Botrytis cinerea in raspberry in Serbia II : Growth rate and virulence of isolates

Growth rate and virulence of 130 isolates of Botrytis cinerea, derived from raspberry fruit originating from six commercial fields in a raspberry growing region of Serbia and classified in two morphological and four genetic groups were studied. The results showed significant differences in mycelial growth rate among the isolates. The highest and lowest recorded growth rates were 24.5 mm/day and 8.4 mm/day, respectively, while the growth rate of most isolates ranged from 15.8 to 21.8 mm/day. The growth rate of isolates that belong to different morphological and genetic subgroups varied similarly. Furthermore, growth rate intervals of all subgroups overlapped, suggesting that the groups cannot be distinguished based on growth rates of the isolates contained. The studied B. cinerea isolates exibited different levels of virulence towards vine, sunflower and raspberry leaves, while an analysis of variance revealed that both the isolates and the inoculated host species were significant sources of variation (P<0.01). Sunflower and raspberry leaves were significantly more sensitive than vine leaves. However, correlation between isolate virulence and different hosts was not found.


INTRODUCTION
Botrytis cinerea is a necrotrophic polyphagous plant pathogen well-known for its great phenotypic and genetic variability.Differences among isolates in colony morphology, sporulation and sclerotia production are usually attributed to the multinucleate and heterocaryotic nature of hyphae or conidia and the aneuploid state of nuclei (Hansen & Smith, 1932;Büttner et al., 1994;Chardonnet et al., 2000;Yourman et al., 2001).It is also believed that the presence of Boty and Flipper transposon elements in the genom (Giraud et al., 1999) could contribute to isolate phenotypic diversity.Thus, Martinez et al. (2003Martinez et al. ( , 2005) ) found vacuma isolates (without transposons) mostly to belong to the mycelial type and have a higher growth rate than transposa isolates (containing both transposons), while Giraud et al. (1999) reported a difference in fungicide resistance frequencies in transposa and vacuma isolates.Our previous study (Tanović et al., 2014) revealed that all B. cinerea isolates originating from raspberry fields in Serbia belonged exclusively to the Group II genetic entity of B. cinerea described by Fournier et al. (2003).The isolates were divided into two main morphological (mycelial and sclerotial) and four genetic groups (transposa, vacuma, boty and flipper).In order to improve our understanding of B. cinerea populations, growth rates and virulence of isolates from different subgroups were determined and analyzed in this paper.

Fungal isolates
A total of 130 B. cinerea isolates, derived from diseased raspberry fruit collected at six locations in a major raspberry growing region in Serbia, identified based on their pathogenic and morphological characteristics and classified in two morphological and four genetic groups (Tanović et al., 2014), were used in this study (Tables 1 and 2).

Maintenance
The isolates were cultured on potato dextrose agar (PDA) medium at 20 o C and stored on slants at 4 o C for short-term or in 20% glycerol at -80 o C for longterm storage.

Mycelial growth in vitro
In vitro growth rate of the isolates was determined by transferring mycelial plugs (Ø 10 mm) from the edge of 4-day-old colonies on PDA plates.Their growth was recorded after 3-day incubation at 20 o C in the dark by measuring two diameters of each colony at right angles.Three replicates per isolate were used and the experiment was repeated twice.All data were pooled together and subjected to analysis of variance and Duncan's multiple range test.

Virulence test
A virulence test was performed on leaf discs of three host species (Vitis vinifera L., cv.Reisling Italico, Helianthus annus L., cv Kolos, and Rubus idaeus L., cv.Willamette) using a method described by Martinez et al. (2003).Primary leaves of sunflower, grown in a greenhouse, or fully developed leaves of vine and raspberry collected from a vineyard and an orchard, respectively, were detached, rinsed in distilled water and air dried.Five leaf discs per host per isolate (total 1950), 25 mm in diameter, were cut using a cork borer and placed on moist filter paper in 90 mm Petri dishes.Mycelial plugs 10 mm in diameter, cut from the edge of 4-day-old colonies grown on PDA, were placed centrally on the upper side of each leaf disc.The inoculated discs were incubated in moist chambers at 20 o C for 3 days.Lesion development on leaf discs was assessed visually on a semi-quantitative graded scale (0 = healthy, 1 = 10% rotten, 3 = 20%, 5 = 40%, 7 = 60%, 9 = 80%, 11 = 90%, and 13 = totally rotten) as proposed by Martinez et al. (2003).A mean disease severity index (DSI) was calculated.To assess the effects of the inoculated host plants and the isolates from each location separately the data were analyzed by ANOVA at 5% probability level with individual pairwise comparisons made using Duncan's multiple range test (Statistika Inc, 2001).In addition, correlation coefficients were calculated using the mean values of virulence for each of 130 isolates.

Mycelial growth rate
The isolates showed significant differences in mycelial growth rate at 20 o C on PDA medium (Figure 1).The highest and lowest growth rates were 24.5 mm/day and 8.4 mm/day, respectively, while the growth rate of most isolates ranged from 15.8 to 21.8 mm/day.These values, as well as the average growth rates of the isolates from each location are shown in Table 3.

Isolates
The results of Duncan's multiple range test showed that both the isolates and isolate locations were statistically significant sources of variation (p<0.01).However, the isolates from a single location were not grouped together, each group rather contained several subgroups of isolates characterized by different growth rates (data not shown).
Growth rates of the isolates from different genetic, morphological and sporulation ability groups are presented in Figure 2. The highest and lowest recorded growth rates of the isolates from each subgroup are summarized in Table 4.
The lowest and highest growth rates, 8.4±2.8 mm/day and 24.5±0.6 mm/day respectively, were both recorded for sclerotial transposa isolates.Among sporulating isolates, the highest growth rate (23.2±1.2 mm/day) was found in a sclerotial isolate belonging to the vacuma genetic group.

Virulence
The studied B. cinerea isolates originating from each location exibited different levels of virulence to each host plant.Furthermore, the analysis of variance revealed that both the isolates and inoculated host species were significant sources of variation (P<0.01).Sunflower and raspberry leaf discs were significantly more sensitive than vine leaf discs (F=60.34;df= 2; F crit =3.02; P<0.0001).The average DSI was 6.2 for both sunflower and raspberry leaves and 2.8 for vine leaves (Table 5).2.8±1.5 a 6.2±2.0 b 6.2±1.5 b 1 Calculated as average for all isolates from a location 2 The same letter in a row indicates non-significant difference 3 The average for all isolates from all locations Differences in virulence were also detected within all four genetic subgroups of B. cinerea as presented in Figure 3.The highest recorded value of DSI for all hosts was 12.6, while the lowest ranged from 0 for vine and raspberry to 0.2 for sunflower.Isolates unable to infect raspberry or vine leaves (DSI=0) were found within transposa, vacuma and boty subgroups (Figure 3).The lowest and highest values of DSI for the isolates belonging to four different genetic subgroups were summarized in Table 6.The calculated correlation coefficients of isolate virulence to the three host plants were as follows: r vine, raspberry = 0.61, r vine, sunflower = 0.27 and r sunflower-raspberry = 0. 26.
Neither of these values was statistically significant at 5% probability level.

DISCUSSION
Our examination of growth rates and virulence of 130 isolates of B. cinerea, originating from six raspberry fields, revealed a great diversity among the isolates concerning both features.The difference between the highest and lowest growth rates was 16.1 mm/ day, suggesting different abilities of the isolates to live and spread saprotrophically, regardless of their morphological or genetic grouping.The results of Martinez et al. (2003) and Samuel et al. (2012) showed that the growth rate of vacuma isolates was higher than that of transposa.However, according to our results, mycelial growth rates of the isolates belonging to vacuma, transposa or boty subgroups varied similarly ( flipper group contained only two isolates and was excluded from the analysis).Although we did not have enough individuals in each subgroup to perform a regular statistical analysis, we noticed that the growth rate intervals of all genetic subgroups overlapped, suggesting that groups cannot be distinguished based on the growth rate of the isolates contained.Therefore, vacuma isolates did not grow faster than the isolates containing any of the transposons, which had been suggested earlier (Martinez et al., 2003).
The growth rate, virulence, fungicide sensitivity, and genetic variability of B. cinerea have been studied extensively over the past decades (Giraud et al., 1997(Giraud et al., , 1999;;Martinez et al., 2003Martinez et al., , 2005;;Pollastro et al., 2007).However, search for relationships between genetic and biological or ecological features of its isolates was generally unsuccessful (Kerssies et al., 1997;Alfonso et al., 2000;Martinez et al., 2003).To investigate the hypothesis that isolates from different genetic subgroups differ in their ability to establish infection, we performed in vitro sensitivity tests using detached leaves of vine, sunflower and raspberry.The results showed similar differences among the isolates in each genetic subgroup, suggesting that vacuma isolates were neither more nor less pathogenic to any of the investigated hosts than the other B. cinerea genetic subgroups.Furthermore, there was no correlation in isolate virulence to different hosts, which supports the idea that hosts may shape the pathogen's population structure (Fournier & Giraud, 2008).This finding confirmed the importance of knowing the pathogen population structure on each attacked host for developing an effective control strategy (Samuel et al., 2012).However, further research is needed to improve our understanding of the genetic structure of the pathogen using more powerful molecular techniques.

Figure 1 .
Figure 1.Growth rate of Botrytis cinerea isolates originating from raspberry fields at different locations in Serbia

Figure 1 .
Figure 1.Growth rate of Botrytis cinerea isolates originating from raspberry fields at different locations in Serbia

Figure 2 .
Figure 2. Growth rate of Botrytis cinerea isolates belonging to different genetic, morphological and sporulation ability groups

Table 1 .
A list of Botrytis cinerea isolates originating from different locations in Serbia and their classification as vacuma (without transposable elements), transposa (containing both Boty and Flipper elements), flipper (containing only Flipper) and boty (containing only Boty element)

Table 2 .
Morphological features and sporulation ability of Botrytis cinerea isolates originating from different locations in Serbia

Table 3 .
Growth rates of Botrytis cinerea isolates from different locations 1 Calculated as average growth rate of all isolates from a location.2Thesame letter in column indicates non-significant difference

Table 4 .
The lowest and highest growth rates of Botrytis cinerea isolates belonging to different genetic, morphological and sporulation ability groups 1 Based on all available isolates from each genetic, morphological and sporulation ability subgroup: 14 vacuma, 58 boty, 56 transposa and 2 flipper; 106 sclerotial and 24 mycelial; 119 nonsporulating and 11 sporulating isolates

Table 5 .
The average disease severity index (DSI) for vine, sunflower and raspberry leaves inoculated with Botrytis cinerea isolates from different locations

Table 6 .
The lowest and highest values of disease severity index (DSI) for vine, sunflower and raspberry leaves inoculated with Botrytis cinerea isolates Host plant Type of isolates 1 Lowest DSI Highest DSI 1 Based on all available isolates from each genetic subgroup: 14 vacuma, 58 boty, 56 transposa and 2 flipper isolates