleri 1 13.056 3.194 1.000 90.278 2.017 0.048 NA
Phytophthora sansomeana 2 58.889 10.007 1.000 94.722 1.308 0.004 NA
Phytopythium helicoides 2 22.500 1.896 1.000 91.944 1.611 0.004 NA
Pythium aff. diclinum 3 78.704 2.501 0.032 32.963 5.282 1.000 NA
Pythium aff. dictyosporum 2 93.333 1.143 0.006 85.139 4.223 0.017 NA
Pythium aphanidermatum 3 53.241 3.823 1.000 99.907 0.093 0.000 NA
Pythium cryptoirregulare 1 99.722 0.278 0.018 95.556 1.002 0.021 NA
Pythium intermedium 3 83.056 4.780 0.015 53.426 6.643 1.000 NA
Pythium irregulare 3 98.889 0.449 0.001 80.463 3.531 0.027 NA
Pythium kunmingense 2 100.000 0.000 0.002 89.306 1.496 0.007 NA
Pythium paroecandrum 3 93.981 1.636 0.002 49.259 4.369 1.000 NA
Pythium spinosum 3 80.556 4.118 0.023 44.722 6.999 1.000 NA
Pythium sylvaticum 3 99.444 0.244 0.001 74.444 2.636 0.083 NA
Pythium terrestris 1 99.167 0.589 0.021 77.778 3.422 0.270 NA
Pythium ultimum 3 99.167 0.298 0.001 99.259 0.322 0.000 NA
Pythium ultimum var. sporangiiferum 3 96.481 1.044 0.001 98.056 0.723 0.001 NA
Pythium ultimum var. ultimum 3 99.815 0.128 0.001 99.630 0.219 0.000 NA
Control NA 2.579 0.310 NA 8.730 0.767 NA

Table 2. Forty-three oomycete species highly aggressive on soybean cv. Sloan in the seedling root rot assay measured as root area, root length and dry weight per root. Only species with significant differences from the non-rice control are represented (Complete table, see Supplementary Table 3). Data were analyzed by multivariate analysis of variance (MANOVA) combining all the parameters: root area, root length and weight per root; and followed by univariate analysis for each of the measured parameters. Plants were grown at 20ºC in a growth chamber for 2 weeks.

Species N mnv.p ar ar.se ar.p ln ln.se ln.p wpr wpr.se wpr.p
Aphanomyces cladogamus 9 0.000 2.330 0.503 0.000 31.646 7.178 0.002 28.667 4.853 0.044
Aphanomyces cochlioides 9 0.019 4.379 0.613 0.019 53.977 9.329 1.000 38.000 6.000 1.000
Phytophthora drechsleri 9 0.000 2.257 0.582 0.000 21.703 5.614 0.000 16.444 2.863 0.000
Phytophthora rosacearum 27 0.000 4.973 0.631 0.000 58.232 8.622 0.031 42.000 2.377 1.000
Phytophthora sansomeana 18 0.000 0.258 0.063 0.000 2.254 0.563 0.000 2.333 0.518 0.000
Phytophthora sojae 27 0.000 2.147 0.389 0.000 28.181 5.324 0.000 20.926 3.647 0.000
Phytopythium aff. vexans 18 0.001 4.813 0.442 0.001 54.681 4.386 0.388 38.333 1.955 0.587
Phytopythium chamaehyphon 27 0.000 4.637 0.346 0.000 54.824 3.552 0.163 42.074 2.074 1.000
Phytopythium helicoides 18 0.000 3.794 0.318 0.000 41.125 2.731 0.016 36.111 1.699 0.251
Phytopythium litorale 27 0.002 5.076 0.352 0.002 63.166 4.454 0.677 53.481 1.730 1.000
Phytopythium mercuriale 27 0.005 5.288 0.362 0.005 65.888 4.230 1.000 54.815 1.758 1.000
Pythium aff. diclinum 27 0.000 1.768 0.426 0.000 18.602 4.557 0.000 28.741 4.584 0.000
Pythium aff. dictyosporum 18 0.000 2.186 0.498 0.000 22.220 5.379 0.000 18.778 4.110 0.000
Pythium aff. dissotocum 27 0.000 2.715 0.301 0.000 38.133 4.282 0.000 27.926 2.545 0.000
Pythium aff. torulosum 27 0.000 2.889 0.352 0.000 38.977 5.004 0.000 28.222 2.536 0.000
Pythium aphanidermatum 27 0.000 4.073 0.350 0.000 51.256 4.599 0.016 56.741 2.906 1.000
Pythium attrantheridium 27 0.000 2.051 0.247 0.000 27.772 3.462 0.000 23.259 2.368 0.000
Pythium coloratum 27 0.000 4.316 0.521 0.000 51.893 6.566 0.003 56.593 3.759 1.000
Pythium conidiophorum 27 0.002 5.518 0.623 0.002 64.866 6.973 0.343 59.481 3.862 1.000
Pythium contiguanum 27 0.019 5.440 0.472 0.019 66.416 4.915 1.000 62.074 2.859 1.000
Pythium cryptoirregulare 9 0.000 1.740 0.215 0.000 17.280 2.335 0.000 19.556 2.375 0.000
Pythium heterothallicum 27 0.000 2.431 0.254 0.000 34.058 3.957 0.000 27.556 2.349 0.000
Pythium hypogynum 27 0.002 5.164 0.379 0.002 62.808 4.852 0.339 53.185 2.230 1.000
Pythium intermedium 27 0.000 3.410 0.632 0.000 42.129 7.779 0.000 41.852 5.690 0.000
Pythium irregulare 27 0.000 1.618 0.291 0.000 19.662 3.980 0.000 20.963 2.325 0.000
Pythium kunmingense 18 0.000 1.301 0.108 0.000 10.641 1.174 0.000 13.667 1.510 0.000
Pythium litorale 9 0.023 4.599 0.618 0.023 53.376 6.658 1.000 38.000 3.986 1.000
Pythium longandrum 27 0.049 5.578 0.401 0.049 67.096 4.512 1.000 59.259 2.697 1.000
Pythium longisporangium 27 0.004 5.844 0.578 0.004 61.178 5.407 0.139 44.667 3.257 0.894
Pythium lutarium 27 0.000 2.535 0.333 0.000 33.427 4.884 0.000 26.148 2.542 0.000
Pythium minus 27 0.001 5.075 0.365 0.001 58.632 3.784 0.451 48.444 2.731 1.000
Pythium nagaii 27 0.026 5.602 0.321 0.026 71.571 3.604 1.000 52.889 2.112 1.000
Pythium nunn 27 0.039 5.760 0.411 0.039 69.767 4.598 1.000 61.556 2.414 1.000
Pythium oopapillum 27 0.000 2.670 0.368 0.000 36.522 4.982 0.000 27.926 2.967 0.000
Pythium periilum 27 0.002 5.543 0.561 0.002 59.440 5.378 0.186 49.556 4.088 1.000
Pythium periplocum 27 0.000 4.998 0.716 0.000 52.741 7.378 0.000 38.489 4.391 0.003
Pythium sylvaticum 27 0.000 1.995 0.241 0.000 26.115 3.424 0.000 28.667 3.027 0.000
Pythium tardicrescens 27 0.000 4.745 0.629 0.000 57.746 7.890 0.001 43.704 4.007 0.104
Pythium terrestris 9 0.000 2.379 0.368 0.000 23.475 4.138 0.000 36.222 4.576 1.000
Pythium ultimum 27 0.000 0.183 0.039 0.000 1.539 0.431 0.000 5.111 1.397 0.000
Pythium ultimum var. sporangiiferum 27 0.000 1.337 0.240 0.000 15.196 3.169 0.000 19.200 2.497 0.000
Pythium ultimum var. ultimum 27 0.000 0.481 0.070 0.000 4.228 0.738 0.000 8.256 1.252 0.000
Pythium vanterpoolii 27 0.022 6.023 0.567 0.022 70.135 7.034 1.000 47.704 2.306 1.000

Supplementary figures

Supplementary Figure 1. Comparison of the non-inoculated controls and inoculated seedlings with the 84 oomycete species combined for the four parameters measured: (A) root area (cm2), (B) root length (cm), (C) weight per root (mg), and (D) weight per shoot (mg). Boxplot represent distribution of data, the line represents the median for each group and dots indicates outliers.

Supplementary Figure 2. Principal Component Analysis (PCA) on seedling parameters measured to determine correlation and contribution for the evaluation pathogenic/non-pathogenic oomycete species. The values on parenthesis indicate the percent of variance explained by the respective axis. The length and the direction of the vectors indicate the contribution of each parameter to the corresponding variance.

Supplementary Figure 3. Distribution of parameters analyzed: (A) root area (cm2), (B) root length (cm), and (C) weight per root (mg), on three groups based on the significance of the MANOVA and univariate analysis: group 1 (non-significant for all analyses), group 2 (significant for MANOVA, but not for all univariate analyses), group 3 (significant for both MANOVA and univariate analyses). Boxplot represent distribution of data, the line represents the median for each group and dots indicates outliers.

Supplementary Figure 4. Effect of media on the frequency of recovery of oomycete species summarized by clade in 2012. Numbers at the end of the bar represent number of total isolates per clade.