Botryotinia fuckeliana (de Bary ex de Bary) Whetzel (anamorph: Botrytis

cinerea Pers.:Fr.) is a cosmopolitan ascomycetous fungus that causes grey

mould on a great number of plants in the temperate zone worldwide by

infecting various tissues (Jarvis, 1980). In grapevine, the frequent

occurrence of B. cinerea prior harvesting results in serious losses of fruits

and deterioration of wine quality. This is also the case in Eger, a major

Hungarian wine region in the North-Eastern part of the country, where B.

cinerea is considered to the third most important grapevine pathogen after

downy mildew (Plasmopara viticola /Berk. and Curt ex de Bary/ Berl. and

de Toni) and powdery mildew [Erysiphe necator Schwein. var. necator

(syn.: Uncinula necator /Schwein./ Burrill var. necator)], with an estimated

annual loss of up to 15-20 %. In contrast, some 100 km eastwards in the

Tokaj wine region, B. cinerea is also responsible for the phenomenon called

‘pourriture noble’ (noble rot). Under certain unique environmental

conditions, mycelia growing on the surface of the uninjured, healthy berry

drains water (but no substrates) via the fine infection hyphae. As a

consequence, the concentration of all the soluble compounds within the

berry significantly increase (Jarvis, 1980). Such berries yield the sweet,

special quality wine called „aszu”

Fekete, E.

Gál, Lajos

Karaffa, Erzsébet Mónika

Karaffa, L.

Kövics, György

Váczy Kálmán, Zoltán

Repository of the Academy's Library

 91DISTRIBUTION OF TRANSPOSONS IN BOTRYTISCINEREA ISOLATES COLLECTED FROM THE WINE REGIONS OF EGER AND TOKAJ, HUNGARY  Kálmán Z. Váczy1 – Levente Karaffa2 – Erzsébet Fekete2 – György J. Kövics3 – Lajos Gál1 – Erzsébet Sándor3  1Research Institute for Viticulture and Enology, Eger, Hungary 2Department of Genetics and Applied Microbiology, Faculty of Science, University of Debrecen, Debrecen, Hungary 3Department of Plant Protection, Faculty of Agriculture, University of Debrecen, Debrecen, Hungary  Botryotinia fuckeliana (de Bary ex de Bary) Whetzel (anamorph: Botrytis cinerea Pers.:Fr.) is a cosmopolitan ascomycetous fungus that causes grey mould on a great number of plants in the temperate zone worldwide by infecting various tissues (Jarvis, 1980). In grapevine, the frequent occurrence of B. cinerea prior harvesting results in serious losses of fruits and deterioration of wine quality. This is also the case in Eger, a major Hungarian wine region in the North-Eastern part of the country, where B. cinerea is considered to the third most important grapevine pathogen after downy mildew (Plasmopara viticola /Berk. and Curt ex de Bary/ Berl. and de Toni) and powdery mildew [Erysiphe necator Schwein. var. necator (syn.: Uncinula necator /Schwein./ Burrill var. necator)], with an estimated annual loss of up to 15-20 %. In contrast, some 100 km eastwards in the Tokaj wine region, B. cinerea is also responsible for the phenomenon called ‘pourriture noble’ (noble rot). Under certain unique environmental conditions, mycelia growing on the surface of the uninjured, healthy berry drains water (but no substrates) via the fine infection hyphae. As a consequence, the concentration of all the soluble compounds within the berry significantly increase (Jarvis, 1980). Such berries yield the sweet, special quality wine called „aszu”. Literatute  Transposable elements (TEs) are fragments of DNA that can insert into new chromosomal locations and often make duplicate copies of themselves in the process (Feschottes et al., 2002). TEs were first discovered in maize (McClintock, 1984) and later have been found in several eukaryotic, eubacteria and archaea genome. In addition to the wide array of ‘hosts’, the variety of transposons described also increased considerably (Finnegan, 1989). Fungal transposons were first identified in the yeast Saccharomyces cerevisiae (Boeke, 1989), though the first indirect evidence for their  92presence in filamentous fungi arose years earlier from conventional genetic studies with Ascobolus immersus mutants (Decaris et al., 1978).  There are two main classes of TEs (Finnegan, 1989). Class I elements are related to retroviruses and they transpose through the reverse synthesis of DNA from template RNA, while class II elements move in the genome through direct DNA to DNA transposition without an RNA intermediary. Class I elements are known as retrotransposons and include TEs with or without ‘long terminal repeated sequences’ (LTRs). Retroelements have been found in a number of fungal species such as Alternaria alternata (Kaneko et al., 2000), Ascobolus immersus (Goyon et al., 1996), Aspergillus fumigatus (Neuvéglise et al., 1996), Aspergillus nidulans (Nielsen et al., 2001), Neurospora crassa (Kinsey and Helber, 1989). DNA transposons are also widespread and have been described among others in Agaricus bisporus (Sonnenberg et al., 1999), Ascobolus immersus (Colot and Rissignol, 1995), Aspergillus niger (Glayzer et al., 1995), Magnaporthe grisea (Kachroo et al., 1994), Nectria haematococca (Enkerli et al., 1997), Neurospora crassa (Yeadon, 1995), and Podospora anserina (Hamann et al., 2000). B. cinerea has been shown to possess two transposons. Boty is a class I LTR-retro-transposon (Diolez et al., 1995), while Flipper is a class II element (Levis et al., 1997). In this paper we will show that at least four genotypes of isolates related to the presence or absence of these transposons occur in the Eger wine region. Potential significance of this finding is discussed.  Materials and Methods  Field strains of B. cinerea were collected from various locations of the Eger and Tokaj wine districts. They were isolated from infected berries between 2003 and 2004 during the vintage period (September-October). Fungal strains from both wine regions are numbered by the chronology of collection, irrespective to the local provenance. DNA was extracted from aerial mycelium of B. cinerea with Plant DNA Purification Kit (QuiaGene). Transposons were detected with PCR reactions described at Munoz et al., 2002. Presence or absence of the two transposons was confirmed by agarose gel electrophoresis (Figure 1) using standard protocols (Sambrook et al., 1989).     93Results  B. cinerea has been shown to possess a highly versatile genome reflected in a considerable metabolic flexibility. Depending on the actual environmental conditions (Martinez et al., 2003), the fungus is able to act both as a saprophyte and a pathogen, and has developed resistance to most of the fungicides used to control it (Faretra and Pollastro, 1991; Leroux et al., 1999). Studies on French and Chilean B. cinerea isolates have revealed the presence of three distinct intrapopulation: (1) transposa, having transposable elements Boty and Flipper, (2) vacuma, having none of the two and (3) boty containing the transposable element Boty alone (Munoz et al., 2002). The flipper intrapopulation, containing the transposable element Flipper alone has thus far only been described in two separate isolates from the United Kingdom and France (Albertini et al., 2002). Boty and Flipper are both specific for B. cinerea. While proteins encoded by transposable elements are supposed to be used exclusively for the purpose of their own reproduction without any interference with the metabolism of the host organism, a more rapid biomass formation in the vacuma intrapopulation relative to boty and transposa was observed (Martinez et al., 2003). It should also be noted that the level of fungicide resistance significantly differed in transposa and in vacuma-type B. cinerea populations in French isolates (Albertini et al., 2002). It is not yet known whether the two events are related to each other, and if they are then it is a cause or a consequence of the altered transposon pattern. In the framework of this project 68 and 17 B. cinerea isolates have been collected from the Eger and Tokaj wine regions, respectively. To the best of 700 bp 1200 bp 600 bp 61 65 67 63 68Flipper Boty 1300 bp Figure 1. Gel electrophoresis picture of transposon elements Samples  94our knowledge, this is the first Central-Eastern European collection of its kind, and only the fourth worldwide. Two French studies from the Champagne and Bordeaux wine regions, respectively analysed a collection of 259 (Giraud et al., 1997) and 121 (Martinez et al., 2003) isolates, while a Chilean study was based on 69 cases. Distribution of the Flipper and Boty transposons in the Hungarian isolates are markedly different to those in French and Chilean collections (Table 1).  Table 1. Distribution of transposons in Botrytis cinerea isolates collected from the Eger and Tokaj vine regions, Hungary, and percentage of the transposa, vacuma, flipper and boty intrapopulations in other countries as found in the literature Type of straina No. of isolatesb Percentage Percentage   Eger Tokaj Eger Tokaj Chile(c) France(d) France(e) transposa 12 0 17.64 0 79.71 75.00 61.34 flipper 51 17 75.00 100 0.00 0.00 0.00 boty 1 0 1.47 0 11.59 0.00 0.00 vacuma 4 0 5.89 0 8.70 25.00 38.66 All 68 17 100 100 100 100 100 a transposa isolates contain both the Boty and Flipper transposable elements. Flipper and boty are isolates containing Flipper or Boty, respectively, while vacuma are isolates without Boty and Flipper. b all the isolates as well as viticultural and geographical details of their provenance are available from the first author of this paper upon request. c Munoz et al.,2002; d Giraud et al., 1997; e Martinez et at., 2003  While genotype transposa is clearly prevalent in the French and Chilean samples with some two-third of the isolates containing both transposons, the percentage of this particular intrapopulation was less than 18 percent in Eger and zero percent in Tokaj. While genotype boty was present in over 10 percent of the Chilean collection, we found only one single strain in Eger (and none in Tokaj) that carried the Boty transposon alone. We note, while the French studies indicated the presence of boty genotype in their collections, no defined values were provided in either case.  Distribution of the genotype vacuma ranged between 8 and 38 percent in the literature. Our investigations yielded only a handful of vacuma isolates in Eger and none in Tokaj. The most striking observation in our study is the appearance of the genotype flipper, an intrapopulation of B. cinerea hitherto considered extremely rare. However, this genotype is obviously prevalent in the two Hungarian wine regions studied, with 75 percent of the isolates containing only flipper  95transposon in Eger and all in Tokaj. None of the three collections cited in this paper have reported on the appearance of this genotype. Transposons are highly mobile genetic elements while B. cinerea is a truly cosmopolitan fungus. Comparative analysis of transposon distributions in B. cinerea isolates collected from all around the world may thus be a worthy method to study fungal population genetics.  There are no tested hypothesises on the physiological role of transposons. Mobility of transposons including those in the filamentous fungi Magnaporthe grisea (Ikeda et al., 2001) and Fusarium oxysporum (Mes et al., 2000) were reported to increase during certain stress conditions such as substrate deficiency, drought, heat and exposure to  -radiation. It remains to be tested whether stress conditions will influence B. cinerea transposons in anyway. Discussion  This study showed that all of the four transposon-related genotypes of B. cinerea ever described in the literature exists in the Eger wine region. Most noteworthly, genotype flipper, considered extremely rare elsewhere in the world is apparently dominant both in the Eger and Tokaj wine regions. In fact in Tokaj, flipper was the only transposon found. It remains to evaluate whether B. cinerea genotypes defined over transposon distribution are relevant to the role the fungus plays in viticulture and enology.  This work was supported by grants from the Ministry of Agriculture and Rural Development (33013/2003 and 46024/2004). Levente Karaffa’s Lab is grant-aided by the OTKA (Hungarian Scientific Research Fund; F 042602). Erzsébet Fekete and Erzsébet Sándor are recipients of an OTKA postdoctoral scholarship (D 048617) and a János Bolyai Scholarship (BO/00446/04), respectively.  References  Albertini, C., Thebaud, G., Fournier, E. and Leroux, P. (2002): Eburicol 14-demethylase gene (CYP51) polymorphism and speciation in Botrytis cinerea. Mycol. Res. 106:1171-1178. Boeke, J.D. (1989): Transposable elements in Saccharomyces cerevisiae, In: Mobile DNA, Berg, D.E. and Howe, M. (Eds) ASM Press, Washington DC, pp. 335-374. Colot, V. and Rossignol, J.L. (1995): Isolation of the Ascobolus immersus spore color gene b2 and study in single cells of gene silencing by methylation induced premeiotically. Genetics 141:1299-1314. Decaris, B., Francou, F., Lefort, C. and Rizet, G. 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Sándor3 1Research Institute for Viticulture and Enology, Eger, Hungary  2Department of Genetics and Applied Microbiology, Faculty of Science, University of Debrecen, Debrecen, Hungary 3Department of Plant Protection, Faculty of Agriculture, University of Debrecen, Debrecen, Hungary  Summary  Analysis of the distribution of the transposable genetic elements Boty and Flipper in Botrytis cinerea (grey mould) isolates collected from the Eger and Tokaj wine regions, North-Eastern Hungary is presented. We demonstrate the prevalence of a rare intrapopulation called Flipper, and discuss the differences among B. cinerea populations isolated from Western European and South American wine regions. 

Distribution of transposons in Botrytis cinerea isolates collected from the wine regions of Eger and Tokaj, Hungary

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