Elsevier Editorial System(tm) for International Biodeterioration & Biodegradation Manuscript Draft Manuscript Number: IBB-D-12-00008R1 Title: Fungal biodeterioration of historical library materials stored in Compactus movable shelves Article Type: Original Paper (max 10,000 words) Keywords: Eurotium halophilicum; Aspergillus halophilicus; book deterioration; cultural heritage; mould contamination; xerophilic fungus Corresponding Author: Dr. matteo montanari, Corresponding Author's Institution: First Author: matteo montanari Order of Authors: matteo montanari; Valeria Melloni, Dr.; Flavia Pinzari, Dr.; Gloria Innocenti, Dr. Abstract: Mould biodeterioration of historical library materials in archives and libraries is a frequent and complex phenomenon, that may have important economic and cultural drawbacks in our society. Compactus shelving is one of the systems utilised for the conservation of library materials, because it allows for a more efficient use of space and protection against dust deposition. However, in the last ten years there have been many reports on monospecific mould infections within compactus shelves, in spite of conventional control of environmental temperature and humidity according to recommended standards. Contamination was commonly characterized by white spots of mycelium, each measuring about 0,5-1 cm in diameter, developing on volume binding, especially when made of leather, parchment or textiles. Till now, the identification of the causal agent at species level was never accomplished, since all attempts to grow it on artificial substrates for subsequent identification were unsuccessful. Using a range of sampling techniques, including adhesive tape and nitrocellulose membrane, and a combination of conventional culturing methods, direct microscopical observations and a molecular approach, we have for the first time successfully identified at species level the fungus causing infections inside compactus shelves.
Transcript
Elsevier Editorial System(tm) for International Biodeterioration & Biodegradation Manuscript Draft Manuscript Number: IBB-D-12-00008R1 Title: Fungal biodeterioration of historical library materials stored in Compactus movable shelves Article Type: Original Paper (max 10,000 words) Keywords: Eurotium halophilicum; Aspergillus halophilicus; book deterioration; cultural heritage; mould contamination; xerophilic fungus Corresponding Author: Dr. matteo montanari, Corresponding Author's Institution: First Author: matteo montanari Order of Authors: matteo montanari; Valeria Melloni, Dr.; Flavia Pinzari, Dr.; Gloria Innocenti, Dr. Abstract: Mould biodeterioration of historical library materials in archives and libraries is a frequent and complex phenomenon, that may have important economic and cultural drawbacks in our society. Compactus shelving is one of the systems utilised for the conservation of library materials, because it allows for a more efficient use of space and protection against dust deposition. However, in the last ten years there have been many reports on monospecific mould infections within compactus shelves, in spite of conventional control of environmental temperature and humidity according to recommended standards. Contamination was commonly characterized by white spots of mycelium, each measuring about 0,5-1 cm in diameter, developing on volume binding, especially when made of leather, parchment or textiles. Till now, the identification of the causal agent at species level was never accomplished, since all attempts to grow it on artificial substrates for subsequent identification were unsuccessful. Using a range of sampling techniques, including adhesive tape and nitrocellulose membrane, and a combination of conventional culturing methods, direct microscopical observations and a molecular approach, we have for the first time successfully identified at species level the fungus causing infections inside compactus shelves.
UNIVERSITÀ DEGLI STUDI DI BOLOGNA
DIPARTIMENTO DI PROTEZIONE E VALORIZZAZIONE AGROALIMENTARE
Illinois, USA; 1 mm thick, n. 745), were gently pressed over spots, to collect mycelium, fruiting
structures and spores. Tapes were aseptically mounted over sterile glass slides and transferred to the
laboratory. Each tape was then bisected, one half was used for optical and electron microscope
*Detailed Response to Reviewers
examination, and the other half for direct DNA extraction; (iii) sterile membranes of nitrocellulose
(0.45 µm pore-size, Millipore; 47 mm in diameter) where gently pressed for ten seconds over
mycelial spots, then immediately transferred on 6 cm Petri dishes containing dichloran 18 per cent
glycerol (DG18) agar (Samson et al. 2002).
5. Page 5, lines 8-20. The whole paragraph is poor described. Please indicate the volume of the PCR
reaction mixture, concentration of primers, of the template DNA and concentration of the BSA in the
reaction mixture.
At page 5 from line 11 the paragraph is modified as follows:
ITS region of ribosomal DNA was amplified by Polimerase Chain Reaction (PCR) using ITS1f and ITS4 as
fungal primers (White et al. 1990, Gardes and Bruns 1993), PCR master mix (Promega, Mannheim,
Germany) and Bovine Serum Albumine (BSA, Sigma) in the reaction mixture.
ITS region of ribosomal DNA was amplified by Polimerase Chain Reaction (PCR) using for each reaction 25
μl of PCR master mix (Promega, Mannheim, Germany), 50 pmole of both ITS1f and ITS4 as fungal primers
(White et al. 1990, Gardes and Bruns 1993), 1 μl of Bovine Serum Albumine (BSA 20 mg/ml, Fermentas), 4
μl of DNA template and water nuclease-free to a final volume of 50 μl.
6. Page 5, lines 28-29. Authors say that the DNA was extracted according to the protocol described by
Schabereiter-Gurtner et al. (2001), but the final purification step is not the one of the cited protocol. In the
protocol of Schabereiter-Gurtner the DNA in the supernatant was cleaned with the QIAamp Viral RNA Mini
Kit Qiagen, following the protocol of the manufacturer. It is ok if you use another purification protocol, but
please clarify in the text that this is a modification made by the authors in the present study.
At page 5 from line 22 the paragraph is modified as follows:
Environmental DNA was extracted directly from three Fungi tape fragments (2 x 3 cm) following
the protocol described by Schabereiter-Gurtner et al. (2001). This step was perfomed to compare
sequences obtained using a culture-based method with those obtained using a culture-independent
method. This method combines enzymatic (lysozyme and proteinase K) and mechanical steps
(freeze and thaw cycles) in the presence of cetyltrimethylammonium bromide (CTAB) and a final
step with chloroform – isoamyl alcohol (24:1).
Environmental DNA was extracted directly from three Fungi tape fragments (2 x 3 cm) following
the protocol described by Schabereiter-Gurtner et al. (2001), modified by using a different kit for
the DNA cleaning step. This analysis was perfomed to compare sequences obtained using a culture-
based method with those obtained using a culture-independent method. DNA extraction combines
enzymatic (lysozyme and proteinase K) and mechanical steps (freeze and thaw cycles) in the
presence of cetyltrimethylammonium bromide (CTAB) and a final step with chloroform – isoamyl
alcohol (24:1).
7. Page 5, lines 32-44. Authors mention in this paragraph that 20 colonies were randomly selected for
"each sample". Do the authors mean as "sample" the three DNA crude extracts derived from the three fungi
tape fragments? If so, please clarify in the text. Does it mean that they screened 20x3 colonies? The whole
paragraph is not enough clear.
At page 5, from line 36 the paragraph is modified as follows:
For each sample, PCR product was purified using NucleoSpin® Extract II kit Protocol
(MACHEREY-NAGEL, Düren, Germany). Purified PCR products (3 μl) were cloned using pGEM-
T Easy Vector System I Kit (Promega), following the manufacturer protocol. Twenty colonies
positive for the recombinant plasmids were randomly selected for each sample...
For each DNA crude extract, PCR product was purified using NucleoSpin® Extract II kit Protocol
(MACHEREY-NAGEL, Düren, Germany). Purified PCR products (3 μl) were cloned using pGEM-T Easy
Vector System I Kit (Promega), following the manufacturer protocol. A total of twenty white colonies
positive for the recombinant plasmids were randomly selected from the three samples (DNA crude
extracts)...
8. Page 5, lines 41-43. Please explain how was performed the screening of the PCR products on the basis
of their different molecular weight on agarose gels? Fragments amplified with the same primers will be
approximately of the same length. Did you perform the PCR using the internal primers of the vector
(SP6/T7) or using directly the ITS1f/ITS4 primers? Please clarify in the text all these details. It would be
difficult to repeat the experiments by reading this section.
Regarding the screening: indeed we didn’t observe any differences in molecular weight between the
bands, for this reason we decided to sequence all the positive bands
So, at pag 5 from line 41 the paragraph is modified as follows:
... using the same protocol used above. PCR products were run by agarose (1.5%) gel
electrophoresis and then bands were screened on the basis of their different molecular weight.
Twenty PCR products were selected and subjected to sequencing at Macrogen Inc. Comparative
sequences analysis was performed as described above.
... using the same primers (ITS1f and ITS4) and protocol used above. PCR products were run by
agarose (1.5%) gel electrophoresis. Products giving positive bands were subjected to sequencing at
Macrogen Inc. Comparative sequences analysis was performed as described above.
Furthermore we added in the results the following sentence (see further too):
“From the twenty selected clones of the clone library derived from environmental DNA we
obtained twenty bands on agarose gel with the same molecular weight”.
Results
9. Page 6, line 2 and line 31 change "between" to among.
“between” is changed to “among” wherever required
10. Page 6, line 11, change "blu" to blue.
At pag 6 line 11 “blu” is changed to “blue”
11. Page 6, line 23, change "appared" to appeared.
At pag 6 line 23 "appared" is changed to “appeared”
12. Page 6, lines 25-33. I miss the accession numbers of the sequenced microorganisms and clones The
resulting sequences must be deposited at the database prior the submission of the manuscript, and the
accession numbers have to be supplied in the manuscript at the section of methods or in the results (on a
Table). This fact is very important in order to control the quality of the obtained sequences, and furthermore,
to allow the comparison of sequences derived from future works with the sequences obtained in this study.
Please supply the accession numbers.
At pag 6 line 26 the paragraph is modified as follows:
ITS sequences derived from three randomly selected colonies developed on DG18 agar after
sampling with nitrocellulose membrane, compared with those available in the online database,
revealed the best match BLAST with Eurotium halophilicum (An. Aspergillus halophilicus)
(GenBank ID: EF652088), at 100% of similarity. The twenty ITS sequences derived from selected
clones of the clone library obtained from adhesive tape samples, were highly similar between each
others. Compared with those available in the database, they also revealed the best match BLAST
with the same Eurotium halophilicum (An. Aspergillus halophilicus) (GenBank ID: EF652088), at
97% of similarity.
ITS sequences derived from three randomly selected colonies developed on DG18 agar after
sampling with nitrocellulose membrane, were identical among each others. Compared with those
available in the online database, they revealed the best match BLAST with Eurotium halophilicum
(An. Aspergillus halophilicus) (GenBank ID: [EF652088]), at 100% of similarity. One of them
(isolate MM373) was deposited at the NCBI database (tab. 1).
From the twenty selected clones of the clone library derived from environmental DNA we obtained
twenty bands on agarose gel with the same molecular weight. Sequencing of all the amplicons
revealed high similarity among each others. Compared with those available in the database, they all
revealed the best match BLAST with the same Eurotium halophilicum (An. Aspergillus
halophilicus) (GenBank ID: [EF652088]), at 97% of similarity. Ten sequences of them were
deposited at the NCBI database (tab. 1).
The following tab of the accession numbers of the deposited sequences was produced and submitted to
IBB together with the revised manuscript as an individual file:
Tab. 1: Nucleotide sequence accession numbers at NCBI database
Code Accession number
Isolate MM373 [JN839940]
Clone 1m1 [JN839949]
Clone 2m1 [JN839950]
Clone 7m1 [JN839951]
Clone 8m1 [JN839952]
Clone 9m1 [JN839953]
Clone 12m1 [JN839954]
Clone 13m1 [JN839955]
Clone 14m1 [JN839956]
Clone 17m1 [JN839957]
Clone 22m1 [JN839958]
13. Page 6 and 7, lines 37 and 20, respectively. Please unify the way to write water activity Aw or aw.
aw is unified in Aw wherever required
Answers to Reviewer #2
Reviewer #2: “An interesting study which highlights potential problems at the "dryer" end of
accepted storage conditions. Good work, but confirms the obvious. It would be good to see a
comment emphasising the value of ventilation, micro-monitoring of humidity, a regime of stack
moving and inspection included as obvious practical measures. At the end of the day, where such
growths occur, humidity is locally still too high for the susceptible bindings in question.”
At page 7, from line 42, the paragraph is modified as follows:
Further surveys and laboratory studies are needed to better define the microclimate conditions that
favour this fungal species, the specific characteristics of the substrates that support its growth and
the mechanisms for its dispersion inside storage environments.
The operations suggested to conservators and librarians to avoid the diffusion of this fungus are as
follows: i) the HVAC (heating, ventilation, and air conditioning), if available in the library, must be
checked for efficacy, and ventilation grilles cleaned and checked to verify their correct functioning;
ii) HVAC filters should be changed more frequently; iii) the side panels of the compactus units are
better to be removed so as to facilitate ventilation, or alternatively it should be established a regime
of daily stack moving.
Frequent visual inspection of materials stored within compactus shelving units is highly
recommended, notwithstanding the presence of a well-functioning HVAC and maintenance of
optimal standard climatic values. Micro-environmental anomalies and localised deterioration
phenomena, if promptly detected can be easily contained and resolved, while invasive
reclamation/rehabilitation of materials should, as far as possible, be avoided and implemented only
as a last resort.
Further studies have now been addressed in Italian libraries, and in our laboratories to the
comprehension of E.halophilicum auto-ecology, its metabolic requirements, and the kind of damage
actually provoked on books.
“Fungal biodeterioration of historical library materials stored in Compactus movable shelves” by M. Montanari, V. Melloni, F. Pinzari and G. Innocenti
HIGHLIGHTS
First report of Eurotium halophilicum as the causal agent of fungal contamination in libraries and archives in Italy
Compactus movable shelves as a new manmade niche for Eurotium halophilicum
identification at species level using a combination of conventional and biomolecular techniques
*Highlights
Montanari M. 1
1
Fungal biodeterioration of historical library materials stored in Compactus movable 1
shelves 2
3
Matteo Montanari a, Valeria Melloni a, Flavia Pinzari b, Gloria Innocenti a 4
5 a Dipartimento di Protezione Valorizzazione Agroalimentare Alma Mater Studiorum 6 Università degli Studi di Bologna, viale Fanin 46, 40127 Bologna, Italy, e-mail 7
b Istituto Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario. 9 Ministero per i Beni e le Attività Culturali, via Milano 76, 00184 Roma, Italy, e-mail 10
Mold deterioration of historical library materials in archives and libraries is a frequent and 2 complex phenomenon that may have important economic and cultural consequences. 3 Compactus shelving is one of the systems utilised for the conservation of library materials, 4 because it allows for a more efficient use of space and protection against dust deposition. 5
However, in the last ten years there have been many reports on single species mould 6 infections within Compactus shelves in spite of conventional control of environmental 7 temperature and humidity to recommended standards. Contamination was commonly 8 characterized by white spots of mycelium, measuring 0.5-1.0 cm in diameter and observed on 9 volume binding, especially those of leather, parchment or textile. Until now, the 10 identification of the causal agent at species level has not been reported since attempts to grow 11
it on media for subsequent identification were unsuccessful. Using a range of sampling 12 techniques, including adhesive tape and nitrocellulose membrane, and a combination of 13
conventional culturing methods, direct microscopic observations and molecular methods, we 14 have for the first time identified to species level the fungus causing infections inside 15
Compactus shelves. 16
17
18
Keywords: Eurotium halophilicum, Aspergillus halophilicus, book deterioration, cultural 19
Storage of books and documents inside structures intended for their preservation has created 2 new manmade environments for microbial species such as fungi and bacteria to inhabit 3 (Kowalik 1980, Zyska 1997, Nittérus 2000). High-density storage systems referred to as 4 "movable shelving” or “Compactus type shelving” are employed by many libraries, archives 5
and conservation institutions suffering from limited space. These systems minimize the 6 amount of space required for storage by compacting blocks of shelves (or cabinets of drawers) 7 tightly together. These blocks slide along tracks and can be moved apart (opened) for the 8 retrieval of items positioned on a particular block and then moved back together (closed). 9 Compactus shelves can also protect against dust deposition (Gallo and Regni 1998) and fire. 10 These characteristics are advantageous for the preservation and storage of materials but can 11
also be problematical when used in conservation environments lacking efficient climate 12 control systems. Resistance to heat and humidity exchange between the micro-environment 13
within the Compactus units and the outer environment, can present risk to stored objects, 14 especially when these objects are composed of hygroscopic materials. International 15 Federation of Library Associations (IFLA) recommends 18-20°C air temperature and 50-60% 16 relative humidity (RH) for effective preservation of documents like books and periodicals in 17 archives and libraries. In practice, it is difficult to maintain a stable temperature and relative 18
humidity level, even with the benefit of air conditioning. 19 In the last decade, several investigations in libraries and archives especially within Compactus 20
shelving blocks have reported mold contamination and growth on volume bindings made of 21 leather, parchment or cotton fibres. Surprisingly these reports consistently observed white and 22
irregular spots of fungal spread mainly on the exposed part of the volumes stored mainly in 23
the lower shelves of the blocks. Optical and scanning electron microscopy examination of 24
samples collected with transparent adhesive tapes revealed the presence of conidiophores 25 typical of the genus Aspergillus. However, due to cultivation challenges and peculiarities of 26
morphology, identification to species level was not accomplished (Pinzari and Montanari 27 2011). In this study, specific identification of this common, putative Aspergillus isolate 28 contaminating books stored inside Compactus shelves is reported. 29
30
2. Material and methods 31
2.1 Case study 32
Our investigation was accomplished in a deposit of an historical library in Rome, containing 33 books and papers stored in Compactus type shelves and contaminated by molds. Mold 34
contamination was spread all over the depository, mainly in the lower shelves of the 35 Compactus blocks, in a large number of volume binding made by fabric or leather, especially 36 on the exposed part of the book such as spine, upper edge and fore edge. Infestation pattern 37 on the books was similar to those detected during previous surveys in other archives and 38 libraries in Italy, consisting of irregular white, mycelial spots of variable diameter (Fig. 1). 39
Observations of these spots at low magnification with a digital microscope (DinoLite pro, 40
AM413TFVW-A, Italy) demonstrated many conidiophores scattered on the mycelium. 41
2.2 Sampling 42
Sampling of fungal elements was performed on contiguous fungal spots of eight contaminated 43 volumes using the following methods: (i) sterile cotton swabs (Cultiplast – LP Italiana SPA, 44 Italy) were wiped across fungal spots then transferred to the laboratory in sterile tubes and 45
used for fungal culturing and identification; (ii) pieces (6 x 2 cm) of a removable transparent 46 adhesive tape specifically intended for microbiological sampling (Fungi Tape; Scientific 47 Device Lab., Glenview, Illinois, USA; 1 mm thick, n. 745), were gently pressed over spots, to 48
collect mycelium, fruiting structures and spores. Tapes were aseptically mounted over sterile 49 glass slides and transferred to the laboratory. Each tape was then bisected, one half was used 50
Montanari M. 4
4
for optical and electron microscope examination, and the other half for direct DNA extraction; 1 (iii) sterile membranes of nitrocellulose (0.45 µm pore-size, Millipore; 47 mm in diameter) 2 where gently pressed for ten seconds over mycelial spots, then immediately transferred to the 3 surface of 6 cm Petri dishes containing dichloran 18% glycerol (DG18) agar (Samson et al. 4
2002). 5
All samples were transferred to the laboratory the same day of collection and immediately 6
processed. 7
2.3 Agar cultures 8
Each swab of fungal growth was immersed in a sterile glass vial containing 5 ml of Ringer’s 9
solution, and homogenized in a sonication bath at 40±2 kHz frequency for 5 minutes 10 (Trampuz et al. 2007). Aliquots (100 µl) of homogenate were spread on each 9 cm Petri 11
dishes (4 plates per sample) containing separately Malt Extract Agar 2% (MEA 2%), Czapek 12 Yeast Agar with 20% sucrose (CY20S) (Pitt and Hocking 1985) and DG18 agar. Plates were 13 incubated for 7-14 days at 20°C in the dark and growth transferred DC18 agar for isolation in 14 pure culture. 15
Nitrocellulose membrane on DG18 plates were incubated at 20°C in the dark. After fungal 16 colony appearance, membranes were removed and portions of mycelium were picked up for 17 isolation in pure culture on DG18 agar plates. 18
Fungal isolates were identified to genus level using biometric and microscopic features (Ellis 19
1971 and 1976, Von Arx 1981, Domsh and Gams 1993). Dominant fungal isolates were 20
identified to species level by DNA analysis. 21
2.4 Optical and scanning electron microscopic observations 22
Two fragments (1.5 x 2 cm) were bisected from half of each fungi tape bearing captured 23 fungal elements. The first fragment was further divided in two parts (1 x 1.5 cm): one part 24
was transferred on a glass slide with a drop of cotton blue stain, the other part on a glass slide 25 with a drop of fluorescein diacetate (FDA) solution (20 µg of FDA in 1 ml of phosphate 26
buffer pH 7.3). Cotton blue staining was used for observations of morphological fungal 27 structures at white light microscope. FDA staining was used for observations of active 28 structures using an inverted epifluorescent microscope (Nikon eclipse T2000) equipped with a 29
FITC filter (blue excitation wave length: 495 nm). Active structures (positive staining) were 30 assessed by the presence of a greenish fluorescence emanating from the cytoplasm of spores 31 and hyphae, due to the liberation of fluorescein by enzymatic (hydrolytic) cleavage. Samples 32
stained with FDA were observed after 20 min of incubation in the dark at 20°C. All slides 33 were examined at 400 and 600x magnification. Micrographs were acquired using a digital 34
camera connected to a PC equipped with NIS Elements software (Nikon). 35
The second fragment was observed using an EVO 50 Scanning Electron Microscope 36 produced by the Carl-Zeiss Electron Microscopy Group (Oxford, UK). Tape fragments 37 measuring 5-10 mm in diameter were cut and mounted on to a 12 mm metal stub using 38 double-sided carbon adhesive tape. Parts of agar cultures were also observed by SEM 39 imaging. Samples were examined using a 20 kV electron beam, both at variable pressure 40
(VPSE) and after metallization with high vacuum mode with gold (Goldstein et al. 2003). 41 Fungal samples supported on adhesive tape were directly metalized without previous fixation. 42 Elements were observed to be dry at the time of the sampling, and fixation would have added 43 artefacts. For observation with the scanning electron microscope (SEM), ascomata at various 44 developmental stages on agar media were excised, placed in phosphate buffer (pH 7.0), and 45
fixed in glutaraldehyde buffer for 2 h, rinsed in distilled water and post-fixed in 2% OsO4 for 46 12 h at 5°C, dehydrated in an ethanol series, taken to amyl acetate, and critical point dried in a 47
Polaron E-3000 dryer (Quorum Technologies, Ringmer, UK) using carbon dioxide. Dried 48 samples were coated at 40mA to obtain a 15 nm-thick layer of gold (Baltec Sputter Coater) 49
and examined using the SEM EVO 50 (Carl Zeiss, Cambridge, UK). 50
Montanari M. 5
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2.5 Molecular analysis 1
2.5.1 DNA analysis of isolated fungal cultures 2
DNA was extracted from mycelium of fungal isolates using the NucleoSpin® Plant II 3 (MACHEREY-NAGEL, Düren, Germany) for fungi, following the manufacturer instructions. 4 ITS region of ribosomal DNA was amplified by Polymerase Chain Reaction (PCR) using for 5 each reaction 25 μl of PCR master mix (Promega, Mannheim, Germany), 50 pmole of both 6 ITS1f and ITS4 as fungal primers (White et al. 1990, Gardes and Bruns 1993), 1 μl of Bovine 7
Serum Albumin (BSA 20 mg/ml, Fermentas), 4 μl of DNA template and water nuclease-free 8 to a final volume of 50 μl. The thermocycling program was as follows: 3 min denaturation at 9 94°C, followed by 35 cycles of 30 sec. denaturation at 94°C, 30 sec. annealing at 55°C, and 1 10 min extension at 72°C. Ten minutes at 72°C were used as a final extension step. Sequencing 11 of amplified ITS regions was performed at Macrogen Inc. (Korea). Taxonomic identifications 12 were performed comparing retrieved sequences with those available in the online databases 13 provided by the National Centre for Biotechnology Information (NCBI) using the BLAST 14
search program (Altschul et al. 1997). 15
2.5.2 Total DNA extraction from adhesive tapes. 16
Environmental DNA was extracted directly from three fungal tape fragments (2 x 3 cm) 17 following the protocol described by Schabereiter-Gurtner et al. (2001), modified by using a 18 different kit for the DNA cleaning step. This analysis was performed to compare sequences 19 obtained using a culture-based method with those obtained using a culture-independent 20
method. DNA extraction combines enzymatic (lysozyme and proteinase K) and mechanical 21 steps (freeze and thaw cycles) in the presence of cetyltrimethylammonium bromide (CTAB) 22
with the final step with chloroform – isoamyl alcohol (24:1). DNA in the supernatant is 23 cleaned with the NucleoSpin® Plant II kit for soil, starting from step 3 of the manufacturer’s 24
protocol. The final elution step was repeated two times with 25 μl of 70°C preheated PE 25 Buffer. Cleaned DNA extract was used for PCR amplification analysis for fungi, using the 26
same protocol used for DNA from fungal culture. 27
2.5.3 Creation of clone libraries and sequence analysis 28
PCR amplification from environmental DNA may produce a mixture of different amplicons, 29 which should be separate to accomplish a detailed phylogenetic analysis on members of the 30
fungal community. To separate single ITS amplified fragments, a clone library containing the 31 ITS fungal regions was realized. For each DNA crude extract, PCR product was purified 32
using NucleoSpin® Extract II kit Protocol (MACHEREY-NAGEL, Düren, Germany). 33 Purified PCR products (3 μl) were cloned using pGEM-T Easy Vector System I Kit 34
(Promega), following the manufacturer protocol. a total of twenty white colonies positive for 35 the recombinant plasmids were randomly selected from the three samples (DNA crude 36 extracts) and then subjected to a denaturation step (6 min at 97°C) and PCR amplification 37 using the same primers (ITS1f and ITS4) and protocol used above. Electrophoresis of PCR 38 products was conducted in agarose (1.5%) gels. Products giving positive bands were 39
subjected to sequencing at Macrogen Inc. Comparative sequences analysis was performed as 40
described above. 41
42
3. Results 43
3.1 Cultivation 44
Sampling procedure using sterile swabs produced on agar media only few colonies typical of 45
Cladosporium and Penicillium spp.. Direct plating nitrocellulose membrane on DG18 plates, 46 produced after 6-7 days, slow-growing white colonies, slightly depressed in the middle (Fig. 47
2). All colonies were of similar morphological and biometric features. After re-isolation on 48 DG18 agar, white colonies were observed to grow at a rate of 4-7 mm per week (Fig. 3). As 49
Montanari M. 6
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agar medium get drier (about 2-3 weeks after inoculation), colonies developed cleistothecia 1 that were globose, white to cream in colour, 150-200 µm in diameter (Fig. 4). The conidial 2
state was not observed in these cultures. 3
3.2 Optical and SEM microscopic observations 4
Optical microscopic observations of adhesive tape samples revealed the presence of fungal 5 structures, conidiophores and conidia, typical of Aspergillus spp. (Fig. 5). Observations of 6 fungal elements trapped on the adhesive tape showed only conidial states. Fungal structures 7
stained with FDA, observed with an epifluorescent microscope using blue filter, fluoresced 8
green revealing an enzymatic activity (Fig. 6). 9
All samples collected directly from books using the adhesive-tape technique examined under 10 Scanning Electron Microscopy, showed fungal elements typical of Aspergillus species, 11
specifically large conidia single or in chain, slightly ovate, echinulate with prominent scars, 12 and conidiophores with narrow vesicles finely covered with a layer of hairy structures (Fig. 13
7). 14
Fungal growth from agar cultures on DG18 plates, observed under SEM, included many 15 ascomata with asci and mature ascospores. Hypertrophic hyphae, slightly covered with bare 16 and short hairs (Fig. 8), were also observed in most of the cultures. Ascomata appeared 17 spherical to subspherical, mostly 100-150 µm in diameter, asci measured 10-15 µm in diam. 18
and are spherical or nearly so. Ascospores by SEM appeared lenticular, with rough surface 19 and sharp furrow bordered by ridges, mostly 5x7 µm. The ascomata appeared characterised 20
by a smooth surface with semi-globose prominent structures, which make the whole structure 21
resembling a “morula” (Fig. 9). 22
3.3 Molecular analysis 23
ITS sequences derived from three randomly-selected colonies developed on DG18 agar after 24 sampling with nitrocellulose membrane were identical and were best match by BLAST with 25
of similarity. One of them (isolate MM373) was deposited at the NCBI database (tab. 1). 27
From the twenty selected clones of the clone library derived from environmental DNA we 28 obtained twenty bands on agarose gel with the same molecular weight. Sequencing of all the 29
amplicons were high similar. Compared with those available in the database, they also 30 revealed best match BLAST with the same Eurotium halophilicum (An. Aspergillus 31 halophilicus) (GenBank ID: [EF652088]), at 97% of similarity. Ten sequences were deposited 32
at the NCBI database (Table 1). 33
34
Tab. 1: Nucleotide sequence accession numbers at NCBI database 35
36
4. Discussion 37
Fungi damaging materials in indoor environments are chiefly primary colonizers capable of 38 rapid growth even when water activity is low (i.e. Aw <0.8). When a substrate is attacked by a 39 fungus, its water activity changes sufficiently to support the growth of other species (fungi 40 and bacteria), such as in natural successions (Samson et al. 1994). Secondary colonisers are 41
species that have a higher resistance to water stress. These species develop thanks to unstable 42 microenvironments characterised by small changes in air temperature or humidity due to 43 night/day alternation. Poor ventilation and surface temperature dynamics can produce foci of 44 water condensation and local micro-climates with localized peaks of Aw greater than the 45 surrounding indoor environment. These circumstances are favourable to some fungal species 46 that are able to proliferate in places where the overall environmental conditions would 47 otherwise appear to be hostile (Pinzari 2011). Findings reported here, are consistent with 48
single species Eurotium halophilicum contamination of materials stored in Compactus 49
Montanari M. 7
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shelves. Identification of this fungus was achieved by a combination of conventional, 1 molecular methods and SEM observations. Isolation of this fungus was achieved only by 2 direct plating on DG18 agar of fungal elements collected directly from volume surfaces. The 3 sterile nitrocellulose membrane technique has shown to be very effective for collection of 4 fungal elements for direct plating. All sequences obtained from pure cultures were highly 5 similar to sequences obtained from total DNA extracted directly from the adhesive tape 6 samples (environmental samples), and all these sequences had the best BLAST matches (97-7 100% similarity) with the unique rDNA sequence attributed to E. halophilicum as published 8
on GenBank Data Base (Peterson 2008). 9
It is important to emphasize that, in the present study, the fungus showed a different life cycle 10 and morphological aspect in vivo and in vitro. On volume bindings the fungus was observed 11
only its anamorphic stage as conidiophores and conidia. When observed at SEM, 12 conidiophore stipes and vesicles were densely coated by curly micro-filaments similar to hairs 13
all over their surface. On agar media, the fungus presented in its teleomorph producing many 14 ascomata and ascospores, SEM observations showed the presence of bare and short hairs on 15 hypertrophic hyphae. These features of anamorphic and teleomorphic structures are consistent 16 with those described by Christensen et al. in the original description of E. halophilicum 17 (1959) and by Samson and Lustgraaf (1978). However, the dense layer of hairy and curly 18
structures observed on the wall of conidiophores and hyphae were never reported before. 19 Hairs might be an adaptation of the fungus to increase its ability to capture water from the 20
surrounding air in a dry environment. Eurotium halophilicum is a xerophilic fungus with a 21 high tolerance to water stress. The minimum observed water activity (Aw) for germination and 22
growth is 0.675, one of the lowest for Eurotium species (Christensen et al. 1959). The 23
occurrence of this fungus is associated with air-dust (Abdel-Hafez 1990) or house-dust in 24
association with mites and A. penicillioides and storage of dry food (Hocking and Pitt 1988). 25 Sequences of Eurotium halophilicum have been associated with library material only by 26
Michaelsen et al. (2010) by DGGE-fingerprinting, without any information about its viability 27 and role on the substrate deterioration. Therefore this is, in our knowledge, the first report 28 where E. halophilicum is isolated and cultivated from book bindings and where its role in the 29
contamination of library materials is demonstrated by a combination of visual, culture and 30 culture-independent methods. Similar contamination patterns and microscopic features 31 observed in several samples collected with the adhesive tape technique during previous 32
surveys performed in other archives and libraries where Compactus type shelving is used 33 (Montanari et al. 2007, Pinzari and Montanari 2011) suggest that E. halophilicum might have 34
a large distribution in this particular environment, at least in Italy. Detection failure in 35 previous surveys may have been due to inadequate sampling procedure and unsuitable agar 36 media. As suggested by Christensen et al. (1959) and Samson and Lustgraaf (1978), the 37 occurrence of this fungus in indoor environments may be underestimated due to inadequacy 38
of classical methods and its very slow growth on typical media. 39
In conclusion the high tolerance to water stress combined with the high affinity to library 40
materials showed by this fungus represent a serious threat for librarians and conservators. 41 Conventional control of temperature and relative humidity in conformity with recommended 42 standards may be insufficient to prevent material colonization, especially if enclosed systems 43
with low ventilation rate, such as Compactus type shelves, are adopted. 44
The operations suggested to conservators and librarians to avoid the diffusion of this fungus 45
are: i) the HVAC (heating, ventilation, and air conditioning), if available in the library, must 46
be checked for efficacy, and ventilation grilles cleaned and checked to verify their correct 47 function, ii) HVAC filters should be changed frequently; iii) to facilitate ventilation, side 48 panels of the Compactus units should be removed or a program of daily stack moving should 49
be established. 50
Montanari M. 8
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Frequent visual inspection of materials stored within Compactus shelving units is highly 1 recommended, notwithstanding the presence of a well-functioning HVAC and maintenance of 2 optimal standard climatic values. Micro-environmental anomalies and localised deterioration 3 phenomena, if promptly detected can be easily contained and resolved, while invasive 4 reclamation/rehabilitation of materials should, as far as possible, be avoided and implemented 5
only as a last resort. 6
Further studies have now been established in Italian libraries and in our laboratories for E. 7
halophilicum ecology, metabolic requirements, and damage actually provoked on books. 8
9
5. References 10
Abdel-Hafez, S.I.I., Moubasher, A.H., Barakat, A., 1990. Keratinophilic fungi and other 11
moulds associated with air-dust particles from Egypt. Folia Microbiologica 35, 311-325. 12 13 Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 14
1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search 15 programs. Nucleic Acids Research 25, 3389-3402. 16 17 Andrews, S., Pitt, J.I., 1987. Further studies on the water relations of xerophilic fungi, 18
including some halophiles. Journal of General Microbiology 133, 233-238. 19 20
Christensen, C.M., Papavizas, G.C., Benjamin, C.R., 1959. A new halophilic species of 21 Eurotium. Mycologia 51, 636-640. 22
23 Domsh, K.H., Gams, W., 1993. Compendium of Soil Fungi. Vol I, IHW-Verlag, Eichin. 24 25
Ellis, M.B., 1976. More Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, 28 Kew. 29 30
Gallo, F., Regni, M., 1998. Conditions microclimatiques dans les bibliothèques italiennes. La 31 climatologie dans les archives et les bibliothèques. Actes des Troisièmes Journées sur la 32 Conservation Préventive. Arles, 2-3 décembre 1998, Centre de Conservation du Livre 69-83. 33 34
Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes 35
application to the identification of mycorrhizae and rusts. Molecular Ecology 2,113-118. 36 37 Goldstein, J., Newbury, D., Joy, D., Lyman, C., Echlin, P., Lifshin, E., Sawyer, L., Michael, 38
J., 2003. Scanning Electron Microscopy and X-Ray Microanalysis. Springer Science + 39 Business Media Inc, USA. 40 41 Hocking, A.D., Pitt, J.I., 1988. Two new species of xerophilic fungi and a further record of 42 Eurotium halophilicum. Mycologia 80, 82-88. 43
44 Kowalik, R., 1980. Microbiodeterioration of library materials. Restaurator 4, 99–114. 45
46 Michaelsen, A., Piñar, G., Pinzari, F., 2010. Molecular and microscopical investigation of the 47 microflora inhabiting a deteriorated Italian manuscript dated from the 13
Montanari, M., Pinzari, F., Ricci, M., 2007. Moulds on book stored on compactus shelves: a 1 case study. In: Padfield, T., Borchersen, K., (Eds.), Proceedings of the Conference 2 Copenhagen Museum Microclimates, The National Museum of Denmark, Copenhagen, pp. 3 14-17. 4 5 Nittérus, M., 2000. Fungi in archives and libraries, a literary survey. Restaurator 21, 25–40. 6 7 Peterson, S.W., 2008. Phylogenetic analysis of Aspergillus species using DNA sequences 8 from four loci. Mycologia 100, 205–226. 9
10 Pinzari F., 2011. Microbial ecology of indoor environments: the ecological and applied 11
aspects of microbial contamination in archives, libraries and conservation environments. In: 12 Sabah, A., Abdul-Wahab, Al-Sulaiman (Eds.), Sick Building Syndrome: Public Buildings and 13 Workplaces, Springer-Verlag Berlin Heidelberg, pp. 153-178. 14 15 Pinzari, F., Montanari M., 2011. Mould growth on library materials stored in compactus-type 16 shelving units. In: Sabah, A., Abdul-Wahab Al-Sulaiman (Eds.), Sick Building Syndrome: 17
Public Buildings and Workplaces, Springer-Verlag Berlin Heidelberg, pp. 193-206. 18 19 Pitt, J.I., Hocking, A.D., 1985. Fungi and Food Spoilage. Academic Press, Sydney. 20 21
Samson, R.A., Flannigan, B., Flannigan, M.E., Verhoeff, A.P., Adan, O.C.G., Hoekstra, E.S., 22 1994. Health Implications of Fungi in Indoor Environments. Air Quality Monographs, Vol. 2, 23
Samson, R.A., Lustgraaf, B.V.D., 1978. Aspergillus penicilloides and Eurotium halophilicum 29 association with house-dust mites. Mycopathologia 64, 13-16. 30
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Mandrekar, J.N., Cockerill, F.R., Steckelberg, J.M., Greenleaf, J.F., Patel, R., 2007. 37 Sonication of removed hip and knee prostheses for diagnosis of infection. The New England 38 Journal of Medicine, 357, 654-663. 39 40 Von Arx, J.A., 1981. The genus of fungi sporulating in pure culture. Cramer, Vaduz. 41
42 White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal 43 ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Shinsky, J.J., White, 44 T.J .(Eds.), PCR Protocols: A Guide to Methods and Application,. Academic Press, San 45
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Figure legends 2 3 Fig. 1. Fungal elements on volume binding 4 Fig. 2. White fungal colonies growing on DG18 agar plate (6 cm diameter) after sampling with sterile 5 nitrocellulose membrane 6 Fig. 3. Pure cultures growing in DG18 agar plate (6 cm Ø): front (left) and reverse (right) 7 Fig. 4. Cleistothecia observed in DG18 agar plate (bar = 200 µm) 8 Fig. 5. Optical micrograph on adhesive tape: conidiophore and conidia stained with cotton blue (bar = 10 µm) 9 Fig. 6. Optical micrograph on adhesive tape: Active conidiophore and conidia stained with FDA (bar = 20 µm) 10 Fig. 7. SEM micrograph of fungal elements recovered on adhesive tape: conidia and hyphae (bar = 2 µm ); B: 11 conidial head, conidia and hyphae (bar = 10 µm ) 12 Fig. 8. SEM micrograph of mycelium growing in pure culture on agar medium, showing a portion of an 13 hypertrophic hypha with bare and short hairs (bar = 1 µm). 14 Fig. 9. SEM micrographs fungal growth from pure culture on agar medium. A: ascomata (bar = 100 µm); B: 15 ascospores (bar = 10 µm). 16
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