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Keywords:

  • Botryotinia fuckeliana;
  • endophyte;
  • geographic variation;
  • host specialization;
  • linkage disequilibrium

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The experimental design was a crossed factor, sampling isolates of B. cinerea from each host under study in three distinct regions of southern England, each 100–150 km from the others. Isolates were obtained from ripening symptomless fruits of wild blackberry (R. fruticosus agg.) and cultivated strawberry (F. ×ananassa) to represent ‘standard’ aggressive forms of the fungus, and roots and leaves of healthy plants of wild primrose and dandelion to represent systemic forms. Thus, all isolates were taken from infections in a latent phase of colonization.

Isolate collection

Isolates characteristic of hosts in which only aggressive necrotic or localized quiescent types of infection were expected, because they were well-studied and no reports of systemic infection had been published, were collected from symptomless fruits of cultivated strawberry and wild blackberry. Isolates characteristic of systemically infected hosts were cultured from symptomless leaves and roots of wild primrose and dandelion. Isolates were collected from all hosts in three different regions of southern England in the vicinity of the cities of Brighton, Bath and Reading, except that no primrose isolates could be obtained from the primrose population sampled near Bath. Reading is approximately 100 km from Brighton and Bath, which are about 150 km apart. Collections were from sympatric hosts where possible. Around Brighton, dandelion, strawberry and 15 of the 35 blackberry isolates were collected from sympatric host populations on a farm site (50°50′N, 0°3′W); primrose is only locally common and was collected from a nature reserve 33 km away (50°49′N, 0°27′W), along with 20 blackberry isolates. At Reading, dandelion and blackberry isolates, and three of the six primrose isolates were collected from the University campus (51°26′N, 0°56′W). The remaining three of the primrose isolates came from about 7 km north of the University campus (51°29′N, 0°59′W, and the strawberry isolates from about 10 km south (51°23′N, 0°50′W). In the Bath area, samples were collected at 51°26′N, 2°17′W. All blackberry isolates were collected during August and September 2005. Strawberry isolates were collected in August and September 2005 and again in June–August 2006. Dandelion and primrose isolates were collected between August and December 2005, and between June and September 2006.

For each plant sampled, one fruit per strawberry plant was collected, and two to three fruits were collected from each blackberry bush. One leaf and a root were collected from each wild primrose and dandelion plant. Plant samples were washed gently with running water to remove any debris on the surface and then rinsed in 95% ethanol for 10 s. Next, each sample was washed with 70% ethanol for 1 min, 20% Domestos (0·1% hypochlorite) solution for 3–5 min and sterilized distilled water three times. Pieces of roots and leaves (c. 1 cm2) of dandelions and primrose were plated on 3% malt-extract agar (MEA; Oxoid) supplemented with streptomycin and chloramphenicol (0·2 g L−1). Strawberry sections and blackberries were plated on Botrytis selective medium (BSM; Edwards & Seddon, 2001). Small portions taken from the growing tips of mycelia similar to B. cinerea were subcultured on to 3% MEA and the purity and identity of the culture confirmed microscopically. Cultures were incubated at 18 ± 2°C under UV light with a photoperiod of 12 h day−1. Medium-term storage was on MEA slants at 2°C.

The isolates studied here were not single-spored because individual conidia of B. cinerea contain many nuclei (2–13: Saleh, 2002), and vegetatively compatible isolates may fuse to produce heterokaryotic mycelium.

Microsatellite and transposon characterization

For each DNA extraction, 100 mL malt-extract broth was inoculated with 5-mm agar discs taken from the edge of an actively growing culture (1–2 weeks) and incubated at room temperature on a shaker at 112 r.p.m. Mycelium was harvested after 7 days by filtering the culture through sterilized muslin cloth in a laminar flow hood. The mycelium was transferred to a sterilized filter paper in a sterile Petri dish which was sealed with parafilm and stored at −20°C until DNA was extracted. Approximately 100 mg of the fungal mycelium was ground to a fine powder in liquid nitrogen using a sterilized mortar and pestle, transferred to a 1·5-mL microtube.

DNA extraction was done using a Qiagen Plant Mini-kit according to the manufacturer’s instructions. DNA was quantified using the Quant-iT Picogreen dsDNA quantitation kit (Invitrogen) and adjusted to 10 ng μL−1.

Nine published microsatellite primers for B. cinerea (Fournier et al., 2002) were used to characterize the isolates, along with the Boty and Flipper transposable elements (Giraud et al., 1999). The PCR programme used was: initial denaturing at 94°C for 2 min followed by 35 cycles of 1 min at 94°C, 1 min at annealing temperature (optimized for each primer) and 30 s at 72°C. A final extension period at 72°C lasted 5 min. Each 10-μL reaction contained 2 μL water, 5 μL PCR master mix (Abgene), 1 μL each of forward and reverse primers and 1 μL template DNA solution. Annealing temperatures were 53°C for primers Bc1, Bc2, Bc3, Bc5, Bc6 and Bc9; and 59°C for primers Bc4, Bc7 and Bc10. Fragment profiles were determined using an ABI 3130 capillary electrophoresis machine (Applied Biosystems) with three labelled primers with well-separated fluorescence peaks. Reaction products Bc1, Bc4, Bc9; Bc3, Bc6 and Bc10; and Bc2, Bc5 and Bc7; were mixed because the expected products had nonoverlapping ranges. Primers Bc1, Bc2 and Bc6 were labelled with 6-carboxyflourescein (FAM-6); Bc3, Bc4 and Bc5 with the proprietary NED (Applied Biosystems); and Bc7, Bc9 and Bc10 with 6-carboxy-2′, 4, 4′, 5′, 7, 7′-hexachlorofluorescein (HEX).

The Flipper transposon was detected using PCR with the primer pair F300 (5′-GCACAAAACCTACAGAAGA-3′) and F1550 (5′-ATTCGTTTCTTGGACTGTA-3′) (Levis et al., 1997). The Boty transposon was detected by PCR with the primer pair Boty F4 (5′-CAGCTGCAGTATACTGGGGGA-3′) and Boty R4 (5′-GGTGCTCAAAGTGTTACGGGAG-3′) (Ma & Michailides, 2005). All primers were synthesized by Sigma Genosys. All reactions were repeated, and isolates for which discrepant or null results were observed were triplicated. The result occurring twice was then included in the data analysed; otherwise the data were discarded.

Statistical analysis

FST statistics measuring genic diversity between populations classified by host or location and linkage disequilibria between loci were calculated with arlequin 3 (Excoffier et al., 2005). Calculations for both canonical variates analysis (CVA) and analysis of molecular variance (amova) population separations were made in mathematica v4–6 (Wolfram Research). CVA was further conducted using genstat v8–10 (VSN International), with similar results. mathematica and genstat code used in this study is available on request from the authors.

To explore differentiation between groupings of isolates more completely, CVA was used, using a trace statistic as a summary of differentiation. CVA is not widely used, so a brief explanation is needed. Let the number of measurements made on each individual be p. These data can be represented as a vector xi for the ith individual, with elements xij,the index j running from 1 to p. The allele frequency at the jth locus in the kth group is denoted fkj. Differences between groups can be visualized best by using coordinates that maximize those differences relative to the differences within groups. The coordinates resulting from this transformation are the canonical variates (Kendall, 1975). CVA will produce a visualization of the data that shows groups as clearly separated, whether the differences are genuine or the result of chance sampling effects. The natural measure of how separate the groups found are is the trace of the matrix ratio W−1B, where B is the matrix of between-group sums of squares and products and W is the matrix of within-group sums of squares and products. This measure and a randomization test (10 000 replicates) were used to determine whether groups were more distinct than expected by chance.

If there are significant differences between groups in the CVA, the eigenvectors of the ratio matrix provide a natural summary of where these lie. There are g1 eigenvectors of W−1B, e1...ei...eg−1, with elements labelled ejk, which specify the transformation of coordinates that maximally separates groupings. A small element in one of these eigenvalues means that measurement or locus contributes little to discrimination between groups. However, if an allele is rare, but found mainly within one group, it may have a high loading but little discriminatory power. Therefore, to understand the ways in which groups differ the relative values of inline image, where an overbar denotes a mean value across groups, were used.

The data in this study were cross-classified by host and region, but were not completely balanced. To test whether differentiations resulting from one factor, e.g. host, were influencing the other, a CVA was performed using host as the classifying factor. The data were then projected onto the residual space, omitting variation in the directions specified by the eigenvectors of W−1B which separate the groups of FA. A CVA was then done on this reduced space, but using the groups of the second factor FB. If this was significant then it was likely that both factors were independently associated with variation in the genetic structure, since the major causes of differentiation between levels of FA did not influence separation according to factor FB. This and the signficance test were verified extensively using simulated data.

Principal components analysis (PCA) has been very illuminating in some studies (Novembre et al., 2008) and was used to examine the overall data. PCA asks the question ‘what projection of the data accounts for as much of the variability as possible?’. If variation within groups occurs on different axes to variation between groups and is substantial, a PCA projection will not show group separations, even though they may be present.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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
HostBrightonBathReadingTotal
Fragaria ×ananassa (strawberry)18293077
Rubus fruticosus agg. (blackberry)353531101
Taraxacum officinale agg. (dandelion)6181135
Primula vulgaris (primrose)240630
Total828378243

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
LocationHostBotyFlipperBoth Boty and FlipperTotal
  1. aHost–location groupings differed (χ= 72·7, 15 d.f. < 0·001 using a generalized linear model with Poisson error and logarithm link). Host and location differences were not significant when compared to the χ2 between host–location groupings (F9,15 = 2·49, = 0·06 and F11,15 = 2·15, = 0·08).

  2. bNo isolates obtained.

BrightonaBlackberry680034
Strawberry078019
Dandelion6006
Primrose102824
BathBlackberry620034
Strawberry703030
Dandelion28283318
Primrose   b
ReadingBlackberry090031
Strawberry083030
Dandelion0100013
Primrose50006

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 (< 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, < 0·001) and sites (compared to sites within hosts as subpopulations, < 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 hostsMean genic diversityaMean pairwise differencesbMean squared allele frequencyMean number of alleles per locus
StrawberryBlackberryDandelion
  1. aProbability that any two alleles from the population differ, averaged over loci.

  2. bMean number of differences between any two haplotypes.

  3. c*P < 0·05; ***P < 0·001

Strawberry   0·863·850·01317·1
Blackberry0·084***c  0·883·960·01020·8
Dandelion0·079***0·015*** 0·883·930·02913·8
Primrose0·083***0·025***0·012*0·873·810·0339·9
 Pairwise FST between locations     
BathBrighton
Bath   0·914·090·01220·4
Brighton0·020***  0·873·940·01219·6
Reading0·031***0·027*** 0·894·000·01320

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, < 0·001). Dandelion and primrose populations were separated on the third axis at < 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; < 0·001).

image

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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image

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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image

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).

image

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 < 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  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.

image

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, > 0·1; black, < 0·0001; grey indicates intermediate levels of significance. P1–P10 refer to microsatellite loci.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
  • Baraldi E, Bertolini P, Cheierici E, Trufelli B, Luiselli D, 2002. Genetic diversity between Botrytis cinerea isolates from unstored and cold stored kiwi fruit. Journal of Phytopathology 150, 62935.
  • Barnes SE, Shaw MW, 2002. Factors affecting symptom production by latent Botrytis cinerea in Primula ×polyantha. Plant Pathology 51, 74654.
  • Barnes SE, Shaw MW, 2003. Infection of commercial hybrid primula seeds by Botrytis cinerea and latent disease spread through the plants. Phytopathology 93, 5738.
  • Beever RE, Weeds PL, 2005. Botrytis taxonomy and genetic variation. In: EladY, WilliamsonB, TudzynskiB, DelenN, eds. Botrytis: Biology, Pathology and Control. Dordrecht, the Netherlands: Kluwer, 2952.
  • Boff P, Kastelein P, De Kraker J, Gerlagh M, Kohl J, 2001. Epidemiology of grey mould in annual waiting-bed production of strawberry. European Journal of Plant Pathology 107, 61524.
  • Calpas JT, Konschuh MN, Toews CC, Tewari JP, 2006. Relationships among isolates of Botrytis cinerea collected from greenhouses and field locations in Alberta, based on RAPD analysis. Canadian Journal of Plant Pathology 28, 10924.
  • Choquer M, Fournier E, Kunz C et al. , 2007. Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphagous pathogen. FEMS Microbiology Letters 277, 110.
  • Debets AJM, 1998. Parasexuality in fungi: mechanisms and significance in wild populations. In: BridgeP, CouteaudierY, ClarksonJ, eds. Molecular Variability of Fungal Pathogens. Wallingford, UK: CAB International, 4152.
  • Decognet V, Bardin M, Trottin-Caudal Y, Nicot PC, 2009. Rapid change in the genetic diversity of Botrytis cinerea populations after the introduction of strains in a tomato glasshouse. Phytopathology 99, 18593.
  • Edwards SG, Seddon B, 2001. Selective media for the specific isolation and enumeration of Botrytis cinerea conidia. Letters in Applied Microbiology 32, 636.
  • Elad Y, Williamson B, Tudzynski P, Delen N, 2004. Botrytis spp. and diseases they cause in agricultural systems – an introduction. In: EladY, WilliamsonB, TudzynskiP, DelenN, eds. Botrytis: Biology, Pathology and Control. Dordrecht, the Netherlands: Kluwer, 18.
  • Excoffier L, Laval G, Schneider S, 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1, 4750.
  • Fournier E, Giraud T, 2008. Sympatric genetic differentiation of a generalist pathogenic fungus, Botrytis cinerea, on two different host plants, grapevine and bramble. Journal of Evolutionary Biology 21, 12232.
  • Fournier E, Giraud T, Loiseau A et al. , 2002. Characterization of nine polymorphic microsatellite loci in the fungus Botrytis cinerea (Ascomycota). Molecular Ecology Notes 2, 2535.
  • Fournier E, Giraud T, Albertini C, Brygoo Y, 2005. Partition of the Botrytis cinerea complex in France using multiple gene genealogies. Mycologia 97, 125167.
  • Giraud T, Fortini D, Levis C et al. , 1999. Two sibling species of the Botrytis cinerea complex, transposa and vacuma, are found in sympatry on numerous host plants. Phytopathology 89, 96773.
  • Holz G, Coertze S, Williamson B, 2004. The ecology of Botrytis on plant surfaces. In: EladY, WilliamsonB, TudzynskiP, DelenN, eds. Botrytis: Biology, Pathology and Control. Dordrecht, the Netherlands: Kluwer, 927.
  • Isenegger DA, MacLeod WJ, Ford R, Taylor PWJ, 2008. Genotypic diversity and migration of clonal lineages of Botrytis cinerea from chickpea fields of Bangladesh inferred by microsatellite markers. Plant Pathology 57, 96773.
  • Jarvis W, 1962. The infection of strawberry and raspberry fruits by Botrytis cinerea. Annals of Applied Biology 50, 56975.
  • Jarvis WR, 1980. Epidemiology. In: Coley-SmithJR, VerhoeffK, JarvisWR, eds. The Biology of Botrytis. London, UK: Academic Press Inc, 21950.
  • Van Kan JAL, 2006. Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends in Plant Science 11, 24753.
  • Karchani-Balma S, Gautier A, Raies A, Fournier E, 2008. Geography, plants and growing systems shape the genetic structure of Tunisian Botrytis cinerea populations. Phytopathology 98, 12719.
  • Kendall MG, 1975. Multivariate Analysis. London, UK: Griffin.
  • Levis C, Fortini D, Brygoo Y, 1997. Flipper, a mobile Fot1-like transposable element in Botrytis cinerea. Molecular and General Genetics 254, 67480.
  • Ma ZH, Michailides TJ, 2005. Genetic structure of Botrytis cinerea populations from different host plants in California. Plant Disease 89, 10839.
  • McNicol RJ, Williamson B, 1989. Systemic infection of black currant flowers by Botrytis cinerea and its possible involvement in premature abscission of fruits. Annals of Applied Biology 114, 24354.
  • Michailides TJ, Elmer PAG, 2000. Botrytis gray mold of kiwifruit caused by Botrytis cinerea in the United States and New Zealand. Plant Disease 84, 20823.
  • Muñoz G, Hinrichsen P, Brygoo Y, Giraud T, 2002. Genetic characterisation of Botrytis cinerea populations in Chile. Mycological Research 106, 594601.
  • Novembre J, Johnson T, Bryc K et al. , 2008. Genes mirror geography within Europe. Nature 456, 98101.
  • Rowe HC, Kliebenstein DJ, 2008. Complex genetics control natural variation in Arabidopsis thaliana resistance to Botrytis cinerea. Genetics 180, 223750.
  • Saleh H, 2002. Studies in the Genetic Variability and Management of Botrytis cinerea. Reading, UK: University of Reading, PhD thesis.
  • Shafia A, 2009. Latent infection of Botrytis cinerea. Reading, UK: University of Reading, PhD thesis.
  • Sowley ENK, Dewey FM, Shaw MW, 2010. Persistent, symptomless, systemic and seedborne infection of lettuce by Botrytis cinerea. European Journal of Plant Pathology 126, 6171.
  • Váczy KZ, Sándor E, Karaffa L et al. , 2008. Sexual recombination in the Botrytis cinerea populations in Hungarian vineyards. Phytopathology 98, 13128.
  • Williamson B, Tudzynski B, Tudzynski P, Van Kan JAL, 2007. Botrytis cinerea: the cause of grey mould disease. Molecular Plant Pathology 8, 56180.