# Comparison of Botrytis cinerea populations collected from tomato greenhouses in Northern Algeria

Ahmed Adjebli, Christel Leyronas, Kamel Aissat, Philippe C. Nicot

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## To cite this version:

Ahmed Adjebli, Christel Leyronas, Kamel Aissat, Philippe C. Nicot. Comparison of Botrytis cinerea populations collected from tomato greenhouses in Northern Algeria. Journal of Phytopathology, 2015, 163 (2), pp.124-132. 10.1111/jph.12289. hal-02631269

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# Comparison of *Botrytis cinerea* populations collected from tomato greenhouses in Northern Algeria

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Ahmed Adjebli¹*, Christel Leyronas², Kamel Aissat¹, Philippe Nicot²

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¹Université Abderrahmane Mira, Faculté des Sciences de la Nature et de la Vie, Laboratoire d'écologie microbienne (L.E.M), Bejaia, 06000, Algérie.

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²INRA-UR407 Pathologie végétale, Domaine St Maurice CS 60094, F-84143 Montfavet cedex, France.

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*Corresponding author: philippe.nicot@avignon.inra.fr

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## Abstract

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To estimate the genetic diversity and population structure for a better understanding of the spread of *Botrytis cinerea*, we genotyped with nine microsatellite markers 174 isolates collected from four greenhouses during three growing seasons in the region of Bejaia. Four of these isolates were detected as *B. pseudocinerea* according to the allele size at locus *Bc6*. For all other isolates further studied, all loci were polymorphic, with the mean number of alleles per locus ranging from 2.77 to 5.22. Considerable genetic variability was detected in all sub-populations (D*>0.87; Hnb>0.40). Based on the standardized index of association analysis, significant but low level of clonality occured, not excluding the possibility of recombination. (rD=0.07, P<0.001). A total of 109 haplotypes were characterized among the isolates, few of which were shared between sub-populations. This, together with moderate genetic differentiation among sub-populations according to the geographic origin (0.080<FST<0.167), suggested a low level of inoculum exchange among greenhouses and little carryover of inoculum from one sampling season to the next. The importance of genetic structure of *B. cinerea* populations is discussed and should be taken into consideration for the management of gray mold.

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**Key words:** Microsatellite makers / Gray mold / Genetic diversity / Inoculum exchange

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## Introduction
Dispersion is a key process in the dynamics and evolution of natural populations. In addition to its primary role in the colonization process, dispersion also affects the process of adaptation of organisms. In plant pathogens, a better understanding of the dispersion process thus appears to be a major issue for better control. The pace at which microbes spread to new niches is a critical determinant of the emergence or re-emergence of infectious diseases in plants (Anderson et al. 2004). Plant pathogens are dispersed by contact, wind, water, animal vectors (e.g. insects and birds) and by humans through seed and infected plant material (Nagarajan and Singh 1990). Dispersal can occur over a distance from a few centimeters or less between roots in soil to hundreds of kilometers between susceptible crops. The transport of a small number of pathogenic spores can result in the eventual infection of entire fields (Mims and Mims 2004; Viljanen-Rollinson et al. 2007).

For many fungal plant pathogens, long-distance dispersal is an important strategy enabling them to colonize new areas or to survive between different seasons (Brown and Hovmøller 2002; Isard et al. 2005).

Local spread of inoculum is also of interest for day-to-day disease management and much effort has been dedicated to studying short-distance gradients of dispersion for various plant pathogenic fungi, including *B. cinerea* (Agrios 2005; Allard and Soubeyrand 2012; Mfegue et al. 2012; Johnson and Powelson 1983). Gradients of dispersion are considered to be influenced by many factors, including the type of cropping system (Qandah and Del Rio Mendoza 2012). Greenhouses, for example, are often regarded as quasi-closed systems, which generate short distance dispersal (Campen and Kempkes 2009). However, depending on the season and the type of greenhouse, substantial exchange of air can occur between the inside of a greenhouse and its outside environment (Boulard et al. 1997; Fatnassi et al. 2009), which could thus result in concomitant exchange of airborne inoculum.
While direct observation of the movements of airborne inoculum is a difficult experimental task, other approaches have been used to study the spread of inoculum within a greenhouse and possible exchanges with the outside environment. This is the case for *Botrytis cinerea*, a fungus known for its capacity to produce massive amounts of anemophilic spores on diseased plant tissue, and considered as a key pathogen on many greenhouse crops (Dik and Wubben 2004). Using selenate-resistant mutants, Korolev *et al.* (2006) clearly demonstrated the exchange of airborne inoculum in both directions (in- and outbound) between a glasshouse and its close environment. Modelling approaches have also been developed for the quantification of such exchanges (Leyronas and Nicot 2013; Leyronas et al. 2011).

In contrast, little is known about the spread of inoculum beyond the vicinity of a greenhouse despite its potential impact on disease management practices, possibly because of experimental difficulties. Nevertheless, molecular typing of isolates and tools of population genetics offer the possibility of an indirect approach to this subject through the comparison of distant sub-populations of the pathogen. In the case of *B. cinerea*, microsatellite markers are available (Fournier et al. 2002) and previous studies have shown a high level of diversity among isolates of this fungus on a variety of crops, allowing for comparison of population structures (Fournier and Giraud 2008; Isenberg et al. 2008a; Karchani-Balma et al. 2008). While populations of *B. cinerea* have been well characterized in various regions of the world (Fekete et al. 2012; Fournier and Giraud 2008; Decognet et al. 2009; Esterio et al. 2011; Ma and Michailides 2005; Mirzaei et al. 2009), information is scant for northern African countries (Karchani-Balma et al. 2008) and lacking for Algeria.

The main objectives of the present study were thus to establish a first detailed record of the genetic characteristics of the *B. cinerea* population in a vegetable growing area of Northern Algeria. A second objective was to identify traits that are compatible with the
hypothesis that exchange of inoculum occurred between farms. For this, isolates were
collected from diseased plants in greenhouses of the Bejaia region during three growing
seasons, and their genetic features were examined to test the hypothesis of a geographical
effect on the structure of the population.

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Materials and methods
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Fungal sampling and isolation

A total of 264 *B. cinerea* isolates were collected from commercial tomato greenhouses in May-June of 2007, 2008, and 2010, in the Bejaia region of Algeria (Fig. 1, Table 1). Access to one of the sampling sites (greenhouse in Souk El Tenine) was not possible after 2007. Using sterile cotton swabs, isolates were taken from diseased plant organs (fruits, leaves, flowers and stems) showing abundant sporulation, brought back to the laboratory and stored at -20°C until use for isolate purification and single spore isolation as described by Leyronas et al. (2012).

To produce mycelial tissue for DNA extraction, the single spore isolates were
cultured on malt extract agar medium at 25°C in the dark. After 10-14 days of incubation,
mycelia and conidia were collected by adding 1 ml of sterile distilled water in the Petri
plates, and by scraping the surface of the colony with a sterile cotton pad. The fungal
material was then lyophilized and stored at -20°C until used for DNA extraction.

DNA extraction
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The DNA of all field isolates of *B. cinerea* was extracted from 15 mg aliquots
of lyophilized fungal material in 96-well plates, using a DNeasy Plant extraction maxi Kit
(Qiagen®). DNA quality and quantity were assessed using agarose gel (1%)
electrophoresis. The plates containing DNA were then stored at -20°C until used for
microsatellite amplification.

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105 Microsatellite amplification and size analysis

106 Amplification reactions for the nine microsatellite loci described by Fournier et al.
107 (2002) were carried out using a QIAGEN® Amplification Multiplex PCR kit. Some of the
108 primers had the same annealing temperature, which allowed the amplifications to be
109 multiplexed. The reaction mixture consisted of 552 µl of PCR Master mix (containing DNA
110 polymerase, MgCl2 Multiplex PCR buffer and triphosphate-deoxyribonucleotides), 242µl of
111 MilliQ water and 22µl of each multiplexed primer. Each well of a PCR plate contained 8 µl of
112 reaction mixture and 2 µl of DNA from a *Botrytis* isolate.

113 The amplifications were carried out using an Eppendorf thermocycler. They entailed an
114 initial preheat for 15 min at 95°C, followed by 25-40 cycles comprising 0.5 min of
115 denaturation at 94°C, 1.5 min of annealing at 50°C (for the microsatellite loci Bc1, Bc3, Bc6,
116 Bc9), 53°C (for Bc2 and Bc5) or 59°C (for Bc4, Bc7, Bc10) and 1 min of extension at
117 72°C. Amplifications were ended by a final extension step of 30 min at 60°C. In order to
118 verify that the amplification occurred, sub-samples were then taken from each well and
119 deposited on 1% agarose gels for electrophoresis.

120 The amplification products were stored at -20°C until used for assessing the sizes of
121 the microsatellites. After a denaturation phase at 94°C for 3 min and addition of size
122 markers, amplification products were scanned with an ABI 3730 x1 DNA sequencer. Data
123 were analyzed with GeneMapper software (Applied Biosystem). Based on the sizes of the 9
124 microsatellite markers, a haplotypic profile could be assigned to each isolate, defining
125 multilocus genotypes (MLG). Genotyping was repeated for some (but not all) of the isolates
126 to confirm the results. Isolates belonging to subspecies *B. pseudocinerea* are morphologically
127 identical to those of *B. cinerea*, but they can be identified on the basis of the allele size at
128 locus Bc6 (Walker et al. 2011).
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**Population genetics analysis**The analysis was carried out only for isolates of *B. cinerea*

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(excluding *B. pseudocinerea*) whose complete 9-microsatellite haplotypic profile could be
obtained.

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The software Genetix 4.05 (Belkhir et al. 1996) was used to compute an index of unbiased allelic diversity (Hnb), the mean number of alleles per locus (based on allele frequencies) and allelic richness over all loci in the total sample and in each sub-population collected in the four greenhouses cited previously. Intra-population diversity was characterized by computing diversity indices using Genclone software (version 2.0). We used the Simpson diversity index (D*), an equitability index (ED*) and the index of genotypic richness (R). The D* index measures the probability that two randomly selected individuals in a population have different MLGs (Arnaud-Haond et al. 2007). The ED* index is equal to 1 when all MLGs represented in a population have the same abundance (equitable distribution of clones) (Arnaud-Haond et al. 2007). Finally, we used the R index of genotypic richness, which varies from 0 when all isolates in a sample possess the same genotype, to 1, when all isolates possess a different genotype (Dorken and Eckert 2001).

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In order to test for genetic recombination, the standardized index of association (r<sub>D</sub>) was
calculated using Multilocus 1.3b (Agapow and Burt 2001). The r<sub>D</sub> index is a measure of the
multilocus linkage disequilibrium (Brown et al. 1980; Haubold et al. 1998), which gives
information on whether two different individuals sharing the same allele at one locus are more
likely to share an allele at another locus. For each pair of isolates, the number of loci with
respect to which they differ is calculated, and the variance of this number is compared with
that expected if there is no linkage disequilibrium. The r<sub>D</sub> index is equal to zero if there is no
linkage disequilibrium, and it increases as linkage disequilibrium increases. The null
hypothesis of complete panmixia (r<sub>D</sub>= 0) was tested with the procedure implemented in the
software, by comparing the observed data set to 1000 randomized data sets in which infinite

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recombination has been imposed by randomly shuffling the alleles among individuals,
independently for each locus.

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## Genetic differentiation between sub-populations

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Estimation of genetic differentiation by pairwise comparison between the sub-populations collected at different locations in a given season was performed using the software ARLEQUIN 3.0 (Excoffier et al. 2005), without repeated MLGs, after 3024 permutations and progressive Bonferroni correction as described by Rice (1989). According to Wright (1978), the genetic differentiation between two populations is low when $0<F_{ST}\le0.05$, moderate when $0.05<F_{ST}\le0.15$, substantial when $0.15<F_{ST}\le0.25$ and very high when $F_{ST}>0.25$.

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## Results

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### Distinction between *B. cinerea* and *B. pseudocinerea*

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After isolation, single-spore purification, DNA extraction and genotyping of the 264 isolates, complete genotypic profiles with the nine microsatellites were obtained for only 174 isolates. No variation of the microsatellite sizes was found after genotyping repetitions of some isolates. Four of them, collected in Tichy in 2007 and 2008, were characterized as *B. pseudocinerea* and excluded from further genetic analysis. We had thus 170 fully genotyped isolates of *B. cinerea*, distributed in nine groups of approximately 20 isolates per sampling site and per year (Table 2; one exception in Tichy in 2007 with only 5 isolates). Each of these groups will be referred to as a "sub-population" in the rest of this paper.

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### Global genetic diversity

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A total of 109 multilocus genotypes (MLG) were distinguished among the 170 isolates of *B. cinerea* and the mean number of alleles per locus was 8.22 (Table 2). The overall level of genetic diversity in the total population was high, as shown by an unbiased genetic diversity (Hnb) of 0.57, an index of genotypic diversity (D*) of 0.98 and a Richness index (R)
179 of 0.64. The high index of equitability (ED*= 0.90) indicated an equitable distribution of
180 clonal haplotypes between the nine sub-populations. Finally, the estimate of linkage
181 disequilibrium was significant but very low (rD=0.07), suggesting the occurrence of limited
182 genetic recombination (Table 2). Although the results obtained for the total sample were
183 interesting in our study, other repetitions in the time for the site of Souk-El-Tenine must be
184 undertaken to confirm these results.

185 Genetic diversity among sub-populations

186 Separate examination of the nine sub-populations confirmed the overall high level of
187 allelic diversity, with an Hnb index ranging from 0.40 to 0.68 (Table 2). This diversity
188 appeared higher in 2010 than in other years. The average number of alleles per locus was 8.22
189 for the total sample and ranged from 2.77 to 5.22 within the nine sub-populations, suggesting
190 that several alleles were not shared among sub-populations. Haplotypic diversity was also
191 high, with a D* index higher than 0.90 in eight of the nine sub-populations. Significant but
192 low multilocus linkage disequilibrium (rD) was observed in all sub-populations (Table 2; one
193 exception: Tichy 2007, for which only 5 isolates were available).

194 Among the 109 MLGs observed within the total sample, 88 were represented by a
195 single isolate and 21 were represented by at least two isolates (Fig. 2). Among those, twelve
196 were shared between sub-populations collected in different greenhouses and seasons (Table
197 3). One MLG was detected 17 times in the total sample and was shared between four sub-
198 populations. The number of shared MLG between greenhouses varies with the year.
199 Therefore, 3 MLGs were found in 2008, only 2 in 2010 and 1 MLG was found in 2007 (Table
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201 Genetic differentiation between sampling sites

202 Pairwise tests of genetic differentiation among sub-populations yielded generally low
203 to moderate FST values (Table 4). The highest values were obtained in 2007 between Tichy
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(site C) and the two other coastal sites, showing significant genetic differentiation between
these sub-populations on that year. Moderate but significant differentiation was also observed
between Tichy and Baccaro (site B) in 2008. No significant differences were observed in
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Discussion

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This study provides the first data on genetic features of *B. cinerea* populations in Algeria. It is also the first report of the detection of *B. pseudocinerea* in North Africa. This species, detected at a frequency of 1.5 % among the 264 isolates collected in our study, was also observed at low frequencies (varying between 0.5 and 15% according to the location and the season) in other parts of the world, in sympatry with *B. cinerea* populations (Fekete et al. 2012; Fournier et al. 2005; Fournier et al. 2003; Martinez et al. 2005; Scagnelli et al. 2010, Walker et al. 2011; Wissels 2012; Ma and Michailides 2005).

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Among the isolates of *B. cinerea*, the high genetic diversity (both allelic and haplotypic) observed in the present study indicates a highly heterogeneous population. Similar situations have been described for populations collected from different hosts around the world (Table 5). Such high levels of diversity could result from genetic recombination, as suggested by the low value of the standardized index of association r<sub>D</sub> found in the present work and in several previous studies (Table 5). High levels of recombination have been reported for most studies of *B. cinerea* populations, with the exception of a study in various field crops in California (Ma and Michailides 2005). Recombination has been mostly attributed to sexual reproduction, and could also be related to other mechanisms of variability known for *B. cinerea* (Van Der Vlugt-Bergmans et al. 1993; Beever and Weeds 2004).

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The overall level of clonality in the *B. cinerea* population, measured by the ratio of the number of distinct MLGs present among the total number of isolates characterized in the present study was 0.64. When considering the sub-populations of individual greenhouses, it

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229 was consistently higher than 0.56. These numbers are overall similar to those reported for 230 studies of *B. cinerea* in various crops and locations (Table 5). However, they contrast sharply 231 with results of a study conducted in tomato greenhouses in experimental conditions (Decognet 232 et al. 2009). In that study, the ratio of the number of distinct MLGs identified among the total 233 number of characterized isolates was 0.70 among isolates sampled from the air spores before 234 the development of the disease in the greenhouses. It decreased sharply to a value of 0.23 235 among isolates sampled from the air 15 days after the inoculation of plants with two known 236 strains of *B. cinerea*, and decreased further to 0.03 among isolates collected from diseased 237 plants 60 days after inoculation. Under the hypothesis that two isolates sharing an identical 238 genotypic profile were clones, these authors concluded that abundant sporulation by the two 239 introduced strains of *B. cinerea* resulted in a displacement of the initially present population 240 (Decognet et al. 2009).

241 In the present study, we observed identical MLGs in different greenhouses during a 242 given year (Table 3). This observation is compatible with the hypothesis that inoculum 243 exchange may have occurred between greenhouses. This hypothesis is supported by the fact 244 that genetic differentiation (Fst values) was not significant on a given year for greenhouses 245 which shared MLGs (one exception in 2008 for Baccaro and Tichy; Table 4). The possibility 246 of inoculum exchange between the greenhouses is also supported by meteorological data 247 showing that prevailing winds in the Bejaia region are west-bound between April and 248 September (Alkama et al. 2007; http://fr.windfinder.com/windstats/ windstatistic_bejaia_ 249 aeroport.htm), a period that covers the time when *Botrytis* epidemics occur in the tomato 250 greenhouses of that region (Aissat et al. 2008). Considering the dominant wind direction 251 during our study and the distance between the sampling sites, genotypic data are generally 252 (but not systematically) in agreement with possible contribution of inoculum between 253 greenhouses from the most eastern to the most western site of a given year.
Earlier studies have reported genotype flows between regions for *B. cinerea*
populations examined in chickpea fields in Bangladesh (Isenegger et al. 2008b) and for two
other species of *Botrytis* in field crops in the Netherlands (Staats 2007). In our study, the
small number of shared MLGs between sampling sites could be due to the fact that
greenhouses are somewhat confined environments, although some level of inoculum
exchange between the inside and outside of a greenhouse has previously been demonstrated
(Korolev *et al.* 2006). However, the occasional presence of shared MLGs in different
greenhouses of the Bejaia region could also result from their presence in multiple copies in
global air masses arriving to that region. A consideration of the trajectories of air masses
across the Mediterranean (Kushta et al. 2012; Khalfa et al. 2013; Lelieveld et al. 2007) makes
it impossible to exclude occasional influx of *B. cinerea* inoculum from more distant sources,
such as agricultural regions in southern France and Spain where *Botrytis* is known to be
abundant (Leyronas and Nicot 2013; Moyano et al. 2003). This hypothesis is further
supported by the detection in Bejaia of *B. pseudocinerea*, a new species formerly described in
France and considered to be migrating to other parts of the world (Isenegger et al. 2008b).
More conclusive evidence for direct exchange of inoculum among farms of the Bejaia region
would thus require additional information on short term evolution of the population structures
during a growing season, together with concomitant information on dominant haplotypes
present in distant potential sources of occasional inoculum. In the meantime, gray mold
management strategies in the Bejaia region should not rule out the possible exchange of
inoculum between farms and thus the spread of particularly aggressive strains or fungicide-
resistant strains.

276 Acknowledgments

This work was supported partially by a grant of the Ministry of Higher Education and
Scientific Research of Algeria, and INRA, UR407 Pathologie végétale, Domaine Saint
279 Maurice Montfavet France. We are grateful to Magali Duffaud and Claire Troulet for their

280 technical participation in the experiments.

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411

412

413

414 **Figure captions:**

<figure><img src="image_3.png" /></figure>

415

416 **Fig. 1:** Location of *Botrytis cinerea* sampling sites in the Bejaia region (North Algeria) (A:

417 Souk-el-Tenine, B: Baccaro, C: Tichy and D: Merdjouamane).

418
<figure><img src="image_4.png" /><figcaption>**Fig. 2:** Frequency distribution of multilocus genotypes (MLGs) among 170 isolates of *B. cinerea* sampled from the Bejaia region.</figcaption></figure>

419

420

421

422

423
**Table 1.** Origin of *Botrytis cinerea* isolates collected from commercial tomato greenhouses in Bejaia region (North Algeria) from 2007 to 2010.

<table><thead><tr><th>Sampling location</th><th>GPS coordinates</th><th>Sampling date</th><th>Number of genotyped isolates</th></tr></thead><tbody><tr><td>A: Souk-el-Tenine</td><td>Latitude: 36.62958<br/>Longitude: 5.29764</td><td>30 May 2007</td><td>23</td></tr><tr><td rowspan="3">B: Baccaro</td><td rowspan="3">Latitude: 36.64683<br/>Longitude: 5.20958</td><td>30 May 2007</td><td>21</td></tr><tr><td>1 June 2008</td><td>19</td></tr><tr><td>5 June 2010</td><td>19</td></tr><tr><td rowspan="3">C: Tichy</td><td rowspan="3">Latitude: 36.6938<br/>Longitude: 5.10217</td><td>30 May 2007</td><td>05</td></tr><tr><td>1 June 2008</td><td>22</td></tr><tr><td>5 June 2010</td><td>19</td></tr><tr><td rowspan="2">D: Merdjouamane</td><td rowspan="2">Latitude: 36.67619<br/>Longitude: 4.94483</td><td>1 June 2008</td><td>23</td></tr><tr><td>5 June 2010</td><td>19</td></tr></tbody></table>
<table><caption>**Table 2.** Genetic diversity in nine *B. cinerea* sub-populations sampled from commercial tomato greenhouses.</caption><thead><tr><th rowspan="2">Sampling year and location</th><th rowspan="2">Number of isolates</th><th rowspan="2">Number of MLG<sup>a</sup></th><th colspan="2">Allelic diversity<sup>b</sup></th><th colspan="3">Haplotypic diversity<sup>c</sup></th><th colspan="2">Index of multilocus linkage disequilibrium<sup>d</sup></th></tr><tr><th>Hnb</th><th>number of alleles per locus</th><th>D*</th><th>ED*</th><th>R</th><th>r<sub>D</sub></th><th>P value</th></tr></thead><tbody><tr><td><strong>Total sample</strong></td><td>170</td><td>109</td><td>0.57 (0.17)</td><td>8.22</td><td>0.98</td><td>0.90</td><td>0.63</td><td>0.07</td><td>&lt;0.001</td></tr><tr><td><strong>2007 samples</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Souk-el-Tenine</td><td>23</td><td>14</td><td>0.45 (0.25)</td><td>3.33</td><td>0.90</td><td>0.53</td><td>0.59</td><td>0.28</td><td>&lt;0.001</td></tr><tr><td>Baccaro</td><td>21</td><td>17</td><td>0.47 (0.21)</td><td>2.77</td><td>0.97</td><td>0.66</td><td>0.80</td><td>0.13</td><td>&lt;0.001</td></tr><tr><td>Tichy</td><td>5</td><td>5</td><td>0.68 (0.14)</td><td>3.33</td><td>1.00</td><td>ND</td><td>1.00</td><td>-0.01</td><td>0.587</td></tr><tr><td><strong>all sites</strong></td><td><strong>49</strong></td><td><strong>35</strong></td><td><strong>0.54 (0.18)</strong></td><td><strong>5.33</strong></td><td><strong>0.97</strong></td><td><strong>0.71</strong></td><td><strong>0.71</strong></td><td><strong>0.12</strong></td><td><strong>&lt;0.001</strong></td></tr><tr><td><strong>2008 samples</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Baccaro</td><td>19</td><td>15</td><td>0.56 (0.11)</td><td>3.44</td><td>0.96</td><td>0.40</td><td>0.78</td><td>0.11</td><td>&lt;0.001</td></tr><tr><td>Tichy</td><td>22</td><td>14</td><td>0.50 (0.17)</td><td>3.44</td><td>0.91</td><td>0.54</td><td>0.62</td><td>0.16</td><td>&lt;0.001</td></tr><tr><td>Merdjouamane</td><td>23</td><td>13</td><td>0.40 (0.20)</td><td>3.55</td><td>0.87</td><td>0.50</td><td>0.55</td><td>0.21</td><td>&lt;0.001</td></tr><tr><td><strong>all sites</strong></td><td><strong>64</strong></td><td><strong>39</strong></td><td><strong>0.54 (0.14)</strong></td><td><strong>4.66</strong></td><td><strong>0.97</strong></td><td><strong>0.85</strong></td><td><strong>0.60</strong></td><td><strong>0.08</strong></td><td><strong>&lt;0.001</strong></td></tr><tr><td><strong>2010 samples</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Baccaro</td><td>19</td><td>16</td><td>0.57 (0.25)</td><td>4.44</td><td>0.97</td><td>0.47</td><td>0.83</td><td>0.12</td><td>&lt;0.001</td></tr><tr><td>Tichy</td><td>19</td><td>17</td><td>0.65 (0.13)</td><td>5.22</td><td>0.98</td><td>0.53</td><td>0.89</td><td>0.12</td><td>&lt;0.001</td></tr><tr><td>Merdjouamane</td><td>20</td><td>15</td><td>0.50 (0.21)</td><td>3.55</td><td>0.95</td><td>0.60</td><td>0.74</td><td>0.15</td><td>&lt;0.001</td></tr><tr><td><strong>all sites</strong></td><td><strong>58</strong></td><td><strong>45</strong></td><td><strong>0.60 (0.20)</strong></td><td><strong>6.55</strong></td><td><strong>0.99</strong></td><td><strong>0.86</strong></td><td><strong>0.77</strong></td><td><strong>0.10</strong></td><td><strong>&lt;0.001</strong></td></tr></tbody></table>

<footnote><sup>a</sup>MLG: multilocus genotypes.</footnote>

<footnote><sup>b</sup>Hnb: nonbiased genetic diversity, calculated with repeated genotypes (standard deviations between brackets),</footnote>

<footnote>ND: undefined.</footnote>

<footnote><sup>c</sup>D*: genotypic diversity index; E: equitability index; R: Richness index.</footnote>

<footnote><sup>d</sup>r<sub>D</sub>: standardized index of association.</footnote>
<table><caption>**Table 3.** Frequency distribution of Multilocus Genotypes (MLG) detected in more than one greenhouse.</caption><thead><tr><th rowspan="2">MLG</th><th colspan="3">2007</th><th colspan="3">2008</th><th colspan="3">2010</th></tr><tr><th>Souk-el-Tenine</th><th>Baccaro</th><th>Tichy</th><th>Baccaro</th><th>Tichy</th><th>Merdjouamane</th><th>Baccaro</th><th>Tichy</th><th>Merdjouamane</th></tr></thead><tbody><tr><td>H20</td><td></td><td>2</td><td></td><td>1</td><td>1</td><td></td><td>2</td><td></td><td></td></tr><tr><td>H32</td><td></td><td>1</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>H89</td><td></td><td></td><td></td><td>2</td><td></td><td></td><td>3</td><td></td><td></td></tr><tr><td>H25</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td><td>2</td></tr><tr><td>H30</td><td>7</td><td>1</td><td></td><td></td><td></td><td>8</td><td></td><td>1</td><td></td></tr><tr><td>H55</td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>H65</td><td></td><td></td><td></td><td></td><td>1</td><td>2</td><td></td><td></td><td></td></tr><tr><td>H80</td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td>1</td></tr><tr><td>H82</td><td></td><td>1</td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td></tr><tr><td>H88</td><td></td><td></td><td></td><td>1</td><td></td><td>3</td><td></td><td></td><td></td></tr><tr><td>H100</td><td></td><td></td><td>1</td><td></td><td>1</td><td></td><td>1</td><td></td><td></td></tr><tr><td>H108</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>1</td><td></td></tr></tbody></table>
<table><caption>**Table 4.** Genetic differentiation (F<sub>ST</sub>) of *B. cinerea* sub-populations sampled from four sites in the Bejaia region of Algeria.</caption><thead><tr><th>Season</th><th>Site A-Site B<sup>a</sup><br>(13 km)</th><th>Site A-Site C<br>(23 km)</th><th>Site B-Site C<br>(10 km)</th><th>Site B-Site D<br>(20 km)</th><th>Site C-Site D<br>(15 km)</th></tr></thead><tbody><tr><td>2007</td><td>0,062<sup>b</sup></td><td><u>0,167</u></td><td><u>0,115</u></td><td>ND<sup>d</sup></td><td>ND</td></tr><tr><td>2008</td><td>ND</td><td>ND</td><td><u>0,095</u></td><td>0,058</td><td>-0,019</td></tr><tr><td>2010</td><td>ND</td><td>ND</td><td>0,011</td><td>0,016</td><td>0,050</td></tr></tbody></table>

<footnote><sup>a</sup> A: Souk-el-Tenine; B: Baccaro; C: Tichy; D: Merdjouamane. Numbers between brackets indicate the distance in km between two sampling sites.</footnote>

<footnote><sup>b</sup> Pairwise F<sub>ST</sub> were calculated following Weir and Cockerham, without repeated MLG. Underlined values are significantly different (<i>P</i> < 0.01) after 3200 permutations and a Bonferroni correction.</footnote>

<footnote><sup>c</sup> ND: not determined.</footnote>
<caption>**Table 5.** Genetic diversity indices of *B. cinerea* populations characterized with microsatellite makers</caption>

<table><thead><tr><th>Origin of study</th><th>Host plant</th><th>Number of microsatellites</th><th>Number of isolates</th><th>Number of MLGs</th><th>Number of MLG/Number of isolates</th><th>Hnb</th><th>D*b</th><th>rD<sup>d</sup></th><th>Reference</th></tr></thead><tbody><tr><td>Algeria</td><td>Tomato</td><td>9</td><td>170</td><td>109</td><td>0.64</td><td>0.57</td><td>0.98</td><td>0.07</td><td>Present study</td></tr><tr><td>France</td><td>Grapevine, bramble</td><td>8</td><td>184</td><td>180</td><td>0.98</td><td>0.75</td><td>0.75</td><td>-</td><td>Fournier et al. 2008</td></tr><tr><td>Tunisia</td><td>Grapevine, faba bean, tomato, strawberry</td><td>9</td><td>153</td><td>120</td><td>0.78</td><td>0.79</td><td>0.99</td><td>0.08</td><td>Karchani-Balma et al. 2008</td></tr><tr><td>Hungary</td><td>Strawberry, rape</td><td>5</td><td>79</td><td>32</td><td>0.40</td><td>0.66</td><td>0.91</td><td>0.08</td><td>Fekete et al. 2012</td></tr><tr><td>Bangladesh</td><td>Chickpea fields</td><td>9</td><td>146</td><td>69</td><td>0.47</td><td>0.25</td><td>0.54</td><td>-</td><td>Isenegger et al. 2008</td></tr><tr><td>South Africa</td><td>Pear orchards</td><td>9</td><td>181</td><td>91</td><td>0.50</td><td>0.14</td><td>0.69</td><td>-</td><td>Wessels 2012</td></tr></tbody></table>

<footnote>ª Hnb: nonbiased genetic diversity, calculated with repeated genotypes (standard deviations between brackets),</footnote>

<footnote>ᵇ D*: genotypic diversity index; E: equitability index; R: Richness index.</footnote>

<footnote>ᶜ rD: standardized index of association.</footnote>