Virulence characteristics of Bremia lactucae populations in Norway
Berit Nordskog, Abdelhameed Elameen, David M. Gadoury & Arne Hermansen
European Journal of Plant Pathology Published in cooperation with the European Foundation for Plant Pathology ISSN 0929-1873 Eur J Plant Pathol DOI 10.1007/s10658-014-0422-9
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Author's personal copy Eur J Plant Pathol DOI 10.1007/s10658-014-0422-9
Virulence characteristics of Bremia lactucae populations in Norway Berit Nordskog & Abdelhameed Elameen & David M. Gadoury & Arne Hermansen
Accepted: 31 March 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2014
Abstract Use of resistant cultivars represent an efficient control measure for lettuce downy mildew (Bremia lactucae), although the durability of presently deployed resistance genes remains uncertain. Our objective was to document the pathogenic diversity of B. lactucae isolates in Norway. A total of 69 isolates of B. lactucae were collected between 2001 and 2006 from 65 commercial fields and four greenhouses in southeastern and southwestern Norway and tested for the presence of one or more of 19 virulence factors (v-factors). Phenotypic diversity was calculated based on presence or absence of vfactors, and as an overall comparison of v-phenotypes for each isolate. Disease severity varied over the years of the study, and epidemics were most consistently severe in southeastern Norway. The most commonly occurring vfactors, in order of frequency, were v5/8, v7, v2, v18, v4, v13, v6, v11, v12, v1 and v10. Virulence factor v17 was B. Nordskog (*) : A. Elameen : A. Hermansen Bioforsk, Norwegian Institute for Agricultural and Environmental Research, Høgskoleveien 7, 1430 Ås, Norway e-mail:
[email protected] A. Elameen e-mail:
[email protected] A. Hermansen e-mail:
[email protected] D. M. Gadoury Department of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA e-mail:
[email protected]
not found, while v36 was found in one isolate only. A total of 44 different v-phenotypes were identified within the population represented by the 69 isolates, yielding an incidence of unique virulence types of 63 %; a relatively high level of pathogen diversity. Four of the identified vphenotypes were identical to races Bl:17, 18, 22 and 24, which have been previously reported in European populations of B. lactucae. The variability of the Norwegian B. lactucae populations verifies the genetic flexibility of this pathogen and its great ability to adapt to changes in host plants and surrounding conditions. Keywords Epidemiology . Lactuca sativa . Lettuce . Lettuce downy mildew . Race-specific resistance . Virulence factors
Introduction Lettuce downy mildew, caused by Bremia lactucae Regel, is a major constraint in lettuce production worldwide (Crute and Dixon 1981). Sporadic epidemics of lettuce downy mildew have occurred on cultivated lettuce in Norway since 1927 (Jørstad 1964), and have become more frequent and severe in some regions of Norway since 1996 (Hermansen and Myrstad 1997; Nordskog et al. 2008a). Since the early 1990s, there has been a shift from production of lettuce in greenhouses towards field production in Norway. Additionally, field production of lettuce itself has become more intensive during this period, with two to three crops per season, often without crop rotation,
Author's personal copy Eur J Plant Pathol
now representing a common commercial standard. The degree to which the severity of downy mildew epidemics reflects a change in the production system, or a shift in pathogen virulence, is poorly understood. Availability of resistant cultivars has made the disease manageable over the last 5 years, but the durability of presently deployed resistance genes in the presence of a pathogen population of undetermined diversity remains uncertain. The species B. lactucae can infect several genera in the Compositae (Crute and Dixon 1981; Lebeda et al. 2002). However, there is a high level of host specificity within the pathogen species, and only a few wild Lactuca spp. have been confirmed as hosts of the formae speciales of B. lactucae that is virulent on cultivated lettuce (Lactuca sativa L.) (Crute and Davis 1977; Lebeda and Syrovátko 1988). In Europe, prickly lettuce (L. serriola L.) is a common weed (Lebeda et al. 2001), and the most studied wild source of B. lactucae (Lebeda et al. 2002; van Treuren et al. 2011). Consequently, many resistance genes from L. serriola have been used in the breeding of commercial lettuce cultivars (Crute 1992; Lebeda et al. 2014). The plant-pathogen interaction between lettuce and B. lactucae has been defined as a gene-for-gene system wherein specificity is determined by dominant resistance genes (Dm-genes) or resistance factors (Rfactors) in the plants, which are matched by dominant factors for avirulence (v-factors) in the pathogen (Crute and Johnson 1976; Crute 1992). Variation in virulence of B. lactucae is characterized as physiological races or virulence phenotypes (v-phenotypes), and can be defined as phenotypes that can be distinguished by the reaction of a specified set of differential cultivars (van Ettekoven and van der Arend 1999). Breeding for resistance to lettuce downy mildew has been continuous since the 1930’s (Lebeda et al. 2014). Since even relatively small infections can result in unacceptable quality of the lettuce, most breeding programs have focused upon race-specific resistance. Such resistance can be short-lived, and necessitates not only the discovery of new resistance genes to compensate for virulence shifts in the pathogen population, but an understanding of the diversity and relative frequencies of virulence factors in the pathogen population over space and time. Several such population studies have been reported from Sweden (Jönsson et al. 2005), Germany (Lebeda and Zinkernagel 2003), France (Maisonneuve et al. 2011), Czech Republic
(Petrželová et al. 2013), Israel (Sharaf et al. 2007), Brazil (Braz et al. 2007; Souza et al. 2011) and Australia (Trimboli and Nieuwenhuis 2011). In addition, isolates derived from wild L. serriola populations have been studied extensively in the Czech Republic (Lebeda 1984, 1986, 2002; Lebeda et al. 2008; Petrželová and Lebeda 2011). Management of lettuce downy mildew in Norway has involved the combined use of fungicides, cultivar resistance, and suppressive cultural practices. Despite this integrated approach, losses increased due to increasingly severe epidemics of downy mildew in Norway beginning in the late 1990s. Our objective was to document the pathogenic diversity of B. lactucae isolates occurring in lettuce production areas of Norway during the period 2001 to 2006, as a necessary step towards more effective deployment of resistant cultivars. Preliminary accounts of this research have been published (Nordskog 2006; Nordskog and Hermansen 2007).
Materials and methods A total of 69 isolates of B. lactucae were collected and virulence tested during the period 2001 to 2006. Sixtyfive of the isolates were collected from conventional lettuce fields in the southeast (Buskerud, Vestfold and Østfold Counties) and the southwest (Rogaland County) of Norway, while four isolates were collected from two greenhouses in Rogaland County in 2002 (Fig. 1). The virulence screening was based on bulk isolates where five leaves with freshly sporulating lesions collected from one or two adjacent plants were characterized as one isolate. Isolates were collected from different locations and cultivars, representative for the incidence of B. lactucae in each region and year. The sample size varied from one to 16 samples per region and year (Table 1). Each isolate was tested for the presence or absence of 19 v-factors using a differential set (EU-A) of lettuce cultivars following the protocol established by van Ettekoven and van der Arend (1999). The virulence tests were carried out at Svalöv Weibull, Sweden (2001, 2002 and 2004) and Seminis Research, The Netherlands (2005 and 2006). Phenotypic diversity was calculated in relation to vfactors and v-phenotypes. The virulence complexity was expressed as the mean number of v-factors per
Author's personal copy Eur J Plant Pathol Fig. 1 Lettuce production regions of Norway where isolates of Bremia lactucae were sampled for virulence testing during 2001 to 2006. The sampling took place in the counties Rogaland (a), in the southwest, and Vestfold (b), Østfold (c) and Buskerud (d) in the southeast of Norway
63º
60º
D C
B
A 6º
9º 0
isolate (Ci) and as the mean number of virulence factors per v-phenotype (Cp) (Andrivon 1995), calculated as C i = Σ j pj. vj , j = 1.... N p, and C p = (Σ jv j )/Np , j = 1 … N p, where pj is the frequency of the jth v-phenotype in the population, vj is the number of virulence factors Table 1 Basic characteristics of the Norwegian population of Bremia lactucae analysed for presence of virulence in 2001 to 2006
Regiona
SE
a
Isolates collected in the southeast (SE) from Buskerud, Vestfold and Østfold Counties; isolates collected in southwest (SW) from Rogaland County
SW
Year
50
12º 100
15º
200 km
represented in v-phenotype j, and Np is the number of v-phenotypes identified in the population. Direct pair-wise comparisons of the populations at each region and year were made using the Rogers index of proportional overlap (HR) (Andrivon 1995; Groth and Sample size
v-factors Analysed
Present
19
16
v-phenotypes (races)
2001
11
7
2002
11
19
16
6
2003
1
19
14
1
2004
8
19
17
8
2005
3
19
15
3
2006
7
19
12
4
2002
12
19
15
7
2004
16
19
15
14
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Roelfs 1987), calculated as: HR =0.5Σj|pj1 −pj2|, where pj1 and pj2 are the frequencies of v-phenotype in samples 1 and 2 respectively, the sum being made over all vphenotypes present in population 1 and/or population 2. HR varies from 0 when all v-phenotypes are present at equal frequencies in both populations, to 1 when the two populations do not share any v-phenotypes. A data matrix of 69 isolates was constructed, standardized and analyzed using the Dice and simple matching coefficients. Both analyses resulted in almost similar results, and only the results obtained by the Dice coefficient are presented. The similarity (S) between isolates (Dice 1945) was calculated as Sxy =2a/(2a+b+c), where a is the number of v-factors present in both isolates, b is the number of v-factors present only in isolate x and c is the number of v-factors present only in isolate y.
Results Disease severity varied over the years of the study. While the pathogen was commonly found in Buskerud County every year, B. lactucae was detected in Vestfold and Østfold only in 2001 and 2004, and in Rogaland County only in 2002 and 2004. A total of 44 unique vphenotypes were identified among the 69 B. lactucae isolates collected during the period 2001 to 2006 (Table 1). The number of v-factors per isolate ranged from four to 16, with an average of 11.75 v-factors present in each isolate. Most isolates (90 %) were virulent to 10 or more of the 19 v-factors that were tested. The most commonly occurring v-factors were v1, v2, v4, v5/8, v6, v7, v10, v11, v12, v13 and v18 (Fig. 2). Virulence factor v17 was not found in any of the tested isolates, while v36 was found in one isolate only. The most variable v-factors were v3, v14, v16 and v37, while v15 was commonly found in isolates collected in the southeast in 2001, 2002 and 2003, but became less frequent and was not present at all in 2006 (Table 2). The opposite was observed for v38, which appeared for the first time in 2004. Four of the identified v-phenotypes were similar to races previously denominated in Europe; Bl:17, 18, 22 and 24 (IBEB n.d.). Bl:17 was found in one isolate from the southeast in 2001, Bl:18 was identified in one isolate in the southeast in 2001 and in three isolates from the southwest (greenhouse) in 2002. A total of seven isolates were identical to Bl:22, of which three isolates
were from the southeast in 2001, and four isolates were collected in 2002, with two from the southwest and two from the southeast, respectively. One isolate from the southwest was identical to Bl:24 in 2004. There were few changes in the diversity of v-factors over time and many v-phenotypes only differed from another by one or two v-factors. The virulence complexity was variable, with a mean number of v-factors per isolate (Ci) ranging from a low of 9.67 in 2004 to a high of 13.18 in 2001 (Table 3). The shifts were however not indicative of any trends towards fewer or more v-factors per isolate during the years of our survey. The similarity between isolates, based on common v-factors was ranging from 0.59 to 1.00 (Table 4). The complexity of vfactors per v-phenotype (Cp) did not expose any differences to the complexity of individual isolates (Table 3). The diversity of v-phenotypes was relatively stable within each region during the years of this survey (Table 3), and there were few overlapping vphenotypes between regions and over years. A total of 11 v-phenotypes were identified more than once (two to seven times), and five of these occurred in successive years. There were some similarities between the southeast and southwest in 2002 (HR =0.83), with one isolate from each region identified as similar v-phenotype. No overlap between regions was identified in the other years of this survey. Overlapping of v-phenotypes over years was most apparent from 2001 to 2002, where two v-phenotypes identified in the southeast in 2001 reappeared in the same region in 2002 (HR =0.64). In addition, two other v-phenotypes found in the southeast in 2001 reappeared in the southwest in the following year (HR =0.74); one in greenhouse lettuce (BL 18) and one in field lettuce (BL 22). One of the v-phenotypes found in the southwest in 2002 reappeared in the southeast in 2005 (HR =0.92). Two v-phenotypes identified in greenhouse lettuce in the southwest in 2002 were compared to the five vphenotypes which were identified in isolates from nearby fields later in the same season. None of the vphenotypes found in the field were identical to those found in greenhouse-isolates. However, only one vfactor (v37) distinguished the most common vphenotype found in greenhouse (3 of 4 isolates; Bl:18) from one of the v-phenotypes in field lettuce. In the remaining v-phenotypes found in the field, the differences in virulence to the greenhouse isolate were in regard to presence or absence of three v-factors (v14, v15 and v16). The second v-phenotype from the
Author's personal copy Frequency (% of isolates tested)
Eur J Plant Pathol 100 90 80 70 60 50 40 30 20 10 0 1
2
3
4
5/8
6
7
10
11
12
13
14
15
16
17
18
36
37
38
Virulence factor
Fig. 2 Frequency of virulence factors in isolates from cultivated lettuce in Norway (N=69) during 2001 to 2006
populations of B. lactucae. The geographical origin and introduction year of Bl:17 was Sweden in 1999, Bl:18 originated from the UK in 1999, while Bl:22 and Bl:24 were first found in the Netherlands in 2000 and 2002, respectively (van Treuren et al. 2011). Further genetic analyses would be required to ascertain if the relationship of the Norwegian isolates extends beyond a phenotypic similarity. Race specific resistance to B. lactucae has generally provided temporary suppression of downy mildew epidemics, with a loss of resistance as virulence changes in the pathogen (Lebeda and Schwinn 1994; Reinink 1999). The frequency of v18 was reported to be generally low in the Czech Republic during 1999–2011 (Petrželová et al. 2013) and in Germany before 1996 (Lebeda and Zinkernagel 2003). However, the frequency of v18 rapidly increased in Germany after 1996 corresponding with the introduction of lettuce cultivars incorporating Dm18 (Lebeda and Zinkernagel 2003). A similar shift in virulence may have occurred in
greenhouse was identified in one isolate only and differed greatly from all other v-phenotypes, with only five v-factors present (v2, v4, v7 and v13).
Discussion The degree of diversity we found in Norwegian populations of B. lactucae, wherein 63 % of the isolates collected represented unique v-phenotypes, is consistent with the magnitude of pathogenic variation reported for several European B. lactucae populations (Jönsson et al. 2005; Lebeda and Zinkernagel 2003; Petrželová et al. 2013; Sharaf et al. 2007). This was not totally expected, as a substantially lesser degree of variability was reported from Sweden in 2000, where only Bl:18 was found, albeit only nine isolates were collected in this year of the study (Jönsson et al. 2005). Four of the identified v-phenotypes were identical to races Bl:17, 18, 22 and 24, which were previously reported in European
Table 2 Observed frequency of v-factors in isolates of Bremia lactucae sampled in Norway during 2001 to 2006 Regiona Year
Sample size v-factor 1
SE
SW
a
2
3
4
5/8 6
7
10
11
12
13
14
15
16
17
18
36
37
38
2001 11
0.9 1.0 0.2 0.9 1.0 0.9 1.0 0.9 0.9 1.0 1.0 0.5 0.9 0.5 0.0 1.0 0.0 0.6 0.0
2002 11
1.0 1.0 0.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.4 1.0 0.4 0.0 1.0 0.1 0.1 0.0
2003
1
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0
2004
8
0.8 0.6 0.3 0.5 0.9 0.3 0.9 0.5 0.3 0.4 0.6 0.4 0.4 0.9 0.0 0.8 0.0 0.1 0.6
2005
3
0.7 0.7 0.0 1.0 1.0 1.0 0.7 1.0 1.0 1.0 1.0 0.7 0.7 0.0 0.0 1.0 0.0 0.3 0.7
2006
7
1.0 1.0 0.0 0.9 1.0 1.0 1.0 0.9 1.0 1.0 0.9 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.9
2002 12
0.9 1.0 0.0 1.0 0.9 0.9 1.0 0.9 0.9 0.9 1.0 0.5 0.3 0.5 0.0 1.0 0.0 0.1 0.0
2004 16
0.5 1.0 0.1 1.0 1.0 1.0 0.9 0.4 0.9 0.6 0.9 0.0 0.0 0.9 0.0 0.9 0.0 0.1 0.9
Isolates collected in the southeast (SE) from Buskerud, Vestfold and Østfold Counties; isolates collected in the southwest (SW) from Rogaland County
Author's personal copy Eur J Plant Pathol Table 3 Virulence complexity expressed as mean number of v-factors per isolate (Ci) and per v-phenotype (Cp) in Bremia lactucae collected in the southeast (SE) and southwest (SW) of Norway during the period 2001 to 2006 Region
Year
Sample size
Ci
Cp
SEa
2001
11
13.18
13.14
2002
11
12.81
13.00
2003
1
14.00
14.00
2004
8
9.00
9.00
2005
3
12.30
12.30
2006
7
11.14
10.5
2002
12
11.92
11.43
2004
16
11.25
11.30
SWb
a
Isolates collected in Buskerud, Vestfold and Østfold Counties
b
Isolates collected in Rogaland County
Norwegian populations of B. lactucae, where v38 was confirmed in 2004, corresponding with loss of field resistance in cultivars incorporating the resistance gene R 38. The survival and initial sources of inoculum for B. lactucae in Norway is still unknown. Oospores have been found in field production of lettuce in Norway (Nordskog et al. 2008b), but their importance relative to other potential sources of inoculum (e.g., airborne sporangia arriving from more southern growing regions) is unknown. Lettuce seedlings are generally produced locally, eliminating the risk of introducing inoculum through transport of plant material from other regions.
Although Lactuca serriola occurs commonly as a weed in lettuce production regions, B. lactucae has not been found on this host in Norway (Nordskog 2006). However, symptoms of B. lactucae on L. serriola is very variable (Lebeda et al. 2008), and the disease may not be recognised if symptoms are expressed as small nectrotic spots with sparse sporulation. Infections of B. lactucae is common on L. serriola in the Czech Republic (Petrželová and Lebeda 2004) and the Netherlands (Hooftman et al. 2007), but only sporadically found on wild Lactucae spp. in other parts of Europe (Lebeda et al. 2001). A clear difference in compositions of vfactors and v-phenotypes between crop and wild pathosystems was demonstrated in the Czech Republic (Lebeda et al. 2008), indicating no direct epidemiological linkage between the different species of host plants. However, some overlap in virulence structure may indicate a potential for migration or gene flow between the pathosystems. Sporangia of B. lactucae have been shown to survive for at least 24 h outdoors when not exposed to direct solar radiation (Nordskog et al., unpublished). Long distance transport of sporangia of Peronospora tabacina and Pseudoperonospoca cubensis has previously been described (Main et al. 2001), but long distance dispersal of sporangia of B. lactucae from Northern Europe to Norway has not yet been demonstrated. However, numerous circumstantial and anecdotal accounts of unexpected outbreaks could be explained by such longdistance transport. For example, a May 2002 outbreak of B. lactucae in a greenhouse in Rogaland County was
Table 4 Comparison of similarities between isolates in regard to v-factors, within each population and between populations over years and regions. Mean of the simple similarity coefficient is calculated for each isolate combination Region
Year
SE 2001
SEa
SWb
SW 2002
2003
2004
2005
2006
2002
2001
0.85
2002
0.88
0.92
2003
0.85
0.88
1.00
2004
0.59
0.60
0.57
0.61
2005
0.79
0.82
0.81
0.60
0.76
2006
0.78
0.82
0.77
0.62
0.82
0.94
2002
0.83
0.81
0.83
0.62
0.79
0.83
0.85
2004
0.70
0.73
0.65
0.65
0.71
0.82
0.76
a
Isolates collected in Buskerud, Vestfold and Østfold Counties
b
Isolates collected in Rogaland County
2004
0.85
Author's personal copy Eur J Plant Pathol
unusually early in the growing season (Nordskog et al. 2008a), and it occurred in an area where there were no records of B. lactucae in the previous year (Nordskog et al. 2008a). Three of the four isolates sampled from this location was identified as Bl:18, a race which was also found in the Netherlands, Germany, UK, France and Sweden in 2002 (A. van der Arend, The International Bremia Evaluation Board (IBEB), pers. comm.). The similarities of v-phenotypes in Norwegian isolates of B. lactucae and the widely occurring races in Europe (Bl:17,18, 22 and 24) could also indicate some long-distance transport of sporangial inoculum, although concurrent selection of the same virulence could just as easily be explained by the deployment of a similar spectrum of resistance genes in commercial cultivars in both regions (Lebeda and Schwinn 1994) thereby selecting a pathogen population with similar virulence factors. In conclusion, the variability of the Norwegian B. lactucae populations verifies the genetic flexibility of this pathogen and its great ability to adapt to changes in host plants and surrounding conditions. A combination of increased grower awareness, disease-suppressive cultural practices, timely fungicide applications, and the introduction of lettuce cultivars incorporating new resistance genes has resulted in few reported losses due to B. lactucae in Norway over the last 5 years. The consequent low disease pressure may extend the useful life of the presently deployed resistance genes in previously released cultivars. Acknowledgments We greatly appreciate the close cooperation with local advisors in the Norwegian Agricultural Extension Service who sampled the lettuce isolates. We also thank A. Hägnefelt, Swalöf Weibull, Sweden and I. Weber, Seminis Research, the Netherlands, and their colleagues for testing the B. lactucae isolates used in this study. Thanks also to T. Rafoss for statistical advice, and A. Stensvand for valuable comments on the manuscript. Figure 1 was kindly provided and adjusted by E. Fløistad. This study was part of a project funded by The Research Council of Norway, Innovation Norway and Norwegian lettuce growers.
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