Preliminary trial of the cultivation of Pleurotus djamor var. fuscopruinosus from southern Thailand using sawdust substrate and applications

doi:10.21203/rs.3.rs-5267286/v1

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Preliminary trial of the cultivation of Pleurotus djamor var. fuscopruinosus from southern Thailand using sawdust substrate and applications

https://doi.org/10.21203/rs.3.rs-5267286/v1

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Pleurotus is an edible mushroom, that has been consumed and cultivated worldwide. Pleurotus djamor var. fuscopruinosus (MFLU24-0016) is a new record in Thailand that provided morphology descriptions with phylogeny analysis and provided valuable information for the possibility of cultivation. The mycelium of P. djamor var. fuscopruinosus strain MFLUCC24-0056 was grown on PDA medium, and sorghum medium showed the best spawn production. Sawdust is suitable for the growing mycelium. The wild strain of P. djamor var. fuscopruinosus produced fruiting bodies at 25–28°C and 70–80% humidity. Primordia formed four weeks after the mycelia fully colonised the substrate and illuminated the conditions for the development of a mature fruiting bodies in 4–5 days. Nutritional analysis of 100 g of dried P. djamor var. fuscopruinosus showed 15.99 ± 0.2% of carbohydrates, 25.00 ± 1.5 (g/100 g) of protein, 30.25 ± 0.5% of moisture, 19.10 ± 0.9% of fibre, 7.59 ± 1.0% of ash, and 2.07 ± 0.5 (g/100 g) of fat content. Antioxidant activities of P. djamor var. fuscopruinosus 50% (v/v), ethyl acetate extract showed the strongest 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity at 694.47 ± 3.92 µg/ml compared to 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) radical cation scavenging activity at 652.92 ± 2.53 µg/ml. In vitro anticancer effects were evaluated using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed the extract of P. djamor var. fuscopruinosus exhibited higher cytotoxicity against lung cancer A549 than colorectal cancer SW480 with IC₅₀ values of 382.03 ± 4.55, and 245.73 ± 7.60 µg/ml, respectively, compared to the positive control doxobrucirin with IC₅₀ values of 14.96 ± 1.58 µg/ml. The exhibition cytotoxicity against RAW 264.7 cells had IC₅₀ values of 213.08 ± 4.08 µg/ml. Antidiabetic properties were investigated. Inhibition of the α-glucosidase enzyme in vitro showed that the hot water extract was higher at 582.91 ± 3.0 µg/ml, followed by ethyl acetate at 473.87 ± 1.4 µg/ml, and less was methanol at 357.63 ± 3.3 µg/ml based on comparison with acarbose (635.70 ± 4.9 µg/ml). Furthermore, 3T3-L1 cells were evaluated, and the glucose consumption assay of this mushroom was shown at 582.91 ± 3.0 µg/ml from hot water extract compared to metformin (IC₅₀ at 99.58 ± 0.59 µg/ml).

Bioactivities

Edible mushroom

Nutritional

Taxonomy

Pleurotoid

Pleurotus belongs to the Pleurotaceae, the genus was identified by Paul Kummer in 1871, and its species has moced to many genera such as Favolaschia, Hohenbuehelia, Lentinus, Marasmiellus, Omphalotus, Panellus, Pleurocybella, and Resupinatus [1]. The identification of Pleurotus based on the morphological characteristics of the basidiomata is complex, such as P. djamor (Rumph. ex Fr.) Boedijn [2], P. eryngii (DC.) Quél. (Zervakis et al. 2001), P. ostreatus (Jacq.ex Fr.) P. Kumm [3], P. ostreatus-sapidus [4] and P. pulmonarius (Fr.) Quél. [5]. Before the taxonomy of Pleurotus species was because of their wide distributions woideworld, and one species was used for multiple names [6]. Later, the combination of both morphological studies and molecular phylogenetic analysis is providing greater results to understand classification of Pleurotus species [2]. There are 758 taxon names listed under Pleurotus in the Index Fungorum (http://www.indexfungorum.org/), but only 25 species were accepted [7]. Pleurotus ostreatus (Jacq.) P. Kumm. is the type species that is to be remembered as edible mushrooms and the most famous species for cultivation worldwide. The main morphological characteristics of Pleurotus are defined by pleurotoid basidiomata, decurrent lamellulae, smooth and elongated to cylindrical basidiospore shapes, dimitic hyphal system, with skeletal hyphae and generative hyphae, and present clamp connections [8, 9]. Pleurotus species are mostly edible and found in both tropical and temperate regions[10], and most species of Pleurotus cause white rot in decayed and hard deadwood [10–12].

The Pleurotus species were considered famous for healthy foods because they have a high nutrient content, such as proteins, carbohydrates, fibres, minerals, and vitamins[13, 14]. There are several important elements included in cultivations, food applications, and the value of medicine [15, 16]. Pleurotus species have several bioactivities such as anticancer, antimicrobial, antiviral, antineoplastic, antioxidant, anti-inflammatory, anti-ageing, anti-tumour, antimutagenic, antilipidemic, hepatoprotective, hyperglycemic, and hypotensive properties [16]. For instance; Pleurotus species was able to inhibit Gramme-negative bacteria such as Vibrio cholera, Escherichia coli, and Pseudomonas sp.; and Pleurotus species were ranked as the second world mushroom market for consumption and industry worldwide[17]. The cultivation of Pleurotus using many agricultural and forest wastes is preferred. It is easy to cultivate using low cost from most of all hardwoods, wood by-products, and agricultural waste [18–20]. In Thailand, eight species of Pleurotus are listed [21], and many species were cultivated such as P. citrinopileatus, P. djamor, P. eryngii, P. floridanus, P. giganteus, P. ostreatus var. columbinus, P. ostreatus, and P. Pulmanarius [19, 22, 23]. Pleurotus sirindhorniae was a new member of Pleurotus that was discovered in northern Thailand [24]. As mentioned above, most species of Pleurotus are suitable for human consumption. Therefore, studying its distribution and cultivation is useful in ensuring source stability for commercial use. This study aims to assign isolates to the new record of Pleurotus using morphology and rDNA sequences. Description, illustrations, and molecular phylogenetic analyses of a multigene DNA sequence data set (ITS and LSU) were provided, the first cultivation was investigated, and its biological activitie are tested.

Collecting and Isolation

Two collections of Pleurotus djamor var. fuscopruinosus were collected from Nakhone Si Thammarat Province, southern Thailand. Pure cultures were isolated from fresh basidiomata by cutting tissue context using sterile forceps plated on Petri dishes with potato dextrose agar (PDA) and incubated at room temperature for 2 weeks. Pure cultures were deposited at Mae Fah Luang University.

Morphology Study

Macromorphology notes were followed by Largent (1986), and the colour notes of the mushrooms were from Kornerup and Wanscher (1978). Fresh basidiomata were dried in a hot air dryer at 40–50°C for 24 hours or until the samples were completely dried and kept in zip-locked plastic bags to deposit the Herbarium of Mae Fah Luang University (MFLU). The micromorphology was examined from dry specimens using standard procedures and terms Vellinga (2011). The examination was carried out with chemical reactions of water, 3–6% KOH, and Congo red. All microcharacters were photographed, measured, and drawn using a compound microscope Nikon Eclipse Ni. 25 basidiospores were measured from the inside view. The notation [50, 3, 3] indicated that the measurement was made on 50 basidiospores in three samples from two collections. At least 25 basidia, cheilocystidia, terminal cells, and pileipellis hyphae were measured per collection of each species. The dimensions of the microscopic structures are given as follows: (a–)b–c–d(− e), in which “c” represents the average, “b” the fifth percentile, “d” the 95th percentile and the minimum and maximum values “a” and “e” are shown in parentheses. “Q” the length/width ratio of the spores in the inside view was calculated. The dried fruiting bodies were deposited in the Herbarium of Mae Fah Luang University. A record was added to the GMS mushrooms database [28].

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from dry specimens using the Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Ltd., Hangzhou), by following the manufacturer’s instructions. The ITS and nrLSU regions were amplified by polymerase chain reaction (PCR). For amplification of ITS, the primers ITS1-F and ITS4 [29, 30], the nrLSU with primers LR0R and LR5 [30, 31]. PCR amplification, purification and sequencing of ITS and LSU was used using PCR primers by Shanghai sangon Biological Engineering Technology & Services Co., Ltd. The PCR cycle for ITS and LSU was set up: 3 mins at 94°C; 35 cycles of 30 s at 94°C, 40–50 s at 50–55°C, 1 min at 72 C; 10mins at 72°C.

Sequence alignment and phylogenetic analysis

Sequences of Pleurotus species were checked using Bioedit Sequence Alignment Editor version 7.0.9.0, and contigs were assembled using SeqMan (DNAstar, Madison, WI, USA). The quality of each sequence of Pleurotus species was checked and compared in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) to verify similarity with Pleurotus sequences from other countries. The reference sequences of GenBank consisting of an outgroup are indicated in Table 1. The ITS and nrLSU datasets were aligned using MAFFT v.7 [32] in online access (http://mafft.cbrc.jp/alignment/server/), and the alignment data sets were performed in TrimAl to eliminate ambiguously aligned positions [33].

The combined data set of ITS-LSU were analysed in Bayesian inference (BI) and Maximum likelihood (ML) analyses. All analyses were done on the web server CIPRES science Gateway version 3.3 [34] online (https://www.phylo.org/). Maximum likelihood (ML) phylogenetic tree inference was performed using RAxML-HPC2 on XSEDE version 8.2.12 [35]. The dataset of two gene regions was analysed in a single analysis (one for each gene) and combined. GTR + I + G for ITS. BI analyses were selected the best model from jModelTest2 in XSEDE version 2.1.6 [36]. BI analyses were performed in MrBayes on XSEDE version 3.2.7a [37]. Two runs of five simultaneous MCMC chains will be run for 1,000,000 generations with trees and parameters sampled every 1,000th generation, for a total of 10,000 samples. The remaining samples will be used to calculate posterior probabilities (PP) and the majority rule consensus tree. Trees will be viewed using FigTree v1.4.0. The new sequences were deposited in GenBank.

Table 1

The GenBank accession numbers and geographical origins of the taxa were used in the phylogenetic analysis.
Species names	Specimen/culture	Locality	GenBank Accession Numbers
Species names	Specimen/culture	Locality	ITS	nLSU
Pleurotus abalonus	HKAS81197	China	MN546043	–
P. abieticola	HKAS46100	China	KP771695	KP867909
P. australis	ICMP 21585	New Zealand	MH395977	MH396002
P. calyptratus	P67	South Korea	KY962483	KY963067
P. calyptratus as P. djamor f. calyptratus	HMAS 63355	–	AY562495	AY562496
P. citrinopileatus	HKAS85965	China	KP867920	KP867911
P. cornucopiae	CBS 283.37	–	MH855911	MH867415
P. cystidiosus	IFO30607	Japan	AY315778	–
P. djamor	FUM-085	Iraq	KY951474	–
P. djamor	CC055	Mexico	KX573926	–
P. djamor	1092014J	Pakistan	KX056435	–
P. djamor	rxsbn-473	China	MW374226	–
P. djamor	SCK21	Thailand	MK026938	–
P. djamor	Brazil	MBsn	KF280325	–
P. djamor var. djamor	ABM464683	Malaysia	KC582635	–
P. djamor var. fuscopruinosus	MFLU24-0015	Thailand	PP192013	PP192011
P. djamor var. fuscopruinosus	MFLU24-0016	Thailand	PP192014	PP192012
P. djamor var. roseus	ABM1049204	–	KC582640	–
P. dryinus	HKAS94448	Finland	MN546046	–
P. eryngii	CCMSSC00480	China	KX836350	–
P. euosmus	CBS 307.29	China	EU424298	–
P. ferulaginis	HIK133	Italy	KF743826	HM998795
P. flabellatus	P35	South Korea	KY962451	KY963035
P. flabellatus	ACCC51447	China	EU424303	–
P. fossulatus	P146	South Korea	MG282485	MG282545
P. fuscosquamulosus	LGAMP50	Greece	AY315789	–
P. giganteus	MFLU14-0637	Thailand	KP135559	–
P. levis	DPL6135	USA	KP026244	–
P. nebrodensis	Italy UPA6	UPA6	HM998816	–
P. ostreatus	TENN 53662	Austria	AY854077	AY645052
P. opuntiae	MA-PO7	Mexico	MK757594	–
P. ostreatoroseus	P94	South Korea	MG282434	–
P. parsonsiae	ICMP 18169	New Zealand	MH395975	MH396000
P. placentodes	HKAS51745	China	KR827693	KR827695
P. populinus	P70	South Korea	KY962486	KY963070
P. pulmonarius	HKAS86009	China	KP867918	KP867906
P. purpureo-olivaceus	ICMP 20713	New Zealand	MH395976	MH396001
P. salmoneostramineus	P60	South Korea	KY962476	KY963060
P. smithii	IE74	Mexico	AY315779	–
P. tuber-regium	CBS 850.95	Nigeria	MH862563	MH874190
P. tuoliensis	CCMSSC03105	Xinjiang, China	KU612906	–
Hohenbuehelia atrocoerulea	AMB 18080	Hungary	KU355304	KU355389
H. petaloides	AMB 18088	Italy	KU355346	KU355402

Preliminary cultivation trial

Effect of grain media for spawn production

The six different grains were used to find the best spawn production: Hordeum vulgare (barley), Zea mays (corn), Oryza sativa (paddy rice), Pennisetum glaucum (L.) R. Br. (millet), Oryza sativa L. (rice berry), Sorghum bicolor (sorghum). Each spawn grain will be washed and soaked overnight, the water will be drained, the mixture will boil for 10–15 minutes and leave to cool. The grains were placed in 30 × 140 mm test tubes to approximately 120 cm depth, autoclaved at 121°C for 15 minutes, and left to cool. Tubes containing the same grain length were inoculated with five mycelial plugs of approximately 5 mm diam. from the mycelia colony on PDA plates under aseptic conditions and test tubes were incubated at 28 \(\:\text{℃}\:\)under dark conditions. The length of the linear mycelium was measured every 2 days for 10 days to calculate the growth rate. All analyses were carried out in five replicates.

Effect of different agricultural wastes on mycelium growth

Ten agricultural wastes including banana leaves, sugarcane bagasse, coconuts sell fibre, corn cob, coffee ground, corn husks, mixed leaves, pine apple, rice husks, rice straw, sawdust, substrate material was mixed with 9% rice bran, 1% sugar, 1% calcium carbonate, 0.03% ammonium chloride, 0.03% magnesium sulfate and 0.036% monopotassium phosphate. Mix the substrate with the supplements and fill with distilled water. Each Petri dish was placed with 20 g of a mixed substrate with 5 replications per substrate. Sterilised in an autoclave at 121 ° C for 1 hour and left to cool, the Petri dish of each substrate was inoculated with a 10 mm mycelium disc under aseptic conditions and incubated at 28 ºC under dark conditions. Diameter measurements for mycelium proliferation every 2 days for 10 days. The protocol follows [38, 39].

Fruiting test

The best substrate from above was selected for the fruiting test. Rubber sawdust was used as the main substrate mixed (w/w) with 9% rice bran, 1% sugar, 1% calcium carbonate, 0.03% ammonium chloride, 0.03% magnesium sulfate, and 0.036% monopotassium phosphate [39]. All substrate supplements were mixed manually with 70% moisture. The mixture (800 g) was packed into polypropylene bags and then capped with a plastic ring and lid. Sawdust bags were sterilised at 121 ° C for 1 hour. After the temperature cooled, the spawns were inoculated in the sawdust bags and, appropriately, 50 g of mushroom spawn was inoculated in the sawdust bags under aseptic conditions. The bags were incubated in darkness at 25 ± 1°C, and 60–75% relative humidity. All analyses were carried out from five replicates. After the mycelial had completely colonised the bag substrates, they were removed from the shelves and opened at the ends. The bags were kept at the mushroom house with 70 to 80% humidity. Watering was carried out two times a day using a tap water sprayer until the fruiting bodies had fully developed.

Statistics Analysis

The mycelia growth rate in the spawning production and the substrate test of the mushroom strains were determined, and the data analysis was statistically in terms of the variance of means using Tukey’s test with a significance of P < 0.05. Yield data and biological efficiency (B.E.) were recorded. Yield data mean the total weight of fresh mushroom per kilogramme of substrate [40, 41], biological efficiency (B.E.) means the weight of harvest/weight of dry substrate) x 100% [42, 43].

Determination of the nutritional content

The basidiomata of P. djamor var. fuscopruinosus were the proximate analysis. Fresh basidiomata were dried at 50ºC for 24 hours or until completely dried and powdered using a blender. Proximate value was optioned on the ash, carbohydrate, fat, fibre, moisture, and protein. The determination of ash, fat, fibre, and moisture was followed by the AOAC (Assoc. of Analytical Chemistry). The protein content of the mushroom samples was determined using the Kjeldahl method following [44] and the conversion factor used for nitrogen to protein was 6.25. Carbohydrate was calculated by following equation [45]:

Carbohydrate (g/100 g sample) = 100- \(\:\frac{\:(\text{m}\text{o}\text{i}\text{s}\text{t}\text{u}\text{r}\text{e}+\text{f}\text{a}\text{t}+\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}+\text{a}\text{s}\text{h}+\text{c}\text{r}\text{u}\text{d}\text{e}\:\text{f}\text{i}\text{b}\text{e}\text{r})\:\text{g}}{100\:\text{g}}\)

Crude Extraction

Mycelium of P. djamor var. fuscopruinosus was subcultured on potato dextrose agar using agar plugs and grown in 100 plates and incubated at 28 ºC for 4 weeks or until the mycelium was covered with plate media. Agar plates with mycelium cultures were soaked with 30 mL HPLC grade ethyl acetate (EtOAc, CH₃ − COO − CH₂ − CH₃), methanol (CH₃OH) using a homogeniser (Ultra-turraxP®), and water (H₂O). The process was carried out in three replicates. The mycelium of our mushroom strains (mycelium grown on media) were soaking three days for the first extraction of each solvent. The liquid layer and the solid layer were separated. The solid layer was added solvents again and the mixture was soaked for three days each time for the second and third extractions. Subsequently, the solid layer was discarded. The liquid layers (EtOAc extracts) were pulled together into the preweighed vial. Then it was air-dried to let the liquid component evaporate. The remaining solid (powdery) component is the crude extract. This will be used for further laboratory analyses.

Antioxidant Assay

2,2-Diphenyl-1-picrylhydrazyl radical-scavenging activity (DPPH)

The DPPH radicals were followed by the protocol of [46]. 4 mL of each sample with various concentrations mixed with 1 mL of a methanolic solution containing DPPH radicals, resulting in the final concentration of DPPH being 0.2 mM. The mixture was shanked and left for 30 min and the absorbance was measured at 517 nm. Ascorbic acid and Troxol were used as a positive control. The amount of sample necessary to decrease the absorbance of DPPH by 50% (IC₅₀) was calculated graphically. The IC₅₀ was calculated.

The percentage inhibition was calculated as follows:

The formula= (A0-A1)/A0] × 100.

Notes = A0 is the absorbance of the control and A1 is the absorbance of the samples (Sudha et al. 2012).

2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) radical cation scavenging activity (ABTS)

The ABTS followed the protocol of [47]. The radicals ABTS produced by the reaction between 7 mM, ABTS in water and 2.45 mM potassium persulfate, incubated in the dark at room temperature for 12 h. Before use, the solution was diluted with ethanol to obtain an absorbance of 0.700 ± 0.025 at 734 nm. 10 µL of the sample with 1.0 mL of ABTS working standard in a microcuvette. Ascorbic acid and Troxol were used as a positive control. The IC₅₀ was calculated. The amount of sample necessary to decrease the absorbance of ABTS by 50% (IC₅₀) was calculated graphically. The percentage inhibition was calculated as follows:

The formula= [(A0-A1)/A0] ×100.

Notes = A0 is the absorbance of the control, and A1 is the absorbance of the sample (Sudha et al. 2012).

Antidiabetic Assay

α-glucosidase Assay

The α-glucosidase inhibitory assay procedure followed a previously described method [48] with modifications. The sample solutions at 200 µm/ml were dissolved with 100% dimethyl sulfoxide (DMSO) in phosphate buffer (pH 6.8), and 50 µl of each sample was pipetted and mixed with 100 µl of alpha-glucosidase enzyme (0.35 unit/ml) in vitro. After pre-incubation at 37 \(\:℃\) for 10 min, 100 µl of 1.5 mM p-NPG was added and the sample was further incubated at 37 \(\:℃\) for 20 min, then 1000 µl of Na₂CO₃ (1M) was added to terminate the reaction. Acarbose was used as a positive control. The absorbance was measured at 405 nm with a microplate reader (PerkinElmer, lnc., USA).

Glucose Consumption Assay

The cell line 3T3-L1 was used for the glucose consumption assay. 3T3-L1 cells were seeded at a density of 4 × 105 cells per well in 96-well plates in 100 µL of culture medium and left to adhere for 24 hours or until cells were growing more than 80% in an incubator (5% CO2, 37°C) before the test. The crude extract of P. djamor var. fuscopruinosus was dissolved in the appropriate solvent (10% DMSO) and 100 µL of extract was added to 96-well plates and left for 24 h. Another 96-well plate was transfed 10 µL of supernatant from each well of the previous prepared plate, and 40 µL of enzymes (GOD (enzyme 25Kv) + POD (enzyme 10kv) + Buffer). The absorbance at 510 nm was measured using a microplate reader (INFINITE M NANO). The difference concentration of glucose (4,2,1,0.5 and 0.25 mg/mL) was prepared for standard. 100 µL of 0.5% MTT was added to 96 well plates incubated for 4 h. Final added sterile DMSO 100 µL. The absorbance at 570 nm was measured. Cytotoxicity was present as the concentration of extracts that inhibit cell growth by 50% (IC₅₀).

Anticancer Assay

Lung cancer cells (A549) and colorectal cancer cells (WS480) were used for the anticancer assay. Cytotoxicity procedures against cancer cells [49] and the resazurin reduction test on 96-well tissue culture plates were followed. Cancer cells were weighed at a density of 4 × 10⁵ cells per well in 96-well plates in 100 µL of culture medium and left to adhere for 24 h or until cell growth increased by more than 80% in the incubator (5% CO₂, 37°C) before the test and the crude extract of P. djamor var. fuscopruinosus strain MFLUCC24-0056 was dissolved in the appropriate solvent (10% DMSO) and 100 µL of extract was added to 96-well plates and left to adhere for 24 h. Then 100 µL of 0.5% MTT was added to 96 well plates incubated for 4 h. Final added sterile DMSO 100 µL. The absorbance at 570 nm was measured using a microplate reader (INFINITE M NANO). Cytotoxicity was present as the concentration of extracts that inhibit cell growth by 50% (IC50). IC₅₀ was calculated from a different amount of the trend line of the concentration. In each case, an appropriate number of concentrations was used.

Cytotoxicity Assay

Raw cells (264.7) were used for the cytotoxicity cytotoxicity test of the extract of P. djamor var. fuscopruinosus was done by the reduction of 3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) to formazan. 100 µL of raw cells were seeded in a 96-well plate of 4 × 10⁵ cells per well and left for adhering 24 hours or until the cells grow more than 80% in the incubator (5% CO₂, 37°C) before being exposed to the mushroom extract, and the mushroom extract was dissolved in the appropriate solvent (10% DMSO) was added 100 µL of extract was added to the 96-well plates and left for 24 hours. 100 µL of 0.5% MTT was added to 96 well plates incubated for 4 h. Final added sterile DMSO 100 µL. The absorbance at 570 nm was measured using a microplate reader (INFINITE M NANO). Cytotoxicity was present as the concentration of extracts that inhibit cell growth by 50% (IC₅₀). The IC₅₀ was calculated from a different amount of trend line of the concentration. In each case, an appropriate number of concentrations were used.

Identification

Phylogenetic Analysis

There are 43 accessions, including 41 representative species of Pleurotus and two species of Hohenbuehelia for the outgroup (Table 1). The combined dataset of ITS + nrLSU consisted of the final alignment was 1,640 characters long including gaps. The Maximum Likelihood (RAxML) analysis is provided in Fig. 1. The Bayesian and ML analyses resulted in similar tree topologies; thus, only the ML tree is shown with both maximum likelihood bootstrap (BS) values and Bayesian posterior probabilities (PP). The phylogenetic tree showed that the clade of P. djamor verities is clustered including synonym species such as P. salmoneostramineus Lj.N. Vassiljeva, P. flabellatus Sacc., P. ostreatoroseus Singer, P. Opuntiae (Durieu & Lév) Sacc. Thai specimens, P. djamor var. fuscopruinosus (MFU24-0015, MFU24-0016), also grouped with other varieties of P. djamor with high bootstrap support (BS 93%, PP 0.98%).

Taxonomy

Pleurotus djamor var. fuscopruinosus Corner.

Faces of Fungi: FoF 15389, Index Fungorum number: IF 117633, Figs. 2.

Pileus 35–80 mm diam., rounded flabelliform to subreniform; greyish to white (colour code), greyish-brown (5B1-5D3), turned pale to white or whitish with age, greyish (5D3) fuliginneo-pruinosu at the base of the proximal part; surface when young covered with fibrillose toward the margin becoming disappear when mature; margin inflexed to straight, sometimes wavy and split when fully mature. Lamellae decurrent, narrow, 0.5–2 mm wide, crowded, with more than 5 lamellulae lengths, white,with smooth concolorous edge. Stipe 10–15 × 10–20 mm, short or nearly absent, eccentric to lateral, smooth, white to pale red (7A3). Context white, and 1–2.5 mm thick in pileus, softly. Taste and odour not recorded. Spore print white to pinkish (9A1, 9A2).

Basidiospores [50, 3, 3] (5.4–)6.0–7–8.0(–8.9) × (3.1–)3.0–4–4.5(–4.8) µm, Q = (1.2–)1.44–1.78–2.18(–2.52), broadly ellipsoid to cylindrical, hyaline, smooth, thin-walled. Basidia (17.9–)18–21–25.2(–25.3) × (4.8–)4.8–5.7–6.7(–6.7) µm, clavate, with 4 sterigmata, hyaline, thin-walled. Cheilocystidia (11.5–)11.9–22.7–31.7(–33.5) × (6.1–)6.4–9.9–12.2(–12.3) µm, short clavate to clavate, thin-walled with brownish walls. Pleurocystidia absent. Hyphal system dimitic similar in the stipe context and pileus context; Skeletal hyphae 5–7 µm wide, very thick-walled, hyaline, septate, elements with 2 to 3 branches; generative hyphae 5–6 µm wide, cylindrical, slightly thick-walled, hyaline. Pileipellis presents a cutis composed, hyphae 2–5 µm wide, slightly thick, some encrusted with annular-like in KOH, brownish walls; clavate to cylindrical terminal elements 17–56 × 5–8 µm, brownish walls; minute fascicles present at the ends of cells and more fascicles as observed at the margin of the pileus. Stipitipellis not observed. Clamp connections present in all observed tissue.

Habitat and distribution

Saprobic on dead decaying wood, growing in small groups of two-four basidiomes.

Materials examined

THAILAND, Nakhone Si Thammarat province, Lan Sa Ka districts, 10 January 2020, Monthien Phonemany (MFLU24-0015). ibidem, Lan Saka district, Nakhon Si Thammarat province, collected on 11 January 2020, Monthien phonemany (MFLU24-0016).

Notes

Five species from different geographical regions have been reported that have salmon-pinkish-coloured basidiomata, such as P. djamor (Rumph. ex Fr.) Boedijn, P. ëous (Berk.) Sacc., P. flabellatus (Berk. and Broome) Sacc., P. ostreatoroseus Singer, P. salmoneostramineus Lj.N.Vassiljeva [8, 50–54]. These species are grouped in the tree topologies in Fig. 1. Pleurotus parsonsiae also belongs to the same clade as P. djamor, but its morphology is totally different from P. djamor because it has the size of (pileus 7–12 cm), flesh creamy white flesh, longer stipe (8 × 8 mm), lager basidiospores (9–1 × 4–4.5 µm) [55].

Pleurotus djamor (Rumph. ex Fr.) Boedijn has been identified with several synonyms and was published, but there are only 9 taxon names listed on the index fungroum. Corner [8] devised P. djamor into six varieties by their characters such as pileus, lamellae colour, the shape of stipe, spore, and substrate. Comparison of P. djamor varieties by Corner [8] found that our specimen was different from that of P. djamor var. cyathiformis Corner has a white to pallid ochraceous, lamellae edge not draker, a larger stipe (30–40 × 7–8 mm) cyathiform and smaller basidiospres; Pleurotus djamor var. djamor (Rumph. ex Fr.) Boedijn has almost lateral imbricate, terricolor, merismatiod, and smaller besidiospores (5.5–6.5 × 2.5×3 µm); Pleurotus djamor var. terricolus has nearly lateral imbricate, lignicolour, not merismatiod; Pleurotus djamor var. fuscoroseus Corner and Pleurotus djamor var. roseus Corner has pink basidiomata, gill pink. Thai specimens (MFLU24-0015 and MFLU24-0016) characteristic of flabelliform greyish to white pileus, greyish fuliginneo-pruinosu at the base of the proximal, cheilocysidia clavate with brownish walls, pileipellis hyphae encrusted with annular-like and minute fascicles present at the end cells. Thai specimen fits well with P. djamor var. fuscopruinosus was originally described from Malaysia, but it has larger cheilocystidia 45 × 5–14 µm [8]. Furthermore, in the phylogenetic tree, MFLU24-0015 and MFLU24-0016 were grouped in the clade of P. djamor with high bootstraps (BS 93%, PP 0.98%). The type species P. djamor var. fuscopruinosus was reported without molecular analysis, but based on its morphological characteristics, there were differences from other varieties of P. djamor. As mentioned above, we confirmed that the Thai specimen should be P. djamor var. fuscopruinosus. However, this variety indicates a more complicated ancestry of P. djamor with sterile hymenium on the upper side of the pileus, or these two varieties may represent intermediate forms of them

Preliminary cultivation trial

Effect of grain media for spawn production

After Ten days of incubation, the mycelium Pleurotus djamor var. fuscopruinosus growth on different cereal grains and is shown in Table 2 and Fig. 3. Sorghum bicolor had the fastest growth rate (11.58 ± 0.51), followed by paddy rice (9.16 ± 0.97), barley rice (8.02 ± 0.92) respectively. Corn had the slowest growth rate (5.96 ± 0.82^d), followed by Millet (6.96 ± 0.34), Rice berry (6.1 ± 0.68) respectively.

Table 2

Effect of cereal grains on mycelium growth of *P. djamor* var. *fuscopruinosus* in 10 days.
Grain Types	Mycelial growth rate (cm)	Mycelium Density
Barley rice	8.02 ± 0.92^c	Compact
Corn	5.96 ± 0.82^d	Compact
Millet	6.96 ± 0.34^d	Compact
Paddy rice	9.16 ± 0.97^b	Thin
Rice berry	6.1 ± 0.68^d	Lightly thin
Sorghum	11.58 ± 0.51^a	Compact

Effect of different agricultural wastes on mycelium growth

The tested substrates significantly influenced the diameter of the mycelial growth of P. djamor var. fuscopruinosus. After ten days of incubation, the growth of mycelium on various substrates was investigated and the analysis shown in Table 3 and Fig. 4. The highest diameter of the mycelial colony was found in sawdust (4.37 ± 0.18) and coconut fibre (4.05 ± 0.10), followed by rice straws (3.17 ± 0.46). The lower diameters are rice husks, corn husks, and pineapple, with non-significant differences, respectively. The lowest diameter was found in banana leaves, bagasse, and corncob, with non-significant differences, respectively.

Table 3

Effect of different sources of agricultural waste on mycelium growth.
Substrate sources	Mycelium colony diameter (cm)	Mycelium Density
Banana leaves	2.58 ± 0.46^b,c,d	Compacted
Bagasse	2.58 ± 0.11^b,c,d	Compacted
Corn cob	2.58 ± 0.37^c,d	Compacted
Corn husks	2.74 ± 0.53^b,c,d	Lightly compacted
Coconut fiber	4.05 ± 0.10^a	Thin
Mix leaves	2.41 ± 0.32^e	Lightly thin
Pineapple	2.57 ± 0.25^c,d	Compacted
Sawdust	4.37 ± 0.18^a	Compacted
Rice husks	2.95 ± 0.35^b,c	Lightly thin
Rice straw	3.17 ± 0.46^b	Lightly compacted

Fruiting test

The primordia of P. djamor var. fuscopruinosus were produced after an open bag 5–10 days at 25–28°C with 70–80% humidity and illuminated conditions. Then the mature mushroom developed in 4 to 5 days (Fig. 5). Production was harvested and weighed daily. In fruiting trials of P. djamor var. fuscopruinosus, the maximum of the average wet weight is 52.29 ± 16.61 g. The overall yield is 12.19 ± 4.78 g. The biological efficiency is 13.56 ± 4.49%. There are 116 fruiting bodies; on average, pileus size is 30–150 × 10–80 mm, and the stipe is 4–21 × 10–56 mm, which the size decreases with time (Table 4).

Table 4

Harvesting results of *P. djamor* var. *fuscopruinosus* for 30 days.
Replication	R 1	R 2	R 3	R 4	R 5	Overall/average
Average wet weight (g)	49.96 ± 0.65	37.9 ± 12.83	72.06 ± 52.54	66.51 ± 11.15	35.01 ± 17.48	52.29 ± 16.61
Yield data (g)	9.99	7.58	18.01	16.62	8.75	12.19 ± 4.78
Biological efficiency (%)	12.49	10.1	18.96	17.5	8.75	13.56 ± 4.49
Number of fruiting bodies	24	17	37	24	17	116
Average pileus size (w × l mm)	40–150 × 20–80	40–85 × 20–55	30–100 × 10–80	40–100 × 16–28	30–115 × 20–74	30–150 × 10–80
Average stipe size (w × l mm)	6–20 × 20–50	4–20 × 20–53	4–21 × 20–56	4–15 × 20–48	4–20 × 10–50	4–21 × 10–56

R = means replication, w = means width, l = means length.

Nutritional analysis

The proximate value obtained from P. djamor var. fuscopruinosus is shown in Table 5. The ash content is recorded at 7.59 ± 1.0 g/100 g, the carbohydrate content is 15.99 ± 0.2%, the fat content is recorded at 2.07 ± 0.5%, the fibre content is 19.10 ± 0.9%, the moisture content is 30.25 ± 0.5 g/100 g, the protein content is 25.00 ± 1.5 g/100 g.

Table 5

The proximate composition of *P. djamor* var. *fuscopruinosus* cultivated in rubber sawdust.
Nutritional Composition	Proximate Value
Ash (%)	7.59 ± 1.0
Carbohydrates (%)	15.99 ± 0.2
Crude fibre (%)	19.10 ± 0.9
Fat (g/100 g DW)	2.07 ± 0.5
Protein (g of/100 g DW)	25.00 ± 1.5
Moisture (g/100 g FW)	30.25 ± 0.5

Antioxidant

Three different extractions, namely ethyl acetate (C₄H₈O₂), methanol (MeOH) and hot water extract (H₂O) from P. dajmor var. fuscopruinosus, were used for the antioxidant assay in terms of the DPPH and the ABTS radical scavenging activities of different extracts. The radical-scavenging activity results are shown in Table 6. The results indicated that the DPPH radical scavenging activity was 694.47 ± 3.92 µg/ml for the ethyl acetate extract and the ABTS scavenging activity was 652.92 ± 2.53 µg/ml. The results for MeOH and the H₂O extract showed that the results were inactive. The standard control ascorbic activity (vitamin C) of ABTS scavenging activity was 8.57 ± 0.43 µg/ml, and the DPPH radical scavenging activity was 13.03 ± 0.48 µg/ml. The troxol (vitamin E) of the ABTS scavenging activity was 12.29 ± 0.37 µg/ml, and the DPPH radical scavenging activity was 12.88 ± 0.43 µg/ml.

Table 6

Antioxidant activities of *P. dajmor* var. *fuscopruinosus* in terms of the DPPH and ABTS radical scavenging activities (IC₅₀µg/ml) of different extracts.
Types of samples	DPPH scavenging activities (IC₅₀µg/ml)			ABTS scavenging activity (IC₅₀µg/ml)
Types of samples	Ethyl acetate	MeOH	H₂O	Ethyl acetate	MeOH	H₂O
P. dajmor var. fuscopruinosus	694.47 ± 3.92	Inactive	Inactive	652.92 ± 2.53	Inactive	Inactive
Ascorbic	8.57 ± 0.43			13.03 ± 0.48
Troxol	12.88 ± 0.43			12.29 ± 0.37

Anticancer

The extract of P. dajmor var. fuscopruinosus shown inhibition of lung cancer cells A549 and colorectal cancer cells (SW480) in Table 7. The ethyl acetate was exacted showed an IC₅₀ values for against of SW480 at 195.54 ± 3.95 µg/ml, and A549 at 217.25 ± 4.53 µg/ml but exaction of MeOH and hot water extract showed the results not active. Doxobrucirin (medicine) used for a positive control showed an IC₅₀ values of inhibition at 14.96 ± 1.58 µg/ml.

Table 7

Inhibition of cancer cells A549 and SW480 from the extract of *Pleurotus dajmor* var. *fuscopruinosus*.
Type of cells	IC₅₀ values of inhibition (µg/ml)
Type of cells	Ethyl acetate	MeOH	H₂O
colorectal cancer cells SW480	382.03 ± 4.55	Inactive	Inactive
Lung cancer cells A549	245.73 ± 7.60	Inactive	Inactive
Doxobrucirin (medicine)	14.96 ± 1.58

Cytotoxicity

Cytotoxicity of P. dajmor var. fuscopruinosus is shown in Table 8. The ethyl acetate showed an IC₅₀ value for against Raw cells 264.7 at 213.08 ± 4.08 µg/ml, and the exaction of MeOH and hot water extract showed the results were not active. Doxobrucirin (medicine) used for a positive control, showed an IC₅₀ value of inhibition at 14.80 ± 0.14 µg/ml.

Table 8

Inhibition of Raw cell 264.7 from the extract of *Pleurotus dajmor* var. *fuscopruinosus*.
Type of cells	IC₅₀ values of inhibition (µg/ml)
Type of cells	Ethyl acetate	MeOH	H₂O
Raw cells 264.7	213.08 ± 4.08	Inactive	Inactive
Doxobrucirin (medicine)	14.80 ± 0.14

Antidiabetic

α-glucosidase inhibitory activity

Inhibition of the α-glucosidase enzyme in vitro was examined, and the values obtained are shown in Table 9. Pleurotus dajmor var. fuscopruinosus showed the highest ability to inhibit hot water extract (582.91 ± 3.0 µg/ml) as compared to ethyl acetate (473.87 ± 1.4 µg/ml) and methanol (357.63 ± 3.3 µg/ml). Acarbose was used as the control, showing an IC50 of 635.70 ± 4.9 µg/ml.

Table 9

α-glucosidase inhibitory activity of *P. dajmor* var. *fuscopruinosus* from differences in solvents extraction.
Species name	IC₅₀ values of inhibition (µg/ml)
Species name	Ethyl acetate	MeOH	H₂O
Pleurotus dajmor var. fuscopruinosus	473.87 ± 1.4	357.63 ± 3.3	582.91 ± 3.0
Acarbose	635.70 ± 4.9

Glucose consumption

In the glucose consumption assay, 3T3-L1 cells were used to evaluate the bioactivity of the formulations with three different extractions, showing an IC₅₀ of hot water extract at 582.91 ± 3.0 µg/ml, whereas ethyl acetate and MeOH are not shown to be active. Metformin was used as a control and showed an IC50 of 99.58 ± 0.59 µg/ml.

Table 10

Glucose consumption activity of *Pleurotus dajmor* var. *fuscopruinosus* from differences in solvents extraction.
Species name	Solvent & IC₅₀ µg/ml
Species name	Ethyl acetate	MeOH	H₂O
Pleurotus dajmor var fuscopruinosus	Inactive	Inactive	582.91 ± 3.0
Metformin	99.58 ± 0.59

Although there are 25 species of Pleurotus, only some species have been studied as having good commercial importance, potential for cultivation, and adaptability of substrate types, such as the variety of agroindustrial lignocellulosic wastes. Pleurotus djamor is an edible mushroom well known as pink oyster mushroom, and its varieties were distributed in many countries such as Argentina, Brazil, Indonesia, Japan as P. salmoneostramineus Lj.N. Vassiljeva, Laos, Malaysia, Mexico, and Thailand [8, 15, 21, 53, 56, 57–59]. P. djamor var. fuscopruinosus was first identified by Corner in 1981 based on the morphology briefly described and without any molecular evidence yet. Thai specimens provided a better description and molecular analysis evidence.

According to the purpose of this study, we identified Thai specimens (MFLU24-0015, MFLU24-0016) as a new recorded variety, and isolated pure cultures were tested for the effect of grain medium on spawn production. We found that sorghum had the fastest growth rate and could be a suitable substrate for the spawning of P. djamor var. fuscopruinosus based on its low cost in Thailand. Our study agreed with many reports that sorghum has the best spawn production, such as Thulasi et al. [60], who tested the best spawn production of P. eous and P. florida, Kumla et al. [22], who tested the best spawn production of P. giganteus, and Thongklang and Luangharn [19], who also tested the best spawn production of P. ostreatus. The previous study by Oei and Nieuwenhuijzen [61] reported that sorghum is used worldwide for the spawn production of Pleurotus species. Another test is the effect of different agricultural wastes on the growth of mycelium. The result in Table 2 showed that the mycelium of this species could be grown on substrates made of different agricultures. According to the results, coconut fibre found that mycelium growth is fastest in sawdust, but its density is thin compared to mycelium growth in a sawdust substrate. Therefore, sawdust was selected as the most suitable substrate source to produce mycelium growth (including the diameter and density). Our study case agreed with Hoa [38] who reported that sugarcane residue, acacia sawdust, and corn cob were suitable sources of lignocellulosic substrates for the growth of the mycelium of P. ostreatus and P. cystidiosus. Moreover, [63] also reported that corn cob, rice straw, and sawdust can be used as substrates for the growth of P. tube-regiun.

The results of the fruiting body test showed that P. djamor var. fuscopruinosus can be grown well in sawdust as a substrate for fruiting body production. This agrees with previous studies of Pleurotus species, which can be cultivated within a wide range of different natural resources and agricultural waste such as logs, sawdust, straw dust, and weed [19, 22, 61]. In addition, Shah et al. [64] reported that sawdust provides a high yield of P. ostreatus. Jegadeesh et al. [65] cultivated the pink oyster mushroom P. djamor var. roseus in various agroresidues using a low-cost technique. Our study is the first to cultivate P. djamor var. fuscopruinosus using sawdust as the main substrate with some additives. These agree with Mandeel et al. [66] reporting that the nutrient content, growth, and yield of Pleurotus cultivated in sawdust depend on the chemical constituents. In future studies, we can modify the substrate or chemical to find low-cost alternative ways to increase mushroom production. It is interesting to obtain suitable techniques for the large-scale development of these mushrooms for their commercial and medicinal properties. The results of this experiment showed that various supplemented cereal grains can be used to promote the growth of P. djamor var. mycelia. fuscopruinosus.

Nutritional information of P. djamor var. fuscopruinosus was shown to be high culinary, as this study showed a high percentage of charbohydrate, proteint, mosture, fibre, ash and low fat. Therefore, the nutrient of this mushroom could be compared with other types of food such as eggs and milk [67, 68]. In general terms, mushrooms provide more protein than most other crops and most wild plants [69]. In the proximate of P. djamor var. fuscopruinosus (Table 5), the protein content is 24.99%. However, the protein content of mushrooms is affected by a number of factors, including the type of mushroom, the stage of development, the sampled part, the level of nitrogen available, and the location [70, 71]. The fat content is 2.07%; the fat content of these mushrooms was shown to be similar to that of P. cystidiosus O.K. Mill. (2.05 ± 0.0%) [62], and could be reported to have a low fat content compared to other species, for example P. eryngii (DC). Quél. (3.4 ± 0.1%) and P. giganteus (3.10 ± 0.0%) [72]. The ash content is 7.59% and the fibre content is 19.10%, compared to the study by [73], which found a lower mount of ash (0.87 ± 0.22%) and fibre (3.10 ± 0.24%). Furthermore, our study is consistent with previous studies by Landingin et al., [74], who published an approximate of P. cornucopiae and found that the species had a lower fat content and a higher protein, ash, and carbohydrate content.

There are more than 270 types of mushrooms that may be beneficial to human health [75]. Many pleurotoid mushrooms have been investigated for bioactive substances that can be useful in the treatment of several diseases [72, 76–78]. Given its extensive culinary and therapeutic benefits, mushroom treatment seems safe, much like other natural regimens [79]. In this study, the bioavailability assay considered the three different types of solvent used to extract mycelium from P. djamor var. fuscopruinosus. 50% (v/v) ethyl acetate was the most active for the antioxidant assay. This study was similar to the results of Cheung et al. [80], who reported that Lentinus edodes (Berk.) Singer at a concentration of 6 mg / ml had a higher DPPH radical scavenging rate of 55.4%. [81] suggested that the ABTS assay is superior to the DPPH assay when applied to a variety of plant foods containing hydrophilic, lipophilic, and high pigmented antioxidant compounds. However, this study applied to mushroom extract on the basis of the result showing that the DPPH assay is higher than the ABTS assay.

Pleurotus species have become more significant due to their ability to resist cancer. Many species of Pleurotus have reported that crude extracts and chemical compounds from fruiting bodies or mycelium of this species exhibited cancer cells of several types, such as cell lines, human hepatoma HepG2 cells, bladder carcinoma, and ovarian cancer [16, 82–85]. In this study, it was confirmed that P. djamor var. fuscopruinosus has anticancer effects in vitro. Ethyl acetate can kill colon cancer cells and lung cancer cells compared to doxobrucirin. On the basis of the results of this study, it was confirmed that P. djamor var. fuscopruinosus has anticancer properties. Furthermore, our study agrees with Xu et al., [86], who reported that ethyl acetate extracted from P. ostreatus exhibited telomerase inhibitory activities and water extracts from the edible lichen Ustilago esculenta Henn. exhibited strong anticancer effects against SNU-1 cells. Raw cell (264.7) used to compare the cytoxicity of exacted from P. dajmor var. fuscopruinosus found that was exactly P. dajmor var. fuscopruinosus has not only toxicity to cancer cells, but also to normol cells. This agrees with the fact that doxobrucirin is a medicine that has side effects on normol cells, or doxorubicin may increase the development of leukemia (cancer of the white blood cells) development [87].

Diabetes is a disorder that is negatively affected by diabetes. This resulted in the creation of numerous medications, and organisations are emerging. Each association has an action mechanism and a side effect. In addition to having the ability to regulate blood sugar levels according to individual and unique characteristics [88]. In vitro antidiabetic activity of P. dajmor var. fuscopruinosus exhibited significant results for its α-glucosidase inhibitory activity (ethyl acetate (473.87 ± 1.4 µg/ml) and methanol (357.63 ± 3.3 µg/ml) in a dose-dependent manner, compared to acarbose, an antidiabetic drug used to treat diabetes mellitus type 2. The extract also showed significant antidiabetic activity in vitro (p < 0.05) at the dose level tested (100 mg/kg b.w.); this was comparable to acarbose, a standard antidiabetic drug. In addition the glucose consumption assay of the wather extract of P. dajmor var. fuscopruinosus is highly active (582.91 ± 3.0 µg/ml) compared to metformin (a drug used to treat diabetes) (99.58 ± 0.59 µg/ml). This agrees with Nayak et al. [89] who reported that P. djamor had diabetes activities based on basidiomata powder. The hot water extract of P. pulmonarius (Fr.) Quél showed a possible in vivo and in vitro antidiabetic effect [90]. Furthermore, extracts of Pluerotus species with high antidiabetic activities, such as P. florida [91, 92], and P. ostreatus [93], Wei et al., [94], who reported that medicinal mushroom extracts (Agaricus blazei Murill) had a substantial effect on lowering blood sugar and showed no difference from metformin.

During the survey of pleurotoid mushrooms in Thailand, two collections of Pleurotus species were identified based on data from multiple gene sequences and detailed descriptions supporting the introduction of a variety, namely P. dajmor var. fuscopruinosus. Currently, a total of eight species of Pleurotus and one variety have been reported in the country, including the ones in this study. More research on Pleurotus species is required, and more new species may be found. Mycelium of P. dajmor var. fuscopruinosus could grow best on sorghum grain and sawdust, which are suitable substrates for mycelium growth. The preliminary cultivation trials of P. dajmor var. fuscopruinosus were successfully carried out in sawdust bags. The nutritional content of P. dajmor var. fuscopruinosus on sawdust substrates was found to have a very low amount of fat but was rich in protein, carbohydrates, moisture, ash, and a large amount of fibre. In this study, the bioactivities assay is active, depending on the polarity of the solvents used for each assay. According to the result of the antioxidant assay, the ethyl acetate extract among three different extracts showed antioxidant activities in both the DPPH and ABTS assays compared to the positive control (ascorbic and troxol). However, antioxidant activities of P. dajmor var. fuscopruinosus provided lower than fruits and vegetables [95]. The anticancer assay showed high cytotoxicity against cancer cells, both lung cancer and colon cancer. It showed that the ethyl acetate extract is toxic to cancer cells and raw cells. This could be acceptable compared to Doxobrucirin or, for example, chemotherapy for the cancer patient, which not only destroys cancer cells but also some normal cells that grow and divide quickly, such as cells lining the digestive tract, blood cells, and hair [96]. The antidiabetic assay showed inhibition and reduction of high blood sugar, and based on the results, it exhibited good a-glucosidase inhibitory activity. Therefore, P. dajmor var. fuscopruinosus has considerable importance in the human diet, as it are rich in protein, non-starchy carbohydrates, dietary fibre and a negligible amount of fat. This mushroom were attractive as sources for the development of new drugs and functional foods. From future perspectives, the cultivation of P. dajmor var. fuscopruinosus is necessary to find out how to increase fruiting body production by changing the ratio and supplementing sawdust with other agricultural wastes to become better substrates. In addition, the bioactivities assay of this species needs to be performed in vivo for further experimental examination. Furthermore, the purification of chemical compounds in P. dajmor var. fuscopruinosus extracts is necessary for further study.

Acknowledgement

Monthien phonemany appreciates the kind support from a Thesis or Dissertation Writing Grant from Mae Fah Luang University, reference no อว 7702(6)/125. Thank you the Thailand Research Foundation (TRF) for the grant “Study of saprobic Agaricales in Thailand to find new industrial mushroom products” (Grant No. DBG6180015) and the Thailand Science Research and Innovation (TSRI) grant 'Macrofungi diversity research from the Lancang-Mekong River basin and surrounding areas' (grant No. DBG6280009). Thank you Dr. Wuttichai Jaidee, who is the protocol assistant and guidance for α-glucosidase assay. Thank you to Miss Chalinee janta who assisted and guided the antioxidant assay.

Author contributions

MP and NT conceptualization and designed the research. MP performed the experiments. MP, PS, and NR formal analysis. MP writing an original draft. NT and RC Funding acquisition. PS, NT, NR, and RC reviewed and edited the manuscript.

Funding

Open Access funding enabled and organized by Thailand Research Foundation (TRF) for the grant “Study of saprobic Agaricales in Thailand to find new industrial mushroom products” (Grant No. DBG6180015).

Availability of data and materials

All of the data that support the findings of this study are available in the main text, or in publicly accessible data repositories, as indicated in the text.

Competing interests

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

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Preliminary trial of the cultivation of Pleurotus djamor var. fuscopruinosus from southern Thailand using sawdust substrate and applications

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Abstract

Figures

Background

Material and method

Collecting and Isolation

Morphology Study

DNA extraction, PCR amplification, and sequencing

Sequence alignment and phylogenetic analysis

Preliminary cultivation trial

Effect of grain media for spawn production

Effect of different agricultural wastes on mycelium growth

Fruiting test

Statistics Analysis

Determination of the nutritional content

Crude Extraction

Antioxidant Assay

2,2-Diphenyl-1-picrylhydrazyl radical-scavenging activity (DPPH)

2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) radical cation scavenging activity (ABTS)

Antidiabetic Assay

α-glucosidase Assay

Glucose Consumption Assay

Anticancer Assay

Cytotoxicity Assay

Results

Identification

Phylogenetic Analysis

Taxonomy

Preliminary cultivation trial

Effect of grain media for spawn production

Effect of different agricultural wastes on mycelium growth

Fruiting test

Nutritional analysis

Antioxidant

Anticancer

Cytotoxicity

Antidiabetic

α-glucosidase inhibitory activity

Glucose consumption

Discussion

Conclusions

Declarations

References

Additional Declarations

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