Morphological and Molecular Identication of Oyster Mushroom [Pleurotus Ostreatus (Jacq.) P. Kumm]

Oyster mushroom (Pleurotus ostreatus) is eshy, edible fungi and used throughout the world. Among the most well-known species of mushrooms, Pleurotus sp. is the third most popularly grown mushroom in the world and rank second in India. The most well-known species of Pleurotus are P. ostreatus, P. orida, P. eryngii, P. cystidiosis, P. abellatus, P. cornucopie, and P. sajor-caju. According to the morphological, molecular character and ITS sequencing, the Pleurotus ostreatus was distinguish from the other species. The cap (pileus) of the oyster mushroom has whitish to grey color and eshy. Stipe cream and smooth surface. Spores are whitish to lilac grey in mass, cylindrical to oblong shape. Spore print was white in color. Whitish mycelium growth in PDA plate. The ITS sequence was submitted to gene bank NCBI and it was accepted by NCBI with accession number MW446165.


Introduction
Oyster mushroom (Pleurotus ostreatus) is also known as 'Dhingri' belongs to the family Pleurotaceae. It is inevitable diet food in India. The word mushroom is derived from the Gallo Roman mussiro which is de ned as a "macro-fungus" that has a eshy and used throughout the world to describe the fruiting bodies of saprophytic, mycorrhizal and parasitic fungi. It is either epigenous or hypogenous and large enough to be seen with the naked eye and to be picked by hand [1]. The fruiting bodies of this mushroom are observed in different shades of white, cream, grey, yellow, pink or light brown depending upon the species. The oyster mushroom has three distinct parts -a ashy shell or spatula shaped cap (Pileus), a short or long lateral or central stalk called stipe and long ridges and furrows underneath the pileus called gills or lamellae. The popularity of oyster mushroom has been increasing due to its ease of cultivation, high yield potential and high nutritional value.
The genetic study of mushroom has been worked out using molecular markers especially polymerase chain reaction (PCR). It is effective tools of plant biotechnology used for the assessment of genetic diversity. oyster mushroom (Pleurotus sp.) was differentiated on the basis of morphological (sporocarp, sporophore, colour, size), cultural, ITS sequencing (accession number) and Phylogenetic analysis [2,3].

Morphological identi cation
The Pleurotus ostreatus was identi ed based on the various morphological characters such as mycelium growth, height of fruiting body, stipe length, stipe diameter, pileus diameter, microscopic view of mushroom spore and spore print.

Growth and pure culture of mushroom
Potato dextrose agar (PDA) medium was poured in sterilized Petri plate and inoculated with 10 mm disc of Pleurotus ostreatus under aseptic condition. Then, the Petri plate were incubated at 25 ± 2°C. The diameter of colony was measured after 6 days of growth. sub culturing was done on PDA slants as and when needed.

Microscopic view of mushroom spore
Mushroom spore studied by observing cotton blue stained slides under compound light microscope by using 40x objective and 10x eyepiece lenses. After calibrating the microscope, the measurement of spore was taken with the help of microscopic measuring slide [4].

Spore print
While a single mushroom spore can't be seen by the naked eye, a pile of many spores and color of a mushroom's spores is an easily identify by obtaining a mushroom's "spore print".
To make a spore print rst remove the stem from smaller mushroom and place the cap, gills or pores downward, on a piece of paper or glass. For white color oyster mushroom using a black paper while white paper used for pink oyster mushroom. In case of large mushroom, slices off a section of the cap and use only the section. Place a cup or glass upside-down on top of mushroom, to keep in air tight condition. After overnight, when remove the cap and lift the mushroom cap, we got a print like the ones illustrated to the left [5].

Molecular identi cation
Mushroom species was identi ed based on molecular method. In molecular method, genome sequencing, was done for ITS region. For genome sequencing the pure culture of Pleurotus sp. was sent to Euro ns Genomics India Pvt. Ltd., Bangalore. The fungal genomic DNA was extracted from mycelia grown in 250 ml of PDB at 28°C for 5 days. The mycelia were harvested from broth and lyophilised and stored at -20°C for further process.

DNA extraction
The genomic DNA for PCR was extracted by using DNA isolation kit. The ITS region of fungi, including ITS1 (5'TCCGTAGGTGAACCTGCGG3') and ITS4 (5'TCCTCCGCTTTATTGATATG3') were ampli ed. The ampli cation was performed in 30 µl reaction volume with 0.1 mM of each dNTP and 100pmol of both forward and reverse primer. ABI Veriti pcr was programmed for initial denaturation at 94°C for 4 min, and 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The ampli cation was completed with a nal extension at 72°C for 5 min. Further it was sequenced by ABI 3730 capillary sequencing [6].

Sequencing
Consensus sequence of the PCR amplicon was generated from forward and reverse sequence data using aligner software. 6. The ITS region sequence was used to carry out BLAST with the database of NCBI GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Based on maximum identity score rst ten sequences were selected and aligned using multiple alignment software program Cluster W. Distance matrix was generated and the phylogenetic tree was constructed using MEGA X [6].

Results
Oyster mushroom (Pleurotus sp.) was distinguished on the basis of their morphological and Molecular characteristics. Morphologically identi ed by mushroom mycelial growth on PDA plate, spore print, spore size and also identi ed by measuring length and diameter of pileus and stipe from different collected samples of oyster mushroom (Fig. 1

Molecular identi cation
The identi cation sequence was BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and similarity with other species computed. The evolutionary history (Fig. 2) was inferred using the UPGMA method [7]. The optimal tree with the sum of branch length = 0.02507224 was shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) shown next to the branches [8]. The evolutionary distances were computed using the Maximum Composite Likelihood method [9] and were in the units of the number of base substitutions per site. This analysis involved 5 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 703 positions in the nal dataset. Evolutionary analyses were conducted in MEGA X [6].
The BLAST analysis of the ITS sequence data, (Table-1) supported the morphological identi cation, whereby the closest match with NCBI sequence number MT261808 (100 %) in the NCBI GenBank database was found to be the Pleurotus ostreatus. Further, the sequence was submitted to gene bank NCBI and it was accepted by NCBI with accession number MW446165. So, based on the morphological and molecular methods the oyster mushroom was identi ed as Pleurotus ostreatus.

Morphological studies
As per the nding of this study, the morphological characteristics of Pleurtous ostreatus was more or less similar with Chhetry and Pfoze [10] who reported that the pileus of Pleurotus spp. was soft, smooth, light yellow with 4.5-11 cm diameter. It possessed white, broad decurrent gills with often lateral stipe, usually elongated from 5-8 cm with white, solid, several fruit bodies joined at the base to form a large common base. The fresh sample was soft, white with mild odour. Ram [11] showed that Pleurotus sp. was morphologically characterized by white spores with an eccentric or lateral stem of eshy texture. Cap was 2-15 cm wide with 3-11 cm long and upper surface smooth with white, spathulae to kidney shaped, margin decurved or inrolled. Stem was usually short or stem poke base, imbricate in groups of 5-20 cm. Gills were 18-20 cm at margin, 5-15 mm wide, decurrent, sometimes uniting to form a net or pore like pattern on the stem, white when fresh, yellowish when dried. Murugesan et al. [12] isolated oyster mushroom from forest region and identi ed Pleurotus sp. based on microscopic view and phylogenetic tree.

Molecular identi cation
The study con rmed that the Pleurotus ostreatus MW446165 sequence was shown 94 per cent similarity with Pleurotus ostreatus MT261808. Similarly, it was 86 per cent match with Pleurotus ostreatus MK603976. Shnyreva and Shnyreva [13] analyzed ten Pleurotus sp. based on Internal Transcribed Spacer (ITS) sequences of rDNA. A phylogenetic tree was constructed on the basis of 31 oyster fungi strains of different origin and 10 reference sequences from Gene Bank to fungal species identi cation. They were revealed the divergence between commercial strains and natural isolates of Pleurotus ostreatus by phylogenetic analysis.
Oyster mushroom (Pleurotus ostreatus) was molecular identi ed by using ITS primer, which was closest match with result of Imtiaz et al. [14]. They were collected twenty strains of Pleurotus spp. from different region and used DNA sequences of the ITS (Internal Transcribed Spacer) region to analyse the genetic diversity of Pleurotus strains. Result indicated that the among the 20 strains, the major one included P. ostreatus, P. cystidiosus, P. eryngii, and P. pulmonarius. Similarly, Liu et al. [15] assessed the genetic diversity of Pleurotus ostreatus strains on the basis of the Internal Transcribed Spacers (ITS) sequence, translation elongation factor (EF1α) and the second largest subunit of RNA polymerase II (RPB2). The polygenetic tree constructed using combined results of the ITS, EF1α and RPB2 sequence analyses showed the genetic relationship between studied strains. They were provided valuable information on its relationship and identi ed Pleurotus ostreatus on molecular basis.

Conclusion
Mushroom is a type of macro fungus that form large fructi cation visible without the help of microscope. The collected samples of mushroom fungus were further identi ed based on various morphological characters and molecular features. On the basis of morphological characters viz., whitish mycelium growth, spore print, length and diameter of stipe and pileus, the fungus was identi ed as Pleurotus ostreatus and it was also matched with sequencing and BLAST results. 10. Data Availability statements ITS sequence was sent to NCBI and it is available with nucleotide database number MW446165.1 Figure 1 Morphological identi cation of Pleurotus ostreatus Figure 2 Phylogenetic relationship of Pleurotus ostreatus isolate with other species