MOLECULAR AND PATHOLOGICAL VARIABILITY ASSOCIATED WITH TRANSPOSABLE ELEMENTS OF BOTRYTIS CINEREA ISOLATES INFECTING GRAPE AND STRAWBERRY IN EGYPT

Grey mold caused by Botrytis cinerea, is known to cause great losses in most vegetable and fruit crops. Fifty-one isolates of B. cinerea were collected from grape (BCG) and strawberry (BCS) grown in different Egyptian locations. Variation among isolates was demonstrated using fenhexamid resistance and genetic approaches. Isolates were classified into various pathogenic groups depending on their reactions towards lettuce leaves. Genetic variability was identified in all isolates using transposable elements (TEs) analysis which revealed either the presence or absence of boty and flipper transposons. Furthermore, TEs typing of B. cinerea isolates demonstrated four TE types, on the basis of TE distribution in B. cinerea populations, namely, transposa (having both boty and flipper), flipper (possessing only flipper), boty (having only boty), and vacuma (lacking both boty and flipper elements). Transposa type was predominant (43.1%) and both transposa and vacuma isolate types showed no specialization with respect to host plant or plant location, while flipper type revealed a geographical preference in (BCG) isolates. Pathogenicity was also correlated to TE type as isolates containing transposa type revealed some degree of correlation with virulence behaviour, suggesting that transposa populations have higher pathogenic potential as compared to vacuma ones. The sensitivity of sampled isolates was tested against fenhexamid as one of the most important botryticides. Sensitivity to fenhexamid was shown in all isolates from strawberry and grape, grown in different locations, with low EC50 values between 0.012-0.084 μg/ml. This finding provided a cue for effective usage of fenhexamid for grey mold management. The present work demonstrated a correlation between the distribution of TEs and some fungal features such as isolate source and virulence, but no correlation was found between morphological characteristics, TE type, and sensitivity to fenhexamid. Cluster analysis based on phylogenetic tree showed that the Egyptian isolates branched as a separate divergent group from the others retrieved from GenBank, reflecting the presence of sequence polymorphism between the current isolates of B. cinerea and those previously identified.


INTRODUCTION
Botrytis cinerea sensu lato, the causal agent of grey mold disease, is a phytopathogenic necrotrophic fungus. It causes extensive pre-and post-harvest damages and infects over 200 plant species worldwide. B. cinerea is not host specific and shows virulence variation towards different host plants (Mirzaei et al., 2009). This fungus is a complex pathogen due to its high flexibility in adapting to various host plants and environmental conditions. Typical symptoms comprising of greyish soft rotten of infected plant part lead to great production loss in affected plants in a broad spectrum of hosts. Morphological characteristics such as fungal mycelia, conidia and sclerotia are useful features in the study of B. cinerea isolates. Generally, variability in fungi is manifested in morphology (Chardonnet et al., 2000), pathogenicity (Wahab et al., 2019), fungicide resistance (Abdel Wahab, 2015) and genomic instability (Dufresne et al., 2006;LÓPez-Berges et al., 2009) including ploidy (Buttner et al., 1994). Genetic variation of fungal pathogen had been detected using molecular techniques such as genomic transposable elements (Dufresne et al., 2006;LÓPez-Berges et al., 2009;Abdel Wahab, 2015) restriction fragment length polymorphism (Giraud et al., 1997), DNA fingerprinting repetitive sequences (Ma and Michailides, 2005), amplified fragment length polymorphism (Moyano et al., 2003) and genomic typing by microsatellite . These methods had made a significant impact on fungal identification, taxonomic and phylogenetic studies (Fournier et al., 2005). More noticeably, the development of specific primers against B. cinerea genomic has permitted fast detection at the genus/species levels (Karakaya and Bayraktar, 2009). Although many studies have been conducted, a regular trend and a positive correlation between biological and ecological features are still in need to be established (Mirzaei et al., 2009). Because of the genetic plasticity of B. cinerea, a high evolutionary potential of resistant strains makes fungicides to be hardly adopted by farmers in order to totally eradicate grey mold disease. The potential of some fungicides had been evaluated on the basis of the phenotypic and genetic diversity among fungal strains (Ahmed and Naim, 1993;Tanovic et al., 2009). However, more evaluations are needed to be studied as these fungicides are always affected by environmental factors. Thus, the purposes of the current study were to (i) discriminate B. cinerea isolates collected from different locations in Egypt on the basis of their morphological, pathogenic, and molecular features; (ii) combine such data to generate a robust characterization of B. cinerea isolates; (iii) establish the phylogenetic relationships among B. cinerea isolates with respect to geographic origin and host plants; and (iv) test sensitivity of B. cinerea isolates towards fenhexamid which is effectively used for the control of this fungus. MATERIALS AND METHODS Collection of plant samples: Plant samples were collected from symptomatic and asymptomatic berries of grape (Vitis vinifera) and fruits of strawberry (Fragaria ananassa), grown in five Egyptian locations during 2015-2016 (Table 1).  Abdel Wahab and Helal (2013). Samples were cut into small pieces (0.5cm), put in sterilized water for 5 min, dried on sterilized filter paper, then put on m1KERS medium and incubated for 3-21 days at 23 o C. A pure culture was obtained by the monospore technique using potato dextrose agar (PDA) medium. Single spore cultures were preserved using sterilized paraffin oil at 4 o C. Cultural and morphological characteristics of B. cinerea isolates were described according to Ellis and Waller (1974). Morphological characteristics of B. cinerea isolates were tested by putting mycelial agar plugs (6 mm in diameter) from the colony margin of each isolate of 4day old cultures on the center of a sterile Czapek's Dox medium plate (9 cm diameter). Three replicates were prepared for each isolate and then incubated for one week at 23°C. Length, width and volume of 200 conidia from each isolate were measured according to Shirane (1989). The conidial volume was determined by measuring conidial dimensions using an objective (40X) of a light microscope, Olympus microscope (BH2), using the following formula: Conidial volume ( m 3 ) = . 2 . π 6 Where L = conidial length ( m), W = conidial width ( m) and π = 3.14159. The conidial shape was examined by a 40X objective of the light microscope and photographed for documentation. Sclerotia were measured for colour, shape, size and number per dish. Mycelial isolate culture (6 mm diameter) of three days was transferred from the colony margin using a sterilized cork-borer to the center of PDA Petri dishes (9 cm); each isolate was measured 21 days after incubation at 23°C.

Pathogenicity test:
Detached leaves of lettuce plant (Lactuca sativa L. cv. 'Baladi') were prepared to test isolate pathogenicity. Three leaves were prepared as replicates for each isolate, excised from the central part of lettuce head and placed in a plastic box containing moist towels. Mycelial colony plug (6 mm diameter) was removed from the colony margin of each isolate using a sterilized corkborer and transferred to each lettuce leaf with the mycelial side of culture plug facing the leaf surface. Each plastic box was covered to maintain humidity and then incubated at 23 o C. The measurement of lesion diameter around culture plug was taken daily for 72h. This test was performed in triplicate. The aggressiveness test was conducted according to Abdel Wahab (2015). Fenhexamid sensitivity assay: Sensitivity of all B. cinerea isolates to the hydroxyanilide fungicide, fenhexamid was determined on Czapeck's Dox agar medium amended with a range of fenhexamid concentrations (0.5, 1.5 and 10 mg/L), allowing the growth of resistant isolate but fully inhibiting the growth of sensitive isolates. Fenhexamid (Teldor SC, 500 g/L, Bayer Crop Science) was suspended in sterile distilled water and added to autoclaved media that had cooled to 50°C. The culture plug (6 mm) of four-day old isolate was excised and put on the center of PDA plate amended with different fenhexamid concentrations, previously prepared. Three replicates were prepared for each isolate and then incubated at 23°C for three days. Colony diameter was measured in two perpendicular directions in each plate and the mean was estimated. This test was conducted twice using three replicates for each concentration. The 50% effective concentration (EC50), 50% mycelial growth inhibition, was determined by regressing the relative growth rate (% control) against the log of the fungicide concentration. Molecular analysis of Botrytis cinerea isolates: Single spore culture with the typical morphological characteristic of each B. cinerea isolate was transferred to potato dextrose broth (PDB) medium in Erlenmeyer flasks and incubated at 23 o C in the dark for 7 days. Genomic DNAs were extracted and purified from harvested mycelia using mini preparation procedure according to Möller et al. (1992). B. cinerea isolates were identified either by PCR using a pair of specific primers, C729+ and C729- (Rigotti, 2002) (Table 2) or by sequencing the internal transcribed spacer (ITS) regions of fungal ribosomal DNA, using the ITS1/ITS4 primers (White et al., 1990) (Table 2). PCR was carried out in a total volume of 25μl of PCR reaction consisting of 2μl genomic DNA (50 ng/μl), 0.5μl of each primer "Bio-search Technologies" (10μM), 12.5μl Red PCR master mix (Bio-line) and 9.5μl H2O. The PCR amplification was performed in a thermocycler (Techne-Progene). The reaction was programmed as follows: Initial denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, terminating with a final run on 1.5 agarose gel, staining with Red Safe dye and visualized under UV light. A 100-bp DNA marker (Bio-line) was used as a molecular length standard. Detection of Transposable Elements (TEs) in Botrytis cinerea isolates: PCR amplification of the two transposable elements (Boty and Flipper) using the specific primers BotyF4/BotyR4 and F300/F1550 for each element as demonstrated by Diolez et al. (1995) and Levis et al. (1997) respectively (Table 2) was conducted as outlined before for Molecular analysis but using the following program: an initial denaturation for 5 min at 95 ˚C, followed by 40 cycles of denaturation at 94 ˚C for 1 min, annealing at 60 ˚C for the primer pair F300/F1550, or 68 ˚C for the primer pair BotyF4/BotyR4, for 1 min, extension at 72 ˚C for 1 min (for boty primers) or 3 min (for flipper primers), and terminated with a final extension at 72 ˚C for ten min. Amplified products were separated by electrophoresis on 1% agarose gel in TBE, stained with Red Safe dye and visualized under UV light. A molecular length standard of 1 kb DNA ladder (Bio-line) was used. Bc-hch amplification and digestion: A primer pair 262/520L (Table 2), described by Fournier (Fournier et al., 2003), was used to amplify the Bc-hch gene to distinguish between B. cinerea and B. pseudocinerea. Amplification was performed in a thermocycler (Techne-Progene). The PCR program was as that outlined before for molecular analysis. Restriction fragment length polymorphism (RFLP) was generated through digestion of the 1171 bp product using 2 units of the restriction enzyme HhaI (Biolabs, New England), incubated for 90 min at 37°C, resolved on a 2% agarose gel along with a 1Kbp DNA standard ladder marker and visualized under UV light. Detection of Transposable Elements (TEs) in Botrytis cinerea isolates: PCR amplification of the two transposable elements (Boty and Flipper) using the specific primers BotyF4/BotyR4 and FlipperF300/ Flipper F1550 for each element as demonstrated by Diolez et al. (1995) and Levis et al. (1997) respectively (Table 2) was conducted as outlined before for Molecular analysis but using the following program: an initial denaturation for 5 min at 95 ˚C, followed by 40 cycles of denaturation at 94 ˚C for 1 min, annealing at 60 ˚C for the primer pair F300 and F1550, or 68 ˚C for the primer pair BotyF4 and BotyR4, for 1 min, extension at 72 ˚C for 1 min (for boty primers) or 3 min (for flipper primers), and terminated with a final extension at 72 ˚C for ten min. Amplified products were separated by electrophoresis on 1% agarose gel in TBE, stained with Red Safe dye and visualized under UV light. A molecular length standard of 1 kb DNA ladder (Bio-line) was used. Bc-hch amplification and digestion: A primer pair Bc-hch262/ Bc-hch520L (Table 2), described by Fournier (Fournier et al., 2003), was used to amplify the Bc-hch gene to distinguish between B. cinerea and B. pseudocinerea. Amplification was performed in a thermocycler (Techne-Progene). The PCR program was as that outlined before for molecular analysis. Restriction fragment length polymorphism (RFLP) was generated through digestion of the 1171 bp product using 2 units of the restriction enzyme HhaI (Biolabs, New England), incubated for 90 min at 37°C, resolved on a 2% agarose gel along with a 1Kbp DNA standard ladder marker and visualized under UV light.

Distinction of B. cinerea and B. pseudocinerea:
The Neurospora crassa vegetative incompatibility locus homo-log, Bc-hch, was amplified as described by Fournier et al. (2003) using the primers 262 (5-AAGCCCTTCGATGTCTTGGA-3) and 520L (5-ACGGATTCCGAACTAAGTAA-3). Restriction fragment length polymorphisms (RFLPs) were generated through digestion of the 1171 bp product with the restriction enzyme HhaI for 90 min at 37°C, resolved on a 2% agarose gel and visualized by ethidium bromide staining under a UV light. Phylogenetic analysis of Botrytis cinerea isolates using three nuclear genes: Identification of the B. cinerea isolates was confirmed by the phylogenetic analysis described by Staats (2004), based on DNA sequencing of the three nuclear genes, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase subunit II (RPB2) genes using the specific primer pairs G3PDHfor/G3PDHrev, HSP60for/HSP60rev and RPB2for/ RPB2rev, respectively (Table 2) (Staats, 2004). PCR amplification was done on the partial DNA sequences of such three nuclear genes and performed in PCR thermocycler (Techne-Progene). Amplification was carried out in total volume 25μl of PCR reaction as described before under molecular analysis using the following program: Initial denaturation at 94°C for 4 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s and a final extension step at 72°C for 10 min for both HSP60 and RPB2 gene segments. The same program with an annealing temperature of 64°C was applied to G3PDH gene fragment. The PCR amplicons were separated by agarose gel electrophoresis (1.2 % in TAE 0.5×), purified and sequenced. The phylogenetic analysis was carried out based on a combined dataset of the partial sequences of RPB2, HSP60 and G3PDH that were obtained from the tested B. cinerea isolates. Molecular and phylogenetic analyses were performed using MEGA6 software, version 6 (Tamura et al., 2013). The missing data and nucleotide gaps in the DNA sequences were deleted. The phylogeny tree was constructed using the Neighbour joining method and individually tested with a bootstrap of 1000 replicates to achieve the reliability of any given branch pattern in each Neighbour joining tree. Statistical analysis: All data that scored from morphological features, mycelial growth rates, pathogenicity and fenhexamid sensitivity assays were tested by analysis of variance (ANOVA) to determine the significant differences. Data means were treated using the least significant difference test at P = .05 level. RESULTS Pathogen isolation from grape and strawberry naturally infected with grey mold: Diseased plant parts were cultured on the selective medium, m1KERS for isolation of the causal agent, microscopically identified as B. cinerea. Data presented in Table 1 showed that B. cinerea was identified in 51 isolates which included 25 and 26 isolates collected from grape and strawberry, respectively. Phenotypic diversity of Botrytis cinerea isolates: Variation among all isolates of B. cinerea was achieved by morphological and microscopical characterization. The results obtained showed that B. cinerea isolates varied in growth texture (Table  3) and were categorized as compact, heavy warty, light warty, fluffy and watery. Conidial dimensions differed significantly among isolates, ranging from 10.6 to 12.7 µm in length, 7.3 to 8.9 µm in width and 297.6 to 491.1µm 3 in volume ( Table 3). Conidia of all isolates were described as oval shape and sclerotia characteristics like size, number and distribution pattern were determined for each plate. Data obtained indicated that sclerotia varied in shape with two types, being cerebriform in all isolates except BCS102, BCS105 and BCS110 whose sclerotia were flat (Table 3). 402.5±91.2 *, All data are means of triplicate measurements ± standard deviation (SD) at LSD.05, ** All data are means of 20 conidial measurements ± standard deviation (SD) at LSD.05.
Four categories of the sclerotial pattern were recognized and these were: large irregular, large in circle, small/scattered and aggregate in the center of culture plate. A different number of sclerotia was observed per culture plate and ranged from 17.7 to 201.3 sclerotia/plate. In addition, the mycelial growth rate ranged from 0.3 to 4.9cm (Table 4). Pathological and molecular pattern of Botrytis cinerea isolates: The degree of virulence of 51 isolates of B. cinerea collected from grape and strawberry was assessed by using detached lettuce leaves (see materials and methods). A significant difference among isolates depending on their aggressiveness in terms of lesion diameter was observed (Table 4). Statistical analysis showed that isolates differed significantly in lesion size with diameter ranging from 0.3 to 5.1 cm. Isolates were divided, depending on their virulence, into three categories: highly virulent with a lesion diameter ranging from >2.5 cm to 5.1 cm (43.1%); moderately virulent with a lesion ranging from >1 to 2.5 cm (43.1%); low virulent with a lesion diameter ranging from 0.3 to 1 cm (13.7%). Results of typing twenty-five isolates of B. cinerea from grape and twenty-six isolates from strawberry for TEs presence using either the specific primers BotyF4 & BotyR4 to amplify boty element or F300/F1550 to amplify flipper element showed four TEs types (Table 4). 2.7 ± 0.4cd 2.1±0.2bcdefg T *, MGR: mycelial growth rate. All data for growth rate of isolates collected from strawberry and grape are means of triplicate measurements ± standard deviation (SD) at LSD.05, respectively. **, data for lesion diameter are means of triplicate measurements ± standard deviation (SD) at LSD.05 after 72h of infection. ***, transposable element type, B, only boty, F, only flipper, T, transposa, V, vacuma. a-m , means in a column followed by a different letter differ significantly (p=.05). Data were means ± SD of triplicate measurements.

B C G 1 0 2 B C G 1 0 1
For boty detection, PCR generated 510 bp, whereas, PCR revealed the expected 1250 bp for flipper element. Among 51 isolates tested, 22 have the two transposable elements, boty and flipper (transposa type), which indicated that transposa isolates were predominant (43.1%) in almost all of B. cinerea populations isolated from grape and strawberry. While, 18 have only boty element (boty type, 35.3%), 8 have only flipper element (flipper type, 15.7%) and 3 have neither of these two elements (vacuma type, 5.9%).

Sensitivity of Botrytis cinerea isolates to fenhexamid:
The results revealed that all B. cinerea isolates were highly sensitive to fenhexamid. EC50 of strawberry isolates was less than 0.1μg fenhexamid ml -1 (Figure 1). EC50 values ranged from 0.012-0.078 and 0.014 -0.084μg fenhexamid /ml for B. cinerea isolates collected from both strawberry and grape, respectively (Figures 2 and 3).   The highest sensitive isolates were BCS113, BCS116 and BCS118 from strawberry where EC50 value was as low as 0.012μg/ml, BCG101 and BCG119 from grape where EC50 value was 0.014μg/ml. The lowest sensitive isolate from strawberry was BCS112 where EC50 value was 0.078μg/ml and BCG117 from grape where EC50 value was 0.084 μg/ml. Molecular divergence of Botrytis isolates using the three nuclear genes: Molecular identification of all isolates by PCR using a pair of specific primers, C729+ and C729- (Rigotti et al., 2002) revealed a single band of 700 bp (data not shown) and this confirmed that all isolates were B. cinerea. The PCR amplification targeting the three nuclear genes of the two isolates, BCS101 and BCG101, using specific primers ( Table 2) revealed amplicons of approximately 1000, 1100 and 1100 bp, respectively (Figure 4), corresponding to a partial sequence of the three housekeeping genes: heat-shock protein 60 (HSP60), glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and DNA-dependent RNA polymerase subunit II (RPB2). DNA sequencing and phylogenetic analysis of the three nuclear genes: Sequence analysis of the amplified products revealed that the two isolates tested, BCS101 and BCG101, were B. cinerea based on the percentage of identity which ranged from 98 to 100% using BLAST search (GenBank accession no. MH752681, MH752682, MH752683, MH752684 and MH752685).

Bc-hch amplification and digestion:
In order to distinguish between B. cinerea and B. pseudocinerea, the current isolates were tested by PCR using Bc-hch specific primers followed by digestion of the Bc-hch locus with HhaI enzyme. PCR analysis with Bc-hch specific primers (262/520L), revealed a fragment of 1171 bp corresponding to the region of the Bc-hch gene, in five grape isolates (BCG103, BCG106, BCG117, BCG121, BCG124) and three strawberry isolates (BCS101, BCS104, BCS112) ( Figure 8A). The isolates under study demonstrated that they were B. cinerea as they revealed a 517 bp amplicon after using the restriction enzyme, HhaI. The PCR amplicons resulted from the amplification of the Bc-hch gene were digested with the restriction enzyme HhaI indicated that they belonged to Group II ( Figure 8B).

DISCUSSION
Variability of B. cinerea isolates was evident among isolates collected from two different host plants, grape and strawberry which were grown in various geographic locations in Egypt. When the variation in virulence was tested, isolates were found to lie in three categories: high, moderate and low virulence. However, no correlation was found between virulence and geographic origin. In contrast, a great variation in aggressiveness was observed among B. cinerea isolates collected from the same location. This could be due to various reasons including the ability of B. cinerea conidia to disseminate over long distances and the phenomenon of gene flow that may circumvent geographical differentiation (Mirzaei et al., 2009). However, no specific pattern was observed between isolate location and morphological features. Results of transposable elements (TEs) typing of B. cinerea isolates from grape and strawberry indicated that transposa type was predominant (43.1%) in almost all of B. cinerea populations studied, a finding that was previously reported by Esterio et al. (2011) followed by boty (35.3%), flipper (15.7%) and vacuma (5.9%) types. This distribution frequency of the four transposon types was in agreement with that reported in a recent study (Abdel Wahab, 2015), but differed from those published elsewhere (Samuel et al., 2012;Kumari et al., 2014). This discrepancy may be due to a number of reasons such as differences in population size, location, time of sampling, plant species, plant growth stage and saprotrophic versus pathogenic fitness. In fact, TEs typing had been used as a qualitative marker to study genetic variation (Giraud et al., 1999;Daboussi and Capy, 2003;Abdel Wahab, 2015), and this led them to become a molecular character of all isolates of B. cinerea from strawberry and grape. The four TE types were recognized on the basis of TE distribution in B. cinerea isolates and had previously been documented (Muñoz et al., 2010;Wahab et al., 2019). The current study demonstrated a correlation between the distribution of TEs and some fungal characters such as the location, host plant and virulence. All BCG isolates having flipper type were collected from grape growing in Alexandria, while the majority of BCS isolates which were collected from EL-Behiera have revealed all TE types. Whereas the mean conidial dimensions were the same in transposa, boty, flipper and vacuma isolates, virulence testing revealed that all BCG isolates with transposa type were highly virulent except BCG101 isolate which demonstrated moderate virulence. Similarly, while all BCS isolates with transposa type were moderately virulent, BCS122 isolate exhibited weak virulence. As to vacuma type, BCG isolates were shown to be weak in virulence, but BCS isolates were found to be moderate in this respect. These results suggested that transposa isolates had higher virulence potential as compared to vacuma ones. These findings are in agreement with those previously reported (Martinez et al., 2005;Schilling et al., 2013;Samuel et al., 2012;Kumari et al., 2014;Abdel Wahab, 2015;Pande et al., 2010). Noticeably, there was no diversity in virulence among isolates on the basis of location. However, variation among isolates collected from different geographical locations were characterized based on their sensitivity towards fenhexamid. Results revealed that all isolates, obtained from both strawberry and grape, were fenhexamid-sensitive with EC50 values ranging between 0.012 -0.084 μg/ml, indicating fenhexamid sensitivity (Rodríguez et al., 1997), and may suggest that all isolates were related to B. cinerea group II Albertini and Leroux, 2004;Leroux et al., 2002;Leroux et al., 2010). However, no correlation was found between sensitivity to fenhexamid and the possession of TE, and this is consistent with previous studies (Giraud et al., 1999;Albertini et al., 2002), but contradictory with others Leroux et al., 2002). The inability to isolate a fenhexamidresistant isolate may suggest that fenhexamid-resistance is absent among Botrytis cinerea populations in Egypt. However, this preliminary observation remains to be confirmed by involving more isolates from more host plants and more geographical locations. This evidence is supported by the fact that fenhexamid is still effective in controlling B. cinerea infections in Egypt. It might be relevant to indicate that RFLP and sequence analyses of all isolates investigated in this study demonstrated no polymorphism among Botrytis cinerea isolated from Egypt. Additionally, the genetic distance reported here between the Egyptian isolates and the GenBank accessions and the fact that they constituted a separate divergent group when phylogenetic analysis was conducted may help to explain the widespread sensitivity to fenhexamid commonly encountered among Egyptian isolates of B. cinerea. Although the results reported here were derived from a large number of samples isolated from various host plants growing in different geographical locations, no divergence events were observed among isolates. Combining the results of fenhexamid sensitive isolates with molecular analysis of population structure could help in planning a suitable control measures in which effective disease management would be achieved by applying fungicides in rotation with less chances of developing resistant variants. However, future studies involving more isolates from new other locations and plant species may still be needed to establish a clearer picture of variation among B. cinerea isolates in Egypt.