DETECTION AND CHARACTERIZATION OF BOTRYTIS CINEREA ISOLATES FROM VEGETABLE CROPS IN EGYPT

Botrytis cinerea is a necrotrophic plant pathogen that causes plenty of crop losses in Egypt and worldwide. Fifteen isolates of B. cinerea were collected from cabbage, pepper and lettuce grown in different locations in Egypt and subjected to investigation. Diversity in phenotypic, pathological and molecular characteristics was detected among isolates, leading to categorizing them into different groups. Molecular variation was demonstrated in all isolates by transposable elements (TEs) analyses. Four TE types, based on the presence or absence of two transposable elements, boty and flipper, were recognized among B. cinerea isolates in which transposa type (having both TEs, boty + flipper) was predominant (40%), while only boty and only flipper types appeared with distribution values of 26.7 and 20%, respectively and vacuma type (Lacking both TEs) showed the lowest distribution value (13.3%). Furthermore, vacuma population demonstrated the lowest pathogenic potential comparing to others. A correlation was found between TE type and virulence level of isolate, but no impact of TE type was observed on phenotypic characteristics of B. cinerea.


INTRODUCTION
Botrytis cinerea Pers.: Fr. (teleomorph, Botryotinia fuckeliana (de Bary) Whetzel) is the most common pathogen causing more destruction on mature and senescent tissues (Williamson et al., 2007). This fungal pathogen causes blossom blight, leaf spots/blight, fruit rot, bud rot, damping-off, stem rot/canker, tuber/root rot and bulb rot. When no application of chemical control is adopted (Villa-Rojas et al., 2012), it could cause post-harvest losses, up to more than 40%, in both field-and greenhouse-grown horticultural crops (Pedras et al., 2011). Worldwide, B. cinerea may cause more than $10 billion worth losses annually (Weiberg et al., 2013). B. cinerea attacks wounded, weakened or senescent tissues of leafy/fruity vegetables, and also healthy tissues of plants in maturity. Infections may be invisible at harvest, but they may develop rapidly at low temperatures and high relative humidity during storage. On pepper, although grey mold starts in the field, infection appears during storage. Additionally, grey mold of white cabbage (Brassica oleracea) causes losses under bad storage conditions and considers as the main disease rapidly colonized by B. cinerea responsible for losses of cabbage (Leifert et al., 1993). Moreover, old leaves and damaged stems of lettuce could be good target tissues to be rapidly colonized by B. cinerea (Davis et al., 1997). Controlling this pathogen is difficult due to its different infection modes, the ability to produce sexual and asexual spores to survive under favourable and unfavourable conditions and its wide host range. Furthermore, it may elicit noticeable disease symptoms either during pre-harvest period or remain latent to induce later severe symptoms during the post-harvest period (Fillinger and Elad, 2016). The conidia of B. cinerea quickly spread by water or wind, but the sclerotia are indispensable for survival under adverse environmental conditions (Brandhoff et al., 2017).
Severe grey-mold occurs in the field after prolonged duration of rain, fog and more dew leading to major crop losses. Furthermore, Botrytis blight is probably the most common disease of greenhouse-grown crops where the cool moist weather and lower ventilation are among the most favourable factors for disease development. Likewise, vegetables and fruits could be infected by grey mold during cold storage and shipment if the conditions in the storage house are not suitable for a particular commodity. Genomic variability in terms of fungal morphology (Chardonnet et al., 2000), genomic instability (Dufresne et al., 2006;LÓPez-Berges et al., 2009), pathogenicity (Aboelghar et al., 2019), and ploidy (Buttner et al., 1994) was previously demonstrated. Additionally, different genomic typing of B. cinerea was conducted based on restriction fragment length polymorphism (Giraud et al., 1997), microsatellite (Fournier et al., 2002), amplified fragment length polymorphism (Moyano et al., 2003), DNA fingerprinting repetitive sequences (Ma and Michailides, 2005), and transposable elements (Dufresne et al., 2006;LÓPez-Berges et al., 2009). The purpose of the current study was to look for a possible relationship between some distinct characteristics of B. cinerea isolates infected different vegetable crops with respect to geographical locations in Egypt.

MATERIALS AND METHODS Plant samples, fungal isolation & identification:
Symptomatic and asymptomatic plant samples were collected from different parts of cabbage (Brassica oleracea), lettuce (Lactuca sativa) and pepper (Capsicum annuum) which were grown in different locations in Egypt during the season of 2015-2016 (Table 1). All samples were cultured on the selective medium, m1KERS, previously developed by Abdel Wahab and Helal (2013) for Botrytis isolation and then identified microscopically. Mycelial agar plug (6 mm in diameter) was cut out from each colony margin of 4 days old culture using a sterilized cork-borer and transfered to the surface of the medium at the center of a 9 cm-diametere Petri plate. Three replicates of each isolate were prepared, and then incubated at 23 °C for seven days. Conidial dimension of each isolate was measured according to the methodology describe by Shirane et al. (1989) at 40X objective of the light microscope (Olympus microscope, BH2) and calculated using the following formula: Conidial volume (μm 3 ) = L.W 2 . π/6 , where W = conidial width (μm), L = conidial length (μm) and π= 3.14159. Isolated sclerotia were also described for their shape, size, and number per plate. Mycelial growth rate (MGR) of each isolate culture was determined by cutting out mycelial plug (6 mm diameter) from the colony margin and placing it onto the surface of potato dextrose agar (PDA medium) at the center of the Petri plate. Three replicates were used for each isolate. Plates were then incubated at 23 °C for three days and the MGR was measured on a daily basis. Molecular identification of B. cinerea isolates: Genomic DNA of each isolate was extracted according to Möller et al. (1992). Molecular identification was done by PCR using the specific primers, C729F and C729R (Rigotti, 2002) and the ITS primers, ITS1 and ITS4 (White et al., 1990) (Table  2). PCR was performed in 25 μl of total reaction mixture made up of 2 μl genomic DNA (50 ng/μl), 0.5 μl of each primer "Bio-search Technologies" (10 μM), Red PCR master mix (Bio-line) (12.5 μl) and 9.5 μl H2O, using the thermocycler, Techne-Progene. The amplification condition was as follows: Initial denaturation for 4 min at 94 °C followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 60 °C, extension for 1 min at 72 °C and terminated with a final extension for 10 min at 72 °C.

Molecular typing of B. cinerea isolates using Transposable Elements (TEs):
The two transposable elements, Boty and Flipper, were detected using specific primers for each according to Diolez et al. (1995) and Levis et al. (1997), respectively (Table 2). PCR was carried out in a total volume of 25 μl reaction mixture consisting of 2 μl genomic DNA (50 ng/ μl), 0.5 μl of each primer "Bio-search Technologies" (10 μM), 12.5 μl of Red PCR master mix (Bioline) and 9.5 μl of H2O. Amplification was done in a thermocycler (Techne-Progene) using the following programme: an initial denaturation at 95 ˚C for 5 min, followed by 40 cycles of denaturation for 1 min at 94 ˚C, annealing during 1 min at 68 ˚C, extension for 1 min (for boty primers) or 3 min (for flipper primers) at 72 ˚C, and terminated with a final extension for ten min at 72 ˚C.

Artificial infection test of B. cinerea isolates:
Pathogenicity test was conducted using detached lettuce leaves (Lactuca sativa L. cv. 'Baladi'). Three replicates were used of leaves taken from the central part of lettuce heads and placed in a moist sterilized plastic box. Discs from the colony margin of each isolate culture aged seven days were cut out using a sterilized cork-borer (6 mm diameter) and placed with the mycelial side facing the lettuce leaf surface. The covered plastic boxes were incubated under humid conditions at 23 °C for three days. The degree of virulence was recorded daily by measuring lesion diameter around each plug. This test was repeated twice. Statistical analysis: Results were analyzed statistically by analysis of variance (ANOVA) to determine the significance in differences within phenotypic and pathogenicity assays. Data means were analyzed at P .05 level using the least significant difference test.

RESULTS
Phenotypic variability of B. cinerea isolates collected from vegetable crops: Morphological characteristics of B. cinerea showed diversity among isolates (Table 3). The growth texture of isolates was classified into three classes: light warty, fluffy, and cottony ( Figure 1). Whereas all conidia were described as ovate, their measurements were significantly varied in dimensions, ranging from 10.7 to 12.6 µm in length, 7.3 to 8.8 µm in width and 297.6 to 498.3 µm 3 in volume. (Table 3). Furthermore, morphological diversity of sclerotia was found in their size, distribution pattern, and number per plate. The size and distribution pattern of the sclerotia were described as either large irregularly (Figure 2A) or numerous small and scattered ( Figure 2B) in the culture plate. The shape of all sclerotia was cerebriform (Table 3, Figure 2C), while their number varied among isolates and ranged from 21 to 65 sclerotia /plate. Moreover, mycelial growth rate (MGR) showed significant variation which ranged from 1 to 2.9 cm/d (Table 4).  region of all isolates using the primer pair, ITS1 and ITS4 which produced an amplicon with the expected molecular length of approximately 509 bp (Figure 3).

Pathological and TE typing of B. cinerea isolates:
Virulence variation was revealed by the pathogenicity assay which was conducted on fifteen B. cinerea isolates collected from cabbage, lettuce and pepper using the detached lettuce leaf technique (Table 4).
Isolates showed statistically significant variation in terms of lesion diameter which ranged from 0.4 to 4.4 cm and the degree of virulence was thus divided into three categories ( Figure 5), namely, highly virulent with a lesion diameter ranging from >2.5 to 4.4 cm (53.3%), moderately virulent with a lesion diameter from >1 to 2.5 cm (33.3%) and low virulent with a lesion diameter ranging from 0.4 to 1 cm (13.3%).  (Table  4). PCR revealed 510 bp for boty element in 10 out of 15 isolates tested ( Figure 6A). Whereas, for flipper detection, PCR generated 1250 bp in 9 out of 15 isolates tested ( Figure 6B). Six isolates demonstrated the two TEs (transposa type: boty and flipper), confirmed that transposa isolates were predominant (40%) among B. cinerea populations which were collected from various plant organs of different vegetable crops. Whereas, four isolates have only boty element (boty type, 26.7%), three have only flipper element (flipper type, 20%) and two have neither of them (vacuma type, 13.3%).   1.9±0.7b 0.5±0.1d V *MGR, mycelial growth rate. Data followed by the same letter are not statistically different. **all data are means of triplicate measurements ± standard deviation (SD) at L.S.D..05. ***transposable elements type: T, transposa; F, flipper; B, boty; V, vacuma. B, PCR amplification product of 1250 bp representing the Flipper element. Right lane (M) in each gel, represents 1Kbp DNA ladder marker; lanes1 to 5 correspond to cabbage isolates (BCC1 to BCC5); lanes 6 and 9, the positive controls; lanes 7 and 8, the negative controls; lanes 10 to 12 represent lettuce isolates (BCL101 to BCL103); lane 13 corresponds to the positive control; lanes 14 to 20 correspond to seven pepper isolates (BCP1 to BCP7); lanes 21 and 22 represent the negative and positive control, respectively.

DISCUSSION
Symptomatic and asymptomatic samples collected from the tested vegetable plants, cabbage, lettuce and pepper revealed their infection with B. cinerea, which showed a phenotypic diversity among isolates regardless the location and host plant. Such divergence was observed in cultural characteristics like mycelial texture, mycelial growth rate, conidial dimensions and sclerotial characteristics such as size, pattern and number per plate. All conidia observed were oval as previously recorded with B. cinerea isolates collected from grape and strawberry (Wagih et al., 2019). Although the same sclerotial shape (cerebriform) was observed in all B. cinerea isolates collected from the vegetable crops under study, the divergence in sclerotial shape was previously found among strawberry isolates (Wagih et al., 2019). Similarly, variability in virulence was detected when isolate aggressiveness was tested, but no relationship was found between isolate virulence and location/host plant. Interestingly, the isolates collected from lettuce like those collected from cabbage and pepper demonstrated different virulence levels and indicated that there is no host preference for grey mold infection by B. cinerea isolates even if the re-inoculation was done on the same original host plant. Molecular analysis showed various distribution values of TEs depending on TE type in agreement with those previously reported (Abdel Wahab, 2015;Wagih et al., 2019). However, this investigation contradicts other documented studies (Samuel et al., 2012;Kumari et al., 2014). Transposa type demonstrated predominance (40%) and this was in agreement with what was reported before (Esterio et al., 2011), followed by boty type (26.7), flipper type (20%) while vacuma type being the lowest distribution value (13.3%) in this respect. A clear correlation was found between virulence and vacuma isolates which possess low pathogenic potential as previously reported (Martinez et al., 2005;Muñoz et al., 2010;Schilling et al., 2013;Kumari et al., 2014;Wagih et al., 2019). Future efforts should, therefore, be focused on investigating the effect of host plant on the severity level of grey mold using the same isolate type. The TE/virulence relationship should also be studied through the hostpathogen interaction.