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Mycobiota associated with strawberry fruits, their mycotoxin potential and pectinase activity

ABSTRACT Forty-three species and variety belonging to 15 genera were collected from 30 strawberry fruit samples on Glucose-Czapek’s agar medium. Among them, Aspergillus flavus, Aspergillus niger and Penicillium citrinum were the most frequent species recovered from 53.3%, 70.0% and 50.0% of the samples, respectively. According to the ITS rDNA sequence, we confirmed the morphological identification result. Moreover, aflatoxin biosynthesis gene omt-A was detected in A. flavus, while Aopks gene was found in A. niger. Interestingly, we could not detect any aflatoxin or ochratoxin biosynthesis genes in the P. citrinum strain. The concentration of detected aflatoxin was 3.5 ppb produced by A. flavus, while A. niger gave 4.1 ppb as ochratoxin. A. flavus was the most pectinase producer among the selected strains, and the highest amount was obtained at 30°C after 6 days of incubation with initial medium pH 8.


Introduction
Strawberries (Fragaria × ananassa), belonging to the Rosaceae family, are considered to be most important fruits worldwide. They contain a big number of phenolic compounds which have antioxidant properties (Hipol et al. 2014). One of the limiting factors that influence its economic value is the relatively short shelf life caused by postharvest spoilage (Al-Najada and Gherbawy 2015). Fungi were considered the main reason for strawberry postharvest spoilage (Lopes et al. 2014). The fungal species causing postharvest spoilage in strawberries vary according to the region, weather and crop management (Maas 1998). In Egypt, the main fungal species that cause postharvest rot in strawberry are Botrytis cinerea and Rhizopus stolonifer (El-Mougy et al. 2008). Other fungi associated with rotting of strawberry fruit at postharvest stage are Alternaria alternata, Aspergillus niger, Cladosporium, Mucor spp., Penicillium spp., Phytophthora cactorum, Sclerotinia sclerotiorum and Trichoderma spp. (Saber et al. 2003;Embaby and Abd-Ellatif 2013;Jensen et al. 2013;Juan et al. 2016).
During food colonisation by fungi, some species may generate different mycotoxins that are carcinogenic, immunosuppressive, genotoxic, nephrotoxic and teratogenic (Petzinger and Weidenbach 2002).
Aspergillus flavus and A. parasiticus are the main aflatoxin producers, while ochratoxins are produced by Aspergillus ochraceus group and A. section Nigri (Roehuck and Maxuitenko 1994;Varga et al. 1996;Heenan et al. 1998). The natural incidence of A. flavus on different crops and quantification of aflatoxins in fruits and fungal cultures were documented in several studies (Singh and Sumbali 2000;Sarkar et al. 2011;Kachapulula et al. 2019).
Several Penicillium species were documented as afla and ochratoxin producers (Patino et al. 2005;Cabanes et al. 2010). Molecular tools have been used to investigate the afla and ochratoxigenicity of A. flavus and A. niger fungi in different food and feed sources (Mayer et al. 2003;Somashekar et al. 2004;Abdel-Hadi et al. 2010;Agriopoulou et al. 2016). In this matter, different specific primers have been designed for several genes involved in the aflatoxin biosynthetic pathways, i.e., aflR, nor, ord, omt and ver (Scherm et al. 2005;Rodrigues et al. 2009;Ahlberg et al. 2015). The presence of four genes: aflR, nor A, omt A and ver 1 in A. flavus toxigenic isolates were evaluated using quadruplex polymerase (Gherbawy et al. 2012;Bandyopadhyay et al. 2016). Anpks and Aopks genes were used for ochratoxin biosynthesis, the detection of these genes was investigated in different A. niger strains (Castella and Cabanes 2011;Reddy et al. 2013;El-Hamaky et al. 2016).
The wall of fruit cell is composed of high per cent of polysaccharides, approximately 90%, divided into cellulose, hemicellulose and three groups of pectin (McNeil et al. 1984;Nathalie 2006). The principal of fungal spoilage depends on secreting of extracellular lytic enzymes such as pectinases that degrade these polymers to release water and the plant's other intracellular components for use as nutrients for their growth and finally lead to developing inedible, unwanted quality and fruit rotting (Miedes and Lorences 2004;Al-Hindi et al. 2011).
This work is aimed to identify the mycobiota associated with strawberry, detect afla and ochratoxin toxin-producing genes in certain species and evaluate their pectinase activity.

Strawberry fruit samples
Thirty samples of strawberry fruits were collected from different markets in Qena city, Egypt. Samples were put separately in a sterile polyethylene bag sealed and kept in other bags which were also sealed. Samples were transported to a mycological laboratory for fungal analysis.

Determination of mycobiota
Dilution plate method used for isolation of fungi. Known weight of the fruit was placed in autoclaved conical flasks containing 100 ml sterile distilled water. From the appropriate dilution, 1 ml was transferred to each sterile plate followed by addition of about 20 ml liquefied Glucose-Czapek's agar medium (Smith and Dawson 1944;Al-Dory 1980). The plates were incubated at 28 ± 2°C for 5-7 days and the developing colonies were identified and counted. The numbers of colonies were calculated/g of fruits fresh weight.

Molecular characterisation of selected isolates
Based on isolation results, three isolates were chosen randomly from the most common species (A. flavus, A. niger and Penicillium citrinum) for molecular identification. ITS1 and ITS4 primers were used for ITS region amplification from selected isolates as mentioned by White et al. (1990). The purified bands were sequenced using the sequencer Gene analyser 3121. The deduced sequence was edited and aligned. The sequencing data were compared with the Gene Bank database (http://www.ncbi.nlm.nih.gov/BLAST/), where a nucleotide blast program was chosen to identify the homology between the PCR fragments and the sequences on the GeneBank database. A phylogenetic tree was constructed by MEGA6 (Tamura et al. 2013) using the Neighbor joining method with 1000 bootstrap repeats.

Quantitative assessment of afla and ochratoxins
The selected isolates (A. flavus, A. niger and P. citrinum) were inoculated into flasks containing yeast extract sucrose medium (YES) containing: sucrose, 40 g; yeast extract 20 g and distilled water, 1000 ml. The cultivation was made erlenmeyer flasks containing 100 ml of the medium. After autoclaving, the flasks were inoculated with a single 6 mm disc cut out from the margin of a 5-day colony of the fungus grown on Glucose-Czapek 's agar medium. Flasks were incubated at 28 ± 2°C for 14 days (Gabal et al. 1994). The mycotoxin levels were estimated by the fluorometric method using aflatoxin or ochratoxin standards to fluorometer calibration before reading the content of sample toxins (Hansen 1993).

Pectinolytic activity of selected fungi
Three isolates were screened for their abilities to produce extracellular pectinase as described by Saleem et al. (2012). Using a sterile cork borer (6 mm diameter) discs from pre-cultured strains were cut to inoculate sterile conical flask containing 50 ml liquid medium of Hankin et al. (1971). After incubation for 7 days at 28°C, the cultures were filtered, and the filtrates were used to determine pectinase activity (Ammar et al. 1995). The average diameter of clear zones (in mm) for selected strains was measured.

Factors affecting pectinase production
A. flavus was found to be the most active pectinase producer. So, they employed to evaluate the effect of different ecological factors on pectinase production.

Effect of temperature and time course
Flasks inoculated with A. flavus were incubated at different temperature degrees (20°C, 30°C and 40°C) for 14 days and the cultures were harvested at 48-hour intervals. Cultures were filtered then centrifuged at 5,000 rpm for 7 min. The clear supernatants were assayed for pectinase activity.

Effect of pH values
Medium flasks were pre-adjusted to different pH levels ranging from 2 to 12 (0.1 N HCl or 0.1 NaOH) before inoculation with A. flavus. The inoculated flasks were incubated at 30°C for 8 days. Three flasks for each pH value were prepared. At the end of the incubation period, cultures were filtered, centrifuged at 4°C for 7 minutes at 15,000 rpm and the clear supernatants were assayed for pectinase activity according to Sherwood (1966).

Statistical analysis
All experiments were carried out in triplicates. Data obtained were expressed as mean ± standard error of the mean (SEM).

Mycobiota from Strawberry fruits
Forty-three species and variety belonging to 15 genera were collected from 30 strawberry fruit samples (Table 1). Aspergillus, represented by 7 species and variety, was the most frequent genus isolated from 96.6% of the samples comprising 52.9% of the total fungi. A. flavus and A. niger were the most prevalent species among Aspergillus. They were recovered from 53.3% to 70% of the samples matching 26.6% and 21.6% of the total fungi, respectively (Table 1).
After Aspergillus, Penicillium (17 species) occupied the second place, isolated from 76.6% of the samples, comprising 26.4% of the total fungi. The most prevalent species was P. citrinum recovered from 50% of the samples with 9% from the total fungal counts. Alternaria and Cladosporium shared the third place with the same frequencies (46.6% from the samples). The other remaining fungal genera and species were isolated in low frequencies and amounts (Table 1).

Molecular identification of selected strains and phylogenetic analysis
The universal primers ITS 1 and ITS 4 were used to amplify the ITS regions of rDNA from selected isolates (A. flavus, A. niger and P. citrinum). The obtained sequences deposited in GenBank with accession numbers MK185434, MK185435 and MK185436 ( Table 2). The phylogenetic analysis of tested strains with different sequences from National Centre for Biotechnology Information (NCBI) confirmed the morphological identification for the tested strains as A. flavus, A. niger and P. citrinum (Figure 1).

Detection of aflatoxin and ochratoxin producing genes
Polymerase chain reaction (PCR) was applied using four sets of primers for the detection of different genes involved in aflatoxin and ochratoxin biosynthetic pathway. Bands of the fragments of omt-A and Aopks genes can be visualised at 300 and 549 bp, respectively (Figure 2). Aflatoxigenic A. flavus strain showed 300 bp DNA fragments that corresponded to the complete of aflatoxin B1 producing gene. In addition, A. niger isolate showed the complete of investigated ochratoxin genes. The obtained data exhibited complete absence of afla or ochratoxin genes in P. citrinum.

Quantitative assessment of afla and ochratoxins
Data in Table 2 illustrate A. flavus was aflatoxigenic producer, while A. niger exhibited ochratoxigenic activity, but P. citrinum failed to be afla or ochratoxigenic producer. The data obtained from fluorometer showed the amount of aflatoxin produced by A. flavus was 3.5 ppb, while the amount of ochratoxin by A. niger was 4.1 ppb.

Pectinolytic activity
The three fungal species were screened for their abilities to produce pectinase enzyme using the cup-plate method. All tested isolates were pectinase producers, but with variable amounts (Table 2). In the first place, come A. flavus (34 mm) as the most active pectinase producer followed by A. niger (28 mm) and P. citrinum (26 mm). The activity of pectinase has been found to be   affected by variable environmental conditions. The amount of pectinase produced by A. flavus was increased by extending the incubation period at different temperatures showing high levels at 6 days (20°C and 30°C) and 8 days (40°C). In general, the maximum production of the enzyme by A. flavus was achieved after 6 days at 30°C as the incubating temperature ( Figure 3). The highest level of pectinase could be synthesised when the culture medium of the fungal species initially adjusted to pH 8. Considerable amounts of the enzyme were also estimated at pH 6 and pH 10, while low amounts of the enzyme were observed in cultures with more alkalinity or acidity ( Figure 4).

Discussion
Strawberry is the most important fruit containing high amount of vitamin C (Embaby et al. 2016). Their decay is caused by several fungi. Our result indicated that A. flavus, A. niger and P. citrinum were the most frequent species that contaminated strawberry fruits. This result conflicts with many studies which indicated that Botrytis cinerea is the main causative agent for strawberry fruit decay (Embaby et al. 2016;Mouden et al. 2016). Abdelfattah et al. (2016) reported that Botrytis and Cladosporium were the most common fungi from mature and immature strawberry fruits. On the other hand, A. niger, B. cinerea, Penicillium digitatum and Rhizopus stolonifera were considered as postharvest pathogenic fungi which could reduce the shelf life of strawberry fruit (Nabigol and Morshedi 2011). A. alternata, A. niger, Penicillium spp. and other fungal species were previously isolated from strawberry (Saber et al. 2003). Based on ITS region, the conventional identification was confirmed by molecular technique. The phylogenetic analysis revealed that our isolates were recognised to be A. flavus, A. niger and P. citrinum. The molecular characterisation of different fungi to  species level using ITS was used by many investigators (Gherbawy et al. 2016;Abd Elhamid et al. 2017;Alsohaili and Bani-Hasan 2018). The appearance of 300 and 549 bp bands with A. flavus and A. niger revealed the presence of aflatoxin B 1 producing gene in A. flavus and ochratoxin gene in A. niger; on the contrary, these genes are absent in P. citrinum. The presence of two targeted genes confirmed the abilities of tested isolates to produce aflatoxins and ochratoxin as previously mentioned by other researchers (Reddy et al. 2013;El-Hamaky et al. 2016;Sireesha et al. 2017). Ochratoxin producing genes were detected in A. ochraceus, A. alliaceus, A. sclerotiorum, A. sulphureus, A. albertensis, A. auricomus, and A. wentii strains. Gherbawy et al. (2012) documented the presence of aflatoxin and ochratoxin coding genes in 38.8% and 25% of A. flavus and A. niger isolates recovered from date palm, respectively. The concentration of aflatoxins in tested A. flavus isolates recovered from strawberry was ranged between 25.8 and 75.2 ng/ml (Saleem 2017). On the other hand, Gherbawy and Shebany (2018) found that 45.5% of P. citrinum isolates were ochratoxin A producers with amounts ranging from 1.3 to 2.5 μg/L.
A. flavus was the most active pectinase producer. These results fully agree with those obtained by Reddy and Sreeramulu (2012), who found that among four different fungi screened for pectinolytic activity, A. flavus was the most active producer of pectinase. Among Aspergillus fumigatus, A. flavus, A. niger, A. ochraceus, A. oryzae, A. sydowii, Trichothecium sp., Penicillium sp., Trichoderma harzianum and T. viride, A. flavus was able to produce pectinolytic enzyme (Usha et al. 2014). The higher amount of pectinase production by A. flavus was after 6 days of incubation at 30°C with pH 8 for the medium. In this respect, Yadav et al. (2007) reported that the highest yield of pectinase by A. flavus and A. phoenicis was obtained after 6 days, while in A. niger and A. wentii it was obtained after 8 days of incubation. Also, Palaniyappan et al. (2009) found the optimum temperature for pectinase production by A. niger MTCC 281 fungi was 30°C. Identical results were obtained by Yadav et al. (2007Yadav et al. ( , 2008 who found that pH 8 is optimum for pectinase production in culture filtrate of A. flavus. Also, the optimum pH for pectinase activity by A. niger was 8.0 (Batool et al. 2013).
In conclusion, A. flavus, A. niger and P. citrinum constituted 26.6%, 21.6% and 9.0% of the total fungi recovered from strawberry fruits in Qena city, respectively. The tested strains of A. flavus and A. niger were afla and ochratoxin producers, respectively, but P. citrinum could not produce afla or ochratoxin. In addition, A. flavus was the most active pectinase producer and the maximum amount was achieved after 6 days of incubation at 30°C and pH of medium equal pH 8.