a challenge for mushroom growers: daldinia eschscholtzii

Chiang Mai J. Sci. 2021; 48(3) : 1-13http://epg.science.cmu.ac.th/ejournal/Contributed Paper

A Challenge for Mushroom Growers: Daldinia eschscholtzii Associated with Oyster Mushroom Growing Substrate Achala J. Gajanayake [a,b], Milan C. Samarakoon [a,c], Digvijayini Bundhun [a,b], Ruvishika S. Jayawardena [a,b], Thatsanee Luangharn [a] and Kevin D. Hyde*[a,d][a] Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand.[b] School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand.[c] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.[d] Institute of Plant Health, Zhongkai University of Agriculture and Engineering, Haizhu District, Guangzhou 510225,

People’s Republic of China.

*Author for correspondence; e-mail: [email protected]: 21 November 2020

Revised: 5 March 2021 Accepted: 12 March 2021

ABSTRACT Mushroom growing substrates are important for the quality, quantity and nutritional value of

mushrooms produced. However, the fungal contamination in substrates is known as a major problem in mushroom cultivation. Therefore, accurate identification of contaminants may be important in order to improve mushroom cultivation to increase mushroom yield. Oyster mushroom grow bags in a mushroom farm at northern Thailand were contaminated with a Daldinia species. Contaminated oyster mushroom grow bags were brought to mycology laboratory of Center of Excellence in Fungal Research. Multi-locus phylogenies from combined LSU, ITS, RPB2 and TUB2 sequence data indicated that the contaminant Daldinia species clusters with Daldinia eschscholtzii. In this paper, D. eschscholtzii is described with a full description, color images and a phylogenetic tree of Hypoxylaceae to show the placement of the taxon. This is the first report of D. eschscholtzii associated with oyster mushroom growing substrate.

Keywords: grow bag contamination, host, mushroom growing, Sordariomycetes, taxonomy

1. INTRODUCTION Mushrooms are fleshy, spore bearing fruiting

bodies which can be epigeous or hypogeous in formation [1]. Due to nutritional and therapeutic values, mushrooms have been used for human consumption since ancient times [2,3]. Mushroom industry has expanded globally and especially in Asia where commercial cultivation has greatly increased [3,4].

Oyster (Pleurotus spp.) mushrooms are one of the main commercialized edible mushrooms in the global market [4] especially in Asia. They are

prolific sources of biologically active compounds [5], important functional foods or nutraceuticals [2] and are used in cosmetics [6]. They have anti-cancer, anti-diabetic, anti-obesity, anti-oxidant and hepatoprotective activities [2,7,8]. China, India, Japan, South Korea, Taiwan, Thailand and Vietnam are the main consumers and producers of oyster mushrooms in Asia [4,9]. Mushroom growers usually choose oyster mushroom cultivation due to the easiness of cultivation and increased profitability, as they convert a higher percentage

Chiang Mai J. Sci. 2021; 48(3)2

of substrate to fruiting bodies as compared to other mushrooms [9].

Grow bag substrates determine nutrition, quantity and quality of mushrooms produced [10,11]. Contaminants however, are a major constraint in oyster mushroom production. Fungal contaminants include green mold disease which results in severe losses worldwide [12]. Trichoderma pleuroti and T. pleuroticola are the main causal agents of green mold disease in oyster mushroom cultivation, while other Trichoderma spp. such as, T. atroviride, T. harzianum and T. longibrachiatum have also been detected [12,13]. Aspergillus, Chaetomium, Fusarium, Mucor, Penicillium and Rhizopus species have also been reported in contaminated substrates used for oyster mushroom cultivation [14-16]. Although there are several fungal contaminants in mushroom production, comprehensive identifications with phylogenetics are generally lacking.

Daldinia eschscholtzii (≡ Sphaeria eschscholtzii) is a stromatic xylarialean species in Hypoxylaceae of Xylariales [17]. Daldinia taxa are unique in having turbinate to placentiform stromata and it is a unique feature of all the daldinoid clades in Hypoxylaceae [18-20]. This stromatic structure is adapted to severe environmental conditions and provides shelter to many arthropods [18,21]. The asexual morph is hyphomycetous with conidiogenous structures with a nodulisporium–like branching pattern [18]. Daldinia eschscholtzii is widely distributed in the tropics, occurring more often on woody substrates [18], such as Acacia, Albizia, Citrus, Eucalyptus, Ficus, Pinus [22] and also in mangroves [23], on marine algae [24], skin scrapings, nail clippings, blood [25] and on a mantis insect [26]. Daldinia species have been commonly studied for their capability of producing various secondary metabolites useful and applicable in different industries [18,27]. Stromata of D. eschscholtzii produce binaphthalene derivatives, concentricols, cytochalasins, dalesconols and spirodalesol [18,26-28].

During investigations of fungal species associated with commercial and wild mushrooms, we discovered D. eschscholtzii associated with oyster

mushroom growing substrate in a mushroom farm at northern Thailand. This is the first report of a xylarialean fungal contaminant of oyster mushroom growing substrate.

2. MATERIALS AND METHODS2.1 Fungal Isolation and Morphological Characterization

The contaminated oyster mushroom grow bags with black, powdery, cottony mycelium in the substrate were collected from a mushroom farm in Phayao province, northern Thailand in February, 2020 (Figure 1). Fungal mycelia in grow bags were transferred to potato dextrose agar (PDA) plates using a sterile needle, incubated at 25 °C for five days and pure cultures were obtained by sub culturing. The contaminated bags were incubated in the shade for further observations and a sexual morph was observed in July, 2020 which was growing out from the mushroom grow bags (Figure 1). Pure cultures were again obtained using single spore isolation of ascospores following the method described in [29]. Macro and micro–morphologies were examined following [18]. A mycological colour chart was used to describe colours [30].

Micro–morphological examination was done by using Nikon ECLIPSE 80i compound microscope and photographs were taken with a Canon 750D digital camera fitted to the microscope. Measurements were made with the Tarosoft (R) Image Frame Work program and images used for figures were processed with Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, USA).

Colony characteristics of cultures were observed and measured after three weeks. Herbarium specimens and living cultures were deposited in Mae Fah Luang University (MFLU) Herbarium and Culture Collection of Mae Fah Luang University (MFLUCC) Chiang Rai, Thailand respectively. Faces of Fungi number (FoF) was acquired according to [31].

Chiang Mai J. Sci. 2021; 48(3) 3

Figure 1. Contaminated oyster mushroom grow bags and development of stromata. a. Black, cottony masses of fungal mycelium. b, c, e, f. Stromata on grow bags (shown by arrow heads). d. Initial patches on the substrate prior to stromatal growth (shown by arrow heads). Scale bars: a–c = 5 cm, d–f = 2 cm.

2.2 DNA Extraction, PCR Amplification and Sequencing

Two fungal isolates obtained from the mycelium grown on the substrate (MFLUCC 20–0232) and single spore isolation using ascospores (MFLUCC 20-0233), were maintained on PDA at 25 °C. Total genomic DNA was extracted from 50 to 100 mg of axenic mycelium scraped from four weeks old cultures. Mycelia were ground to a fine powder with liquid nitrogen and fungal DNA was extracted using the OMEGA E.Z.N.A.® Forensic DNA Kit following the manufacturer’s instructions. Four loci, internal transcribed spacer (ITS), partial 28S large subunit (LSU), partial beta–tubulin (TUB2) and partial second largest subunit of the DNA directed RNA polymerase II (RPB2) were

amplified using ITS5/ITS4, LR0R/LR5, T1/T2, fRPB2–5f/fRPB27cR primers respectively. PCR products were obtained according to optimized PCR protocols and were visualized on 1% agarose electrophoresis gels stained with ethidium bromide as described in [32]. Verified PCR fragments were purified and sequenced by Biomed Co. LTD, Beijing, China. Successfully obtained nucleotide sequences were deposited in GenBank.

2.3 Sequence AlignmentThe sequences were subjected to BLAST

search in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The BLAST search results and initial morphological studies revealed that our isolates belong to Hypoxylaceae. Other reference


sequences used in the analyses were obtained from GenBank based on recently published data [18,20,32,33] (Table 1). Single gene alignments were obtained by using MAFFT v. 7.036 with default settings and, refined where necessary in BioEdit v. 7.0.5.2 as described in [34].

2.4 Phylogenetic AnalysesMaximum likelihood (ML) and Bayesian

Inference (BI) analyses were performed for the single and concatenated alignments. The ML trees were generated using RAxML-HPC2 on XSEDE (8.2.8) [35,36] in the CIPRES Science Gateway platform [37]. For the model of evolution GTRGAMMA was used and bootstrap support values were obtained by running 1000 replicates. Bayesian analysis was conducted using MrBayes v. 3.1.2 [38]. Two parallel runs of four simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 100th generation. The first 25% of generated trees representing the burn-in phase of the analyses were discarded and the remaining 75% of trees were used to calculate posterior probabilities (BYPP) in the majority rule consensus tree. Phylograms were visualized with FigTree v1.4.0 program [39] and reorganized in Microsoft power point 2010.

3. RESULTS3.1 Phylogenetic Results

The combined LSU, ITS, RPB2 and TUB2 matrix comprised 79 epithets including selected genera in Hypoxylaceae and three outgroup taxa (Figure 2). Single and concatenated alignments resulted similar topologies for both ML and BI analyses. The value of average standard deviation of the split frequencies of BI analysis was 0.009485. The best scoring RAxML tree is shown in Figure 2 with a final ML optimization likelihood value of –38238.950864. The matrix had 1670 distinct alignment patterns, with 33.46% of undetermined characters or gaps. Estimated base frequencies were as follows: A=0.244750, C=0.248411, G=0.267787, T=0.239051; substitution rates

AC=1.388948, AG=4.391836, AT=1.223794, CG=1.130114, CT=7.088423, GT=1.000000; proportion of invariable sites I=0.455562; gamma distribution shape parameter α=0.848129. Our isolates (MFLUCC 20-0232 and MFLUCC 20-0233) clustered in D. eschscholtzii clade.

3.2 Taxonomic Description Daldinia eschscholtzii (Ehrenb.) Rehm,

Annls mycol. 2(2): 175 (1904) Figures 3 and 4.Facesoffungi Number: FoF02990 Growing on oyster mushroom grow

substrate. Sexual morph: Stromata 1.2-2 × 1-1.5 cm, placentiform to turbinate, rarely depressed–hemispherical, sessile to short-pedicellate, perithecial outlines on surface inconspicuous; dark brick (60), greyish sepia (106), or vinaceous grey (116) when immature, black and smooth on maturity, brown granules underneath the surface, fresh and young stromata producing livid purple (81) or vinaceous purple (101) with KOH-extractable pigments; tissue between perithecia brown, pith like to woody, tissue beneath perithecial layer comprising alternate dark and light zones, darker area made of dark brown, pithy to woody tissue 0.1-0.3 mm thick, lighter area grey to greyish brown, initially soft, becoming hard when dry, persistent, 0.2-0.3 mm thick. Perithecia 0.9-1.2 × 0.3-0.4 mm (

stromata producing livid purple (81) or vinaceous purple (101) with KOH–extractable pigments; tissue between perithecia brown, pith like to woody, tissue beneath perithecial layer comprising alternate dark and light zones, darker area made of dark brown, pithy to woody tissue 0.1–0.3 mm thick, lighter area grey to greyish brown, initially soft, becoming hard when dry, persistent, 0.2–0.3 mm thick. Perithecia 0.9–1.2 × 0.3–0.4 mm (x̅ = 1.05 × 0.35 mm). Asci 96–124 × 5 – 8 µm (x̅ = 110 × 6.5 µm, n = 15), 8–spored, long–pedicellate, amyloid, with discoid apical ring, 2–3 × 0.4–0.6 µm. Ascospores 8.2–12.7 × 3.8–6.1 µm (x̅ = 10.4 × 4.9 µm, n=30), uniseriate, dark brown, unicellular, ellipsoid-inequilaterally with narrowly rounded at the ends, comprising a straight germ slit spore length on convex side; perispore dehiscent in 10% KOH smooth, at times showing conspicuous transverse striations, epispore smooth. Asexual morph: Observed on the ten weeks old culture, hyphomycetous. Conidiophores 2–3 × 0.8–1.2 μm (x̅ = 2.5–1 μm, n = 10), hyaline, mononematous, synonymous, with dichotomous or trichotomous conidiogenous apparatus bearing nodulisporium–like branching pattern, 1–3 conidiogenous cells originating from each end. Conidiogenous cells 2.4–4.5 × 1.8–5 μm (x̅ = 3.45–3.4 μm, n = 10), hyaline, holoblastic, terminal or intercalary, cylindrical, with rounded apices, collaret or opening width. Conidia 3.2–5 × 2.5–4.5 μm ( x̅ = 4.1–3.5 μm, n = 40), hyaline, obovoid to ellipsoid, aseptate, smooth, often with a truncate base. Mycelium 1.5–3.5 µm (x̅ = 2.5 µm) wide, superficial, septate, branched, have melanized hyphae with brownish exudates in old cultures.

Culture characteristics – Colonies on PDA reaching 90 mm diam. with a diffused margin after 14 days at 25 oC, colony circular. Front, initially white, turning into gray with olive green to dull green patches with time; reverse black at the center and whitish gray at the periphery.

Material examined: THAILAND, Phayao province, growing out of the substrate of oyster mushroom grow bags, 10 July 2020, AJ. Gajanayake, AJ 061 (MFLU 21–0021), living cultures (MFLUCC 20–0232) and (MFLUCC 20–0233).

Notes: Daldinia eschscholtzii (MFLU 21–0021, MFLUCC 20–0232 and MFLUCC 20–0233) reported in this study shares similar sexual and asexual morphologies with the epitype of D. eschscholtzii (MBT177380) described in Stadler et al. [18]. The stromata, perithecia, asci and ascospores of MFLU 21–0021 are comparatively smaller than the D. eschscholtzii described in [18] as (1.2–2 × 1–1.5 vs 1–7 × 1–4.5 cm, stromata; 0.9–1.2 × 0.3–0.4 vs 0.9–1.8 × 0.3–0.6 mm, perithecia; 96–124 × 5–8 vs 160–210 × 7–10 µm, asci; 8.2–12.7 × 3.8–6.1 vs (10–)11–13(–14) × 5–6.5 µm, ascospores). These dimensional differences are probably due to the environmental and substrate variations.

4. DISCUSSION

In this study, D. eschscholtzii is reported for the first time associated with oyster mushroom growing substrate in northern Thailand. Usually the growers discard the grow bags due to visual observations such as, black mycelium growth on the substrate and lack of mushroom growth. In our study, we isolated fungal mycelia from blackened grow bags and allowed that mycelium to grow further. With time, stromata were observed on contaminated substrate (Figure 1). Morphological and phylogenetic evidence revealed that the fungus growing on grow bag substrate was D. eschscholtzii.

Previous studies have reported that Daldinia species are commonly found on woody substrates [18,25]. Stadler et al. [18] examined D. eschscholtzii specimens associated with dead rubber tree, while Seephueak [40] reported D. concentrica and D. eschscholtzii from old rubber wood logs in Thailand. Oyster mushroom grow bags used in this study was filled with rubber saw dust, lime, gypsum salts, mushroom supplement, rice bran, molasses and spawn made from sorghum and were sterilized (personal communication with the grower). Daldinia spores or hyphal fragments probably entered the grow bags with rubber sawdust during inoculation of Pleurotus spawn. The other possibility is, bags may not have been properly sterilized. Such occurrence causes fungal

= 1.05 × 0.35 mm). Asci 96-124 × 5-8 µm (





4. DISCUSSION



= 110 × 6.5 µm, n = 15), 8-spored, long-pedicellate, amyloid, with discoid apical ring, 2-3 × 0.4-0.6 µm. Ascospores 8.2-12.7 × 3.8-6.1 µm (





4. DISCUSSION



= 10.4 × 4.9 µm, n=30), uniseriate, dark brown, unicellular, ellipsoid-inequilaterally with narrowly rounded at the ends, comprising a straight germ slit spore length on convex side; perispore dehiscent in 10% KOH, smooth at times showing conspicuous transverse striations, epispore smooth. Asexual morph: Observed on the ten weeks old culture, hyphomycetous. Conidiophores 2-3 × 0.8-1.2 μm (





4. DISCUSSION



= 2.5-1 μm, n = 10), hyaline, mononematous, synonymous, with dichotomous or trichotomous conidiogenous apparatus bearing nodulisporium–like branching pattern, 1-3 conidiogenous cells


Table 1. Names, strain numbers and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses.

Species Strain numberGenBank accession numbers

ITS LSU RPB2 TUB2

Annulohypoxylon michelianum CBS 119993 KX376320 KY610423 KY624234 KX271239

A. moriforme CBS 123579 KX376321 KY610425 KY624289 KX271261

A. nitens MFLUCC 12-0823 KJ934991 KJ934992 KJ934994 KJ934993

A. stygium MUCL 54601 KY610409 KY610475 KY624292 KX271263

A. truncatum CBS 140778 KY610419 KY610419 KY624277 KX376352

Daldinia andina CBS 114736T – KY610430 KY624239 KC977259

D. bambusicola CBS 122872T KY610385 KY610431 KY624241 AY951688

D. brachysperma BCC33676 MN153854 MN153871 – MN172205

D. caldariorum MUCL 49211 AM749934 KY610433 KY624242 KC977282

D. chiangdaoensis BCC 88220T MN153850 MN153867 MN172208 MN172197

D. concentrica CBS 113277 AY616683 KY610434 KY624243 KC977274

D. dennisii CBS 114741T JX658477 KY610435 KY624244 KC977262

D. eschscholtzii MUCL 45435 JX658484 KY610437 KY624246 KC977266

D. eschscholtzii CBS 113042 JX658497 – – –

D. eschscholtzii CBS 113047 AY616684 – – –




D. eschscholtzii CBS 116037(2) JX658499 – – –







D. eschscholtzii KC 1616 JX658496 – – –

D. eschscholtzii KC1699 JX658490 – – –

D. eschscholtzii MUCL 38740 JX658493 – – –





D. eschscholtzii MFLUCC19-0153 MK587661 MK587748 MK625012 MK636691

D. eschscholtzii MFLUCC 20-0232 MW672318 – – MW682336

D. eschscholtzii MFLUCC 20-0233 MW672319 MW672325 – MW682337


Table 1. (Continued).


ITS LSU RPB2 TUB2

D. flavogranulata BCC 89363T MN153856 MN153873 MN172211 MN172200

D. korfii STMA14089T KY204020 – – KY204016

D. kretzschmarioides TBRC 8875 MH938531 MH938540 MK165425 MK165416

D. loculatoides CBS 113279T MH862918 KY610438 KY624247 KX271246

D. macaronesica CBS 113040T KY610398 KY610477 KY624294 KX271266

D. petriniae MUCL 49214T – KY610439 KY624248 KC977261

D. phadaengensis BCC 89349T MN153852 MN153869 MN172206 MN172195

D. placentiformis MUCL 47603 AM749921 KY610440 KY624249 KC977278

D. pyrenaica MUCL 53969T KY610413 – KY624274 KY624312

D. steglichii MUCL 43512T KY610399 KY610479 KY624250 KX271269

D. subvernicosa TBRC 8877 MH938533 MH938542 MK165430 MK165421

D. theissenii CBS 113044T KY610388 KY610441 KY624251 KX271247

D. vernicosa CBS 119316T KY610395 KY610442 KY624252 KC977260

Entonaema liquescens ATCC 46302 KY610389 KY610443 KY624253 KX271248

Graphostroma platystomum CBS 270.87 JX658535 DQ836906 KY624296 HG934108

Hypomontagnella monticulosa BCC58592 MN153864 MN153881 MN172219 MN172204

H. monticulosa MUCL 54604T KY610404 KY610487 KY624305 KX271273

Hypoxylon crocopeplum CBS 119004 KC968907 KY610445 KY624255 KC977268

H. fragiforme MUCL 51264T KC477229 KM186295 KM186296 KX271282

H. fuscum CBS 113049T KY610401 KY610482 KY624299 KX271271

H. griseobrunneum CBS 331.73T KY610402 KY610483 KY624300 KC977303

H. haematostroma MUCL 53301T KC968911 KY610484 KY624301 KC977291

H. hypomiltum MUCL 51845 KY610403 KY610449 KY624302 KX271249

H. investiens CBS 118183 KC968925 KY610450 KY624259 KC977270

H. lateripigmentum MUCL 53304T KC968933 KY610486 KY624304 KC977290

H. papillatum ATCC 58729T KC968919 KY610454 KY624223 KC977258

H. petriniae CBS 114746T KY610405 KY610491 KY624279 KX271274

H. porphyreum CBS 119022 KC968921 KY610456 KY624225 KC977264

H. pulicicidum CBS 122622T JX183075 KY610492 KY624280 JX183072

H. rubiginosum MUCL 52887T KC477232 KY610469 KY624266 KY624311

H. samuelsii MUCL 51843T KC968916 KY610466 KY624269 KC977286

H. trugodes MUCL 54794T KF234422 KY610493 KY624282 KF300548

Jackrogersella minutella CBS 119015 KY610381 KY610424 KY624235 KX271240

J. multiformis CBS 119016T KC477234 KY610473 KY624290 KX271262

Pyrenopolyporus hunteri MUCL 52673 KY610421 KY610472 KY624309 KU159530

P. laminosus TBRC 8871 MH938527 MH938536 MK165424 MK165415


Table 1. (Continued).


ITS LSU RPB2 TUB2

P. nicaraguensis CBS 117739 AM749922 KY610489 KY624307 KC977272

P. symphyon TBRC 8873 MH938529 MH938538 MK165428 MK165419

Rhopalostroma angolense CBS 126414 KY610420 KY610459 KY624228 KX271277

Rostrohypoxylon terebratum CBS 119137T DQ631943 DQ840069 DQ631954 DQ840097

Ruwenzoria pseudoannulata MUCL 51394T KY610406 KY610494 KY624286 KX271278

Thamnomyces dendroidea CBS 123578 FN428831 KY610467 KY624232 KY624313

Xylaria hypoxylon CBS 122620T KY610407 KY610495 KY624231 KX271279

X. polymorpha MUCL 49884 KY610408 KY610464 KY624288 KX271280

The strain numbers of type strains are superscripted with T and newly generated strains are in red bold. Abbreviations: ATCC: American Type Culture Collection, Virginia, USA: CBS: Centraal bureau voor Schimmel cultures, Utrecht, The Netherlands, KC: Kew Culture Collection, United Kingdom, MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand, MUCL: Université Catholique de Louvain, Belgium, STMA: Personal Herbarium and Culture Collection of M. Stadler, TBRC/ BCC: Thailand Bio–resource Research Center.

originating from each end. Conidiogenous cells 2.4-4.5 × 1.8-5 μm (





4. DISCUSSION



= 3.45-3.4 μm, n = 10), hyaline, holoblastic, terminal or intercalary, cylindrical, with rounded apices, collaret or opening width. Conidia 3.2-5 × 2.5-4.5 μm (





4. DISCUSSION



= 4.1-3.5 μm, n = 40), hyaline, obovoid to ellipsoid, aseptate, smooth, often with a truncate base. Mycelium 1.5-3.5 µm (





4. DISCUSSION



= 2.5 µm) wide, superficial, septate, branched, have melanized hyphae with brownish exudates in old cultures.


Material examined: THAILAND, Phayao province, growing out of the substrate of oyster mushroom grow bags, 10 July 2020, AJ. Gajanayake, AJ 061 (MFLU 21-0021), living cultures (MFLUCC 20-0232) and (MFLUCC 20–0233).

Notes: Daldinia eschscholtzii (MFLU 21-0021, MFLUCC 20-0232 and MFLUCC 20-0233) reported in this study shares similar sexual and asexual

morphologies with the epitype of D. eschscholtzii (MBT177380) described in Stadler et al. [18]. The stromata, perithecia, asci and ascospores of MFLU 21-0021 are comparatively smaller than the D. eschscholtzii described in [18] as (1.2-2 × 1-1.5 vs 1-7 × 1-4.5 cm, stromata; 0.9-1.2 × 0.3-0.4 vs 0.9-1.8 × 0.3-0.6 mm, perithecia; 96-124 × 5-8 vs 160-210 × 7-10 µm, asci; 8.2-12.7 × 3.8-6.1 vs (10-)11-13(-14) × 5-6.5 µm, ascospores). These dimensional differences are probably due to the environmental and substrate variations.

4. DISCUSSIONIn this study, D. eschscholtzii is reported for

the first time associated with oyster mushroom growing substrate in northern Thailand. Usually the growers discard the grow bags due to visual observations such as, black mycelium growth on the substrate and lack of mushroom growth. In our study, we isolated fungal mycelia from blackened grow bags and allowed that mycelium to grow further. With time, stromata were observed on contaminated substrate (Figure 1). Morphological and phylogenetic evidence revealed that the fungus


Figure 2. RAxML tree based on the analysis of LSU, ITS, RPB2 and TUB2 combined data set. Bootstrap support values for ML values equal to or >60% and BYPP values equal to or >0.95 are shown as ML/BYPP above the nodes. The isolates used for the present study is shown in red bold. Type strains are indicated in black bold. The tree is rooted using Graphostroma platystomum (CBS 270.87), Xylaria hypoxylon (CBS 122620) and X. polymorpha (MUCL 49884). The scale bar represents the expected number of nucleotide substitutions per site.


Figure 3. Daldinia eschscholtzii (MFLU 21–0021). a. Stromata. b. Stroma in longitudinal section showing internal concentric zones and perithecial layer. c. Close up of internal concentric zones and perithecial layer with ostioles. d-f. Asci. g,h. Apical ring bluing in Melzer’s reagent. i-k. Ascospores (j in 10% KOH, showing dehiscing perispore, k germ slit). l. KOH–extractable pigments. Scale bars: a,b = 1 cm, c = 1 mm, d–f = 50 μm, g,h,k = 10 μm, i,j = 5 μm.


Figure 4. Daldinia eschscholtzii (MFLUCC 20–0232). a,b. Colony on PDA after four weeks. c. Greenish patches on PDA after two weeks. d. Dark patches on PDA after four weeks. e. Sterile stromatic structures on PDA after ten weeks. f. Sporulation of the colony on PDA. g. Conidial attachments and conidiogenous cells showing nodulisporium-like branching pattern. h–l. Conidial attachments and conidiogenous cells. m. Conidia. Scale bars: a,b = 30 mm, c,d,f = 500 μm, e = 1 mm, g,l = 30 μm, h–j = 10 μm, k = 25 μm, m = 4 μm.

Chiang Mai J. Sci. 2021; 48(3) 11

growing on grow bag substrate was D. eschscholtzii. Previous studies have reported that Daldinia species are commonly found on woody substrates [18,25]. Stadler et al. [18] examined D. eschscholtzii specimens associated with dead rubber tree, while Seephueak [40] reported D. concentrica and D. eschscholtzii from old rubber wood logs in Thailand. Oyster mushroom grow bags used in this study was filled with rubber saw dust, lime, gypsum salts, mushroom supplement, rice bran, molasses and spawn made from sorghum and were sterilized (personal communication with the grower). Daldinia spores or hyphal fragments probably entered the grow bags with rubber sawdust during inoculation of Pleurotus spawn. The other possibility is, bags may not have been properly sterilized. Such occurrence causes fungal contamination of grow bags resulting loss of productivity in the mushroom cultivation and is a challenge to overcome. 5. CONCLUSIONS

Selection of quality substrates and following the processing guidelines strictly are important to prevent substrate contaminations in mushroom production. A useful spin–off of this study is, we are now aware that Daldinia can be grown in a similar way of Pleurotus growing. Since Daldinia are high in valuable chemical compounds, this methodology is useful in artificial production of Daldinia and using them in industrial application.

ACKNOWLEDGEMENTSThe authors would like to thank Mae Fah

Luang University grant, “Identification of fungal pathogens on commercial and wild mushroom in northern Thailand and their biological control” (Grant number 631A15002) and the grant “Macrofungi diversity research from the Lancang–Mekong watershed and surrounding areas” (Grant number DGB6280009). Ms. Siriporn Luesuwan is thanked for providing samples and Mr. Phongeun Sysouphanthong for his support.

CONFLICT OF INTEREST STATEMENT The authors declare that there is no conflict

of interest.

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