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El gen de la resistencia Pl2 diferencia la patogenicidad en las razas 304, 704, 314 y 714 de Plasmopara halstedii (mildiu del girasol).

Pl2 resistance gene differentiates the pathogenicity in four Plasmopara halstedii (sunflower downy mildew) races 304,704,314 and 714

INTRODUCTION

Downy mildew is one of the major diseases for sunflower cultivation (Helianthus annuus L.). The causal agent is Plasmopara halstedii, an obligate biotroph oomycete from the Peronosporaceae family, diploid, homothallic, and can reproduce sexually and asexually. This disease is found in most parts of the world where the crop is cultivated and where a co-evolution between H. annuus and P halstedii has taken place. Downy mildew causes dwarfing plants and infertile capitulum which reduces productivity (Viranyi & Spring 2011). Two categories of P halstedii resistance exist: qualitative resistance caused by single major Pl loci (Tourvieille de Labrouhe et al. 2000) and quantitative resistance which is controlled by several genes with minor effects (Tourvieille de Labrouhe et al. 2008).

P halstedii is characterized by a high level of evolutionary potential expressed by high virulence, aggressiveness and a great potential in developing new races (Tourvieille de Labrouhe et al. 2000, 2010; Delmotte et al. 2008, Sakr 2011,2012, 2014; Ahmed et al. 2012). Virulence has been defined as specific disease-causing abilities and aggressiveness as non-specific disease-causing abilities (Vander Plank 1968). It displays a gene-for-gene interaction with its host plant and shows physiological races (pathotypes) capable of infecting a variable range of sunflower genotypes. It must be pointed out that all these races are characterized upon the phenotypes (resistant or susceptible) of a set of differential sunflower lines carrying different Pl genes (Tourvieille de Labrouhe et al. 2000). Indeed, it has been possible to identify up to 35 races, with different virulence patterns (Delmotte et al. 2008, Ahmed et al. 2012, Sakr 2014). Fourteen different reference races of this pathogen have now been characterized in France, nine of which emerged in the last ten years. Eight of the races (100, 703, 710, 300, 700, 707, 717, and 730) are also found in other countries, but the six endemic races (304, 307, 314, 334, 704, and 714) provide evidence for evolution in situ (Delmotte et al. 2008, Ahmed et al. 2012, Sakr 2014). To date, the genetic background for avirulence Avr genes in P. halstedii correspondent to Pl sunflower resistance genes has not been investigated (Viranyi & Spring 2011).

Genes that confer resistance to downy mildew are dominant and often form clusters. Pl genes are located on complex loci containing several genes tightly linked. Because of the complexity of the loci, no Pl gene has been cloned yet (Miller & Gulya 1991). The Pl genes have been localized on four clusters in sunflower. Pl1, Pl2, Pl6 and Pl7 genes are clustered on LG8, Pl5 and Pl8 are clustered on LG13, PlArg is found on LG1, and two newly mapped genes (Pl13 and Pl14) on LG1 independent of the PlArg gene (Radwan et al. 2011). Concerning the importance of Pl genes, Pl2 gene has been the object of several studies. Mouzeyar et al. (1993) observed in histological studies, that incompatible interaction H. annuus/P halstedii is due to a hypersensitive reaction for Pl2. Vear et al. (1997) mapped Pl2 and found that its locus is located in LG8. Furthermore, Pl2 resistance gene is mainly used in breeding material (Vear et al. 2003). Recently, Sakr (2012) reported that sunflower differential line D3 (RHA-274) carrying Pl2 gene is important for studying virulence cost in P. halstedii. In this study, phenotypic analyses (morphological, pathogenic and genetic characteristics) were carried out in four pathotypes which have never been documented outside of France: two isolates of races 304 and 314 that do not overcome Pl2 gene, and isolates of races 704 and 714 that can. Hence an attempt was made to generate information about the importance of the Pl2 resistance gene to differentiate the pathogenicity in sunflower downy mildew.

MATERIALS AND METHODS

Oomycete isolates and race identification. The four P. halstedii isolates used in this study were collected in France and maintained at INRA, Clermont-Ferrand (Table 1). Manipulation of this quarantine parasite respected European regulations (No 2003/DRAF/70). Pathogen isolates were isolated in 2005 from naturally infected sunflower plants. Their races identity (Table 1) was determined using the method reported by Tourvieille de Labrouhe et al. (2000): DU 1767 (race 304); DU 1943 (race 314); DU 1734 (race 704) and DU 1915 (race 714). For each P halstedii isolate, five single zoosporangium isolates were obtained according to the method described by Sakr et al. (2007). This study dealt with five single zoosporangium isolates per pathogen isolate, giving a total of 20 single zoosporangium isolates. The characterization of the race for 20 single zoosporangium isolates (Table 2) was determined using the same method adapted in the study by Tourvieille de Labrouhe et al. (2000). There were three replications for each differential line (10 plants in each replication) and the entire experiment was repeated twice for four P. halstedii isolates and 20 P. halstedii single zoosporangium isolates.

Virulence spectrum for P. halstedii isolates and single zoosporangium isolates. To characterize virulence spectrum in P halstedii isolates and single zoosporangium isolates, four quasi-isogenic hybrids differing only in their downy mildew resistance genes were used, obtained from crosses of 2 forms (Tourvieille de Labrouhe et al. 2010): L1a, carrying resistance gene Pl2; L1b, carrying resistance genes Pl2 and Pl8; L2a, carrying no known resistance gene; L2b, carrying resistance gene Pl6. The four hybrids were produced as follows: H1 =L1a x L2a; H2 = L1a x L2b; H3 = L1b x L2a and H4 = L1b x L2b. Two sunflower lines were also used to analyze virulence spectrum for 19 P hasltedii isolates: XRQ (INRA, resistant to all French pathotypes except pathotype 334, carrying Pl5) and RHA340 (USDA, resistant to all known pathotypes, carrying Pl8).

Measurement of agressiveness in P. halstedii single zoosporangium isolates. To characterize aggressiveness criteria: percentage infection, latent period, sporulation density and reduction of hypocotyl length for P. halstedii single zoosporangium isolate (Sakr 2011,2012, 2014), one INRA inbred line 'FU' was used. It carried no Pl gene, but is known to a have high level of quantitative resistance (Tourvieille de Labrouhe et al. 2008). The index of aggressiveness of P halstedii single zoosporangium isolate was calculated as the ration of (percentage infection x sporulation density) / (latent period x dwarfing). All the pathogenic tests were carried out in growth chambers regulated at 18hrs of light, 18[degrees]C [+ or -] 1 and RH of 65-90%.

Morphological observations. After 13 days of infection of the sunflower inbred line 'FU', the zoosporangia and sporangiophores suspensions for 20 single zoosporangium isolates were obtained by grouping all sporulated cotyledons in a small container and adding 1 ml of physiological water for each cotyledon (9g NaCl+1L sterilized water). This slowed zoosporangia maturation to facilitate observations before liberation of zoospores (Sakr et al. 2007). Identification of form and measurement of size was carried out on 50 zoosporangia per treatment under a light microscope (magnification x 400) with 2 replications. Zoosporangia size was calculated from an oval [pi] x a x b, a=1/2 length, b=1/2 width. Furthermore, sporangiophore dimensions were observed by measuring 50 fresh sporangiophores in physiological water under a light microscope (magnification X400) with 2 replications.

DNA extraction and molecular typing. The 12 EST- derived markers were sequenced by Giresse et al. (2007). In the current study, these markers were used because the other molecular markers were non-specific, insufficiently polymorphic within P. halstedii, and no genetic structure in P. halstedii populations was identified by using these markers (Giresse et al. 2007). GenBank accession numbers for 12 EST-derived genomic markers (Giresse et al. 2007) were presented as following: Pha6 CB174585, Pha39 CB174648, Pha42 CB174650, Pha43 CB174680, Pha54 CB174708, Pha56 CB174714, Pha74 CB174642, Pha79 CB174692, Pha82 CB174573, Pha99 CB174703, Pha106 CB174676, and Pha120 CB174660. For 20 single zoosporangium isolates tested, DNA was isolated from infected plant tissues, the 12 polymorphic EST-derived markers were used to genotype P. halstedii single zoosporangium isolates. The polygenetic relations between the 15 isolates were obtained by building a Neighbour-Joining (NJ) tree (Jin & Chakraborty 1993) using Populations 1.2.28 Software (Librado & Rozas 2009). A Bootstrap analysis was performed on 10.000 replicates.

Statistical analysis. All statistical analyses were performed using StatBox 6.7[R] (GimmerSoft) software. Before statistical analysis, the percentages were transformed using the Arcsines function. A normality test showed that the transformed variables were normaly distributed, so the values obtained were submitted to a one-way analysis of variance (ANOVA). The Newman-Keuls test (Snedecor & Gochran 1989) was used to compare the means at P = 0.05.

RESULTS

Analysis of virulence spectrum. Table 2 shows that all sunflower hybrids were resistant to isolates of two races 314 and 304 and sensitive to isolates of races 714 and 704. Moreover, the two sunflower inbred lines XRQ and RHA340 were resistant to all P halstedii isolates tested.

Analysis of aggressiveness criteria. Percentage infection. Intra-isolate variability (Table 3): few plants escaped infection (89 out of 3750 plants) but these very high levels of infection (95%-100%) showed differences between single zoosporangium isolates. All pathogen isolates were uniform for the criterion <<percentage infection>>. Enter-isolates variability (Table 4): the analysis of variance indicated highly significant differences (p=0.0005; F-test= 10.230). The Newman-Keuls test showed that the pathogen isolates formed two very distinct groups. The first group containing the isolates DU 1767 and DU 1943 showed a higher infection level than the second group containing the isolates DU 1734 and DU 1915.

Latent period. Intra-isolate variability (Table 3): although the differences were highly significant for the four isolates of races 314, 304, 704 and 714, they were small for those with a short latent period (DU 1943 and DU 1767). Deviations were slightly larger for the isolates with the longest incubation periods DU 1915 and DU 1734. Analysis of the relation between sporulation percentages based on incubation period (Fig. 1) showed differences in behaviour among P halstedii single zoosporangium isolates. There were two main groups from day 8 onwards: single zoosporangium isolates of P halstedii isolates DU 1943 and DU 1767 sporulated faster than single zoosporangium isolates of P halstedii isolates DU 1915 and DU 1734. All infected plants with single zoosporangium isolates of races 3xx showed more than 80% sporulation 9 days after incubation, whereas pathotypes 7xx needed 11 days after incubation to reach the same level of sporulation. Enter-isolates variability (Table 4): there were highly significant differences between P halstedii isolates (p=0.0001; F-test=38.928). The Newman-Keuls test classified the isolates into two distinct groups. Those with the shortest length of latent period (< 9 days) were the isolates DU 1767 and DU 1943. The isolates DU 1734 and DU 1915 were grouped together and showed longer latent periods (> 10 days).

[FIGURA 1 OMITIR]

Sporulation density. Intra-isolate variability (Table 3): only single zoosporangium isolates for races 314 and 714 showed variation for sporulation density. Table 2 revealed that the single zoosporangium isolate DU1943M4 showed a significant difference, producing more than 18 x [10.sup.5] zoosporangia per cotyledon as compared to a mean of 12 x [10.sup.5] zoosporangia per cotyledon for the other four isolates. Similarly, the two single zoosporangium isolates DU1915M1 and DU1915M6 showed twice as much sporulation as compared with the other three isolates (7 x [10.sup.5] zoosporangia per cotyledon compared to 4 x [10.sup.5]). Fig. 2 shows that the quantities of zoosporangia produced increased with time. There were two main groups from day 9 onwards: single zoosporangium isolates of P halstedii isolates DU 1943 and DU 1767 produced more zoosporangia than single zoosporangium isolates of pathogen isolates DU 1915 and DU 1734 (Fig. 2). The quantity of zoosporangia produced was at a maximum 12 days after incubation. Enter-isolates variability (Table 4): there were large differences between the isolates; sporulation density varied from 5 x [10.sup.5] zoosporangia per ml for DU 1915 to 14 x [10.sup.5] for DU 1842. Differences were highly significant (p = 0.0; F-test = 30.332). The Newman-Keuls test classified the isolates into two very distinct groups. The isolates DU 1734 and DU 1915 showed the lowest sporulation density (< 7 x [10.sup.5] zoosporangia per cotyledon). Isolates DU 1767 and DU 1943 showed the highest sporulation density (< 13 x [10.sup.5] zoosporangia per cotyledon).

[FIGURA 2 OMITIR]

Reduction of hypocotyl length. Intra-isolate variability (Table 3): all pathogen isolates showed variability within isolates for this criterion of aggressiveness. The length of P. halstedii-free sunflower inbred line 'FU' varied between 87.7 to 92.3 mm. Plants diseased had hypocotyls with only one third the mean lengths of P. halstedii-free sunflower inbred line 'FU' (30.85 [+ or -] 0.6 mm and 90.0 [+ or -] 2.3 mm respectively) whatever the single zoosporangium isolate of P. halstedii. The single zoosporangium isolate that caused greatest reduction in length was DU1915M6 with a mean length of 25.6 mm. Single zoosporangium isolate DU1943M5 gave the least reduction (44.9 mm). In all cases, infected plants were smaller than healthy plants. Enter-isolates variability (Table 4): The analysis of variance with five replications per pathogen isolate corresponding to the five single zoosporangium isolates showed highly significant differences between pathogen isolates (p = 0.001; F-test = 25.683). However, these results were mainly due to pathogen isolate DU 1943 which caused less reduction in hypocotyl length than the other three pathogen isolates.

The index of aggressiveness varied 7.0: 1.0 for DU1915M5 and 6.8 for DU1767M4 (Table 3). There were significant differences (p = 0.0001, F-test = 46.566) for single zoosporangium isolates of four races (Table 4). Isolates DU 1767 (race 304) and DU 1943 (race 314) were more aggressive with an index of 5.9 and 3.58 respectively, than isolates DU 1734 (race 704) and DU 1915 (race 714) with an index of 2 and 1.58 respectively.

Morphology of zoosporangia and sporangiophores. The results showed that the two most observed forms of zoosporangia were oval and round (Fig. 3). All morphological data were presented in Table 5. The proportion of oval form varied from 56 to 93% and the zoosporangia size from 302.2 to 505.2 [[micro]m.sup.2]. The proportion of oval zoosporangia varied within the races, for example for race 314 it ranged from 56% to 93%, for race 304 it ranged from 63% to 91%, for race 714 it ranged from 86% to 93% and for race 704 it ranged from 68% to 89%. Mean sporangiophore length was the highest in DU1767M3 isolate (800.0 Im). The sporangiophore length ranged between 299.0 and 800.0 Im. Mean sporangiophore width was the largest in DU1767M1 isolate. The sporangial width varied from 5.0 [micro]m to 16.0 [micro]m.

[FIGURA 3 OMITTED]

Molecular analysis. The combination of 12-EST derived markers revealed four multilocus haplotypes (MLH) among 20 P halstedii single zoosporangium isolates (Table 6). There was no intra-race genetic variation for all pathotypes tested. Single zoosporangium isolates of four races were similar only for three genomic markers Pha39, Pha54 and Pha79. The Neighbour-joining tree showed that isolates DU 1915 and DU 1734 had an intermediary genetic position between isolates DU 1767 and DU 1943 (Fig. 4).

DISCUSSION

For the first time, Flor (1971) propagated the gene-for-gene resistance concept and observing specificity between single host resistance (R) genes and single pathogen avirulence (Avr) genes. However, in sunflower downy mildew, there are no studies about the genetic background for avirulence Avr genes correspondent to Pl resistance genes (Viranyi & Spring 2011). Since tools for analyzing pathogenicity of obligate parasite Plasmopara halstedii are very limited, it appears desirable to use sunflower resistance genes to better analyze the differentiation of virulence and aggressiveness. With bearing this in mind, two isolates of races (304, 704) and two isolates of races (314 and 714) similar in reaction for all sunflower differential lines except for D3 carrying Pl2 gene were used.

Sunflower hybrids H1 to H4 differing only in their downy mildew resistance genes were used to analyze the virulence spectrum in P halstedii isolates. Table 2 demonstrates that all sunflower hybrids were resistant to single zoosporangium isolates and isolates of races 314 and 304, as those sunflower hybrids carrying effective Pl genes were resistant to the two races 304 and 314. On the other hand, all sunflower hybrids were sensitive to the isolates of races 714 and 704. The races 704 and 714 can overcome Pl2 and Pl6 present in H1 and H2 as observed from its behavior on sunflower differential lines carrying the same genes (Table 1). The two sunflower hybrids H3 and H4 came from L1b, which may carry Pl8. Since Pl8 confers resistance to all known races and Pl2 and Pl8 segregate independently in L1b (Tourvieille de Labrouhe et al. 2010), it was not possible to determine whether L1b carried either resistance gene, Pl2 or only Pl8. Moreover, in virulence seedling tests to isolates of races 714 and 704, certain sunflower plants (1-3) per replication for H3 and H4 produced no sporulation on cotyledons and leaves. However, H3 and H4 generated effective resistance to races 714 and 704 in field conditions (Tourvieille de Labrouhe et al. 2010). Our results confirmed that the two sunflower lines XRQ and RHA340 were resistant to all P. halstedii isolates used in this study because they carry effective Pl genes against all races tested in the present study. This type of resistance may be controlled by non-TIR-NBS-LRR (Toll/interleukin-1 receptor (TIR) nucleotide-binding site leucine-rich repeat class) which clustered and linked to the Pl5/Pl8 locus for resistance to downy mildew in sunflower (Radwan et al. 2003). Our results (Table 2) confirmed that isolates of races 714 and 704 were more virulent than isolates of races 314 and 304. In accordance with our data, Sakr (2011, 2012, 2014) reported that isolates of races 7xx more virulent than isolates of races 3xx using sunflower differential line D3 (carries Pl2 gene) for virulence reaction.

High percentage infection, short latent period, high sporulation density, and significant reduction in the length of the hypocotyl represent high aggressiveness (Sakr 2011, 2012, 2014). The frequency of sporulated plants based on incubation period reflected the speed of appearance of symptoms on the plants (Fig. 1) (latent period), and the number of zoosporangia produced by cotyledons reflected the level of invasion of infected tissues (Fig. 2). Analysis of five single zoosporangium isolates of each pathogen isolate showed variability for aggressiveness criteria studied within P halstedii isolate, but not for all pathogen isolates (Table 3). The difference in aggressiveness within P halstedii isolates may be due to the variability in aggressiveness within a population of zoosporangia, of which a single zoosporangium isolate is only one preventative. Our results show that there were significant differences among pathogen isolates for all aggressiveness criteria. Indeed, the index of aggressiveness revealed the presence of significant differences between the isolates of races 304 and 314 and isolates of races 704 and 714 (Table 4). These results are comparable with those found by Sakr (2011, 2012, 2014), who reported that isolates of races 3xx more aggressive than isolates of races 7xx. Our results (Tables 2 and 4) confirmed that isolates of races 714 and 704 were more virulent and less aggressive than isolates of races 314 and 304.

Table 5 showed that there were significant morphological differences for 20 pathogen single zoosporangium isolates of four isolates dU 1943 and DU 1767, DU 1915 and DU 1734. The proportion of zoosporangia of different forms and their sizes and the morphology of sporangiophores do not appear to be useable to differentiate 20 single zoosporangium isolates of three races 300, 314 and 304 as defined by Tourvieille de Labrouhe et al. (2000). The results also showed that zoosporangia morphology did not distinguish single zoosporangium isolates according to their aggressiveness (Table 3). There was no intra-race genetic variation (Table 6), but four genetically-identified groups were detected among 20 P halstedii single zoosporangium isolates of four races: group of isolates of race 304, group of isolates of race 714, group of isolates of race 704 and group of isolates of race 314 (Table 6, Fig. 4). However, the present association was not identified in the previous works (Delmotte et al. 2008, Ahmed et al. 2012). Indeed, Delmotte et al. (2008) and Ahmed et al. (2012) grouped race 304 in a genetic clade and race 314 in another cluster. Also, Delmotte et al. (2008) and Ahmed et al. (2012) grouped races 704 and 714 together in the same genetic clade. Our results underlined non correlation between EST genotypes (Table 5, Fig. 4) and pathogenic traits (Tables 2, 4).

[FIGURA 4 OMITIR]

The current study helps us to underline the role of Pl2 gene in differentiation the pathogenicity in P halstedii. Races 704 and 714 that accumulate a large number of virulence genes might never be the most aggressive on sunflower genotypes as compared with non virulent pathotypes 304 and 314. Regarding avirulence Avr gene correspondent to Pl2 gene in P halstedii, it seems that Avr gene stimulates less virulent spectrum and more aggressive characteristics in races 304 and 314 than races 704 and 714. There was phenotypic variation (morphological and genetic characteristics) for the four P halstedii races without a correlation with pathogenic diversity. It will be necessary to analyze avirulence Avr genes in on a large collection of P halstedii isolates to provide a better insight into interactions between this obligate parasite and its host.

http://dx.doi.org/10.5209/rev_BQCM.2015.v39.49130

ACKNOWLEDGEMENTS

This study was done at INRA Clermont-Ferrand; we thank all persons who helped to perform this work. We gratefully thank Jalal Al-Attar for statistical helping.

BIBLIOGRAPHY

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Nachaat Sakr (1)

(1) Department of Agriculture, Syrian Atomic Energy Commission, Damascus, P.O. Box 6091, Syria. Previous address INRAUBP, UMR 1095, 234 Avenue du Brezet, 63100 Clermont-Ferrand, France. snachaat@hotmail.com, snachaat@gmail.com

Recibido: 8 mayo 2014. Aceptado: 28 octubre 2014.
Table 1
Virulence of four Plasmopara halstedii isolates on nine
sunflower differential lines. S: susceptible, sporulation
on cotyledons. R: resistant, no sporulation. Pl(?): Pl
gene not identified, data from Tourvieille de Labrouhe
et al. (2000)

                                     Differential lines

Isolates                       D1
                             Ha-304      D2        D3       D4
                    Year     without   Rha-265   Rha-274   PMI3
           Race   Isolated   Pl gene     Pl1       Pl2     Pl(?)

DU1943     314      2005        S         S         R        S
DU1767     304      2005        S         S         R        R
DU1915     714      2005        S         S         S        S
DU1734     704      2005        S         S         S        R

                                  Differential lines

Isolates                      D5      D6      D7      D8
                    Year     PM-17   803-1   HAR-4   QHP1
           Race   Isolated   Pl(?)   Pl(?)   Pl(?)   Pl(?)

DU1943     314      2005       R       R       R       R
DU1767     304      2005       R       R       R       R
DU1915     714      2005       R       R       R       R
DU1734     704      2005       R       R       R       R

                             Differential
                                lines
Isolates
                                  D9
                    Year        Ha-335
           Race   Isolated       Pl6

DU1943     314      2005          S
DU1767     304      2005          S
DU1915     714      2005          S
DU1734     704      2005          S

Table 2
Virulence of 24 Plasmopara halstedii isolates and single
zoosporangium isolates on four sunflower hybrids differing
only in their downy mildew resistance genes

Isolates    Race     Year     H1   H2   H3   H4   XRQ   RHA340
                   isolates                       Pl5    Pl8

DU1943      314      2005     R    R    R    R     R      R
DU1943 M1   314      2006     R    R    R    R     R      R
DU1943 M2   314      2006     R    R    R    R     R      R
DU1943 M3   314      2006     R    R    R    R     R      R
DU1943 M4   314      2006     R    R    R    R     R      R
DU1943 M5   314      2006     R    R    R    R     R      R
DU1767      304      2005     R    R    R    R     R      R
DU1767 M1   304      2006     R    R    R    R     R      R
DU1767 M2   304      2006     R    R    R    R     R      R
DU1767 M3   304      2006     R    R    R    R     R      R
DU1767 M4   304      2006     R    R    R    R     R      R
DU1767 M5   304      2006     R    R    R    R     R      R
DU1915      714      2005     S    S    S    S     R      R
DU1915 M1   714      2006     S    S    S    S     R      R
DU1915 M2   714      2006     S    S    S    S     R      R
DU1915 M3   714      2006     S    S    S    S     R      R
DU1915 M5   714      2006     S    S    S    S     R      R
DU1915 M6   714      2006     S    S    S    S     R      R
DU1734      704      2005     S    S    S    S     R      R
DU1734 M1   704      2006     S    S    S    S     R      R
DU1734 M2   704      2006     S    S    S    S     R      R
DU1734 M3   704      2006     S    S    S    S     R      R
DU1734 M7   704      2006     S    S    S    S     R      R
DU1734 M8   704      2006     S    S    S    S     R      R

Table 3
Aggressiveness within pathogen isolate for 20 Plasmopara
halstedii single zoosporangium isolates measured on the
sunflower inbred line 'FU'. According to the Newman-Keuls
test, means followed by the same letter are not significantly
different at P = 0.05, ns = not significant, P: probability,
VC: variation coefficient. Index of aggressiveness
= (percentage infection x sporulation density) /
(latent period x dwarfing) (Sakr 2011, 2012, 2014)

                                                        Sporulation
                                                       density Mean
                         Percentaje                     ([10.sup.5]
Zoosporangium             infection    Latent period   zoosporangia
isolates          Race    Mean (%)      Mean (days)     cotyledon)

DU1943 M1         314       100.0         8.61 b          13.25 b
DU1943 M2         314       100.0         8.53 b          12.75 b
DU1943 M3         314       99.4          8.89 a          11.30 b
DU1943 M4         314       98.9          8.20 c          18.27 a
DU1943 M5         314       98.3          7.88 d          12.10 b
                         P = 22.6 ns      P = 0.0         P = 0.0
                         VC = 2.00%     VC = 1.51%      VC = 5.50%
DU1767 M1         304       100.0         7.94 b           13.04
DU1767 M2         304       98.9          8.74 a           13.60
DU1767 M3         304       100.0         8.01 b           16.26
DU1767 M4         304       100.0         8.59 a           15.31
DU1767 M5         304       100.0         7.97 b           13.32
                         P = 45.0 ns      P = 0.0       P = 43.5 ns
                         VC = 1.77%     VC = 1.67%      VC = 15.02%
DU1915 M1         714       95.9          10.56 b         6.20 a
DU1915 M2         714       95.5          11.85 a         3.33 b
DU1915 M3         714       95.0          10.15 b         3.91 b
DU1915 M5         714       98.3          11.54 a         4.07 b
DU1915 M6         714       91.1          11.17 a         7.62 a
                         P = 8.1% ns      P = 0.0         P = 0.0
                         VC = 4.69%     VC = 2.89%      VC = 3.42%
DU1734 M1         704       95.0          10.97 c          4.37
DU1734 M2         704       98.3          10.88 c          7.72
DU1734 M3         704       95.6          10.51 c          5.84
DU1734 M7         704       97.8          12.48 a          5.58
DU1734 M8         704       95.6          11.61 b          8.07
                         P = 9.3% ns      P = 0.0      P = 21.5% ns
                         VC = 3.22%     VC = 2.88%      VC = 22.66%

                          Hypocotyl        Index
Zoosporangium              length            of
isolates          Race    Mean (mm)    aggressiveness

DU1943 M1         314      42.1 b           3.7
DU1943 M2         314      40.9 b           3.7
DU1943 M3         314      35.6 d           3.5
DU1943 M4         314      39.7 c           3.6
DU1943 M5         314      44.9 a           3.4
                           P = 0.0
                         VC = 1.69%
DU1767 M1         304      35.2 a           4.7
DU1767 M2         304      27.9 b           5.5
DU1767 M3         304      27.6 b           7.4
DU1767 M4         304      26.2 b           6.8
DU1767 M5         304      27.4 b           6.1
                         P = 0.00008
                         VC = 4.42%
DU1915 M1         714      28.3 b           2.0
DU1915 M2         714      28.2 ab          1.0
DU1915 M3         714      29.6 a           1.2
DU1915 M5         714      26.7 b           1.3
DU1915 M6         714      25.6 c           2.4
                         P = 0.0005
                         VC = 2.67%
DU1734 M1         704      26.6 b           1.4
DU1734 M2         704      26.5 b           2.6
DU1734 M3         704      26.8 b           2.0
DU1734 M7         704      28.3 b           1.5
DU1734 M8         704      31.0 a           2.5
                         P = 0.00017
                         VC = 2.68%

Table 4
Mean and standard deviation of aggressiveness among four
Plasmopara halstedii isolates (five replications per
pathogen isolate that correspond to five single
zoosporangium isolates) on the sunflower inbred line
'FU'. According to the Newman-Keuls test, means followed
by the same letter are not significantly different at
P = 0.05), index of aggressiveness = (percentage infection
x sporulation density) / (latent period x dwarfing) (Sakr
2011, 2012, 2014)

                         Percentage
Isolates                 infection           Latent period
                            (%)                  (days)

DU 1943 (race 314)   99.3 a [+ or -] 0.6   8.42 b [+ or -] 0.35
DU 1767 (race 304)   99.8 a [+ or -] 0.4   8.25 b [+ or -] 0.34
DU 1915 (race 714)   95.2 b [+ or -] 2.3   11.06 a [+ or -] 0.62
DU 1734 (race 704)   96.4 b [+ or -] 1.3   11.29 a [+ or -] 0.69
Probability                0.0005                0.0001
F isolates                 10.23                 38.928

                     Sporulation density        Hypocotyl
Isolates              (105 zoosporangia           length
                        per cotyledon)             (mm)

DU 1943 (race 314)   13.53 a [+ or -] 2.46   40.6 a [+ or -] 3.1
DU 1767 (race 304)   14.10 a [+ or -] 1.46   28.9 b [+ or -] 3.2
DU 1915 (race 714)   5.02 b [+ or -] 1.62    27.7 b [+ or -] 1.4
DU 1734 (race 704)   6.31 b [+ or -] 1.39    27.9 b [+ or -] 1.7
Probability                 0.0001                0.001
F isolates                  30.332                25.683

                          Idex
Isolates                   of
                     aggressiveness

DU 1943 (race 314)        3.58
DU 1767 (race 304)        6.1
DU 1915 (race 714)        1.58
DU 1734 (race 704)        2.0
Probability              0.0001
F isolates               46.566

Table 5
Morphological characters of zoosporangia and sporangiophores
obtained on sunflower genotype FU for 20 isolates of
Plasmopara halstedii. (a) 50 zoosporangia per replication,
(b) 50 zoosporangia per replication, (c) 50 sporangiophores
per replication, (d) 50 sporangiophores per replication,
F-tests (P = 0.01), Probability (P = 0.05)

Isolates      Race      % of oval       Size of zoosporangia in
                     zoosporangia (a)    [[micro]m.sup.2] (b)

DU1943 M1     314         93                   424.8
DU1943 M2     314         86                   425.4
DU1943 M3     314         80                   387.6
DU1943 M4     314         56                   372.0
DU1943 M5     314         56                   380.4
DU1767 M1     304         86                   394.0
DU1767 M2     304         78                   422.3
DU1767 M3     304         90                   505.2
DU1767 M4     304         91                   478.7
DU1767 M5     304         63                   344.7
DU1915 M1     714         90                   477.8
DU1915 M2     714         90                   477.8
DU1915 M3     714         93                   734.6
DU1915 M5     714         86                   374.6
DU1915 M6     714         87                   358.9
DU1734 M1     704         74                   505.4
DU1734 M2     704         68                   357.1
DU1734 M3     704         68                   314.3
DU1734 M7     704         74                   302.2
DU1734 M8     704         89                   436.9
Probability                0.0003                0.0001
F isolates                 5.163               312.86

Isolates      Race       Sporangiophore          Sporangiophore
                     length ([micro]m) (c)   width ([micron]m) (d)

DU1943 M1     314          663.5                     9.7
DU1943 M2     314          489.3                     6.3
DU1943 M3     314          356.2                     8.2
DU1943 M4     314          689.3                    10.9
DU1943 M5     314          299.0                    14.3
DU1767 M1     304          478.6                    16.0
DU1767 M2     304          559.6                     8.3
DU1767 M3     304          800.0                     6.5
DU1767 M4     304          456.3                     8.1
DU1767 M5     304          765.3                     9.6
DU1915 M1     714          700.9                     8.5
DU1915 M2     714          456.3                     7.6
DU1915 M3     714          552.1                     6.2
DU1915 M5     714          456.3                     5.0
DU1915 M6     714          558.2                     6.8
DU1734 M1     704          335.6                     7.4
DU1734 M2     704          663.3                     6.8
DU1734 M3     704          712.3                     8.1
DU1734 M7     704          322.2                     9.6
DU1734 M8     704          455.9                     6.0
Probability                    0.0001                0.9853 ns
F isolates                   816.325                 0.358

Table 6
Multilocus haplotypes (MLH) characterized using 12
EST-derived genomic markers on 20 isolates of Plasmopara
halstedii

Isolates                    EST-derived markers

            Pha6   Pha39   Pha42   Pha43   Pha54   Pha56   Pha74

DU1943 M1   1/1     2/2     1/1     2/2     1/1     2/2     2/2
DU1943 M2   1/1     2/2     1/1     2/2     1/1     2/2     2/2
DU1943 M3   1/1     2/2     1/1     2/2     1/1     2/2     2/2
DU1943 M4   1/1     2/2     1/1     2/2     1/1     2/2     2/2
DU1943 M5   1/1     2/2     1/1     2/2     1/1     2/2     2/2
DU1767 M1   2/2     2/2     1/1     1/1     1/1     1/1     1/1
DU1767 M2   2/2     2/2     1/1     1/1     1/1     1/1     1/1
DU1767 M3   2/2     2/2     1/1     1/1     1/1     1/1     1/1
DU1767 M4   2/2     2/2     1/1     1/1     1/1     1/1     1/1
DU1767 M5   2/2     2/2     1/1     1/1     1/1     1/1     1/1
DU1915 M1   1/1     2/2     1/2     1/1     1/1     1/1     1/1
DU1915 M2   1/1     2/2     1/2     1/1     1/1     1/1     1/1
DU1915 M3   1/1     2/2     1/2     1/1     1/1     1/1     1/1
DU1915 M5   1/1     2/2     1/2     1/1     1/1     1/1     1/1
DU1915 M6   1/1     2/2     1/2     1/1     1/1     1/1     1/1
DU1734 M1   2/2     2/2     2/2     1/1     1/1     1/1     2/2
DU1734 M2   2/2     2/2     2/2     1/1     1/1     1/1     2/2
DU1734 M3   2/2     2/2     2/2     1/1     1/1     1/1     2/2
DU1734 M7   2/2     2/2     2/2     1/1     1/1     1/1     2/2
DU1734 M8   2/2     2/2     2/2     1/1     1/1     1/1     2/2

Isolates               EST-derived markers

            Pha79   Pha82   Pha99   Pha106   Pha120

DU1943 M1    3/3     2/2     1/1     2/2      1/1
DU1943 M2    3/3     2/2     1/1     2/2      1/1
DU1943 M3    3/3     2/2     1/1     2/2      1/1
DU1943 M4    3/3     2/2     1/1     2/2      1/1
DU1943 M5    3/3     2/2     1/1     2/2      1/1
DU1767 M1    3/3     2/2     2/2     1/1      2/2
DU1767 M2    3/3     2/2     2/2     1/1      2/2
DU1767 M3    3/3     2/2     2/2     1/1      2/2
DU1767 M4    3/3     2/2     2/2     1/1      2/2
DU1767 M5    3/3     2/2     2/2     1/1      2/2
DU1915 M1    3/3     2/2     1/1     2/2      1/1
DU1915 M2    3/3     2/2     1/1     2/2      1/1
DU1915 M3    3/3     2/2     1/1     2/2      1/1
DU1915 M5    3/3     2/2     1/1     2/2      1/1
DU1915 M6    3/3     2/2     1/1     2/2      1/1
DU1734 M1    3/3     1/1     1/1     2/2      1/1
DU1734 M2    3/3     1/1     1/1     2/2      1/1
DU1734 M3    3/3     1/1     1/1     2/2      1/1
DU1734 M7    3/3     1/1     1/1     2/2      1/1
DU1734 M8    3/3     1/1     1/1     2/2      1/1
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Author:Sakr, Nachaat
Publication:Botanica Complutensis
Date:Jan 1, 2015
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