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• AIMS AND PLANT MATERIALS • 1 – A preliminary step : to check the level of aggressiveness

AGGRESSIVENESS AND PECTINOLYTIC ACTIVITIES WITHIN FOUR EUROPEAN POPULATIONS OF OROBANCHE CUMANA COLLECTED IN SUNFLOWER FIELDS Christophe V É RON É SI and collaborators GPPV, Nantes University (France).

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• AIMS AND PLANT MATERIALS • 1 – A preliminary step : to check the level of aggressiveness

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  1. AGGRESSIVENESS AND PECTINOLYTIC ACTIVITIES WITHIN FOUR EUROPEAN POPULATIONS OF OROBANCHE CUMANA COLLECTED IN SUNFLOWER FIELDS Christophe VÉRONÉSI and collaborators GPPV, Nantes University (France) P. SIMIER, S. THOIRON, H. BENHARRAT, C. THÉODET, S. CONSTANT, P. THALOUARN (dir), A. FER, P. DELAVAULT K. PAGEAU, C. VIEIRA DOS SANTOS, C. VÉRONÉSI, M-C. ARNAUD, P. LABROUSSE, D. BOZEC.

  2. Sunflower lines Resistance “gene” Resistance to “races” • AIMS AND PLANT MATERIALS • 1 – A preliminary step : to check the level of aggressiveness of the four european broomrape populations used in this study 2603 K41 Ha89 Record PH-BC2-56 LC1093A Kremnij None Or1 Or2 Or3 Or4 Or5 Or6 Control of susceptibility A A+B A+B+C A+B+C+D A+B+C+D+E A+B+C+D+E+F Methods. Typical differential screening experiments in pots with 7 sunflower lines kindly provided by André Berville (INRA, Montpellier) Sunflower lines were infested by 4 populations of broomrape • population collected in Romania • Population collected in Bulgaria • two populations collected in Spain Co-cultures in greenhouse (10 mg broomrape seeds per plant)

  3. • AIMS AND MAJOR RESULTS • 1 – A preliminary step : to check the level of aggressiveness of the four european broomrape populations used in this study • 70 days after sunflower sowing, infestation was estimated • by counting attached broomrapes. • Broomrapes were divided into three developmental stages : • Young tubercles carrying neither adventitious roots nor a stem • (stages 1 and 2, Labrousse et al. 2001 and 2004 ), • Tubercules carrying adventitious roots and a growing stem • (stage 3, 4) • - Emergences (stage 5) Stage 1 Stage 2 Stage 4 Stage 3 Stage 5 2603 infested 2603 healthy

  4. • AIMS AND MAJOR RESULTS • 1 – A preliminary step : to check the level of aggressiveness of the four european broomrape populations used in this study Aggresiveness of the Romanian population is high against the line PHBC2-56. Resistance seems to be total for the lines LC1093A and Kremnij (described as bearing respectively Or4, Or 5 and Or6 « genes ») This suggests that this population belongs to the race D and that resistance is only partial for the line PHBC2-56 Only the lines LC1093A and Kremnig display a strong resistance to the Bulgarian population. Bulgarian population is more aggressive that the Romanian population and hence may belong to the race E. Resistance seems to be total for the both lines.

  5. • AIMS AND MAJOR RESULTS • 1 – A preliminary step : to check the level of aggressiveness of the four european broomrape populations used in this study The spanish population 1 displays the same level of aggressivness against the seven sunflower lines than the Bulgarian population. This spanish population belongs also to the race E. This other spanish population is the most aggressive population tested in this study since only the Kremnij line, described as carrying Or6, is strongly resistant. Some broomrapes however succeed in emerging, suggesting that Kremnij is only a partially resistant line to this broomrape population. This spanish population might belong to the race F.

  6. • AIMS AND MAJOR RESULTS • 2 – To follow the kinetic of broomrape development through hydroponic co-cultures with the pathosystems showing the strongest responses of resistance  Hypersensitive response ? Examples / spanish populations Rock wool • Methods. Preconditioned seeds (around 100) are placed 1-2 mm from roots of a 10d-old sunflower seedling and broomrape germination is induced by GR24. Petri dishes are covered with aluminium foil. • Kinetic of broomrape development is followed weekly during 8 weeks : living and necrotic broomrapes are counted and divided into two developmental stages : • Young tubercles carrying neither adventitious roots nor a stem (Stages 1 and 2, Labrousse et al., 2001, 2004) • Older tubercles carrying adventitious roots and a stem Coïc neutrophile solution Glass fiber paper Susceptible line (2603) Resistant line

  7. • AIMS AND MAJOR RESULTS • 2 – To follow the kinetic of broomrape development through hydroponic co-cultures with the pathosystems showing the strongest responses of resistance  Hypersensitive response ? Examples / spanish populations Positive control (21-d infestation) : Roots carry a high number of broomrapes of the race E. Few of them are necrotic because of trophic competition. On the other hand, few fixations are observed on the line LC1093A, however they do not succeed in surviving over 4 weeks. Resistance previously observed in pots is not total since living young tubercles are observed before turning necrotic from 14-d of infestation. Any hypersensitive reaction is not observed.

  8. • AIMS AND MAJOR RESULTS • 2 – To follow the kinetic of broomrape development through hydroponic co-cultures with the pathosystems showing the strongest responses of resistance  Hypersensitive response ? Examples / spanish populations Positive control (21-d infestation) : Roots carry a high number of broomrapes of the race F. Few of them are necrotic because of trophic competition. On the other hand, few fixations are observed on the line Kremnij. As previously observed in pots, resistance is not total in this pathosystem since few tubercles are living after 8 weeks of infestation. Any hypersensitive reaction is not observed.

  9. • AIMS AND MAJOR RESULTS • 3 – Response to the 4 broomrape populations studied to the root exsudates collected from the 7 sunflower lines 35-d old sunflower Aseptic culture: Sunflower seed is sown on glass balls (2 mm diameter) soaked in sterile disstilled water. Growth is maintained for 5 weeks with a weekly supply of Coïc nutrient solution. Collection of root exsudates: Root exsudates of the 35-day old plant are eluted with 40 mL distilled water. Stimulatory capacity of the roots exsudates : Tests are carried out in Petri dishes containing 5 mg DW preconditionned broomrape seeds and 500 µL of root exsudates. Percentage of germination and viability tests (Evans blue staining) are estimated following 7-day incubation in darkness. Glass balls Root exsudates eluted

  10. • AIMS AND MAJOR RESULTS • 3 – Response to the 4 broomrape populations studied to the root exsudates collected from the 7 sunflower lines • As expected, the stimulating power differs strongly according to the sunflower line, the most stimulating being the susceptible one, showing no difference with GR24–related stimulation. • On the other hand, the 4 broomrape populations display an equivalent sensitivity to a kind of root exsudate. • Differential aggressiveness of the broomrape populations for a specific sunflower line cannot be related to the stimulating power of the root exsudate

  11. • AIMS AND MAJOR RESULTS • 4 – Pectinolytic capacity of the 4 broomrape populations • Methods. • Enzyme assays are performed with culture filtrates of 10d-preconditionned and 7-d germinated seed. Polygalacturonase activity (PG) and PME-coupled PG are measured using polygalacturonic acid and methylated pectin as the substrates respectively. • Major results. • A strong increase in the specific activities of PG and PME-associated PG occurs during germination and growth of broomrape seedlings. • Enhanced pectinolytic capacity is observed from the population D to the F one. Spanish and Bulgaria races E display both similar pectinolytic capacity and similar level of aggressiveness. •  Increase in aggressiveness might be related to an enhanced pectinolytic capacity within the broomrapes populations

  12. • CONCLUSIONS • • While the pectinolytic capacity could be a quantitative trait of aggressiveness, some other enzymes could be also involved in cell wall degradation, e.g suberinases, cellulases, peroxydases .. •  In this topic, we are starting on a course of study on the « secretome » of Orobanche seedlings with the first aim to identifiy the major proteins excreted following germination (« proteomic analysis ») : proteins active in cell wall degradation or in signalling. • Resistance of sunflower to O. cumana is partial and does not show any hypersensitive response. • This opinion is shared to date by an increasing number of researchers (Labrousse et al., 2004 in • Crop Protection 23(4) ; Rubiales et al., 2003 in Weed Sciences 51 ). • Labrousse et al. (2004) : Several resistance responses can be observed at different stages of O. cumana development with a same sunflower genotype. Using recombinant inbred lines derived from a cross between a susceptible (line 2603) and a resistant parent (line LR1), they demonstrate that these resistance responses can be separated in the progeny and that they are quantitative. • What about the actual role of Or resistant genes and the Avr genes in the pathosystem sunflower - O. cumana ? • As a result of those results, we think that indeed new populations appear in sunflower fields showing enhanced aggressiveness, attesting of a genetic adaptation in response to the new sunflower lines. This needs likely the mutation of several genes, notably some genes involved in cell wall degradation.

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