DataSheet_1_Wild Helianthus species: A reservoir of resistance genes for sustainable pyramidal resistance to broomrape in sunflower.pdf

Digital.CSIC. Repositorio Institucional del CSIC
Digital.CSIC. Repositorio Institucional del CSIC
  • Chabaud, Mireille
  • Auriac, Marie-Christine
  • Boniface, Marie-Claude
  • Delgrange, Sabine
  • Folletti, Tifaine
  • Jardinaud, Marie-Françoise
  • Legendre, Alexandra
  • Pérez-Vich, Begoña
  • Pouvreau, Jean-Bernard
  • Velasco Varo, Leonardo
  • Delavault, Philippe
  • Muños, Stéphane
Suppl. File 1. List of wild Helianthus accessions used in this study and associated phenotyping assays. Seventy one accessions were phenotyped in 6 L pots: 36 wild H. annuus accessions and 35 wild Helianthus accessions from other species than annuus, including 21 annual accessions from 8 species (12 taxa) and 14 perennial accessions from 7 species. Eighteen of these accessions were phenotyped for the exudate activity on broomrape seed germination and in rhizotrons. Suppl. File 2. Timeline of the culture and phenotyping in rhizotrons. Phenotyping in rhizotrons of the wild Helianthus species and the control cultivated H. annuus was performed at the attachment stage (14 days after inoculation-dai), tubercle stage (21 dai) and necrotic tubercle stage (28 dai). At 14 dai, samples (fragments of roots with compatible or incompatible attachments) were prepared for cytological studies. D0: Day 0 is the day of inoculation. Suppl. File 3. Numbers of rhizotrons and independent experiments/ accessions. Rhizotrons were inoculated with the race E-BOU (2017). At least 2 independent experiments were performed for each accession. The numbers of Non-Treated (NT) and Treated (T) rhizotrons (with GR24 + DCL) were variable depending on the germinating ability of the accession. At 14 dai and 21 dai, the number of attachments and tubercles were counted respectively. As one rhizotron/ experiment was used for cytological sampling at 14 dai, the number of rhizotrons differed between 14 and 21 dai. Suppl. File 4. Raw data of the number of attachments (at 14 dai), tubercles (at 21 dai) and necrotic tubercles (at 28 dai) / rhizotron for each accession. Raw data are detailed in the specific joined file. NA: data Not Available. Wild Helianthus plantlets were inoculated with conditioned Orobanche seeds (race E-BOU), following 15 days of culture in soil and 6 days in rhizotrons (see Materials and Methods and Suppl. File 2) except for the following experiments: I15: 17 days of culture in soil, and inoculation the day of transfer in rhizotron; I16 and I18: inoculation the day of transfer in rhizotron; I19: plantlets were grown 27 days in soil and 6 days in rhizotron before inoculation. Suppl. File 5. Germination dose-response curves of various O. cumana populations to various germination stimulants. Germination dose-response curves were modelled after normalization of the germination activity (bar : ± SD) thanks to the germination percentage of each populations obtained with 1 μM GR24 (Fig. 1a) using a four parameter logistic curve. For each compound, (±)-GR24; DCL and costunolide, and equimolar mixtures, a range of concentrations from 1 μM to 0.1 pM were applied to conditioned seeds of five O. cumana populations. Suppl. File 6. Half-maximal effective concentration (EC50) of various germination stimulants on various O. cumana populations. EC50 was determined for every compounds and mixtures, and for the five O. cumana populations thanks to the generated dose-response curves presented in the Suppl. File 5. Bar : ± SE. Different letters indicate significant differences at p < 0.05 (Student-Newman-Keuls Methodtest) between germination stimulants for a population (corresponding colored letters) or between population for a germination stimulant (black letters). Due to poor germination in response to DCL and costunolide, the EC50 of the G-RO population could not be determined. Suppl. File 7. Kinetic of GS activity exuded by a set of Helianthus accessions. Germination activities were normalized thanks to the germination percentage of both population obtained with GR24:DCL (equimolar, 1μM). For each accessions (except #774a and #826a), root exudates collected 3, 4, 5 and 6 weeks after sowing were applied to conditioned seeds of the two populations, E-BOU and G-RO. For #774a and #826a root exudates were collected at 3 and 4 weeks of culture only. Bar : ± SE. Suppl. File 8. Quantitative analysis of the phenotyping data at early stages (attachments and tubercles) in rhizotrons inoculated with the race E-BOU. Data were analysed taking into account only GS-treated rhizotrons, except for the cultivated susceptible controls XRQ and 2603. 8a. There was no significant statistical difference in the number of attachments at 14 dai between the wild resistant accessions except for the wild H. annuus #826a. 8b. Ordering the accessions by the efficiency of the development of attachments into tubercles (% of tubercle/ attachment) revealed a significant difference between phenotyping classes I and III, compared to the classes II and IV. Suppl. File 9. Quantitative analysis of the percentage of necrotic tubercles at 28 dai in rhizotrons inoculated with the race E-BOU. Data were analysed taking into account only GS-treated rhizotrons, except for the cultivated susceptible controls XRQ and 2603 (non-treated rhizotrons). Accessions without tubercle development were not taken into account (Class I and Class III, except #833a which develop few tubercles). Statistical analysis was performed using the Kruskal-Wallis test (α = 0.05). Suppl. File 10. List of wild Helianthus accessions used for cytological studies and number of observed samples for each accession. A few accessions from each phenotyping class in rhizotrons were used, inoculated with the race E-BOU. Whole root segment with attachment were cleared with chloral hydrate, or sectioned, imbedded in technovit system and stained with toluidine blue O. Attachments of accessions from classes I and III were all Incompatible (IA). Attachments of accessions from class IV were all Compatible (CA). For the accessions from Class II there were a mixture of IA and CA. As accessions from Classes I and III did not induce seed germination, attachments were sampled only from GS-treated rhizotrons for these accessions. Suppl. File 11. Summary of the the cellular phenotypes observed by cytology on attachments (14 dai). Common cellular resistant mechanisms led to incompatible attachments (IA) independently of the phenotyping classes in rhizotrons. Rarely, some defence reactions were observed in compatible attachments (CA). Suppl. File 12. Defence reactions at proximity of compatible attachments revealed by cytological study of Class II accessions. At 14 dai, accessions from class II, such as H. praecox, developed a mixture of incompatible attachments and compatible attachments. In some cases, defence reactions at proximity of these compatible attachments were observed as green staining suggesting phenolic compounds (a. accession #677 H. preacox; arrowheads), or gumlike substance in xylem vessels (b. accesion #679 H. praecox; red asterisks). Bar = 100 μ. Suppl. File 13. Phenotyping in 6 L pots of wild Helianthus using the most virulent broomrape races G. Boxplots of 7 accessions phenotyped in pots following inoculation with 4 races G (assay No 3) The accessions #2601 H. exilis and #325 H. tuberosus were from class I, #584 H. bolanderi, #677 H. praecox, #761 H. petiolaris and #786 H. debilis tardiflorus from class II and #833 H. annuus from class III. The 4 races G were from Romania (G-RO), Russia (G-RU), Spain (G-GV) and Turkey (G-TK). Five to 8 pots were cultured for each accession/ race. In each pot, the number of broomrape emergences was counted at the time of sunflower flowering. For each accession, Kruskal-Wallis test was performed (α=0.05). The p values were respectively: #2601ex: p=0.29; #325t (no p value); #584b: p=0.52; #677pr: p=0.12; #761p: p=0.004; #786d: p=0.03; #833a: p=0.06; B117: p=0.29; LP2:p=0.008., Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species., Peer reviewed
Digital.CSIC. Repositorio Institucional del CSIC

Digital.CSIC. Repositorio Institucional del CSIC
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Digital.CSIC. Repositorio Institucional del CSIC