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This study tested the hypothesis that aggressive, localized infections and asymptomatic systemic infections were caused by distinct specialized groups of Botrytis cinerea, using microsatellite genotypes at nine loci of 243 isolates of B. cinerea obtained from four hosts (strawberry (Fragaria×ananassa), blackberry (Rubus fruticosus agg.), dandelion, (Taraxacum officinale agg.) and primrose (Primula vulgaris)) in three regions in southern England (in the vicinities of Brighton, Reading and Bath). The populations were extremely variable, with up to 20 alleles per locus and high genic diversity. Each host in each region had a population of B. cinerea with distinctive genetic features, and there were also consistent host and regional distinctions. The B. cinerea population from strawberry was distinguished from that on other hosts, including blackberry, most notably by a common 154-bp amplicon at locus 5 (present in 35 of 77 samples) that was rare in isolates from other hosts (9/166), and by the rarity (3/77) of a 112-bp allele at locus 7 that was common (58/166) in isolates from other hosts. There was significant linkage disequilibrium overall within the B. cinerea populations on blackberry and strawberry, but with quite different patterns of association among isolates from the two hosts. No evidence was found for differentiation between populations of B. cinerea from systemically infected hosts and those from locally infected fruits.
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The motivation for this study arose from the observation of frequent systemic, asymptomatic, infection by Botrytis cinerea in cultivated primula (Primula×polyantha; Barnes & Shaw, 2002, 2003) and lettuce (Lactuca sativa; Sowley et al., 2010). Botrytis cinerea causes grey mould, which is a widespread disease leading to substantial losses in numerous crops including grape, tomato, sunflower, chickpea, soft fruit such as strawberry, and cut flowers (Elad et al., 2004). In the generally accepted life cycle, a spore, distributed by air currents or insects, lands on a plant surface and infects tissues directly, causing a spreading necrotic lesion in which plant tissues are killed ahead of the fungal mycelium by a variety of phytotoxic excretions from the fungus (van Kan, 2006). This will be referred to as an ‘aggressive’ infection. If plant defences are more active than the pathogen’s attack mechanisms, an infection may fail or be contained within a few cells as a ‘quiescent’ lesion until the plant tissue senesces or ripens (Holz et al., 2004). Both aggressive and quiescent infections are local in the sense of spreading from an initial infection within a well-defined zone on the plant containing host cell damage. This life cycle is well established in soft fruits such as blackberry and strawberry, and quiescent infections which develop into symptomatic infections as the fruits ripen are common; however, the pathogen is not distributed through these plants in the absence of symptoms (Jarvis, 1962; McNicol & Williamson, 1989; Boff et al., 2001). By contrast, in cultivated primula and lettuce, identical genotypes of B. cinerea can be isolated from surface-sterilized host tissue sampled from widely separated parts of plants with no visible damage from disease, or from airborne and soilborne disease sources. This genetic evidence suggests that the infection is systemic rather than local. Both quiescent infections and these systemic infections are symptomless, but inside the plant, and will be therefore be described as endophytic. The genome of B. cinerea has been sequenced and there is intensive study of the molecular mechanisms by which the fungus enters plants and causes damage (Williamson et al., 2007). Systemic infection could be of considerable fundamental interest, as it indicates an alternative mode of growth in which either fungal growth just keeps up with marginally activated plant defences, or the fungus grows without triggering defences, or the plant defences are dampened as in a biotrophic attack.
In unpublished pilot studies of various wild hosts, B. cinerea was isolated from surface-sterilized leaves and, more commonly, roots of both healthy dandelion plants, Taraxacum officinale agg. and wild primrose, Primula vulgaris. Because airborne inoculum is unlikely to infect roots and a similar pattern of isolations is seen in symptomless lettuce and P. ×polyantha grown in sterilized soil, this may represent systemic infection. In both P. ×polyantha and lettuce, although the fungus was distributed throughout symptomless plants, inoculation of detached leaves showed the isolates recovered to be capable of aggressive necrotic infections (A Shafia & M W Shaw, University of Reading, unpublished data). It is surprising to find a well-studied aggressive necrotroph to have an extended phase of systemic but asymptomatic growth within its host. The primary objective of this study was therefore to test whether there was genetic differentiation between systemic forms of the fungus (i.e. those infecting primrose and dandelion) and the aggressive forms infecting rosaceous soft fruit. At one extreme, the differences in life-cycle between the two types of infection might indicate cryptic species, as hypothesised by Giraud et al. (1999). An alternative hypothesis is that populations in a single region might form a panmictic epidemiological unit, well-mixed across hosts. This would be expected from the weak specialization shown when B. cinerea was used in cross-host test inoculations (Jarvis, 1980), and the mixing scale of at least several kilometres expected for aerially dispersed pathogens.
Previous molecular work showed a number of genetic subdivisions within B. cinerea. Fournier et al. (2005) used sequence data from several genes to show that the morphological species B. cinerea contains two quite independently evolving lineages distinguished by a private allele of 86 bp at a microsatellite locus Bc6 (Karchani-Balma et al., 2008). Fournier & Giraud (2008), using both sequencing and microsatellite loci (Fournier et al., 2002), showed that B. cinerea populations on R. fruticosus from sites across France differed from those on adjacent grapevines, suggesting a degree of host or ecological specialization. This is in accord with the earlier finding that the population of B. cinerea from vineyards in the Champagne region of France contained at least two ecologically distinct groups (Giraud et al., 1999). However, the characterization of these as distinct subspecies (vacuma and transposa) based on joint presence or absence of the transposons Boty and Flipper, has not been confirmed. Differentiation between virulence-associated genes in isolates from different hosts also suggests at least some degree of host specialization (Choquer et al., 2007). In Chile, populations of B. cinerea on kiwifruit were clearly separated from populations isolated from symptomless tomato leaves (Muñoz et al., 2002). Both were separated from what appeared to be a single population on grape and blueberry. However, RAPD primers were used in that study, with small samples of isolates. In contrast, based on clustering in phenograms derived from microsatellite markers, studies in California found no evidence of separation between isolates of B. cinerea collected from tissues of kiwifruit, grape, fig, pea and squash showing symptoms (Ma & Michailides, 2005). Similarly, using RAPDs, Calpas et al. (2006) found no evidence of host separation, although sample sizes were small (2–7), but did find distinguishable populations in spatially separated glasshouses. More recently, Karchani-Balma et al. (2008) used microsatellite data to characterize isolates of B. cinerea obtained from sporulating lesions, mainly on tomato, sampled from locations along the coast of Tunisia. They found evidence for some separation between populations, but were unable to reliably distinguish differentiation resulting from geography from that resulting from host because different hosts occurred in different locations and most of the isolates came from one group of glasshouses near each other; where more than one host species occurred in a single location, no evidence was of genetic separation between B. cinerea populations on different hosts, suggesting free mixing and a lack of host specialization. Váczy et al. (2008) found little evidence of differentiation between populations of sporulating B. cinerea on grape in the Czech Republic. All of these studies found substantial genetic variability within the population of B. cinerea at a single location or on a single host.
The aim of the study reported here was therefore to test the hypothesis that B. cinerea isolates from rosaceous soft fruits comprise a distinct population from apparently systemic infections in sympatric host species.
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Isolates of B. cinerea were obtained at all three locations from all four hosts, except primrose at Bath (Table 1). No isolates were obtained from 90 and 105 surface-sterilized leaves of strawberry and blackberry, respectively.
Table 1. Sources and sizes of Botrytis cinerea samples used for genetic characterization
|Fragaria ×ananassa (strawberry)||18||29||30||77|
|Rubus fruticosus agg. (blackberry)||35||35||31||101|
|Taraxacum officinale agg. (dandelion)||6||18||11||35|
|Primula vulgaris (primrose)||24||0||6||30|
The B. cinerea population was very variable. The nine primers generated 222 amplicons from the 243 isolates tested. None of the primers generated a regular series of amplicon lengths interpretable as numbers of repeats. No identical haplotypes were found in these populations when no amplification in three replicate runs with successful controls was taken as a null allele. Isolates were usually (86%) haploid at all loci. There were 22 isolates with two alleles present at a single locus, five isolates with two alleles present at two loci and one isolate with two alleles present at three loci. Where multiple alleles were present at a locus, analyses of population diversity described below were repeated taking each allele in turn. The outcome was essentially unchanged by the choice of allele in these isolates.
The transposons Boty and Flipper were common (Table 1). They were usually found singly. Isolates with both transposons were common in dandelion at Bath and in primula in Brighton, but otherwise absent. Both transposons were found in all three locations and all four hosts. At a single location, the two transposons were often restricted to isolates from characteristic hosts. However, these associations were not consistent across locations (Table 2). For example, 90% of isolates from strawberry at Reading contained Flipper, but only 3% at Bath.
Table 2. Frequency of transposons Boty and Flipper in isolates of Botrytis cinerea from four hosts and three locations in southern England
|Location||Host||Boty||Flipper||Both Boty and Flipper||Total|
|Primrose|| || || ||–b|
Seven of the isolates (two each from blackberry at Bath and Brighton, two from Dandelion at Bath and one from strawberry at Reading) had an 84-bp allele at Bc6, similar to the 86-bp allele which characterizes group-1 isolates. However, four of these 84-bp isolates contained Boty or Flipper, which have not been reported to date in group 1. It was therefore concluded that these isolates were probably members of group 2. The conclusions from the analysis which follows were unchanged whether these seven isolates were included or excluded.
Pairwise FST calculations confirmed genetic differences among isolates from different hosts, locations and host–location subgroups (P < 0·001), but not whether the differences were affected by confounding between hosts and locations as a result of unequal sample sizes (Table 3). Using amova, differences were found among hosts (compared to sites within hosts as subpopulations, P < 0·001) and sites (compared to sites within hosts as subpopulations, P < 0·001).
Table 3. Within-population diversity measures and pairwise FST estimates between populations of Botrytis cinerea taken from four host species at three locations in southern England, using microsatellite loci
| ||Pairwise FST between hosts||Mean genic diversitya||Mean pairwise differencesb||Mean squared allele frequency||Mean number of alleles per locus|
|Strawberry|| || || ||0·86||3·85||0·013||17·1|
|Blackberry||0·084***c|| || ||0·88||3·96||0·010||20·8|
| ||Pairwise FST between locations|| || || || || |
|Bath|| || || ||0·91||4·09||0·012||20·4|
|Brighton||0·020***|| || ||0·87||3·94||0·012||19·6|
Using PCA the strawberry isolates formed a group distinct from isolates from all other hosts (Fig. 1a). Isolates from different locations grouped together weakly (Fig. 1b). Using CVA, populations from different hosts clearly separated along the first two canonical axes into three groups: strawberry, blackberry and dandelion+primrose (Fig. 2a, P < 0·001). Dandelion and primrose populations were separated on the third axis at P < 0·001. After removing the effect of geographic location, fungal isolates from all four host species were clearly separated on the first two canonical axes; strawberry and blackberry remained the most distinct groups (Fig. 2b). Similarly, isolates from different locations were distinct from each other, both in the original data and the reduced dataset with the effect of host removed (Fig. 3; P < 0·001).
Figure 1. Principal components analysis of Botrytis cinerea isolates from southern England, based on presence/absence of each allele at nine microsatellite loci. (a) Points labelled by host: 1, Rubus fruticosus agg. (blackberry); 2, Fragaria ×ananassa (strawberrry); 3, Taraxacum officinale agg. (dandelion); 4, Primula vulgaris (primrose). (b) Points labelled by geographic locality: 1, Brighton; 2, Bath; 3, Reading.
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Figure 2. Separation of Botrytis cinerea isolates according to host species based on presence/absence of alleles at nine microsatellite loci. Axis scaling adjusted to the eigenvalue associated with the axis. (a) Original data. (b) Dataset after removal of linear combinations of loci which maximally separate sampling locations. Numbers refer to host species from which isolate was obtained: 1, Rubus fruticosus agg. (blackberry); 2, Fragaria ×ananassa (strawberrry); 3, Taraxacum officinale agg. (dandelion); 4, Primula vulgaris (primrose).
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Figure 3. Separation of Botrytis cinerea isolates according to sampling location based on presence/absence of alleles at nine microsatellite loci. Axis scaling adjusted to the eigenvalue associated with the axis. (a) Original data. (b) Dataset after removal of linear combinations of loci which maximally separate hosts and rescaling the CVA axes to produce real coordinates. (In this set, the CVA transformation of the reduced data produced complex coordinates, but with real and imaginary parts exactly proportional in each axis, so that rescaling by a complex number, i.e. a rotation in the complex plane, resulted in real numbers). Numbers refer to regions from which isolates came: 1, Brighton; 2, Bath; 3, Reading.
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Comparing the lists of 20 alleles contributing most to separating isolates from different sites and hosts (Fig. 4), 12 alleles appeared in both lists. For example, allele 139 at primer 9 was absent from all isolates sampled from Brighton and from R. fruticosus isolates, but common in F. ×ananassa (except at Brighton) (Fig. 4).
Figure 4. Proportions of Botrytis cinerea isolates belonging to (a) each host of origin (b) each sampling location which carried each of the five microsatellite alleles most informative to separate (a) hosts and (b) sampling locations. The alleles shown are those most informative in the original dataset, without first removing the data features discriminating best between groups classified by the alternative factor. Alleles are labelled by primer[allele length].
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Linkage disequilibrium was low between any individual pairs of alleles. There were about 50 000 possible allele pairs between which disequilibrium could occur. The expected number of individually significant linkage disequilibria at P < 0·001 was 50, against 13 observed. However, the overall disequilibrium among pairs of loci, tested by a Fisher exact test on the whole table, was significant at P ≤ 0·001 for 30 of the 36 possible pairs in the whole dataset. The patterns of loci between which there was significant disequilibrium differed between each host species (Fig. 5). In strawberry, locus Bc2 was largely in linkage equilibrium with all the other loci, whereas all other loci were in significant linkage disequilibrium. In blackberry, all loci were in disequilibrium with some other loci. In dandelion, Bc4 and Bc9 were close to linkage equilibrium with all other loci, whereas in primrose, Bc7 and Bc10 each had only one other locus with which there was significant linkage disequilibrium. Because of the great diversity of alleles at any one locus, it was not possible to assign this disequilibrium to particular associations or dissociations between alleles at particular pairs of loci.
Figure 5. Significance of linkage disequilibrium across loci in (a) Rubus fruticosus agg. (blackberry) N = 101; (b) Fragaria ×ananassa, (strawberrry) N = 77; (c) Taraxacum officinale agg. (dandelion); N = 35; (d) Primula vulgaris (primrose). N = 30. Significance based on Fisher’s exact test across all microsatellite alleles at both loci. White, P > 0·1; black, P < 0·0001; grey indicates intermediate levels of significance. P1–P10 refer to microsatellite loci.
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The population of B. cinerea sampled in this study was extremely diverse, and repeated recovery of the same haplotype was rare. Most individuals were haploid and diversity was very large, confirming the findings of other population surveys (Giraud et al., 1999; Michailides & Elmer, 2000; Baraldi et al., 2002; Muñoz et al., 2002; Beever & Weeds, 2005; Fournier et al., 2005; Ma & Michailides, 2005; Fournier & Giraud, 2008; Isenegger et al., 2008; Karchani-Balma et al., 2008; Váczy et al., 2008; Decognet et al., 2009). It remains surprising that sexual fruiting bodies are still apparently hard to find in field settings.
The number of isolates with multiple alleles at a locus is an upper bound on the number of heterokaryotic or partially heterokaryotic isolates. For example, some of the 14% of isolates with multiple bands could be mixtures of vegetatively incompatible isolates, although it would be surprising if such mixtures were stable in culture. Isolates in the sample in this study were extremely diverse, so true mixtures of independent isolates would usually differ at many loci. In contrast, the majority (23/32) of isolates heterozygous at any locus were heterozygotic at a single locus. One explanation for this finding is a parasexual cycle with elimination of much DNA from one partner (Debets, 1998); another is mutation in a particular nucleus during normal colony growth (Beever & Weeds, 2005).
The aim of this work was to test two related hypotheses. The first was that populations of B. cinerea are divided into distinct aggressive (strawberry and blackberry) and systemic (primrose and dandelion) forms. This was not supported by the data. The greatest distinction in the data was between the populations found on the rosaceous fruits strawberry and blackberry. This was very surprising, since both are infected by airborne spores and blackberry is a ubiquitous and frequent hedgerow species in all farming areas in southern England. This distinction was present both in the alleles present and in the patterns of linkage disequilibrium in the distinct populations. Since the populations of isolates on different host species with aggressive and localized B. cinerea were as different from each other as the populations on hosts with systemic endophytic infection, it is plausible that differences between these types of infection are physiological rather than genetically conditioned. If true, this would in turn mean that infection resulting from contact between B. cinerea and a host depends on host condition and the density and environment of the attacking fungus (Barnes & Shaw, 2002). This was supported by evidence that systemic isolates from both lettuce and primrose can cause aggressive infections when inoculated as concentrated spore suspensions on detached plant organs (Shafia, 2009).
The second hypothesis was that the population of B. cinerea would be panmictic. This was not true on the scales probed here; there were distinctive features of the isolates found on each host species which sufficed to separate the population on that host from the population on other hosts, across several hundred kilometres. Furthermore, the transposon data suggested that the populations were not panmictic across hosts on scales of a few hundred metres. Not unexpectedly, there was also regional differentiation. This is consistent with the findings of Fournier & Giraud (2008) and Karchani-Balma et al. (2008). The divergent findings of Ma & Michailides (2005) in California may be explained by smaller sample sizes, a different population structure, or a chance selection of hosts sharing a common pool of pathogen genotypes.
Not all host species could be found sympatrically in all the regions. Both host and regional separation remained significant when the influence of the other was removed. However, two features of the sampling warrant discussion. First, the other host populations were isolated from the strawberry populations at Reading. However, the alleles best differentiating regions were a distinct set from those differentiating host species (Fig. 5), so the spatial separation of these populations did not account for the host differentiation observed. Secondly, the primrose sample was very unbalanced, with the bulk of the isolates from the Brighton region and from a nature reserve separated from other hosts in the same region. However, the interesting feature of the data is that the primrose and dandelion populations were the least separated in the data, so again the main inferences made are secure.
Because the populations were regionally differentiated, it is hard to interpret the substantial linkage disequilibrium found. This could reflect the mixture of local populations on particular hosts which are individually random, as in the Wahlhund effect, but this is hard to test, since the power of tests is small with the sample sizes taken for individual host–locality combinations. Given that asexual reproduction is at least very common in B. cinerea (Jarvis, 1980), it seems likely that the disequilibrium was the result of local differences in the success of particular allele combinations at selected loci altering the frequencies of linked microsatellites. This would be consistent with infection of particular hosts being favoured by the presence of specific alleles at loci related to infection processes; in particular groups of clones, these would be associated with different microsatellite loci. Such an explanation would be consistent with the differentiation between hosts found here, and is consistent with work showing the complexity of the genetic architecture of resistance to B. cinerea (Rowe & Kliebenstein, 2008).
In conclusion, populations of B. cinerea were not well mixed across their hosts. The population on strawberry fruit was quite distinct from that on blackberry. This was unexpected, as the two hosts are both rosaceous, infection mechanisms and development in fruit ripening appear similar, and the two are often sympatric. This suggests that the disease cycles on separate hosts in a single location may be only weakly linked. However, there was no clear differentiation between populations on immature soft fruit and those growing distributed within vegetative tissues of two wild hosts. This suggests that individual B. cinerea isolates may be able to form either symptomless, systemic associations with hosts or aggressive necrotising lesions, depending on environmental circumstances and host condition.