studies on the Rapid and simple DNA extraction Method, Antibacterial Activity and enzyme Activity involved in Plant Biomass Conversion by Cookeina sulcipes and C. tricholoma (Cup Fungi)

Cookeina sulcipes and C. tricholoma are a cup fungi ( Ascomycota ) collected in saraburi, thailand. the fungi have been isolated, cultured and confirmed as respective species. For morphology, both Cookeina sp. are white mycelium and the growth rate on potato dextrose agar (PDA) result present C. sulcipes is faster than C. tricholoma . the molecular characterization from a rapid and simple DNA extraction method that is modified based on thermolysis method, the DNA extraction is finish in thirty minutes and efficiency to continuous with polymerase chain reaction (PCR) amplification to fungi species level identification. the DNA sequence from internal transcribed spacer (its) gene regions by universal primer pairs its5/its4 is effective to confirm Cookeina species level that C. sulcipes has 616 bp and C. tricholoma has 570 bp. including, DNA sequence of large subunit (lsU) gene regions by universal primer pairs lROR/lR5 is generate that C. sulcipes has 912 bp and C. tricholoma has 906 bp. the cultures are screened for antibacterial activity by agar plug diffusion method and found that both isolates have been no activity against test strains ( Bacillus subtilis, Escherichia coli, Kocuria rhizophila ( Micrococcus luteus ), Pseudomonas aeruginosa, Staphylococcus aureus and S. epidermidis ). in a preliminary screening test of enzymes involved in plant biomass breakdown by agar plate method, both Cookeina sp. show cellulolytic and hemicellulolytic enzymatic activity, and manganese peroxidase (MnP) productivity. in contrast, only C. sulcipes had additional laccase activity. Neither isolate generate pectinolytic and lignin peroxidase (liP) activities. thus, Cookeina spp. proved the potentiality to break down lignocelluloses.

step.Pilo, et al. 10 presented a method for rapid genomic DNA extraction from filamentous fungi without buffer, using a sonication bath, which required only an hour to accomplish.Therefore, a rapid and simple method for fungal DNA extraction would have significant benefits.
C. sulcipes and C. tricholoma are reported to be edible. 14However, to date, there has been no report on their biological activity.In nature, Cookeina species grow on dead bark or wood indicating that one major function is as decomposer and aid in lignocellulose breakdown.Primary plant cell wall components are 25-50% hemicellulose, 10-35% pectin, 9-25% cellulose and 10% proteins and the secondary cell wall is 40-80% cellulose, 10-40% hemicellulose and 5-25% lignin. 15Involving, lignocellulosic biomass can be obtained from various sources or industries as a wastes product such as agricultural industry waste, plantations waste, organic waste, etc. 16,17 Therefore, the enzymes involved in plant biomass breakdown or cell wall degradation are cellulolytic, hemicellulolytic and ligninolytic from brown-, white-and soft-rot fungi, 16,18,19 and even pectinolytic enzymes from soft-rot fungi. 18,20his study focused on a more simple, rapid, and efficient method for identifying fungal species and determining antibacterial and enzymatic activities for future study.

Collection and Fungal isolation
Specimens of C. sulcipes and C. tricholoma were collected in Saraburi, Thailand, and brought to the laboratory in a plastic box.Fresh fruiting bodies (ascomata) were divided into two parts.The first part was used to study the morphology and the second to isolate the fungus in culture.The fungi were isolated by spore shooting and single spore isolation methods. 21Spores were placed in a Petri dish with water agar (WA) medium, and the dishes were incubated at room temperature (30 ± 2°C) overnight.Germinated spores were placed on a potato dextrose agar (PDA) plate, and colonies were purified by the hyphal tip transfer method.The pure cultures were subcultured into PDA plates and used to study morphological characterization, molecular identification and as stock culture to maintain at the Thailand Mushroom Culture Collection (TMCC), Biotechnology Research and Development Office, Department of Agriculture, Thailand, for future study.

Morphological identification
Fresh fruiting bodies of C. sulcipes and C. tricholoma were cut with a razor blade and thin sections were observed under a light microscope.Morphological identification is done by observing the macroscopic and microscopic characters, together with colony characteristics, colour and growth pattern.The colony dimensions of the fungi were evaluated by transferring 5-mm diameter agar plugs from the periphery of 5-day-old cultures grown at room temperature.The colony characteristics and colour were determined after 7 days of incubation on PDA at room temperature in triplicate.The colony growth rate was determined at 4 and 7 days by measuring the colony diameter (ø) on PDA with a digital vernier caliper and weighing the dry mycelium of each isolate grown on potato dextrose broth (PDB).

Rapid DNA extraction
The genomic DNA extraction was done based on the thermolysis method. 22Both isolates were cultured on PDA plates for 5-7 days and incubated at room temperature.The mycelial mass was harvested using a flat-tipped needle and transferred into 50 µl of Tris-EDTA (TE) buffer, pH 8 in a 1.5 ml microcentrifuge tube and incubated at 65°C in a water bath or heat block for 20 min.Subsequently, they were incubated at -80°C for 10 min and finally, centrifuged at 13,000 rpm for 1 min.The supernatant containing genomic DNA was directed to polymerase chain reaction (PCR) amplification or stored at -20°C until use (Figure 1).

Antibacterial Activity by Agar Plug Diffusion Method
The agar plug diffusion method for preliminary screening of antibacterial activity. 27hat both Cookeina sp.isolates were screened for six human pathogenic bacteria (Gram-positive: Bacillus subtilis TISTR 1248, Kocuria rhizophila (Micrococcus luteus) TISTR 2374, Staphylococcus aureus TISTR 746 and S. epidermidis TISTR 2141, Gram-negative: Escherichia coli TISTR 074 and Pseudomonas aeruginosa TISTR 2370) obtained from the Thailand Institute of Scientific and Technological Research (TISTR) culture collection, Bangkok, Thailand.The bacteria were activated in nutrient broth (NB) for 24 hours at 37°C.Then, the bacterial solutions were diluted with 0.85% normal saline to an optimum concentration (0.08-0.13) monitoring absorption at 625 nm as a starter. 28A sterile cotton swab was dipped into the bacteria solution and swabbed on the PDA Petri dish.The fungal isolate was cultured for 5 days on a PDA Petri dish at room temperature and a 5 mm diameter fungal agar plug was cut and transferred to the surface of PDA Petri dish previously smeared with bacteria.WA media and sterile fungal plug on PDA Petri dish were used as negative and positive controls.All antibacterial activity analyses were run in duplicates of four plugs in two Petri dishes.After incubating the co-culture plate at room temperature for 24-72 h, the antimicrobial activity was estimated by measuring inhibition zone or clear zone diameters.

enzyme Activities by Agar Plate Method
The fungal isolates were subcultured on a PDA plate and incubated at room temperature for 5 days.Then 5 mm diameter plugs were transferred to agar plates for preliminary screening of enzyme activity in plant biomass degradation.

Cellulolytic Activity
For cellulolytic activity, a modified method was used as follows: Each fungal plug was placed on the plates of carboxymethylcellulose Following incubation, CMCA plates were flooded with 5 ml of Gram's iodine (1% KI and 0.5% I 2 ) stain for 5 min. 29Then, the agar surface was washed with distilled water.The cellulolytic activity of the

hemicellulolytic Activity
Hemicellulolytic activity used a modified method similar to cellulolytic activity: a fungal plug was transferred onto xylan agar (0.2% NaNO 3 , 0.1% K 2 HPO 4 , 0.05% MgSO 4 •7H 2 O, 0.05% KCl, 0.2% peptone, 0.2% xylan and 1.7% agar) plates in Petri dishes, in triplicate, and incubated at room temperature for 4 days.The Petri dish was flooded with 5 ml Gram's iodine, stained for 5 min and surface-washed with distilled water. 29The hemicellulolytic activity of the fungi was observed as a clear zone of hydrolysis and analyzed as in the cellulolytic activity method.

Pectinolytic Activity
For pectinolytic activity, a modified method was as follows: The fungal plug was transferred onto a pectin agar medium (0.5% pectin, 0.1% yeast extract, and 1.5% agar, pH 5) in triplicate and the Petri dishes were incubated at room temperature for 4 days.Following, the Petri dish was flooded with 5 ml of 1% Cetyltrimethylammonium bromide (CTAB), stained for 10 min and surface-washed with distilled water. 31The pectinolytic activity of the fungi was observed as a clear zone of hydrolysis and analyzed as in the cellulolytic activity method.The laccase activity was measured by a modified method following Shrestha et al. 32 : In triplicate, fungal plug of each isolate was placed on a PDA plate supplemented with 1-naphthol (0.005%, Merck), in a Petri dish and incubated at room temperature, in the dark, for 4 days.After incubation, the appearance of a purple colour zone around the colony indicated laccase enzyme production.For lignin peroxidase activity, the method was modified from Delila et al. 33 : In triplicate, the fungal plug was transferred to a PDA plate, supplemented with methylene blue (0.0065%, Sigma-Aldrich), and incubated at room temperature, in the dark, for 14 days.A clear zone around the colony, caused by the disappearance of the blue colour of the dye, confirmed the presence of peroxidase or decolorization of methylene blue dyes.

Manganese Peroxidase (MnP)
For manganese peroxidase activity, the modified method was as follows: In triplicate, an inoculated fungal plug on a PDA plate was supplemented with phenol red indicator (0.008%, Merck), 34 and incubated at room temperature, in the dark, for 18 days.A colour change from yellow to red confirmed the presence of the peroxidase.

statistical Analysis
The triplicates for growth rate and HC in diameter values were analyzed using SPSS, version 28.Means ± standard error (SE) were reported.Statistical analysis used one-way ANOVA, followed by Duncan's test, p < 0.01 was considered significant.

Molecular identification and Phylogenetic Analyses
The result from PCR product and DNA sequences on both gene regions (ITS and LSU) shows that the genomic DNA from rapid extraction, which has not been tested for DNA purity or concentration and agarose gel electrophoresis was efficiently used directly for PCR amplification.DNA sequence length (without primers) of C. sulcipes NTKMITL20-049 and C. tricholoma NTKMITL20-047 on ITS was 616 bp and 570 bp and LSU were 912 bp and 906 bp, respectively.Further, the ITS phylogenetic tree was analyzed and constructed to support identification compared to morphology characteristics.The data analysis on ITS of Cookeina sp. with 615 characters that included alignment gap were parsimony informative with ingroup and one outgroup (Microstoma floccosum).Phylogeny presents C. sulcipes NTKMITL20-049 in the same clade as C. sulcipes, whereas C. tricholoma NTKMITL20-047 in C. tricholoma clade (Figure 2).
Cookeina tricholoma (Mont.)Kuntze, Revisio generum plantarum 2: 849 (1891) (Figure 4) 40).Colonies on PDA medium are white or cream above and reverse of Petri dish, flat elevation, and filamentous form and margin.After 4 days at room temperature, on PDA plates, the average diameter was 28 mm and, after 7 days, 45 mm and, on PBD medium, the average of dry mycelial mass was 0.04 mg after 4 days and 0.04 mg after 7 days.

Antibacterial Activity
The preliminary result of antibacterial activity shows that both isolates of Cookeina present no activity.There are no clear zones seen with six bacterial strains (B.subtilis, E. coli, K. rhizophila, P. aeruginosa, S. aureus, and S. epidermidis) that represented as Gram-negative and Gram-positive bacteria in this study (Figure 6).

enzyme Activities
The result of screening for enzyme activities on the agar plate method is presented as in Table 3 and Figure 7.Both isolates of Cookeina sp.showed similar activity on cellulolytic and hemicellulolytic enzymes by the sight of the clear zone.On manganese peroxidase, C. sulcipes was stronger than C. tricholoma by display of red and orange colour, respectively.However, the fungi exhibited no activity on pectinolytic (no clear zone) and lignin peroxidases (no decolorization).There was no decoloration of media from blue to purple colour.For laccase activity, C. sulcipes showed purple colouration on the reverse of Petri dish.

DisCUssiON
C. sulcipes and C. tricholoma are commonly found in the Central, Northeast, Northern and Southern parts of Thailand, besides other tropical and subtropical countries.C. sulcipes has earlier been described from a material collected from Prachuap Khiri Khan province of Thailand. 1 C. tricholoma has been recorded in Brazil, China (Yunan province), Puerto Rico and Venezuela, besides Thailand. 1,2,4The rapid genomic DNA extraction method used in this study is unique in that it used only a very little quantity of mycelia and without liquid nitrogen 13 or homogenizer (e.g., sonication, 9,10 FastPrep-24™, 11 pestle motor mixer or micro pestle, 12 etc.) to breaking fungal cell wall.This method is faster, simple and does not demand many materials or equipment.Compared with standard fungal DNA extractions such as CTAB method, SDS method, lysis buffer method, etc. 7,8,13,22 or DNA extraction/isolation kit, the method followed in this study is simple, cost saving, and quicker.Both species of Cookeina have been reported to be edible. 14On screening against six bacterial strains, the Cookeina species studied showed no activity.Genus Cookeina is generally found on dead wood.This study confirmed the presence of hydrolytic hemi-and cellulolytic enzymes and laccase and manganese peroxidase that involve in the degradation of plant cell wall.
The most widely studied cellulolytic fungi are Aspergillus sp., Humicola sp., Penicillium sp., and Trichoderma sp. of these, Trichoderma sp. is the most suitable candidate for cellulase production and utilization in the industry. 35Similar is with pectinase production in agro-industrial waste inhabitant fungi such as Aspergillus spp.and Lentinus edodes.Whereas P. chrysogenum, T. harzianum and Schizophyllum commune are important pectinase producing fungi. 36Laccase and manganese peroxidase useful in biodegradation are present in Irpex lacteus, Marasmius sp., Mucor circinelloides, and Pleurotus ostreatus.Similarly, decolorization of wood is carried out by Coprinopsis cinerea, Geotrichum candidum, Phanerochaete chrysosporium, and Trametes species. 37Reports of fungi from the same family Sarcoscyphaceae include Phillipsia sp.SXS629 (on the dead tree trunk, Brazil) in polyphenol oxidase and carboxymethyl cellulase (CMCase) activity.Some of these fungi can grow with increase of biosolids or wastewater treatment plant (WWTP) concentration. 38

CONClUsiON
The rapid DNA extraction is appropriate for filamentous fungi for species identification by PCR, based on ITS, LSU and other regions.This method is simple, convenient, inexpensive, and saves time.In this study, C. sulcipes and C. tricholoma are no bacterial activity.Nevertheless, the Cookeina sp. also a source of lignocellulolytic enzyme, deserves further attention in the future.

Figure 1 .
Figure 1.Schematic representation of rapid genomic DNA extraction based on thermolysis.Created in BioRender.com

Figure 5 .
Figure 5. Growth rate chart at 4 days on PDA, CMCA, xylan agar, pectin agar, and PDA with 1-naphthol or methylene blue or phenol red

Figure 7 .
Figure 7. Colony character of Cookeina sulcipes and C. tricholoma on PDA and medium for screening enzyme activity

table 1 .
GenBank accession numbers of Cookeina in this study blastn) program.Finally, sequence datasets were done by Phylogeny.fr(http://www.phylogeny.fr/index.cgi),following the workflow of advance mode as MUSCLE multiple alignments, Gblocks alignment curation and maximum likelihood (ML) method with 100 bootstrapping procedures to construct the phylogenetic tree.