DETERMINING THE RELATIONSHIP BETWEEN VIRULENCE AND AGGRESSIVENESS IN PLASMOPARA HALSTEDII: MATERIALS AND METHODS

FUNGAL ISOLATES

The P. halstedii isolates used in this study were collected in France and maintained at INRA, Clermont-Ferrand. Manipulation of this quarantine pathogen followed European regulations (No 2003/DRAF/70). Isolate MIL 001 (race 100) was sampled in 1966 and isolate MIL 002 (race710) in 1988. The progeny isolates originated from an initial mixture of pathotypes 100 and 710 (Tourvieille de Labrouhe et al. 2010) and their races (Table 1) were determined using the method reported by Tourvieille de Labrouhe et al. (2000): isolate DU 1842 (race 300); isolate DU 1767 (race 304); isolate 1943 (race 314); isolate 1734 (race 704) and isolate 1915 (race 714).

Table 1: Virulence of seven Plasmopara halstedii isolates on nine sunflower differential lines

Isolates Race Yearisolated Differential lines
D1Ha-304 D2Rha-265 D3Rha-274 D4PMI3 D5PM-17 D6803-1 D7HAR-4 D8QHP1 D9Ha-335
MIL001 100 1960 S R R R R R R R R
DU1842 300 2005 S S R R R R R R R
DU1943 314 2005 S S R S R R R R S
DU1767 304 2005 S S R R R R R R S
MIL002 710 1988 S S S S R R R R R
DU1915 714 2005 S S S S R R R R S
DU1734 704 2005 S S S R R R R R S

R = resistant = incompatible interaction; S = susceptible = compatible interaction ; data from Tourvieille de Labrouhe et al. (2000), identification of virulence for seven P. halstedii isolates was presented by Sakr (2009).

MEASUREMENT OF AGGRESSIVENESS IN P. HASLTEDII PATHOTYPES

To characterize aggressiveness of P. halstedii isolates, one INRA inbred line FU was used. It carried no Pl gene, but is known to have a high level of quantitative resistance (Tourvieille de Labrouhe et al. 2008). The index of aggressiveness of the P. halstedii isolate was calculated as the ratio of (Percentage infection x sporulation density) / (latent period x reduction of hypocotyl length). Percentage infection was considered as successful when the seedlings showed sporulation of the pathogen on the shoot surface. Latent period was defined as the number of days of incubation necessary to obtain sporulating pathogen on 80% of the plants. Sporulation density was defined as the number of zoosporangia of the pathogen produced on a cotyledon. Reduction of hypocotyl length (dwarfing) corresponds to the distance from the stem base to cotyledon insertion and was measured after 13 days of infection on diseased plants showing sporulation of the pathogen on the shoot (Sakr 2009, Sakr et al. 2011). All the pathogenic tests were carried out in growth chambers regulated at 18h light, 18 °C ±1 and RH of 65 – 90%. All statistical analyses of the aggressiveness data were performed using Stat Box 6.7® (GimmerSoft) software. The values obtained were submitted to a one-way analysis of variance (ANOVA).

DNA EXTRACTION AND MOLECULAR TYPING

For each isolate, DNA was isolated from infected plant tissue as previously described for Plasmopara viticola by Delmotte et al. (2006). Then the 12 polymorphic EST-derived markers (Giresse et al. 2007) were used to genotype the P. halstedii isolates. The polygenetic relations between the seven isolates were obtained by building a Neighbour-joining (NJ) tree (Jin and Chakraborty 1993) using Populations 1.2.28 Software (Langella 1999). A Bootstrap analysis was performed on 10.000 replicates.

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