Plant material
A total of 42 D. sophia populations were collected from winter wheat fields of Henan (H1-11) and Xinjiang (X1-31) in China during 2015–2017. The geographical origin and collection year of D. sophia populations were provided in Table S1 and Fig. S1. A tribenuron-methyl susceptible population of D. sophia (X13) was collected from Urumqi, Xingjiang.
Seeds were soaked with 15% H2O2 for 30 min to break dormancy and rinsed thoroughly by water. The treated seeds were placed in moist petri dishes and then transferred to artificial climate chamber at 15 ℃, light/dark, 16/8 h for germination. After a week, the seedlings were transplanted to 10-cm diameter plastic pots containing loam soil (12 plants per pot) and then cultured in artificial climate chamber at 25/15 ℃, light/dark, 16/8 h with light intensity of 15,000 Lux. The seedlings were used in the following procedures.
Single dose resistance assay
The discriminating dose of tribenuron-methyl at 18 g ai ha−1 was sprayed to the plants at 4-leaf stage using a potter precision laboratory spray tower (Burkard Scientific, UK) delivering 600 L ha−1 water at the pressure of 0.3 MPa. The fresh weight of aboveground of the plants were determined after tribenuron-methyl application for 21 days and the fresh weight reduction rate were calculated. The susceptibility of D. Sophia populations to tribenuron-methy was identified according to Moss et al.23 and populations classified as high resistance (RRR) were selected for further mutation type determination.
Detection of ALS isozymes mutation
Genomic DNA of RRR D. sophia populations were extracted from the survived plant using Wizard® Genomic DNA Purification Kit (Promega, Madison, WI). Primer pairs, PCR reaction and program cycle in Xu et al.1 were used to detect the eight resistance mutation sites in ALS isozymes. PCR products were purified with Wizard® SV Gel and PCR Clean-Up System (Promega) and inserted to pLB vector using Lethal Based Fast Cloning Kit (Tiangen, Beijing, China). The mixture was transformed to TOP10 competent E. coli (Tiangen) and finally sequenced by Shanghai Sangon Biological Engineering and Technology Service Co. (Shanghai, China). Ten individual plants and three clones of each were selected for ALS mutation detection.
Generation of RRR homozygous subpopulations
Plants of RRR population with same mutation type in ALS isozymes were cultured to generate seeds. Homozygous subpopulation of susceptible population X13 with wild type of ALS isozymes were also obtained by inbred. In this way, six purified subpopulations (SX13, SX30, SX31, SH5, SH6, SH7) homozygous for wild type, Pro197Ser, Pro197His, Pro197Ala, Pro197Thr mutations, were obtained and used for the following experiments.
Dose response of RRR D. sophia subpopulations to tribenuron-methyl
Whole-plant dose response experiment was employed to identify the GR50 of the RRR homozygous subpopulations. Seeds of subpopulation were cultured as mentioned previously. Tribenuron-methyl was applied to SX13 (0, 0.08, 0.16, 0.33, 0.66, 1.32, 2.64, 5.28, 10.56, 21.12 g ai ha−1), SH7 (0, 5, 10, 20, 40, 80, 160, 320 g ai ha−1) and SH5, SH6, SX30, SX31 (0, 50, 100, 200, 400, 800, 1600, 3200, 6400, 12,800, 25,600 g ai ha−1) subpopulations at 4-leaf stage using a potter precision laboratory spray tower delivering 600 L ha−1 water at the pressure of 0.3 MPa. The aboveground of the plants were harvested after treated for 21 days and the fresh weight was recorded. Each herbicide dose was conducted with three replications and repeated twice. GR50 was calculated by log-logistic equation24:
$${text{y}} = {text{C}} + {{left( {{text{D}} – {text{C}}} right)} mathord{left/ {vphantom {{left( {{text{D}} – {text{C}}} right)} {left[ {1 + left( {{text{x}}/{text{GR}}_{50} } right)^{{text{b}}} } right]}}} right. kern-nulldelimiterspace} {left[ {1 + left( {{text{x}}/{text{GR}}_{50} } right)^{{text{b}}} } right]}}$$
where C and D are the lower limit and upper limit, b is the slope, x is the herbicide dose, and y represents plant fresh weight as percentage of the control. RI, the ratio of GR50 of resistant populations to that of the susceptible population, was used to represent the resistance level.
Cross-resistance patterns of RRR D. sophia subpopulations to other ALS-inhibiting herbicides
Other ALS-inhibiting herbicides, including flucarbazone-sodium (SCT), bensulfuron-methyl (SU), flumetsulam (TP), florasulam (TP), pyroxsulam (TP), imazapic (IMI) and bispyribac-sodium (PTB) were applied to D. sophia at 4-leaf stage with 1×, 5× and 10× fold of the recommendation doses. The herbicides and recommendation doses were listed in Table S2. The survival plant was recorded after treated for 21 days and each dose was replicated with three plastic pots containing 36 plants. Cross-resistance was confirmed as more than 50% individuals survived in the resistant population and less than 10% plants survived in the susceptible population6,25.
Determination of RGR, LAR, NAR and RCC in susceptible and RRR subpopulations
RGR, LAR and NAR were used to indicate the nutritional growth level of susceptible and resistant homozygous subpopulations of D. sophia. RCC was used to evaluate their relative competition ability. RGR, LAR, NAR and RCC were determined according to Zhang et al. with a little modification17.
Under monoculture condition, seeds of each subpopulation were planted separately with three replications and repeat twice. The aboveground tissues of D. sophia without herbicide treatment were sampled at 28, 35 and 42 days after transplant (DAT) to compare the nutritional growth between susceptible and resistant subpopulations. All leaves of the harvested plant were placed on A4 paper drawing with 1 cm2 square and photoed to calculate the leaf area by Photoshop CS3 extended (Adobe Systems Inc., USA). The dry weight was measured after the sample oven dried 96 h at the temperature of 60 ℃. RGR was estimated by the formula RGR = (ln W2 − ln W1)/(t2 − t1)26. LAR and NAR were calculated by the formula LAR = [(ln W2 − ln W1)(LA2 − LA1)]/[(W2 − W1)(ln LA2 − ln LA1)] and NAR = [(W2 − W1) (ln W2 − ln W1)]/[(LA2 − LA1)(t2 − t1)]27. W1 and W2 indicated dry weight per plant at times t1 and t2, respectively. LA1 and LA2 means leaf area per plant at t1 and t2, respectively.
Under admixture condition, plants of susceptible and resistant subpopulations were cultured at a series ratio of S:R = 1:0, 3:1, 1:1, 1:3, 0:1 at a constant density of 644 plants m−2 (24 plants per tray, 23.3 cm × 16.0 cm × 6.0 cm) according to Reboud et al.28. The experiment was conducted with three replications and repeat twice. The aboveground shoots of each plant were harvested at 50 DAT and the leaf area and the dry weight were measured. RCC was calculated according to the formula: RCC = ({(DBS1:3/DBR1:3) + (DBS1:1/DBR1:1) + (DBS3:1/DBR3:1)}/N)/(DBS1:0/DBR1:0)29,30. DBSn:n and DBRn:n means the dry weight or the leaf area of each plant in susceptible and resistant subpopulations planted at ratio of n:n. N is the number of mixed ratio; here N = 3. RCC value greater than 1.0 suggested a superior competition ability of susceptible population. While RCC value less than 1.0 indicated lower competition ability of susceptible population.
Statistical analysis
The data of bioassay was analyzed with SigmaPlot 12.0 (Systat Software, San Jose, CA). The statistical difference of the leaf area, dry weight, RGR, LAR, NAR of D. sophia populations with different ALS mutation were subjected to one-way analysis of variation (ANOVA) followed by Tukey’s multiple comparisons test using SPSS 16.0 (SPSS, Chicago, IL, USA). The criterion for statistical significance was P < 0.05.
Ethics declarations
Descurainia sophia is a notorious weed widely distributed in the wheat fields or wild field of Henan and Xinjiang Province in China. Farmers need to spend a lot of money and manpower to get rid of them in their fields each year. In fact, common weed seeds collection in China is allowed and frequently used in weed research. Thus, collection of D. sophia seeds is permitted naturally and had no influence on the environment. Besides, we have got the oral permission of the field owners when D. sophia seeds were collected from wheat fields.
Source: Ecology - nature.com
