Skip to main content
PMC home page
Mycology logoLink to Mycology
. 2020 May 13;11(2):158–166. doi: 10.1080/21501203.2020.1759719

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

Mohamed A Hussein 1,✉, Ahmed HM El-Said 1, Asmaa S Yassein 1
PMCID: PMC7448860  PMID: 32923023

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.

KEYWORDS: Strawberry, aflatoxin, ochratoxin, pectinase activity

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.

Materials and methods

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.

Detection of aflatoxin and ochratoxin producing genes

Four documented primers (omt-A and Aopks) were used for detection of afla and ochratoxin, producing genes. The sequences of primers were as following: omt-AF “5-GACCAATACGCCCACACAG-”3, omt-AR “5-CTTTGGTAGCTGTTTCTCGC-”3 (Shweta et al. 2013) and Aopks-F “5-CAGACCATCGACACTGCATGC-”3, Aopks-R “5-CTGGCGTTCCAGTACCATGAG-”3 (Reddy et al. 2013). Twenty-five μl as volume was used for PCR reaction by mixing 0.5 μl of DNA with 0.5 μM of each primer, 2.5 μl buffer, 1.5 μl MgCl2, 0.5 μl dNTPs, 0.5 μl Taq polymerase (Jena Bioscience, Germany) and water was added to make the volume up to 25 μl. The reactions were done in a C1000TM Thermo Cycler BioRad, Germany, with initial denaturising at 94°C for 5 min, followed by 35 cycles of 1 min at 94°C, 1 min as annealing temperature at 59°C and 1 min at 72°C for omt-A (Deabes et al. 2018) and 30 cycles of 1 min at 94°C, 1 min at 58°C and 1 min at 72°C for Aopks (El-Hamaky et al. 2016). Ten minutes at 72°C was used as the final extension for two reactions. PCR products were checked on a 1.4% agarose gel, stained with ethidium bromide.

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).

Results

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).

Table 1.

Average total counts (ATC, calculated per g fresh fruit), percentage counts (%C, calculated per total fungi), number of cases of isolation (NCI, out of 30 samples) and percentage frequency (%F, calculated per 30 samples) of various fungal genera and species recovered from strawberry fruits on glucose-Czapek’s agar at 28°C.

Genera and species ATC %C NCI %F
Acremonium 140 1.6 3 10
 A. cerealis 20 0.2 1 3.3
 A. rutilum 100 1.6 1 3.3
 A. strictum 20 0.2 1 3.3
Alternaria 550 6.4 14 46.6
 A. alternata 530 6.1 14 46.6
 A. brassicicola 10 0.1 1 3.3
 A. citri 10 0.1 1 3.3
Aspergillus 4580 52.9 29 96.6
 A. flavus Link 2300 26.6 16 53.3
 A. fumigatus 150 1.7 4 13.3
 A. niger 1870 21.6 21 70
 A. ochraceus 120 1.4 5 16.6
 A. sydowii 60 0.7 3 10
 A. terreus 40 0.5 4 13.3
 A. terrus var. aureus 20 0.2 2 6.6
 A. versicolor 20 0.2 2 2.6
Cladosporium 710 8.2 14 46.6
 C. cladosporioides 700 8.1 14 46.6
 C. sphaerospermum 10 0.1 1 3.3
Emericella nidulans 200 2.3 8 26.6
Epicoccum purpurascens 10 0.1 1 3.3
Mucor hiemalis 10 0.1 1 3.3
Mycosphaerella tassiana 20 0.2 1 6.6
Myrothecium rordium 10 0.1 2 3.3
Penicillium 2290 26.4 23 76.6
 P. aurantiogriseum 160 1.8 5 16.6
 P. camembretii 10 0.1 1 3.3
 P. citrinum 780 9.0 15 50
 P. corylophilum 10 0.1 1 3.3
 P. dendriticum 400 4.6 3 10
 P. duclauxii 20 0.2 1 3.3
 P. echinulatum 130 1.5 4 13.3
 P. funiculosum 370 4.3 5 16.6
 P. implicatum 20 0.2 1 3.3
 P. oxalicum 20 0.2 1 3.3
 P. pinophilum 40 0.5 1 3.3
 P. purpurogenum 110 1.3 6 20
 P. spinulosum 70 0.8 4 13.3
 P. variabile 20 0.2 2 6.6
 P. verruculosum 10 0.1 1 3.3
 P. vinaceum 10 0.1 1 3.3
 P. waksmanii 50 0.6 3 10
Phoma glomerata 10 0.1 1 3.3
Pleospora herbarum 20 0.2 1 3.3
Scytalidium state of hendersonula toruloidea 10 0.1 1 3.3
Sterile mycelia 50 0.6 2 6.6
Trichoderma koningii 20 0.2 2 6.6
Ulocladium 30 0.3 3 10
 U. botrytis 20 0.2 2 6.6
 U. atrum 10 0.1 1 3.3
 Average total count 8660
 Number of genera 15
 Number of species 43 + 1 var.
Open in a new tab

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).

Table 2.

Accession numbers, mycotoxin potential of selected fungal strains (in ppm) and their pectinolytic activity (in mm).

Fungal species Accession No. Aflatoxin Ochratoxin Pectinase activity
Aspergillus flavus MK185434 3.5 ND 34
A. niger MK185435 ND 4.1 28
Penicillium citrinum MK185436 ND ND 26
Open in a new tab

ND = not detected.

Figure 1.

Figure 1.

Open in a new tab

Phylogenetic relationship among three strains belonging to genus Aspergillus and Penicillium compared with some strains at NCBI.

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.

Figure 2.

Figure 2.

Open in a new tab

PCR amplification of omt-A and Aopks genes (300 and 549 bp) for tested isolates, whereas, Lane 1 = P. citrinum, 2 = A. niger and 3 = A. flavus.

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).

Figure 3.

Figure 3.

Open in a new tab

Effect of time course and temperatures on pectinase enzyme production by A. flavus.

Figure 4.

Figure 4.

Open in a new tab

Effect of different pH values on pectinase enzyme production by A. flavus.

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 B1 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. (2007, 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.

Disclosure statement

The authors declare no conflict of interest.

References

  1. Abd Elhamid MI, Ismail IM, Alshishtawy HM, Sedik MZ.. 2017. Identification and molecular characterization of Egyptian Trichoderma isolates. Biosci Res. 14(4):1156–1166. [Google Scholar]
  2. Abdelfattah A, Wisniewski M, Nicosia MGLD, Cacciola SO, Schena L. 2016. Metagenomic analysis of fungal diversity on strawberry plants and the effect of management practices on the fungal community structure of aerial organs. PLoS One. 4:1–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Abdel-Hadi A, Carter D, Magan N. 2010. Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels. J Appl Microbiol. 109(6):1914–1922. doi: 10.1111/j.1365-2672.2010.04820.x. [DOI] [PubMed] [Google Scholar]
  4. Agriopoulou S, Koliadima A, Karaiskakis G, Kapolos J. 2016. Kinetic study of aflatoxins’ degradation in the presence of ozone. Food Control. 61:221–226. doi: 10.1016/j.foodcont.2015.09.013. [DOI] [Google Scholar]
  5. Ahlberg SH, Joutsjoki V, Korhonen HJ. 2015. Potential of lactic acid bacteria in aflatoxin risk mitigation. Int J Food Microbiol. 207:87–102. doi: 10.1016/j.ijfoodmicro.2015.04.042. [DOI] [PubMed] [Google Scholar]
  6. Al-Dory Y. 1980. Laboratory medical mycology. London. P: Kimpton Publishers. Lea & Febiger Philadelphia; p. 410. [Google Scholar]
  7. Al-Hindi RR, Al-Najada AR, Mohamed SA. 2011. Isolation and identification of some fruit spoilage fungi: screening of plant cell wall degrading enzymes. Afr J Microbiol Res. 5(4):443–444. [Google Scholar]
  8. Al-Najada AR, Gherbawy YA. 2015. Molecular identification of spoilage fungi isolated from fruit and vegetables and their control with chitosan. Food Biotechnol. 29(2):166–184. doi: 10.1080/08905436.2015.1027222. [DOI] [Google Scholar]
  9. Alsohaili SA, Bani-Hasan BM. 2018. Morphological and molecular identification of fungi isolated from different environmental sources in the northern eastern desert of Jordan. Jordan J Biol Sci. 11(3):329–337. [Google Scholar]
  10. Ammar MS, Louboudy SS, Azab MS, Afifi MM. 1995. A new method for the estimation of fungal pectinase (s) using the pectin clearing zone (P.C.Z.) technique and its application in food industries. Al-Azhar Bull Sci. 6:325–339. [Google Scholar]
  11. Bandyopadhyay R, Ortega-Beltran A, Akande A, Mutegi C, Atehnkeng J, Kaptoge L. 2016. Biological control of aflatoxins in Africa: current status and potential challenges in the face of climate changes. World Mycotoxin J. 9(5):771–789. [Google Scholar]
  12. Batool S, Javaid Asad M, Saqlan Naqvi SM 1, Mahmood RT, Guffar A, Gulfraz M, Hadri SH. 2013. Production and partial purification of pectin lyase by Aspergillus niger grown on orange peels. Afr J Microbiol Res. 7(13):1144–1149. [Google Scholar]
  13. Cabanes FJ, Bragulat MR, Castella G. 2010. Ochratoxin A producing species in the genus Penicillium. Toxins. 2(5):1111–1120. doi: 10.3390/toxins2051111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Castella G, Cabanes FJ. 2011. Development of a real time PCR system for detection of ochratoxin A-producing strains of the Aspergillus niger aggregate. Food Control. 8(8):1367–1372. doi: 10.1016/j.foodcont.2011.02.014. [DOI] [Google Scholar]
  15. Deabes MM, Khalil WKB, Attallah AG, El-Desouky TA, Naguib KM. 2018. Impact of silver nanoparticles on gene expression in Aspergillus flavus producer Aflatoxin B1. Open Access Maced. J Med Sci. 6(4):600–605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. El-Hamaky AM, Hassan AA, Abo El Yazeed H, Refai MK. 2016. Prevalence and detection of toxigenic A. flavus, A. niger and A. ochraceusby traditional and molecular biology methods in feeds. Int J Curr Res. 8(1):25621–25633. [Google Scholar]
  17. El-Mougy NS, El-Gamal NG, Abdalla MA. 2008. The use of fungicide alternatives for controlling postharvest decay of strawberry and orange fruits. J Plant Prot Res. 48(3):385–395. doi: 10.2478/v10045-008-0048-z. [DOI] [Google Scholar]
  18. Embaby EM, Abdel-Galil MM, Aiat NM. 2016. Control strawberry fruit decay caused by some mold fungi. Int J PharmTech Res. 9(10):184–193. [Google Scholar]
  19. Embaby EM, Abd-Ellatif AA. 2013. Species identification of Colletotrichum the causal agent of strawberry anthracnose and their effects on fruit quality and yield losses. J Appl Sci Res. 9(6):3845–3858. [Google Scholar]
  20. Gabal MA, Hegazi SA, Hassanien N. 1994. Aflatoxin production by Aspergillus flavus field isolates.. Vet Hum Toxicol. 36(6):519–521. [PubMed] [Google Scholar]
  21. Gherbawy Y, Elhariry H, Bahobial A. 2012. Mycobiota and mycotoxins (aflatoxins and ochratoxin) associated with some Saudi date palm fruits. Foodborne Pathog Dis. 9(6):561–567. doi: 10.1089/fpd.2011.1085. [DOI] [PubMed] [Google Scholar]
  22. Gherbawy Y, Shebany Y. 2018. Mycobiota, total aflatoxins and ochratoxin a of cardamom pods. Food Sci Tech Res. 24(1):87–96. doi: 10.3136/fstr.24.87. [DOI] [Google Scholar]
  23. Gherbawy Y, Shebany YM, Alharthy HA. 2016. Characterization of aflatoxigenic aspergilli contaminated poultry and animal feedstuff from the western region of Saudi Arabia. Ital J Food Sci. 28:32–42. [Google Scholar]
  24. Hankin L, Zucker LM, Sands DC. 1971. Improved solid medium for the detection and enumeration of pectolytic bacteria. Appl Microbiol. 22(2):205–209. doi: 10.1128/AEM.22.2.205-209.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hansen AA. 1993. Quantitative testing for mycotoxins (VICAM). AACC. 38:5–8. [Google Scholar]
  26. Heenan CN, Shaw KJ, Pitt JI. 1998. Ochratoxin A production by Aspergillus carbonarius and A. niger isolates and detection using coconut cream agar. J Food Mycol. 1:67–72. [Google Scholar]
  27. Hipol RM, Magtoto LM, Tamang SMA, Damatac AM. 2014. Antioxidant activities of fungal endophytes isolated from Strawberry Fragaria x ananassa fruit. eJBio. 10(4):107–112. [Google Scholar]
  28. Jensen B, Knudsen IMB, Andersen B, Nielsen KF, Ulf T, Jensen D, Larsen J. 2013. Characterization of microbial communities and fungal metabolites on field grown strawberries from organic and conventional production. Int J Food Microbiol. 160(3):313–322. doi: 10.1016/j.ijfoodmicro.2012.11.005. [DOI] [PubMed] [Google Scholar]
  29. Juan C, Oueslati S, Mañes J. 2016. Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition. Food Addit Contam Part A. 33(5):861–868. doi: 10.1080/19440049.2016.1177375. [DOI] [PubMed] [Google Scholar]
  30. Kachapulula PW, Bandyopadhyay R, Cotty PJ. 2019. Aflatoxin contamination of non-cultivated fruits in Zambia. Frontiers Microbiol. 10:1840. https://doi.org/10.3389/fmicb.2019.01840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lopes UP, Zambolim L, Pinho DB, Barros AV, Costa H, Pereira OL. 2014. Postharvest rot and mummification of strawberry fruits caused by Neofusicoccum parvum and N. kwambonambiense in Brazil. Trop Plant Pathol. 39(2):178–183. doi: 10.1590/S1982-56762014000200009. [DOI] [Google Scholar]
  32. Maas JL. 1998. Compendium of strawberry diseases. St Paul, MN, USA: APS Press. [Google Scholar]
  33. Mayer Z, Bagnara A, Färber P, Geisen R. 2003. Quantification of the number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the CFUs of Aspergillus flavus in foods. Int J Food Microbiol. 82(2):143–151. doi: 10.1016/S0168-1605(02)00250-7. [DOI] [PubMed] [Google Scholar]
  34. McNeil M, Darvill AG, Fry SC, Albersheim P. 1984. Structure and function of the primary cell walls of plants. Annu Rev Biochem. 53(1):625–663. doi: 10.1146/annurev.bi.53.070184.003205. [DOI] [PubMed] [Google Scholar]
  35. Miedes E, Lorences EP. 2004. Apple (Malus domestica) and tomato (Lycopersicum esculentum) fruits cell-wall hemicelluloses and xyloglucan degradation during Penicillium expansum infection. J Agri Food Chem. 52(26):7957–7963. doi: 10.1021/jf048890f. [DOI] [PubMed] [Google Scholar]
  36. Mouden N, Al Batnan A, Benkirane R, Ouazzani Touhami A, Douira A. 2016. Diversity and distribution of fungi from strawberry plants grown in Gharb-Loukkos (Morocco). Int J of Recent Sci Res. 7(10):13630–13641. [Google Scholar]
  37. Nabigol A, Morshedi H. 2011. Evaluation of the antifungal activity of the Iranian thyme essential oils on the postharvest pathogens of strawberry fruits. Afr J Biotechnol. 10(48):9864–9869. [Google Scholar]
  38. Nathalie J. 2006. Plant protein inhibitors of cell wall degrading enzymes. Trends Plant Sci. 11(7):359–367. doi: 10.1016/j.tplants.2006.05.006. [DOI] [PubMed] [Google Scholar]
  39. Palaniyappan M, Vijayagopa V, R V, Viruthagiri T. 2009. Screening of natural substrates and optimization of operating variables on the production of pectinase by submerged fermentation using Aspergillus niger MTCC 281. Afr J Biotechnol. 8(4):682–686. [Google Scholar]
  40. Patino B, Gonzalez-Salgado A, Gonzalez-Jaen MT, Vazquez C. 2005. PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species. Int J Food Microbiol. 104(2):207–214. doi: 10.1016/j.ijfoodmicro.2005.02.011. [DOI] [PubMed] [Google Scholar]
  41. Petzinger E, Weidenbach A. 2002. Mycotoxins in the food chain: the role of ochratoxins. Lives Sci. 76(3):245–250. doi: 10.1016/S0301-6226(02)00124-0. [DOI] [Google Scholar]
  42. Reddy KV, Naveen K, Reddy IB. 2013. Incidence and molecular detection of ochratoxigenic fungi from Indian cereal grains. Int J Pharma Bio Sci. 4(3):31–40. [Google Scholar]
  43. Reddy PL, Sreeramulu A. 2012. Isolation, identification and screening of pectinolytic fungi from different soil samples of Chittoor district. Int J Life Sci Biotechnol Pharma Res. 1(3):186–193. [Google Scholar]
  44. Rodrigues P, Venâncio A, Kozakiewicz Z, Lima NA. 2009. Polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic isolates of Aspergillus Section Flavi isolated from Portuguese almonds. Int J Food Microbiol. 129(2):187–193. doi: 10.1016/j.ijfoodmicro.2008.11.023. [DOI] [PubMed] [Google Scholar]
  45. Roehuck BD, Maxuitenko YY. 1994. Biochemical mechanisms and biological implication of the toxicity of aflatoxins as related to aflatoxin carcinogenesis. In: Eaton DL, Groopman JD, editors. The toxicology of aflatoxins: human health, veterinary and agricultural significance. San Diego (CA): Academic Press; p. 27–41. [Google Scholar]
  46. Saber MM, Sabet KK, Moustafa-Mahmoud SM, Khafagi IYS. 2003. Evaluation of biological product, antioxidants and salts for control of strawberry fruit rots. Egypt J Phytopathol. 3(1–2):31–43. [Google Scholar]
  47. Saleem A. 2017. Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of strawberry (Fragaria ananassa Duch.) fruits. Arch Phytopathology Plant Protect. 50(19–20):982–996. doi: 10.1080/03235408.2017.1411155. [DOI] [Google Scholar]
  48. Saleem A, AHM E-S, Maghraby TA, Hussein MA. 2012. Pathogenicity and pectinase activity of some facultative mycoparasites isolated from Vicia faba diseased leaves in relation to photosynthetic pigments of plant. J Plant Pathol Microbiol. 3(6):1–7. [Google Scholar]
  49. Sarkar S, Shilpa P, Girisham S, Reddy SM. 2011. Incidence of toxigenic fungi in rotting fruits of banana. BTAIJ. 5(1):1–4. [Google Scholar]
  50. Scherm B, Palomba M, Serra D, Marcello A, Migheli Q. 2005. Detection of transcripts of the aflatoxin genes afld, aflo, and aflp by reverse transcription-polymerase chain reaction allows differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and Aspergillus parasiticus. Int J Food Microbiol. 98(2):201–210. doi: 10.1016/j.ijfoodmicro.2004.06.004. [DOI] [PubMed] [Google Scholar]
  51. Sherwood RT. 1966. Pectin lyase and polygalacturonase production by Rhizoctonia solani and other fungi. Phytopathology. 56:279–286. [Google Scholar]
  52. Shweta S, Madhavan S, Paranidharan V, Velazhahan R. 2013. Detection of Aspergillus flavus in maize kernels by conventional and real-time PCR assays. Int Food Res J. 20(6):3329–3335. [Google Scholar]
  53. Singh YP, Sumbali G. 2000. Natural incidence of toxigenic Aspergillus flavus strains on the surface of pre-harvest jujube fruits. Indian Phytopath. 53(4):404–406. [Google Scholar]
  54. Sireesha Y, Vimal Keerthana J, Sarathchandra G, Velazhahan R. 2017. Analysis of aflatoxin B1 and aflatoxigenic mold in commercial poultry feeds in Tamil Nadu, India. JANS. 9(1):456–460. doi: 10.31018/jans.v9i1.1213. [DOI] [Google Scholar]
  55. Smith NR, Dawson VT. 1944. The bacteriostatic action of rose bengal in media used for the plate count of soil fungi. Soil Sci. 58(6):467–471. doi: 10.1097/00010694-194412000-00006. [DOI] [Google Scholar]
  56. Somashekar D, Rati ER, handrashekar A. 2004. Isolation, enumeration and PCR characterization of Aflatoxigenic fungi from food and feed samples in India. Food Microbiol. 21(6):809–813. doi: 10.1016/j.fm.2004.01.012. [DOI] [Google Scholar]
  57. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 30(12):2725–2729. doi: 10.1093/molbev/mst197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Usha DK, Kanimozhi G, Panneerselvam A. 2014. Isolation and screening of pectin lyase producing fungi from soil sample of dead organic matters. World J Pharm Res. 3(10):563–569. [Google Scholar]
  59. Varga J, Kevei E, Rinyu E, Teren J, Kozakiewicz Z. 1996. Ochratoxin production by Aspergillus species. Appl Environ Microbiol. 62(12):4461–4464. doi: 10.1128/AEM.62.12.4461-4464.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: a guide to methods and applications. San Diego, USA: Academic Press; p. 315–322. [Google Scholar]
  61. Yadav S, Yadav PK, Yadav D, Yadav KDS. 2008. Purification and characterization of an alkaline pectin lyase from Aspergillus flavus. Process Biochem. 43(5):547–552. doi: 10.1016/j.procbio.2008.01.015. [DOI] [Google Scholar]
  62. Yadav S, Yadav PK, Yadav KDS. 2007. Pectin lyases of a few indigenous fungal strains. J Sci Ind Res. 66:601–604. [Google Scholar]

Articles from Mycology are provided here courtesy of Taylor & Francis

RESOURCES