Mechanism and Control of Black Spot Deterioration on Lacquered Architectural Components of Dajue Temple

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Improve the introduction and discussion with new references on cladosporium in cultural heritage.
2. Add a time and location figure on a map.
3. Increase the resolution and size of the figures.
4. Explain the inhibition halos in the text better.
5. What was the criterion for defining sensitive or resistant?
6. I suggest adding the chemical information of the essential oils to the article and not to the supplementary material.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript submitted by Ai et al investigates microbial black spot deterioration affecting lacquered wooden architectural elements in Dajue Temple (China). The authors combine ATP bioluminescence assays, culture-based fungal isolation, ITS identification, enzymatic assays, and antifungal screening to identify biodeteriogenic fungi and propose conservation strategies. Although the manuscript has scientific merit and the topic would easily reach broader readership, I am against publication in the Journal “Microorganisms” in present form:

Major shortcomings of the Manuscript:

The authors conclude that Cladosporium spp. are the primary agents responsible for black spot deterioration. However, isolation of a dominant culturable fungus does not prove ecological dominance or deterioration responsibility.

No microscopy analyses of deteriorated wooden surface is provided by the authors, and these analyses are very helpful in order to better to understand biodererioration mechanisms.

Identification stops at genus level (Cladosporium spp.), which is problematic because, the genus Cladosporium contains many ecologically distinct species. Many of Cladosporium species are saprobes or plant pathogens and their spores are very common in the air, and are very easily isolated from variety of substrata. Maybe authors isolated just transient organism. But if we have identification to species level maybe we could check if there are other reports of that particular species on deteriorated wooden objects.

Also, for proper molecular identification of Cladosporium species apart from ITS, elongation factor 1α is required al molecular marker.

I am not that convinced that performed enzymatic assays (cellulolytic and ligninolytic activity) demonstrate degradation of lacquer or wood.

No pictures of investigated temple are provided. Also, the manuscript lacks the description of ecological factors present at the temple surroundings (temperature/humidity, light exposure, air flow, human presence). And these informations are kinda must in biodeterioration studies.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses an important and timely issue in the conservation of wooden cultural heritage by investigating microbial-driven black staining on lacquered architectural components, combining microbiological identification with evaluation of potential eco-friendly antifungal treatments.

 

Comments on the Manuscript

Abstract

• (Lines14–20, 21–24) The sentences are overly complex (objective + methodology + results in a single sentence). It is recommended to split them and reduce methodological details.
• Strengthen the formulation of the main findings and practical implications (essential oils and nanoparticles as biocides, monitoring).

 

Introduction (Lines35–88)

• (Lines 35–43) The historical context is overly extensive and should be shortened.
• (Lines 70–75) The claim “first systematic investigation” is additionally supported by references [11,12], which seems contradictory.
• (Lines 83–89) The objectives should be formulated more concisely and in a more experimental-oriented manner.
• Information on the type and condition of the lacquer is missing (g., whether the same type was used across all architectural components, its age, and state of preservation).

 

Results

• (Lines 96–99) ATP (RLU) values are presented without controls/benchmarks. Either add reference values or moderate the interpretation (use “suggests”).
• (Lines 118–119) It is unclear whether other microorganisms were present (why write “dominant”?; only Cladosporium spp. were present?). “Dominant fungus” is not quantified. Provide quantitative data (%, number of isolates, ITS identity).
• (Lines 160–165) Quantitative data are missing, e.g. inhibition zone diameter in mm, mean ± SD.
• Move table S1, Fig. S1 from supplements to the Results part.
• (Lines 169–176) Interpretations (volatility, cost) should be moved to the Discussion.
• A clearer comparative presentation of all tested antifungal agents is recommended. Results should not be unnecessarily separated into supplementary files.

 

Methods

• Sampling details should be added (e.g., sampled area, number of replicates, height from the floor).
• Table S2 should be moved from the Supplementary Materials to the Methods section.
• It should also be clarified whether the data in Table S3 originate from the literature or from the authors’ own experiments.
• Quantification of inhibition zones (in mm) should be included.
• Criteria for effectiveness should be clearly defined (e.g., % inhibition thresholds).

 

Overall Evaluation
Overall, the study is relevant and methodologically sound; however, it requires clarification and refinement in several sections. Improvements are particularly needed in the presentation of quantitative data and in ensuring alignment between the results and their interpretation in the context of existing literature.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

General comments:

The paper investigates pervasive black staining on lacquered wooden components at Dajue Temple, confirming a microbial origin using ATP assays and isolating a dominant Cladosporium strain. Functional tests show the fungus can degrade cellulose and modify lignin, posing a structural threat to lacquer and wood. The authors screen antifungal agents and identify thymol, clove essential oil, and silver‑ion gel as promising, relic‑friendly control options and propose targeted conservation strategies. Overall, the paper offers valuable, actionable insights for heritage professionals and researchers.

In methods there are a lot of key chemicals listed without catalog numbers such as phenol/chloroform, K100 (isothiazolinone), biocides and essential oils (thymol, clove oil, garlic oil, tea tree oil, nano‑silver gel, boric acid, etc.) are described by name and concentration, but for most no catalog number or exact manufacturer is given in the main text. Please correct this throughout the experiment.

Why did the colonisation occure? The authors should give relative humidity and substrate (wood) humidity readings. Its important, if possible, to decrease humidity levels in the insides othervise even if using biocides the re-colonisation may re-occure with years of time.

The study did not test biocides on wood surfaces; antifungal agents were evaluated in vitro on agar plates. Threfore manuscript does not include preliminary compatibility or aging data showing the long‑term effects of these agents on historic lacquer and wood. Biocides may damage wood surfaces. Authors must elaborate on this issue in discussion section and in the abstract then must state thay efects of best biocides on wood surface will be studied in their future research

Move detailed methodological rationale and sampling justification (currently in the latter part of the Introduction where sampling approach and conservation-material parallels are discussed) to Materials and Methods.

Specific commetns:

 

 »Better to write:« To address this unstudied threat, we confirmed the microbial origin of black spots using ATP bioluminescence assays, then characterized microbial communities via culture-dependent methods and ITS sequencing, identifying Cladosporium spp. as the dominant biodeteriogen driving lacquer deterioration. [Abstract]

»Better to write:« Dajue Temple, situated on the eastern slopes of Yangtai Mountain in Beianhe Township, Haidian District, Beijing, traces its origins back to the Tang Dynasty and subsequently underwent successive developments through the Liao, Jin, Yuan, Ming, and Qing dynasties, bearing witness to a rich historical continuum. [Introduction]

»Better to write:« For instance, Pan Jiao’s lab analyzed the microbial communities present on the lacquered surfaces of the Nanhai I shipwreck, while Hala A. M. Afif and colleagues investigated the mold infestation affecting the decorated, gilded wooden boards in the funerary complex of Sultan Al‑Ashraf Qaitbay in Sudan. [Introduction]

»Better to write:« The adenosine triphosphate (ATP) bioluminescence assay was employed to assess microbial activity on the stained wooden surfaces. [Results — In Situ Investigation and Confirmation of Microbial Activity]

»Better to write:« Laboratory cultivation of samples collected from the stained areas yielded predominantly pure cultures with no significant contamination. [Results — Microbial Isolation, Purification, and Identification]

»Better to write:« On CMC agar plates stained with Congo red, distinct clear zones were observed surrounding the fungal colonies. [Results — Assessment of the Wood-Degrading Potential of the Isolated Cladosporium spp.]

»Better to write:« To identify effective antifungal agents against DJSC, plates were inoculated with 106 spores and incubated for four days. [Results — Biocide Susceptibility of Fungal Strains]

»Better to write:« The PCR program consisted of initial denaturation at 98 °C for 3 minutes; followed by 33 cycles of denaturation at 98 °C for 10 s, annealing at 57 °C for 10 s, and extension at 72 °C for 50 s; a final extension at 72 °C for 5 minutes; and holding at 4 °C. [Materials and Methods — Sequencing of Purified Colonies]

»Better to write:« Sampling was conducted within areas of Dajue Temple exhibiting deterioration of the lacquer-painted components. [Materials and Methods — On-site Investigation and Sample Collection]

»Better to write:« The assembled chamber was sterilized by autoclaving at 121 °C for 30 minutes and then dried for subsequent use. [Materials and Methods — Wet Chamber Experiment]

»Better to write:« DNA was precipitated by adding two volumes of pre-chilled anhydrous ethanol and 0.1 volumes of 3 mol/L potassium acetate solution to the supernatant, followed by incubation at −20 °C for 30 minutes. [Materials and Methods — Sequencing of Purified Colonies]

»Better to write:« add reference - A robust body of research has established microbial infestation as a core and long‑term deterioration driver for above‑ground wooden cultural heritage. Kosel et al. Overview of fungal isolates on heritage collections of photographic materials and their biological potency. Journal of Cultural Heritage 2021. [Discussion]

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I express my satisfaction with the incorporated revisions in the manuscript and have no further comments. The article can be published in its current form.

Reviewer 4 Report

Comments and Suggestions for Authors

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