NEW STRAINS OF FILAMENTOUS FUNGI ISOLATED FROM CONSTRUCTION MATERIALS
DOI:10.30825/5.EJPAU.169.2019.22.1

Natalia Kobiałka, Malwina Mularczyk, Katarzyna Kosiorowska, Kinga M. Pilarska, Wojciech Łaba, Michał Piegza, Małgorzata Robak
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

Filamentous fungi are a large group of eukaryotic organisms capable of growth in different environmental niches. The variety of filamentous fungi occurring on building materials is still increasing. In this paper, 19 strains were isolated from deteriorated walls and floor coverings of houses in Opole and Lower Silesia, Poland. The identification of  fungi growing on construction materials was based on microscopic assessment (according the presence or absence of particular, characteristic structures) and molecular identification by  sequence homology of highly diverse regions of ITS1 and ITS2. Obtained isolates were classified into Penicillium, Lecanicillium and Candida genera. Most of the strains (17), have been assigned to the genus Penicillium according to sequence fragments from GenBank Database. The length of the ITS1 region of most isolates was closely related to the length of this region of P. chrysogenum species. The PCR products sequencing confirmed that isolates NK1 and NK2 were P. chrysogenum, the isolate NK3 L. lecanii and theisolateNK12 P. griseoroseum. The isolates Dyw1b, Dyw2, Dyw4, Dyw5b, Dyw6, Dyw7 belonged to the Penicilium genus, however exhibited some differences. The presence of L. lecanii in deteriorated materials was rather surprising but the isolated strain revealed interesting properties. Its spores and biomass are potential biological insecticide agents.

Key words: filamentous fungi, PCR, ITS-region, identification, construction materials.

INTRODUCTION

Filamentous fungi  are a large group of  eukaryotic organisms capable to growth in a variety of environmental niches. Their growth could be either beneficial to humans, as in case of ripped cheeses production, citric acid and enzymes biosynthesis, or detrimental mainly due to the  production of  mycotoxins and deterioration of utility objects.

Biodeterioration, also known as microbial corrosion, is a complex and energy-consuming process in which microorganisms, mainly filamentous fungi, decompose various materials. It is associated with the loss of mechanical properties, crumbling and discoloration, leading to eventual degradation of materials [9, 26]. This process is often accompanied by odor formation, that originates from volatile compounds, such as sesquiterpens and hydrogen sulfide [29]. All materials, both organic and mineral, used in construction are susceptible to microbial corrosion, being a result of chemical and mechanical action of microbial cells and their metabolites on porous structures of these objects [10, 15].

Filamentous fungi in buildings are most commonly found in corners of outer walls, close to floors or ceilings, in basements, ceiling slabs, near doors and windows, at points specifically called “thermal bridges” [8]. Their presence is also common in specific housing conditions and frequently depends on the weather season [1]. The greatest damage in construction is caused by filamentous fungi that attack wood of all deciduous and coniferous species. These fungi exhibit enzymatic potential to decompose cellulose and hemicellulose, while leaving lignin intact [22]. The most commonly occurring species in Europe is Serpula lacrymans, known as the home fungus [8, 21]. It frequently occurs on wood ceilings, wall paneling, frames and floor elements. It belongs to most harmful fungi, that after 6 months is capable of causing the 50% loss of wood dry mass, and residual mechanical strength at 3% of that typical for fresh wood. It does not occur outside buildings, as it is sensitive to sunlight and airflow. Another abundant building-associated fungus is Coniophora puteana, known as the cellar fungus. It is often found on humid and shaded parts of buildings, like basements, ceilings, window and door frames, floors and sanitary fittings [5, 12, 14]. Poria vaporaria is another example of house fungus of significant wood-degradative abilities, comparable to the former examples, but incapable of fast propagation. It typically occurs in wood that has a contact with ground or in humid wooden ceilings.

Besides the discussed species of filamentous fungi, building interiors can be colonized by a wide variety of fungi posing a direct threat to human health. According to the type of the influence, harmful effects can be divided into allergies, mycoses, intoxications with mycotoxins and the so called “sick building syndrome” [3, 15]. These ailments are connected with the production of mycotoxins that exhibit toxic effect, as well as allergenic properties. The presence of fungi can also lead to migraines, irritation of mucosa and gastrointestinal disorders. In terms of mycotoxins biosynthesis, fungi of the genera Aspergillus, Penicillium, Fusarium, Cladosporium and Alternaria are known to be most dangerous [13].

The aim of the present study was to recognize strains of filamentous fungi occurring on building walls and construction materials at specified locations in Poland, with a special regard for the detection of S. lacrymans and S. humantiodes. To identify microscopic characterization to the genus and to the species by PCR ITS product sequencing with ITStest fungi and BLAST alignments with specific rDNA fragments available in the Gene Bank Database and Saccharomyces Genome Database were performed.

MATERIALS AND METHODS

The materials used in the study were four samples obtained from interior walls and wooden baseboard in Lower Silesia residential buildings interior walls of unheated cellars of  detached houses (Złotniki, Wrocław and Laskówka) designated as NK and basement of a 45-years old building (Karłowice, Wrocław) designated as Dyw. Potato Dextrose Agar (Difco) media was used and more than 3 passages were performed to isolate strains. Finally 19 strains were obtained as pure cultures and identification procedure was performed, based on classical macroscopic and microscopic characterization and on molecular techniques.  

Classical identification was performed on pure culture of strains on potato dextrose agar (Difco) after 48–72 hours of growth at 25°C. Macroscopic and microscopic characterization was done by classical observation of colonies and hypha aspects on Petri disches and the presence or absence of particular structures (septa, branches, conidia, ascospores, sporangia, rhizoides,) in microscope slide observation [11]. 

 Molecular identification was done by sequence alignment of  highly diverse regions of ITS1 and ITS2 in rDNA clusters. Biomass of pure culture on potato dextrose broth during 48 hours at 25°C was used as source of template for PCR. Isolation of genomic DNA was performed according to a procedure described by Piegza et al. [17]. Three pairs of primers were used: (a) ITS1 and ITS4 universal primers specific for filamentous fungi, (b) ITS1 and SL, (c) ITS1 and SH (Tab. 1). The SL and SH primers are considered as species-specific to identify Serpula lacrymans and  S. himantioides, respectively  [21]. 

Table 1. Primers sequences

Primer	Sequence
ITS1 (F)	5’-TCCGTAGGTGAACCTGCGG-3’
ITS4 (R)	5’-TCCTCCGCTTATTGATATGC-3’
SL (R)	5’- ATGTTTCTTGCGACAACGAC-3’
SH (R)	5’-TCCCACAACCGAAACAAATC-3’

Bioinformatic confirmation of identification. For 10 strains PCR product of ITS amplification were sequenced and identified with “ITStest fungi” at Centrum Informacji Genetycznych DNAi (Kraków, Poland). Also, sequences of the obtained PCR products were compared with corresponding rDNA fragments available in the GenBank Database (http://www.ncbi.nlm.nih.gov/genbank/).

RESULTS

The procedure of isolation and identification was targeted mainly towards filamentous fungi from genera causing development of brown and white rot. Finally 19 strains were obtained and classified to the genera or species. Based on the microscopic assessment, the isolates were classified to Penicillium and Lecanicillium genera (Tab. 2, Fig. 1). One of isolates (Dyw3) required further purification and finally was  identified as Candida yeast.

Table 2. Isolated fungi strains and size of rRNA fragments

Sample localisation	Isolated strains	Morphological identification	Fragment ITS1-SL [bp]	Fragment ITS1-SH [bp]	Fragment ITS1-ITS4 [bp]	Size of sequenced fragment by “ITStest fungi”  [bp]	Size of ITS1 [bp]	Size of ITS2 [bp]
Room wall in Opole region country house	NK1	Penicillium	363	–	561	792	172	463
	NK2	Penicillium	–	–	544	758	127	477
	NK3	Lecanicillium	–	293	533	772	167	445
Wooden baseboard in Laskówka house (Lower Silesia) Samples Serpula sp. suspected	NK4	Penicillium	–	–	544	–	–	–
	NK5	Penicillium	–	–	547	–	–	–
	NK6	Penicillium	–	–	551	–	–	–
	NK7	Penicillium	–	209	551	–	–	–
	NK8	Penicillium	–	–	551	–	–	–
	NK9	Penicillium	–	–	541	–	–	–
	NK10	Penicillium	–	–	554	–	–	–
	NK11	Penicillium	–	–	556	–	–	–
Cellar wall in Złotniki-Wrocław house	NK12	Penicillium	–	–	594	724	181	385
Cellar wall in Karłowice-Wrocław house	Dyw1b	Penicillium	439	–	558	799	178	462
	Dyw2	Penicillium	427	–	510	792	172	462
	Dyw3	Candida	–	–	570	693	96	439
	Dyw4	Penicillium	411	–	552	823	189	475
	Dyw5b	Penicillium	419	235	550	798	178	466
	Dyw6	Penicillium	–	–	539	797	172	467
	Dyw7	Penicillium	–	–	532	799	177	463

Fig. 1. Lecanicillium lecanii: spores (a),  fragment of  mycelium (b) and  spores accumulation around mycelium in an air buble (c)

The first step of molecular identification of isolates (by sequence homology of  highly diverse regions of ITS1 and ITS2) involved the presence of fragments specific for the genus Serpula, notably S. lacrymans and S. hymantioides, because thisgenus was suspected to occur  in the taken samples (Fig. 2).In PCR with S. lacrymans specific primer (SL), paired with ITS1, single product was obtained for five isolates: NK1 – 363 bp, Dyw1b – 439 bp, Dyw2 – 427 bp, Dyw4 – 411 bp and Dyw5b – 419 bp (results not shown).  However, the length of the rDNA product amplified with ITS1 – SL primers for S. lacrymans is 586 bp. So, none of isolated strains belong to S. lacrymans species, which excluded further speculation on its presence in collected deteriorated materials. Analogous results were achieved with the SH primer specific for S. hymanoides. Amplification products were obtained for only 3 of 18 isolates. The length of product for NK3 isolate was 293 bp, while for isolates NK7 and Dyw5b it was 209 bp and 235 bp, respectively (results not shown). Despite the occurrence of the products, none of the isolates was identified as S. himantioides, as the length of the products with primers pair ITS1-SH should be 429 bp.

Fig. 2. Electrophorograms of products of PCR amplification with ITS1 and ITS4 primers

Since the isolation of S. lacrymans and S. humantiodes was unsuccessful, further PCR with primers ITS1-ITS4 was conducted (Fig. 2) and combined with macroscopic and microscopic evaluation suggested that most of the obtained isolates belonged to the genus Penicillium (NK1, NK2, NK4–NK12, Dyw1b, Dyw2, Dyw4–Dyw6, Dyw7). Nevertheless, Lecanicillium (NK3) and Candida (Dyw3) were also present. The PCR with ITS1–ITS4  primers demonstrated the presence of two amplification products for part of the tested Penicillium isolates (NK2–NK5 and NK8–NK9). Small products, of approximately 300 bp  visible in the Figure 3 for some strains, probably were the results of amplification of mitochondrial DNA.

Fig. 3. Selected fragment exhibiting dissimilarities between tested isolates and reference strains

The length of ITS1-ITS4 rDNA products obtained for the isolates morphologically assigned to the genus Penicillium varied in the range from 541 bp to 594 bp. After comparison of the size of this rDNA fragments of 84 Penicillium species acquired from the GenBank database and those obtained for isolated Penicillium strains, it was established that the length of the obtained PCR products was close to those of P. citrinum, P. sartoryi and P. chrysogenum.

In order to verify the correctness of the assignment isolates to genus and species, for ten selected strains the “ITStest fungi” was used, which is based on the sequence evaluation of ITS1-ITS2 fragments. High homology of the obtained sequences to representants of Penicillium genus, confirmed minimal differences between species. After alignment of the sequence from Dyw1b,  Dyw4, Dyw 5b,  Dyw 6, Dyw7, NK1  and  NK2 to P. chrysogenum sequence a high similarity was shown (Fig. 3).Although more than 100 sequences identical to NK1 were found in  NCBI resources for  Penicillium strains.

The conserved region of 5.8S rRNA was present in all sequences fragments (Fig. 4). Whole sequence has 157 nucleotides, as the human one and  only 43 nucleotides are different. C. albicans sequence of 5.8S rRNA differ by 11 nucleotides from filamentous fungi and of L. lecanii only by 5 nucleotides from Penicillium sequence.

Fig. 4. Alignment of 5.8S rRNA (BioEdit)

For the NK3 isolate the sequencing of PCR product finally allowed to identified it as Lecanicillium lecanii. Specially for alignment of nucleotides observed at specific positions (Fig. 2) The same sequence was found in Genbank (NCBI) for L. lecanii strain ARSEF No. 6543 (Id: EF026005).

For the NK12 isolate, that could not be clearly identified by microscopic and morphological characteristics, the PCR product length was 594 bp and the sequenced product in ITStest fungi had 724 bp and was very similar in sequence to Penicillium fragments. The most important similarity (99%) was found to P. griseoroseum IF 2SW-F2fragment (ID: KY218687.1). On the basis of sequence comparison, the isolate Dyw3 was assigned to the yeast species Candida albicans SC5314-PO (ID: CPO25165.1) with 100% homology.

DISCUSSION

Filamentous fungi pose a significant threat to residential and inventory buildings, not only because of deterioration of construction elements, but also due to the negative influence on human health. In the study, isolation of filamentous fungi colonizing building interiors in Lower Silesia region was conducted, followed by their identification. Despite the choice of wood deterioration places described as specific for the development of Serpula species, mainly S. lacrymans and S. himantoides, none of isolated strains belong to these species.

The initial morphology-based identification of the obtained isolates allowed to assign most of the strains to specific genera. From among 19 isolates, 17 were classified as Penicllium, and the remaining were presumably Lecanicillium and Candida. All strains were tested according to methodology described by Pilarska et al. [19] for the ability to biosynthesis extracellular hydrolases. The results revealed a wide spectrum of enzymatic activities, which confirmed that strains belonging to Penicillium genus were not identical. It is surprising that despite the different location of samples sources, the predominance of Penicillium isolates was observed. Benammar et al. [2] confirmed that in different location, specifically a public bath in Algieria, the presence of fungal genera is very similar and the dominant one is Penicillium. They also indicated the presence of Aspergillus, Mucor, Alternaria and Fusarium [2]. According to Pottier et al. [20] among moulds isolated from wood, grout plastic and wallpaper, the most frequently occurring species represented following genera: Aspergillus Cladosporium, Penicillium, but also Rhizopus, Chaetomium and Trichoderma. Comparable results were obtained in the research of Nabrdalik and Latała [13], who inquired filamentous fungi colonizing building objects in Opole city, in Poland. They confirmed that the dominating fungal microflora comprised following genera: Aspergillus (37.11%), Cladosporium (28.15%) and Acremonium (27.34%). The genera Penicillium and Paecilomyces represented a lesser fraction, 3.64% and 3.03%, respectively, while Alternaria, Stachybotrys and Scopulariopsis occurred at the level below 1%. Fungi from genera Mucor and Rhizopus were not isolated. In this study 89% of strains were assigned to Penicillium and none to Aspergillus, which is surprising. Among the genus Penicillium, following species were isolated: P. griseoroseum and  P. chrysogenum. Nabrdalik and Latała [13], as well as Pottier et al. [20], described the most frequently observed species among Penicillium as: P. notatum, P. chrysogenum, P. brevicompactum, P. corylophilum and P. fellutanum.

The attempt of molecular differentiation of fungal isolates obtained in our study, based on the length of the PCR fragment, located between genes of small and large subunit of ribosome, was partially successful. Using species-specific primers, the absence of S. lacrymans and S. himantoides was confirmed. However some similarity in sequence of starters was detected by the presence of products for five isolates (NK1 – 363 bp, Dyw1b – 439 bp, Dyw2 – 427 bp, Dyw4 – 411 bp and Dyw5b – 419 bp) and 3 isolates (NK3-293 bp, NK7- 209 bp and Dyw5b- 235 bp), for S. lacrymans and S. himantioides,  respectively.

Supplementary application of the ITS1-ITS2 analysis for part of the strains allowed to partially succeed in the identification. Non-coding DNA regions of rDNA, especially ITS1 and ITS2 regions, are far more divergent than 18S rRNA, 26SrRNA and 5.8S rRNA sequences, hence their use in the taxonomic studies of filamentous fungi is common [4]. The length of the ITS1-ITS4 fragments, obtained in PCR for some strains, was of two sizes: a small product of approximately 300 bp and a large product within a range between 533 bp and 594 bp. The identification was performed with respect to the larger product. The presence of smaller product was intriguing, but observed in previous research on Geotricum sp. [18] and  Penicillium [25]. The larger product length of the ITS1-ITS2 region in fungi ranges from 450 to 700 bp [2]. For the fungi morphologically classified as Penicillium, the length of the large product was between 541 bp and 594 bp and the conserved region of 5.8S rRNA (157 nucleotides) was present in all sequence fragments. Skouboe et al. [25] and Tuthil et al. [26] in a PCR reaction with ITS1 and ITS2 primers, obtained products of approximately 600 bp for different Penicillium species. In our studies, the products were characterized by smaller size because of the application of the ITS4 primer instead of the ITS2. In the 90-s a large-scale project was run in order to sequence rDNA genes of all known species of Penicillium [16]. The ITS region of these fungi is insufficiently variable, therefore ineffective in differentiation of closely related species [7, 23–25]. The GenBank Database hosts a number of improperly identified sequences, which results in difficulties in the identification of Penicillium species with the BLAST tool [27]. Due to the limitations of the ITS region application as a species-specific marker of the genus Penicillium, an additional marker is required. Also  Vu et al. [28] noticed that 18–19% of fungal strains could not be identified by ITS1-5.8S and D1/D2 region of 26S rRNA sequences and thresholds predicted for  identi?cation at the genus levels were 94.3%  (based on ITS)  and 98.2% (based on D1/D2 region of 26S rRNA).

Visagie et al. [27] put forward the beta-tubulin (BenA) marker, as most convenient for secondary identification of Penicillium, however, this does not allow do determine several species with identical BenA sequence, i.e. P. camemberti, P. caseifulvum and P. commune. As an alternative to the BenA marker, Visagie et al. [27] suggest the application of markers based on the gene of either calmodulin (CaM) or the largest subunit of polymerase II (RPB2). Both genes exhibit a comparable applicability for the identification to BenA gene, however, sequences in databases are incomplete in this regard. Therefore, for the task of routine identification it is recommended to use all four markers (ITS, BenA, CaM and RPB2). During characterization of new fungal isolates it is a good practice to submit into databases at least sequences of ITS and BenA, to allow for an effective identification [27]. However identification by ITSTest fungi in some case was sufficient. The most attractive isolate obtained in this study was NK3 identified by ITSTest as Lecanicillium lecanii, aspecies known as insect pathogen [6].

CONCLUSIONS

Fungal isolates in the number of 19 were isolated from construction materials. Most of the strains were identified to the genus level based on morphological features and molecular analysis of rDNA. Analyzing the length of the ITS region, a part of the Penicillium isolates were identified as P. chrysogenum, according to the “ITSTest fungi”. Three strains were classified at the specieslevel: Lecanicillium lecanii (NK3), P. griseoroseum (NK12)and Candida albicans (Dyw3).

ACKNOWLEDGEMENTS

This work was partially supported by Wrocław Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018. Work partially realized in the framework of the Biotechnology Students Circle at Wrocław University of Environmental and Life Sciences in Wrocław

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Received: 23.11.2018
Reviewed: 15.02.2019
Accepted: 25.02.2019

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Natalia Kobiałka
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland

Malwina Mularczyk
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland

Katarzyna Kosiorowska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland

Kinga M. Pilarska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland

Wojciech Łaba
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland

Michał Piegza
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
phone +48 71 320 7737
fax +48 71 320 7794
51-630 Wrocław
Poland
email: michal.piegza@upwr.edu.pl

Małgorzata Robak
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland
email: malgorzata.robak@up.wroc.pl

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Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

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