UNIVERSITY OF CATANIA FACULTY OF AGRICULTURE INTERNATIONAL Ph.D. COURSE IN “PLANT HEALTH TECHNOLOGIES AND PROTECTION OF AGRO-ECOSYSTEMS” (XXV CYCLE: 2009-2012) ANNA PANEBIANCO Study on Fungicide Sensitivity and Resistance in a Population of Botryotinia fuckeliana Collected from Table Grapes in Sicily (Southern Italy) Ph.D. Thesis COORDINATOR TUTOR Prof. Carmelo Rapisarda Prof. Giancarlo Polizzi CONTENTS 1. Viticulture in Sicily ........................................................................................... 4 2. Botrytis cinerea ................................................................................................. 7 2.1. Taxonomy .................................................................................................. 8 2.2. Morphology ............................................................................................. 10 2.3. Life cycle and epidemiology ................................................................... 11 2.4. Pathogenesis of Botrytis ........................................................................... 16 2.5. Symptoms of gray mould ......................................................................... 20 2.6. Disease management ................................................................................ 22 2.6.1. Cultural practices ............................................................................ 22 2.6.2. Biological control ........................................................................... 27 2.6.3. Chemical control ............................................................................. 34 3. Fungicide resistance ........................................................................................ 42 4. Objectives of research ..................................................................................... 56 5. Materials and methods .................................................................................... 59 5.1. Pathogen isolates .................................................................................... 59 5.2. Fungicides ............................................................................................... 61 5.3. Sensitivity tests ....................................................................................... 61 5.4. Assays on bean seedling ......................................................................... 62 5.5. Assays on leaves of grapevine ................................................................ 63 5.6. Molecular analysis .................................................................................. 64 5.7. Assays on detached grape berries ........................................................... 65 5.8. Assays on apple fruits ............................................................................. 66 5.9. Statistical data analysis ........................................................................... 67 6. Results ............................................................................................................. 68 6.1. Sensitivity tests ....................................................................................... 68 6.2. Assays on bean seedling ......................................................................... 77 6.3. Assays on leaves of grapevine ................................................................ 79 6.4. Molecular analysis .................................................................................. 81 2 6.5. Assays on detached grape berries ........................................................... 83 6.6. Assays on apple fruits ............................................................................. 88 7. Discussion ..................................................................................................... 90 References 3 1. Viticolture in Sicily Grapevine (Vitis vinifera) is certainly one of the most important crops, with great economic importance in all the areas in the Mediterranean Basin for the several ways of the use of its product (fresh fruit, musts, wine, dry grape, etc). Vineyards cover a total area of 10 million hectares and produce a total yield of about 65 million tons, of which 17 million tons are for table grapes (OIV International Organization of Vine and Wine, 2010). Vines can be cultivated from temperate to tropical climates, but most vineyards are planted in temperate zones. The most concentrated cultures are in Europe. Italy, with its 1.3 million tons, is the largest table grape producer in the Europe (Istat, 2011). Despite the most of this production is destined for the domestic market, a substantial proportion goes to EU countries. The traditional countries, France, Germany, Belgium, Switzerland, have been joined by other Eastern European countries, most notably Russia and Poland. Occasionally table grapes are even exported to neighboring Arab countries and to Canada. According to OIV estimates, Italy ranks 6th in the world table grape production and 3rd as an exporter behind Chile and the United States. Most table grapes are grown in the south, especially in Apulia and Sicily, which account for nearly 65% and 25% of the total area respectively. The cultivars Italia, Victoria and Red Globe are the most widespread varieties in Italy, covering approximately 66% of the table grape area. In Sicily the most vineyards of table grapes are located in the geographic area of Canicattì and Mazzarrone. The production zone of Canicattì includes numerous town districts in the provinces of Agrigento and Caltanissetta. Instead, the geographical area where Mazzarrone table grapes are grown lies on either side of the border between the provinces of Catania and Ragusa and comprises the municipalities of Caltagirone, Licodia Eubea and Mazzarrone in the province of Catania and the municipalities of Acate, Chiaramonte Gulfi and Comiso in the province of Ragusa. The origins of table-grape cultivation in the Mazzarrone area can be traced back far into the past. In the 1930s and 1940s, various varieties of table grapes were grown in the areas mentioned. In the 1950s, major innovations in grape-growing concerned 4 both the range of varieties cultivated and the cultivation techniques, which, together with the land reforms carried out, contributed to the development of grape-growing throughout the district. At the same time, this district became much more specialised in grape-growing, in terms of both the use of innovative vine-training methods and the techniques used to advance or retard the ripening process. The production of table grapes has been a significant factor in the economic development and the commercial activity of the whole district. The production of Mazzarrone table grapes accounts for more than 90% of local agricultural production. The climatic and soil conditions, which are peculiarly suited to growing table grapes, combined with the district's specialization in this activity, gives the end product the quality, organoleptic and commercial characteristics that set it aside from table grapes from other areas. The training system used in Sicily for table grapes is that of “tendone”, consisting of a continuous overhead canopy under which the bunches are disposed (Fig. 1). Figure 1. Sicily pergulate known as `tendone'. 5 In the “tendone” training system, the bunches receive some protection against wind and excessive light intensity and benefit from a microclimate characterized by moderate air temperature and diffuse solar radiation, thus favoring berry development and a more uniform ripening and skin colour. Moreover, this system allows a good separation between the vegetative and reproductive zones, that forms a continuous belt on each side of the vine “row”. The specialised nature of production and the characteristics both organoleptic and commercial of Mazzarrone table grapes have given the product a confirmed reputation on Italian markets. In Italy vineyards can be infected with a variety of temperate-climate fungal diseases, many of which are facilitated by each other or other vineyard pests. Downy mildew (caused by Plasmopara viticola Berliner & de Toni.), powdery mildew (caused by Uncinula necator (Schw.) Burr.) and botrytis rot (caused by Botrytis cinerea Persoon ex Fries) are the most common diseases, each of which can cause total crop loss in the absence of control (Agrios, 2005). To a lesser extent, phomopsis (Phomopsis viticola Sacc.) and black spot (Elsinoe ampelina de Bary) can also be important (Agrios, 2005). Often the development of botrytis rot in the bunches is associated by the presence of other diseases. The causative agents of these secondary rot are opportunist, weakly or not pathogenic fungi, belonging to the genera Aspergillus, Penicillium, Cladosporium, Alternaria and Rhizopus (Hellman, 2004). Among these fungi, A. carbonarius is particularly considered since it was identified as the largest producer of ochratoxin A (OTA) (Cabanes et al., 2002; Abarca et al., 2003, Hocking et al., 2007). A new disease causing vine canker of table grapes was first observed in the San Joaquin Valley, California, in 1989 on vigorous 1-year-old cv. Redglobe vines (Vitis vinifera) (Michailides et al., 2002). Subsequently, in 2003, vine cankers were observed in Italy (Sicily) on vigorous 1-year-old cv. Black Rose vines (Vitale et al., 2008). Based on molecular characterization and pathogenicity tests, the pathogen was identified as Aspergillus spp.(Vitale et al., 2012). Estimated losses for fungal disease development in Italy vineyards amount to 15-40% of harvests depending on climatic conditions. The major economic losses are caused by B. cinerea. Its ability to attack a wide range of crops in a variety of modes of 6 infection and its ability to develop under conditions prevailing during storage, shipment and marketing make its control a challenge. 2. Botrytis cinerea Botrytis cinerea, the anamorph of Botryotinia fuckeliana (de Bary) Whetzel, is an ubiquitous fungus which can attack a wide number of host plants without showing any apparent specialization. At the present, it is conceivable that B. cinerea parasitizes well over 200 host, including fruit trees, grapevine, horticultural plants like strawberry, tomato, pepper as well as ornamentals plants (Leroux, 2007). In many of these hosts the pathogen may infect flowers, leaves, buds, shoots, stems and/or fruits, often limiting plant development, fruit-set, yield and fruit quality in fruit crops (Maude, 1980; Nicholas et al., 1994) and yield and crop quality in vegetables (Maude, 1980; Alfonso et al., 2000). It may also attack seedlings, reducing establishment and so plant density in a new crop. As concerning grapevine, it can affect the plant at different stages of development and the infections by fungal conidia can occur during the whole growing season: inflorescence, flowering, ripening, vegetative stage and cluster (Kretschmer et al., 2007). It is also a saprophyte on senescent and dead plant material. It can be found in open field so as in greenhouses, causing direct losses of production and determining the increase of production costs due to need to control the fungus. B. cinerea is a fungus ubiquitous and its high capacity of adaptation to different climatic conditions allows it to develop in different periods in the year and in different countries. Coley-Smith (1980) referred to Botrytis spp. as temperate area pathogens perhaps because of the vast research that has been carried out in such areas or due to its importance on vineyard grapes. Nevertheless, species of the genus Botrytis occur wherever their host are grown, ranging from tropical and subtropical to cold areas. For example Anderson (1924) recorded B. cinerea in Alaska and Yunis and Elad (1989) dealt with this pathogen in warm and dry areas. A rapid rate of conidial germination, infection, mycelium growth and conidiation occur in many 7 Botrytis spp. under a wide range of microclimate conditions and pose severe disease management problem all around the world (Elad et al., 2007). The fungus must be considered always present in the vineyards. Several biological event, like sporulation of the pathogen, dispersal and germination of conidia, infection, variability, virulence and survival are involved in arising of epidemics of grey mould. Each of these biological events is more or less influenced by climatic and cultural factors. These include temperature, rain, relative humidity, wind, fertilization, phonological state of the host, density of plantation and cultivar susceptibility (Jarvis, 1980). 2.1. Taxonomy Botrytis and its sexual form Botryotinia comprise 22 species and one hybrid (Hennebert, 1973; Yohalem et al., 2003) and are classified within the family Sclerotiniaceae Whetzel (Inoperculate Discomycetes). The species of Botrytis have to date been delimited primarily on the basis of morphological and cultural characteristics coupled with host specificity (Hennebert, 1973; Jarvis, 1977, 1980). Features such as sclerotial size and form and conidium size are useful in delimiting some species, but many species are morphologically similar and growing conditions significantly influence variation. No key to all recognised species is available and identification of species based on traditional criteria can be fraught (Nielsen et al., 2001). Some species have been also distinguished based on sexual crosses between them (Bergquist and Lorbeer, 1972). However, homothallism (self-fertilization) is not uncommon in Botrytis, which makes it difficult to ensure if progeny had two parents. Other Botrytis species apparently entirely lack sexuality, which further limits the use of the biological species concept for species discrimination (Staats, 2007). Rather than on morphological and biological traits, species may be identified by phylogenetic analyses of variable nucleic acid sequences. In this approach, an evolutionary tree is used to model the relationships of a group of individuals. Phylogenetic species can be identified as terminal monophyletic clades. The internal transcribed spacer (ITS) region of the ribosomal DNA has been widely used for 8 species-level discrimination of fungal species, but variation in the ITS region within Botrytis is low, limiting its use in this genus (Holst-Jensen et al., 1998; Nielsen et al., 2001). The intergenic spacer region (IGS) rDNA region may offer better prospects, although its usefulness may be limited by recombination (Giraud et al., 1997). On the basis of the recent phylogenetic analysis, B. cinerea was proposed to be a species complex (Giraud et al., 1997, 1999; Albertini et al., 2002, Munoz et al., 2002; Fournier et al., 2003). Initially, two sympatric sibling species or transposon types were described: 1) transposa, that contained two transposons Boty and Flipper and 2) vacuma, which contained no transposons (Diolez et al., 1995; Levis et al., 1997; Giraud et al., 1997). Recently, Fournier et al., (2005) showed that genetic differentiation determined from multiple gene sequences was not concordant with either of the previously described transposon types (transposa or vacuma) and revised partitioning of B. cinerea into Group I and Group II phylogenetic cryptic species. These cryptic species have also been shown to coincide with resistance to the fungicide fenhexamid, and synonymously known as FenR (resistant) = Group I and FenS (sensitive) = Group II (Albertini et al., 2002). Diagnostic molecular markers for these groups have been developed based on cleaved amplified polymorphic sequence (CAPS) profiles of the Bc-Hch gene, a homologue of the Neurospora crassa vegetative incompa-tibility hch locus (Albertini et al., 2002; Fournier et al., 2003). To date, vacuma, flipper-only, and boty only transposon types have been detected with no transposa types in Group I and all transposon types have been detected in Group II (Giraud et al., 1999; Albertini et al., 2002; Fournier et al., 2003; Ma and Michailides, 2005). In grapevine pathology studies, transposa isolates were shown to be more virulent than vacuma isolates and changes in transposon type frequencies during crop development were possibly due to differences in their saprotrophic and pathogenic fitness (Martinez et al., 2003, 2005). Thus, these observations supported the possibility of genetic differentiation between transposon types (Martinez et al., 2003, 2005). Recently, other phenotypic differences between the two types 'vacuma' and 9 'transposa' have been demonstrated: the transposa isolates are small if compared with the macroconidia of vacuma isolates and they are often resistant to vinclozolin and diethofencarb (Giraud et al., 1999); moreover, the transposa isolates grow slowly when inoculated on a medium rich in nutrients (Martinez et al., 2003). 2.2. Morphology The Botrytis species produce colonies effuse, at first white to grayish, then dark brown (Fig. 2a). The mycelium of B. cinerea is olive brown in colour with cylindrical, septate hyphae, 11-23 μm in diameter (Pearson and Goheen, 1988). Macroconidia, usually called conidia, are produced in clusters from enlarged apical cells at the end of branched, slender, conidiophores (1-3 mm long) (Pearson and Goheen, 1988), which originate from enlarged basal cells (Jarvis, 1977, 1980) (Fig. 2b). They are smooth, single-celled, faintly ash-coloured structures, quite large (8-14 × 6-9 μm) and oval in shape (Willetts, 1997). a b Figure 2. Morphological characteristics of colony (front side) (a), conidia and conidiophores (b) of eight isolates of B. cinerea. Conidia of B. cinerea survive only for a short period in the vineyard. Their survival is influenced strongly by temperature, moisture, activity microbial and by exposure to sunlight. The conidia, when stored in a dry place, can survive for up to 14 months (Salinas et al., 1989), but their survival in the conditions of the vineyard is 10