Nematode counts
Nematode levels in the soil were significantly less when the resistant ‘Freedom’ was used instead of susceptible ‘O39-16’ as the rootstock (Mann-Whitney U = 73.000; P = 0.038) (Fig. 1A). Fewer nematodes were counted in the soil in 2015 than 2016 (Mann-Whitney U = 50.500; P = 0.003). There were no differences in soil nematode counts between sampling done in week 6 compared with week 12 (Mann-Whitney U = 104.500; P = 0.375).
Nematode counts in the roots were greater in the susceptible ‘O39-16’ rootstocks than resistant ‘Freedom’ (Mann-Whitney U = 60.500; P = 0.009) (Fig. 1B). Nematode counts in root samples also were less in 2015 than 2016 (Mann-Whitney U = 62.500; P = 0.011). There was no a significant difference in root nematode counts when samples were collected at 6 versus 12 weeks (Mann-Whitney U = 113.500; P = 0.574).
These results confirm previous observations, as it was observed that ‘Freedom’ rootstocks possess high levels of resistance to nematode infections whereas ‘O39-16’ rootstocks were susceptible [7, 13]. However, little was known about the mechanisms of resistance, that is, whether chemical or physical barriers prevented nematode infections. The analyses of stilbenoids in this study provide evidence that resistance could be imparted by differences in host defense compound levels between these two rootstocks.
Root stilbenoid levels
A total of eleven stilbenoids were putatively identified in this study, and each compound was analyzed by week and year separately (Table 1). The stilbenoid compounds quantified in this study were similar as those found in other studies (Table 1), albeit the resveratrol glycoside piceid was not observed in quantifiable amounts, with only trace characteristic ions observed by LC-MS, despite being observed previously [10, 14]. However, piceid also was not present in sufficiently quantifiable amounts in V. vinifera by Lambert et al. [15] or in many of the wild Vitis spp. studied by Pawlus et al. [16]. The putatively identified miyabenol C, hopeaphenol, and ε-viniferin were the most prevalent stilbenoids observed in this study, which was like previous observations [10, 14–15].
For most analyses, there were no significant differences due to infection status, with a few exceptions (Table 2). Nematode infections increased levels of piceatannol, ampelosin D/quandrangularin A, and ɑ-viniferin in week 12 of 2016. By contrast, ampelopsin A, ω-viniferin, and vitisin B were present in lower levels in nematode infected plants compared to controls during week 12 of 2016. Pallidol had lower levels in nematode infected plants compared to controls in week 6 of 2016. Potentially, a greater number of nematodes present in soil in 2016 (Fig. 1a) resulted in these observations.
Table 2
Mean (± SE) concentrations of individual stilbenoids (µg/g FW) in healthy or RKN-infected roots.
| | | | O39-16 | Freedom | | F-statistic | |
Stilbenoid Type | Putative Name | Year | Week | Control | RKN | Control | RKN | Cultivar | Inoculation | Interaction |
Monomer | piceatannol | 2015 | 6 | 17.6 ± 6.5 | 37.3 ± 17.0 | 12.5 ± 3.6 | 19.0 ± 4.0 | 1.521 | 1.912 | 0.490 |
| | | 12 | 15.5 ± 2.7 | 75.6 ± 27.8 | 11.9 ± 2.9 | 17.6 ± 0.8 | 4.839* | 5.502* | 3.756 |
| | 2016 | 6 | 17.5 ± 4.0 | 35.0 ± 8.5 | 36.7 ± 6.0 | 30.2 ± 18.5 | 1.588 | 0.954 | 4.509 |
| | | 12 | 26.4 ± 3.0 | 36.2 ± 11.8 | 31.8 ± 1.1 | 39.9 ± 3.7 | 0.337 | 1.709 | 0.001 |
| resveratrol | 2015 | 6 | 301 ± 99 | 1170 ± 660 | 105 ± 29 | 131 ± 43 | 3.383 | 1.770 | 1.567 |
| | | 12 | 100 ± 27 | 738 ± 486 | 106 ± 13 | 111 ± 40 | 1.625 | 1.737 | 1.686 |
| | 2016 | 6 | 216 ± 27 | 323 ± 151 | 299 ± 79 | 192 ± 22 | 0.078 | 0.000 | 1.520 |
| | | 12 | 281 ± 88 | 490 ± 226 | 204 ± 25 | 252 ± 14 | 1.659 | 1.112 | 0.434 |
Dimer | ampelopsin A | 2015 | 6 | 250 ± 51 | 265 ± 62 | 204 ± 44 | 281 ± 72 | 0.055 | 0.512 | 0.241 |
| | | 12 | 255 ± 51 | 314 ± 67 | 235 ± 28 | 228 ± 28 | 1.211 | 0.293 | 0.469 |
| | 2016 | 6 | 451 ± 59 | 480 ± 45 | 408 ± 77 | 304 ± 36 | 3.804 | 0.449 | 1.393 |
| | | 12 | 578 ± 47 | 307 ± 22 | 288 ± 26 | 324 ± 29 | 17.527** | 13.049** | 22.154*** |
| ampelopsin D /quadrangularin A | 2015 | 6 | 11.9 ± 3.8 | 24.6 ± 9.5 | 21.8 ± 4.6 | 27.8 ± 4.3 | 1.206 | 2.435 | 0.309 |
| | | 12 | 10.2 ± 1.8 | 38.3 ± 13.0 | 23.3 ± 1.7 | 26.1 ± 1.4 | 0.005 | 5.356* | 3.620 |
| | 2016 | 6 | 26.7 ± 7.5 | 32.0 ± 9.8 | 51.1 ± 3.7 | 51.7 ± 1.3 | 11.509** | 0.209 | 0.133 |
| | | 12 | 28.3 ± 5.4 | 30.0 ± 6.3 | 52.1 ± 6.9 | 58.6 ± 17.6 | 6.430* | 0.156 | 0.054 |
| ε-viniferin | 2015 | 6 | 350 ± 33 | 212 ± 88 | 218 ± 63 | 174 ± 56 | 1.833 | 2.092 | 0.567 |
| | | 12 | 267 ± 91 | 223 ± 23 | 131 ± 9 | 181 ± 40 | 3.024 | 0.004 | 0.836 |
| | 2016 | 6 | 387 ± 55 | 372 ± 47 | 274 ± 49 | 234 ± 27 | 7.544* | 0.360 | 0.076 |
| | | 12 | 379 ± 9 | 214 ± 51 | 248 ± 18 | 331 ± 73 | 0.022 | 0.828 | 7.445* |
| pallidol | 2015 | 6 | 69.5 ± 10.5 | 84.6 ± 20.7 | 182 ± 6 | 184 ± 40 | 20.386*** | 0.135 | 0.077 |
| | | 12 | 61.3 ± 10.2 | 80.5 ± 18.4 | 141 ± 19 | 177 ± 32 | 17.415*** | 1.737 | 0.165 |
| | 2016 | 6 | 86.0 ± 18.6 | 127 ± 15 | 122 ± 13 | 149 ± 6 | 4.433 | 5.890* | 0.255 |
| | | 12 | 125 ± 18 | 104 ± 17 | 162 ± 20 | 197 ± 43 | 6.069* | 0.070 | 1.112 |
| ω-viniferin | 2015 | 6 | 247 ± 49 | 208 ± 26 | 83.4 ± 16.6 | 97.0 ± 21.7 | 20.006*** | 0.175 | 0.739 |
| | | 12 | 358 ± 30 | 304 ± 43 | 103 ± 9 | 105 ± 13 | 68.739*** | 0.930 | 1.029 |
| | 2016 | 6 | 363 ± 51 | 459 ± 28 | 298 ± 75 | 204 ± 16 | 11.045** | 0.001 | 3.911 |
| | | 12 | 528 ± 46 | 312 ± 17 | 196 ± 14 | 241 ± 12 | 57.218*** | 10.148** | 23.936*** |
Trimer | α-viniferin | 2015 | 6 | 33.1 ± 2.4 | 27.9 ± 5.6 | 32.7 ± 5.8 | 36.8 ± 8.1 | 0.532 | 0.008 | 0.631 |
| | | 12 | 41.2 ± 5.8 | 42.1 ± 5.1 | 62.9 ± 2.9 | 63.4 ± 4.2 | 21.407*** | 0.026 | 0.003 |
| | 2016 | 6 | 78.8 ± 5.5 | 63.2 ± 2.9 | 82.6 ± 6.9 | 99.5 ± 16.2 | 4.615 | 0.005 | 3.036 |
| | | 12 | 53.3 ± 2.4 | 61.2 ± 5.5 | 74.5 ± 6.5 | 96.1 ± 5.5 | 28.269*** | 7.844* | 1.661 |
| miyabenol C | 2015 | 6 | 6.39 ± 1.42 | 11.2 ± 7.4 | 148 ± 22 | 169 ± 24 | 53.106*** | 0.411 | 0.165 |
| | | 12 | 6.62 ± 0.90 | 19.2 ± 4.9 | 187 ± 9 | 189 ± 29 | 101.094*** | 0.171 | 0.096 |
| | 2016 | 6 | 69.8 ± 52.5 | 11.1 ± 2.8 | 190 ± 60 | 219 ± 18 | 16.031** | 0.128 | 1.155 |
| | | 12 | 8.67 ± 1.30 | 11.3 ± 2.1 | 216 ± 15 | 235 ± 1 | 805.294*** | 1.923 | 1.094 |
Tetramer | hopeaphenol | 2015 | 6 | 246 ± 45 | 176 ± 19 | 875 ± 224 | 1032 ± 300 | 15.514** | 0.053 | 0.362 |
| | | 12 | 352 ± 36 | 266 ± 41 | 884 ± 112 | 879 ± 167 | 30.134*** | 0.189 | 0.149 |
| | 2016 | 6 | 596 ± 207 | 439 ± 19 | 1100 ± 230 | 1260 ± 120 | 16.355** | 0.000 | 0.913 |
| | | 12 | 423 ± 39 | 282 ± 20 | 1140 ± 70 | 1290 ± 30 | 388.179*** | 0.159 | 16.672** |
| vitisin B | 2015 | 6 | 81.6 ± 17.3 | 69.7 ± 13.1 | 8.79 ± 1.36 | 9.70 ± 2.08 | 50.634*** | 0.345 | 0.468 |
| | | 12 | 131 ± 11 | 104 ± 18 | 9.81 ± 1.12 | 12.2 ± 1.9 | 104.258*** | 1.334 | 1.910 |
| | 2016 | 6 | 113 ± 32 | 162 ± 10 | 57.0 ± 38.1 | 17.3 ± 2.7 | 15.611** | 0.033 | 3.044 |
| | | 12 | 185 ± 19 | 105 ± 5 | 17.2 ± 1.4 | 20.1 ± 1.3 | 167.010*** | 15.505** | 17.961** |
*P < 0.05; ** P < 0.01; *** P < 0.001 |
Figure List |
The susceptible ‘O39-16’ rootstocks consistently possessed greater levels of ω-viniferin and vitisin B than the resistant ‘Freedom’ rootstock (Table 2). By contrast, ‘Freedom’ rootstocks consistently possessed greater levels of miyabenol C and hopeaphenol (Table 2). Previously, Lambert et al. [15] observed vast differences in stilbenoid concentration among many V. vinifera cultivars, including the presence or virtual absence of certain compounds such as miyabenol C and vitisin B. Furthermore, Pawlus et al. [16] observed differences and presence or absence of certain stilbenoids among wild Vitis spp. as well. Unlike this study, Pawlus et al. [16] did not examine currently available commercial rootstock cultivars. Furthermore, although Lambert et al. [15] and Pawlus et al. [16] observed chemistry of stem tissues, this study determined similar differences when comparing stilbenoid levels in the roots of different species-derived rootstocks, namely large differences in certain specific compounds.