Botrytis cinerea in raspberry in Serbia I: morphological and molecular characterization.

Morphological and molecular characterisation of 130 isolates of Botrytis 
 cinerea, derived from raspberry fruit originating from six commercial fields 
 in a raspberry growing region of Serbia (locations: Požega, Prilike, Arilje, 
 Ivanjica, Sabac and Valjevo) was performed. The results showed that all 
 isolates formed white, uniform, aerial mycelia with entire margin on PDA 
 medium. First morphological differences among the isolates appeared after six 
 days of incubation. Three-week old isolates were grouped into eight distinct 
 morphological types - four mycelial and four sclerotial. Mostly, they were of 
 sclerotial type (81.5%) and the most frequently found was an S3 type, which 
 formed large irregularly placed sclerotia. This type was dominant in five of 
 six investigated locations and represented 45-65% of the isolates. The least 
 frequent was the mycelial type M3 (0.7% of the isolates) characterized by 
 mycelial masses. The presence of Boty and/or Flipper transposons was detected 
 in isolates originating from all investigated locations. It was discovered 
 that the B. cinerea population in raspberry in Serbia, besides the 
 well-described genetically isolated sympatric species transposa (43.1%) and 
 vacuma (10.8%), contains also another two, boty (44.6%) and flipper (1.5%) 
 species with only one transposon (either Boty or Flipper) in the genome. In 
 addition, it was revealed that all isolates from raspberry collected in 
 Serbia, transposa, vacuma, boty or flipper, are sensitive or weakly resistant 
 to fenhexamid and therefore belong to the B. cinerea genetical Group II. 
 [Projekat Ministarstva nauke Republike Srbije, br. III 46008: Development of 
 integrated management of harmful organisms in plant production in order to 
 overcome resistance and improve food quality and safety]


INTrODuCTION
Over the last decade, Serbia has been one of the top world producers and exporters of raspberry (Rubus idaeus L.) (Nikolić & Tanović, 2012). The average production over a 10-year period (2002-2012) was 89.476 tons/year (FAOSTAT, 2014) from an area of about 11.041 ha (Anonymous, 2013), mostly concentrated in the western and southwestern parts of the country. Botrytis fruit rot, caused by the phytopatogenic fungus Botrytis cinerea Pers. Fr., is one of major factors limiting raspberry production. Yield losses in commercial fields have been found to exceed 50%, especially during periods of rainy or humid weather right before harvest. In addition, the fungus causes significant losses during shipping and marketing, which makes it one of the most important pathogens of raspberry worldwide (Nikolić et al., 2008). B. cinerea (anamorph of Botryotinia fuckeliana), a pathogen that causes grey mould on a wide variety of plants worldwide, has been extensively studied on many major crops, including grapes, strawberry, kiwifruit, tomato and bulb crops, while its characterization on raspberry is still limited and based on a small number of isolates (Tanović et al., 2009). Well-documented phenotypic diversity of the pathogen is usually explained by the multinucleate and heterocaryotic nature of hyphae or conidia and aneuploid state of nuclei (Hansen & Smith, 1932;Büttner et al., 1994;Chardonnet et al., 2000;Yourman et al., 2001). Initial molecular investigation of French and Chilean populations of B. cinerea had shown that the species is composed of two sympatric species, transposa and vacuma, characterized by the presence of two transposable elements, Boty and Flipper, or by absence of both of them (Giraud et al., 1997(Giraud et al., , 1999Muñoz et al., 2002). Afterwards, boty (containing only Boty) (Giraud et al., 1999;Muñoz et al., 2002;Vaczy, 2009;Fekete et al., 2012) and flipper (containing only Flipper) (Albertini et al., 2002;Beever & Weeds, 2004;Isenegger et al., 2008;Vaczy, 2009;Fekete et al., 2012) isolates were found, suggesting a more complex population structure of B. cinerea than it was previously recognized. Additional molecular studies of different nuclear genes have shown that B. cinerea population is grouped in two genetic entities -Group I and Group II. The described Group I isolates are exclusively vacuma type, belong to one vegetative compatibility group (VCG) and are naturally resistant to fenhexamid, while Group II contains transposa, vacuma, boty, and flipper isolates that belong to several VCGs and are sensitive to fenhexamid (Fournier et al. 2003;Leroux, 2004;Fournier et al., 2005). Giraud et al. (1997) hypothesized that a certain degree of host specialization exists within B. cinerea population. Further studies have revealed significant differences regarding the prevalence of vacuma and transposa isolates on different host plants (Giraud et al., 1999). In addition, Martinez et al. (2003Martinez et al. ( , 2005 found that vacuma isolates were mostly of mycelial type and with higher growth rate than transposa isolates, while Giraud et al. (1999) reported difference in fungicide resistance frequencies in transposa and vacuma isolates. Since successful management of grey mould disease is based on the depth of our knowledge of the pathogen, information on the genetic structure of its population could be a useful tool for developing effective control strategies. Therefore, the aims of this study were: a) to determine the presence and distribution of transposa, vacuma, boty, and flipper isolates of B. cinerea on raspberry fruit in Serbia; b) to characterize some biological features of the isolates from all described subpopulations in terms of colony morphology, virulence, growth rate, sporulation, sclerotia production and pigment release; c) to evaluate the sensitivity of isolates to fenhexamid using a qualitative sensitivity test. The first part of the study focusing on pathogen isolation, morphological and molecular characterization, and sensitivity to fenhexamid is presented in this paper, while the growth rate and virulence, as important fitness parameters of the isolates, will be investigated afterwards and presented in a followup paper.

Fungal isolates
Ripe raspberry fruits expressing grey mould symptoms were randomly collected from commercial orchards from six locations in the raspberry growing region in Serbia. In order to potentiate overgrowth of the fungi the diseased fruits were incubated individually on two layers of moist paper towels in plastic containers for seven days at 20 o C, 97% RH (relative humidity). The isolates were derived by placing a small fragment of developed mycelia on Potato Dextrose Agar (peeled potato -200 g, dextrose -20 g, agar -17 g, distilled water -1 L, PDA) and allowing a 48 h incubation at 20 o C. The obtained isolates were then purified by monospore isolation and cultured on PDA medium at 20 o C.

Maintenance
The isolates were stored on slants at 4 o C for shortterm or in 20% glycerol at -80 o C for long-term storage.

Pathogenicity test
A pathogenicity test was performed as described by Vignutelli et al. (2002) with slight modifications. Apples cv. Golden Delicious were surface sterilized with ethanol (70%) and wounded using sterile nail (4-mm diameter and 3-mm depth) at three position. Mycelial plugs on PDA (Ø 3 mm) were placed into the wounds, while sterile PDA disks were used for inoculation in the control. The isolates were considered pathogenic if fruits showed soft rotting around wounds after two to four days of incubation at 20 o C in the dark.

Identification
The isolates were identified based on pathogenic characteristics, morpholog y of the colony and microscopic observations of conidiophores and conidia and their comparison with the available literature data (Ellis & Waller, 1974).

Morphological characterization
The isolates were grown on PDA medium at 20 o C in darkness for three weeks. Afterwards, phenotypic observations were performed based on mycelial aspect, sporulation and sclerotia production. The isolates were classified into eight morphological types described by Martinez et al. (2003) and used earlier by Tanović et al. (2009) (Figure 1).

Molecular characterization
DNA isolation: DNA was obtained directly by scraping mycelia with a pipette tip from 4-day-old culture on PDA. The mycelia was transferred into 50 µl of PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, CA, USA), vortexed briefly, incubated for 30 min at 56 o C, followed by 10 min incubation at 100 o C, and stored at -20 o C until use (Harrington & Wingfield, 1995). The DNA quality of each isolate was confirmed to be suitable for polymerase chain reaction (PCR) by generation of a single band with the universal primers ITS1 and ITS4 (White et al., 1990).
Detection of transposable elements Flipper and Boty: The presence of the transposable elements Flipper, a 1872-bp class II element , and Boty, a 6-kb retrotransposon (Diolez et al., 1995), previously sporulation, M2 -aerial mycelium with sporulation, M3 -mycelial masses, and M4 -thick and woolly mycelium) and sclerotial (row below -left to right: S1-sclerotia at the edge of Petri dish, S2 -sclerotia large and arranged in a circle, S3 -sclerotia large, placed irregularly, and S4 -sclerotia small and scattered) identified in the B. cinerea genome, was tested in all obtained isolates using PCR methods described by Levis et al. (1997) and Ma & Michailides (2005). The primers used for detection of the Flipper element (F300: 5'-GCA CAA AAC CTA CAG AAG A-3' and F1550: 5'-ATT CGT TTC TTG GAC TGT A-3') amplify a 1250-bp product, corresponding to a major part of the Flipper element. The presence of the Boty element was tested using BotyF4: 5'-CAG CTG CAG TAT ACT GGG GGA-3' and BotyR4: 5'-GGT GCT CAA AGT GTT ACG GGA G-3' primers that amplify a 510-bp product (Ma & Michailides, 2005

Sensitivity of the isolates to fenhexamid
Sensitivity of the isolates to fenhexamid was determined on PDA medium amended with discriminating concentrations of 1, 5 and 10 mg/L of fenhexamid, allowing the growth of resistant but fully inhibiting the growth of sensitive isolates (Stehman & De Waard, 1996). Fenhexamid (Teldor SC, 500 g/l, Bayer CropScience) was suspended in sterile distilled water and added to autoclaved media that had cooled to 50 o C. Petri plates were inoculated with inverted mycelial plugs (10 mm), cut at the edge of 4-day-old colonies on PDA, and incubated for 48 hours at 20 o C. The experiment was conducted in four replicates and repeated twice. Isolates that did not grow at the lowest concentration (1 mg/L) were designated as sensitive, while those able to grow at the higest fungicide concetration (10 mg/L) were considered as highly resistant. The remaining two groups, those that grew at 1 mg/L but not at 5 mg/L, and those that grew at 5 mg/L but not at 10 mg/L, were considered as weakly or moderately resistant, respectively.

Disease simptoms
The most common disease symptom was fruit decay in the form of spreading lesions, typically at the stem end of ripening fruit, accompained by profuse sporulation of the pathogen (Figure 2). In total, 130 isolates were derived by transfering the developed mycelium onto PDA medium. The isolates were marked by a combination of letters indicating the origin of isolates and seriatim numbers as presented in Table 1.

Pathogenicity of the isolates
All tested isolates caused soft rotting of apple fruits after two to four days of incubation (Figure 3). Pathogenicity of the isolates was confirmed by reisolation of the pathogen from inoculated fruits.

Morphological characterization
At the beginning of mycelial development on PDA medium at 20 o C, all isolates formed white uniform aerial mycelium with entire margin (Figure 3).  . Botrytis cinerea -differences in morphology of the isolates: 10-day old mycelial isolate with homogeneous mycelium (left); 7-day old sclerotial isolate with zones of compact areal mycelium and white beginnings of sclerotia (middle); and 10-day old sclerotial isolate containing fully developed black sclerotia (right) Morphological differences among the isolates were noticable after six-day incubation. Mycelia of the isolates producing no sclerotia remained homogeneous, aerial or substrate, while sclerotia-producing isolates formed zones of compact areal mycelium containing white beginnings of sclerotia. Fully developed sclerotia were black, hitched to the medium and appeared in cultures after incubation of 10 days (Figure 4). Final observation of the morphological characteristics of the isolates was performed after three-week incubation and the isolates were classified into eight morphological types (Figure 1).
Under the given experimental conditions, most isolates formed colonies of the sclerotial type (81.5%) ( Figure 5). Among them, the most frequently found were isolates forming large irregularly placed sclerotia, corresponding to the S3 type of isolates. That type was dominant in five of the six investigated locations and represented 45-65% of isolates (Table 2).

Detection of transposable elements Flipper and Boty
Transposable elements were detected in 89.2% of the isolates (Figure 6). Besides transposa (featuring both transposons) and vacuma (without either transposon), subpopulations, i.e. two additional types of isolates containing only one transposable element, either Boty or Flipper, were found. These isolates were designated as boty and flipper, respectively. The resulting molecular determination of the elements at each location is presented in Table 1.

Sensitivity of the isolates to fenhexamid
The results of the sensitivity test revealed a high level of sensitivity to fenhexamid. Moderately or highly resistant isolates were not found in any of the populations, while the presence of weakly resistant isolates varied depending on population. In two of six populations all the isolates were sensitive to fenhexamid. The highest precentage of weakly resistant isolates was recorded in the population originating from Šabac (Figure 7).

DISCuSSION
Our study of the biological traits of 130 isolates of B. cinerea, originating from six raspberry fields, revealed a great phenotypic variability of this species in raspberry, confirming previous findings in other host plants (Grindle, 1979;Di Lena et al., 1981;Faretra et al., 1988;Leone, 1990;Kerssies et al., 1997;Alfonso et al., 2000;Chardonnet et al., 2000;Yourman et al. 2001;Baraldi et al., 2002;Vaczy et al., 2006;Decognet et al., 2007). Based on colony morphology on PDA medium, the isolates were sorted into eight groups, described by Martinez et al. (2003). Among the isolates from all locations, those forming large, irregularly placed sclerotia were prevalent, while the mycelial type isolates with mycelial masses were the least frequent. However, Paul (1929, cited by Lorbeer, 1980 had described only three morphological types of B. cinerea isolates -sclerotial, sporulating and mycelial. Van der Spek (1965, cited by Lorbeer, 1980 investigated isolates from different host plants including: flax, strawberry, raspberry, pea, and tomato and found the same three types that had been recognised by Paul (1929, cited by Lorbeer, 1980. In addition, he noticed that different types of isolates had not occurred on different host plants to equal extent. For example, the mycelial type isolates were the most frequent isolates on flax, while sclerotial isolates were found only occasionally, whereas the sporulating type was not detected at all. On the contrary, high frequency of sclerotial isolates was found in vine, tomato, strawberry, blueberry and rose in Uruguay (Gepp et al., 2007), as well as in vine in Austria (Achleitner, 2008).
In the present study, transposable elements were detected in 89.2% of the isolates originating from raspberry from Serbia. The most frequent were those containing only the Boty element (44.6%), followed by transposa isolates (43.1%), while vacuma was less present (10.8%). The least frequent were flipper isolates with a share of 1.5%. The highest percentage of transposa isolates was detected in Prilike and Arilje locations (50%). Such distribution of subpopulations had not been usually observed in B. cinerea. In most studies only two subpopulations, transposa and vacuma, have been found (McDonald, 1993;Levis et al., 1997;Giraud et al., 1997;1998;1999;Coarer, 2003;Martinez et al., 2003;Ben Ahmed & Hamada, 2005). The most comprehensive study of B. cinerea population structure so far, which included 840 isolates from 23 hosts originating from 15 countries, has revealed that transposa isolates prevailed (69.2%) in all countries and all hosts, followed by vacuma (14.4%) and boty (13.8%) isolates (Pollastro et al., 2007).
Besides vacuma and transposa isolates, those containing only the Boty element have been found in B. cinerea populations in Chile (Muñoz et al., 2002), California (Ma & Michailides, 2005), Croatia (Topalovec-Pintarić et al., 2004) Bangladesh, India, and Australia (Isenegger et al., 2008). In addition, populations from Europe, Bangladesh and India have been shown to contain isolates with the Flipper transposon only (Albertini et al., 2002;Beever & Weeds, 2004;Isenegger et al., 2008). However, none of these populations had a high percentage of boty isolates as observed in our present study. In addition, unexpectedly high percentages of flipper isolates have been found in Bangladesh (70%) and Nepal (22%) (Isenegger et al., 2008). Our results showed that the percentage of vacuma isolates in raspberry in Serbia ranged from a complete abscence in Ivanjica and Prilike to 40% detected in Šabac. A fact that deserves some attention is that the sampling at the locations Ivanjica and Prilike was conducted at the end of July, while samples were collected in Šabac a month earlier, i.e. at the end of June. This may support an observation of Giraud et al. (1997) that the share of vacuma isolates decreases during the growing season. Significant decreases in the frequency of vacuma isolates during the vegetation period have also been recorded in France, Italy and Austria (Martinez et al., 2005;De Miccolis Angelini et al., 2006;Achleitner, 2008). However, further investigation with a more appropriate sampling design is needed in order to make a final conclusion about the dynamics of frequency of vacuma isolates over the growing season.
In order to determine whether the derived vacuma isolates belonged to the described genetic Group I B. cinerea, they were tested for sensitivity to fenhexamid. The results showed that all examined isolates of B. cinerea from raspberry fields in Serbia were sensitive or weakly resistant to fenhexamid and consequently belonged to the genetic Group II of the species.

aCkNOwLEDgEMENT
The study was carried out as part of Project III 46008 "Development of integrated management of harmful organisms in plant production in order to overcome resistance and improve food quality and safety", funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia.