Fungal Genomics & Biology
Open Access

PCR Detection of Heat-resistant Fungi

Hiroyuki Nakagawa¹, Satoshi Yamashita², Nobuhiro Tagashira¹, Toshi-Hide Arima^2* and Yutaka Kikoku^{1* *}Correspondence: Toshi-Hide Arima, Faculty of Bioresource Sciences, Prefectural University of Hiroshima, 5562 Nanatsuka, Shobara, Hiroshima, Japan, Tel: +81-824-74-1856, Email: Yutaka Kikoku, Aohata Corporation, 1-1-25 Tadanoumi-naka, Takehara, Hiroshima, Japan, Tel: +81-846-26-0115, Email:

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Introduction

Fungi are universally present in soil, water, and air and can contaminate and spoil agricultural products and foods. According to FAO’s “The State of Food and Agriculture 2019,” around 14% of all food produced in the world is discarded postharvest on farms or during storage and transportation [1]. Microbial decay accounts for a large amount of these food losses. Molds and yeasts have greater tolerance for desiccation and acidity than most bacteria and can grow at lower temperatures [2]. Mold growth on foods not only results in the loss of good taste and nutritional value but may also produce harmful secondary metabolites. Some of these metabolites are subject to global regulation as mycotoxins and are a problem in international food distribution [3].

Several ascomycetes produce large numbers of ascospores with strong heat-resistance [4]. Occasionally, they contaminate foods during heat-processing by adhering to raw materials and containers. Some species of Byssochlamys and Talaromyces can survive heat-processing of bottled fruit juice and canned fruit, sometimes causing spoilage, because their ascospores can tolerate heating at 85°C [4]. Moreover, Byssochlamys fulva and Byssochlamys nivea are known to produce mycotoxins, such as patulin and byssochlamic acid [5]. Talaromyces macrosporus produces duclauxin, and Talaromyces trachyspermus produces spiculisporic acid [6].

We identified two heat-resistant fungal isolates (BFF228 and BFF75) from quick-frozen low-bush blueberries [7]. BFF228 and BFF75 were identified as Devriesia thermodurans and Hamigera striata by morphological characterizations and phylogenetic analyses of internal transcribed spacer regions, respectively. Seifert et al. [8] isolated four species of Devriesia described as heat-resistant, and BFF228 exhibited the morphological and growth characteristics of Devriesia spp. Hamigera spp have previously been isolated from spoiled canned blueberries and semi-finished strawberry products [9]. Heat-resistant fungi belonging to the genera Byssochlamys, Eupenicillium, Neosartorya, Talaromyces and Thermoascus have also been detected in various fruits and fruit products, such as blueberries, lemon peels, orange juice, and sweetened beverages [7,9-11].

The detection and identification of fungi are performed mainly by phenotypic methods, including macroscopic analysis and microscopy. Morphological identification is often timeconsuming and requires profound mycological knowledge and trained staff with considerable expertise to be performed properly. Moreover, it does not invariably provide adequate identification results because growth and sporulation depend on proper incubation conditions and media to prevent misidentifications. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Fourier transform infrared spectroscopy have recently been applied to efficiently identify fungi [12,13]. However, both techniques have high set-up and consumable costs and require highly skilled personnel capable of operating the systems on a routine basis.

Polymerase Chain Reaction (PCR) methods have advantages over phenotypic methods for identifying fungi that cause food spoilage. Nakayama et al. [14] designed genus-specific primer sets based on the β-tubulin gene sequences of the Byssochlamys and Hamigera genera and identified Byssochlamys and Hamigera at the genus level by PCR. The detection limits of the genus-specific primer sets were 1 ng and 10 pg of template DNA by conventional and nested PCR methods, respectively [14]. Based on the β-tubulin gene sequences of the genus Byssochlamys, species-specific primer sets were designed for B. fulva, B. nivea, Byssochlamys lagunculariae, and Byssochlamys zollerniae, allowing for accurate species-level detection using conventional PCR methods. The detection limit for each species-specific primer set was 100 pg and 10 pg of template DNA by conventional and nested PCR, respectively [15]. Moreover, genus- and speciesspecific PCR methods proved useful for detecting heat-resistant fungi, including Thermoascus and Neosartorya species, respectively. Using conventional and nested PCR methods, the detection limits for each primer set were 100 pg and 10 pg of Thermoascus genus DNA and 40 pg and 4 pg of Neosartorya genus DNA, respectively [16,17]. The sensitivity of detection was improved 10-fold using nested PCR methods.

We developed species-specific PCR methods to detect the heatresistant fungi T. macrosporus, and T. trachyspermus using primer sets targeting the isocitrate lyase gene, which codes for a unique enzyme in the glyoxylate cycle and hydrophobin gene sequences [18,19]. Hydrophobin genes are suitable targets for the species-specific detection of fungi because hydrophobins are unique to fungi and exhibit considerable variability in their overall sequences [20]. Other heat-resistant fungi that cause food spoilage, including Byssochlamys, Hamigera, Neosartorya, and Thermoascus species, were not detected using the Talaromyces-specific primer sets. The detection limit for each species-specific primer set was 50-100 pg of template DNA without using nested PCR. These methods also detected fungal DNA extracted from blueberries inoculated with T. macrosporus [18,19]. Furthermore, conventional PCR methods using crude DNA extract as the template DNA yielded amplicons specific to T. macrosporus and T. trachyspermus [19]. The crude genomic DNA templates were prepared using small colonies and a microwave irradiation protocol [21]. The microwave irradiation protocol is a simple, rapid method for extracting genomic DNA, requiring no specialized techniques. Therefore, this protocol shortens the time required for fungal genomic DNA extraction and provides crude genomic DNA that is a suitable PCR template.

Recently, quantitative PCR (qPCR) and Loop-Mediated Isothermal Amplification (LAMP) assays have been designed for specific detection of the DNA replication licensin factor gene of heat-resistant fungus Talaromyces flavus [22,23]. The specificity of these assays was confirmed by the use of 5 T. flavus strains and 35 other fungal isolates. The detection limits of the qPCR and LAMP assays were 200 fg and 1 fg of template DNA, respectively [22,23]. Both the qPCR and LAMP assays exhibited higher sensitivity than other PCR methods. However, there are several problems with these two assays. The apparatus and reagent kits for qPCR assays are expensive and the system optimization for LAMP assays makes it difficult to completely avoid false positives.

For the routine application of PCR methods on-site in the food and beverage industry, they must be cheap, simple, fast, and reliable compared with other available methods of fungal identification. Conventional PCR methods require only basic PCR reagents, thermocyclers, and electrophoresis apparatus. This equipment is relatively cheap, inexpensive to maintain, and simple to operate.

Discussion and Conclusion

The conventional PCR method using specific primer sets and crude genomic DNA extracts from mycelia cultivated for a short period was sufficient for specific detection of heat-resistant fungi. Thus, conventional PCR methods for detecting heatresistant fungi possess numerous advantages for use in the food and beverage manufacturing environment. This method would be useful for quality control of raw materials and processed products

Acknowledgement

We thank Edanz Group (https://en-authorservices. edanzgroup.com/ac) for editing a draft of this manuscript.

References

1. FAO. The state of food and agriculture 2019. Moving forward on food loss and waste reduction. 2019.
2. Pitt JI, Hocking AD. The ecology of fungal food spoilage. In Fungi and Food Spoilage (3rd edn), Springer, Singapore. 2019;pp:3-9.
3. Dijksterhuis J. Heat-resistant ascospores. In: Food Mycology: A Multifaceted Approach to Fungi and Food (1st edn). CRC Press, Boca Raton, Florida, United States. 2007;pp:101-106.
4. Reddy KRN, Salleh B, Saad B, Abbas HK, Abel CA, Shier WT. An overview of mycotoxin contamination in foods and its implications for human health. Toxin Rev. 2010;29(1):3-26.
5. Samson RA, Houbraken J, Varga J, Frisvad JC. Polyphasic taxonomy of the heat resistant ascomycete genus Byssochlamys and its Paecilomyces anamorphs. Persoonia. 2009;22:14-27.
6. Frisvad JC, Filtenborg O, Samson RA, Stolk AC. Chemotaxonomy of the genus Talaromyces. Antonie van Leeuwenhoek. 1990;57(3):179–189.
7. Kikoku Y, Tagashira N. Nakano H. Heat resistance of fungi isolated from frozen blueberries. J Food Prot. 2008;71(10):2030-2035.
8. Seifert KA, Nickerson NL, Corlett M, Jackson ED, Louis-Seize G, Davies RJ. Devriesia, a new hyphomycete genus to accommodate heat-resistant, Cladosporium-like fungi. Can J Bot. 2004;82(7):914–926.
9. Scaramuzza N, Berni E. Heat-resistance of Hamigera avellanea and Thermoascus crustaceus isolated from pasteurized acid products. Int J Food Microbiol. 2014;168:63-68.
10. Tranquillini R, Scaramuzza N, Berni E. Occurrence and ecological distribution of heat resistant moulds spores (HRMS) in raw materials used by food industry and thermal characterization of two Talaromyces isolates. Int J Food Microbiol. 2017;242:116-123.
11. dos Santosa JLP, Samapundoa S, Biyiklib A, Impec JV, Akkermansc S, Höfted M, et al. Occurrence, distribution and contamination levels of heat-resistant moulds throughout the processing of pasteurized high-acid fruit products. Int J Food Microbiol. 2018;281:72-81.
12. Lima N, Santos C. MALDI-TOF MS for identification of food spoilage filamentous fungi. Curr Opin. Food Sci. 2017;13:26-30.
13. Shapaval V, Schmitt J, Møretrø T, Suso HP, Skaar I, Asli AW, et al. Characterization of food spoilage fungi by FTIR. J Appl Microbiol. 2013;114(3):788-796.
14. Nakayama M, Hosoya K, Matsuzawa T, Hiro Y, Sako A, Tokuda H, et al. A rapid method for identifying Byssochlamys and Hamigera. J Food Prot. 2010;73(8):1486–1492.
15. Hosoya K, Nakayama M, Matsuzawa T, Imanishi M, Hitomi J, Yaguchi T. Risk analysis and development of a rapid method for identifying four species of Byssochlamys. Food Control. 2012;26(1):169–173.
16. Hosoya K, Nakayama M, Tomiyama D, Matsuzawa T, Imanishi Y, Ueda S, et al. Risk analysis and rapid detection of the genus Thermoascus, food spoilage fungi. Food Control. 2014;41:7–12.
17. Yaguchi T, Imanishi Y, Matsuzawa T, Hosoya K, Hitomi J, Nakayama M. Method for identifying heat-resistant fungi of the genus Neosartorya. J Food Prot. 2012;75(10):1806–1813.
18. Yamashita S, Nakagawa H, Sakaguchi T, Arima T-H, Kikoku Y. Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus. Lett Appl Microbiol. 2018;66(1):86–92.
19. Yamashita S, Nakagawa H, Sakaguchi T, Arima T-H, Kikoku Y. Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene. Lett Appl Microbiol. 2019;68(5):415-422.
20. Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latge J-P. Hydrophobins-unique fungal proteins. PLoS Pathog. 2012;8:e1002700.
21. Ferreira AVB, Glass NL. PCR from fungal spores after microwave treatment. Fungal Genet Reports. 1996;43(1):25-26.
22. Panek J, Frąc M. Development of a qPCR assay for the detection of heat-resistant Talaromyces flavus. Int J Food Microbiol. 2018;270:44-51.
23. Panek J, Frąc M. Loop-Mediated Isothermal Amplification (LAMP) approach for detection of heat-resistant Talaromyces flavus species. Sci Rep. 2019;9(1):5846.