INTERACTIONS BETWEEN MUSHROOMS AND FUNGI IN DUAL-CULTURE EXPERIMENTS

Rohina Chaudhary. The current study was undertaken to measure the interaction between five wild mushrooms collected from natural habitat and mycoparasitic fungi such as Fusarium sp. Pythium sp. and Aspergillus sp. Study showed that replacement was 53.32% and deadlock (40.00%). Therefore in 53.32% of pairings the mushrooms replace effectively the pathogenic fungi. Complete replacement and partial growth were take place in equal frequency (6.66%, 6.66%). The results revealed that 93.32% of pairings led to prevention and limited the fungal growth. Identification of wild mushroom strain based on DNA Sequencing by ITS1and ITS4. Through molecular taxonomy sample identified as 8/12 (Clitopilus scyphoides), 33/12 (Agrocybe pediades), 45/12 (Agaricaceae sp.), 48/12 (Trametes ochracea) and (112/12) Irpex lacteus.

484 several changes of sterile distilled water and 0.001% Mercuric Chloride and aseptically break lengthwise exposing the inner tissue (trama) with the aid of a sterile blade. A small piece of 2 x 2mm of the sterile tissue will aseptically transfer onto plates of (2%) malt extract agar and the plates were incubated at 25°C to 30°C temperature for 7 days. Sub sculturing for pure tissue mycelial production was prepared by transferring a small square of 5 x 5mm from the mother plate onto a fresh solid media plates.
Antagonistic activity of wild mushrooms against pathogenic fungi:-Testing antagonistic activity:-The pathogenic fungus tested were collected from molecular biology lab from Shoolini university, India. Competitive interaction between wild mushrooms and soil born plant pathogenic microorganisms was evaluated by dual culture experiments on Petri dishes (90 mm diameter containing 20 ml PDA. In each Petri dish 2 mm diameter mycelial disks, one each from mushroom and fungal colonies was placed on the agar surface 30 mm apart. Immediately after inoculation, the plates were sealed with plastic film and incubated in darkness at 24°C for 10 days. Colony growth and type of interaction was examined regularly. The presence of dense zones of mycelium, aggregated structures such as mycelial cords, pigmented hyphae, exudate droplets and dark pseudo sclerotial lines. The antagonistic activity of the various mushroom cultures was tested by the method described by 15 .

Antagonistic rating scale:-
The antagonistic ability of each mushroom was determined by using antagonistic rating scale given by 16 with slight modifications.
The following score was assigned to each type or sub-type of reaction: A=1; B=2; C=3; CA1=3.5; CB1=4; CA2=4.5; CB2=5, as shown in (Table 1). Table 1:-The antagonism index (AI) The antagonism index (AI) was then calculated for each fungal species using the following formula: AI=A(n1)+B(n2)+C(n3)+CA1(n3.5)+CB1(n4)+CA2(n4.5)+CB2(n5) where n= frequency of each type or sub-type of reaction. Molecular Identification of mushroom:-Molecular identification was done to identify the unknown samples of mushroom. DNA isolated and PCR amplification: Genomic DNA isolated from HIMEDIA kit. Then PCR amplification was performed using a pair of universal primers ITS-1(5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'TCCTCCGCTTATTGATATGC-3') for the region containing ITS1, ITS4 and 5.8S rDNA. The reaction mixture was followed by the kit (HIMEDIA X-PERT). Amplifications will be performed in 50-μl reactions of PCR buffer and PCR reactions will be consists of an initial denaturation at 94°C for 1 min 25 s, 35 cycles of amplification, and a final extension at 72°C for 10 min; each cycle of amplification will be consisted of denaturation at 95°C for 35 s, annealing for 55 s (at 55°C for reactions with ITS1 and ITS4 and at 60°C for reactions with ITS1 and ITS4), and extension at 72°C for 1 min. Amplification products was electrophoreses in 0.8% agarose gel, stained with ethidium bromide and amplicans will be observed under UV light 17 .

Sequencing:-
The PCR product was subjected for sequencing and the DNA sequences was used for. The DNA sequences were used in NCBI (National Center for Biotechnology Information) BLAST (Basic Local Alignment Search Tool) search for bioinformatics analysis, to look for homologus nucleotide sequences. On the basis of this information, species status was assigned.

Subtypes
Interaction category Score A deadlock with mycelial contact 1 B deadlock at a distance 2 C overgrowth without initial deadlock 3 CA1 partial replacement after initial deadlock with contact 3.5 CA2 complete replacement after initial deadlock with contact 4.5 CB1 partial replacement after initial deadlock at a distance 4 CB2 complete replacement after initial deadlock at a distance; * overgrowth of the mushroom by the

Results:-
The frequency of each type and sub-type of reaction is shown in (Table 2, 3). Study revealed that replacement was more frequent (53.32% + 6.6%) than deadlock (40.00%). In 53.32% of pairings the mushrooms replace effectively the pathogenic fungi. Complete replacement and partial growth were take place in equal frequency (6.66%, 6.66%) except overgrowth (40.00%) as shown in (Figure 1, 2 and 3). Overall 93.32% of pairings led to prevention and limited the fungal growth. This clearly indicated that the mushrooms were competetive against the pathogenic fungi. Table 2:-Antagonism index values (in brackets) and competitive reactions between mycelium mushrooms and pathogenic fungi in pairings on potato dextrose agar medium.
A, deadlock with mycelial contact; B, deadlock at a distance; C, overgrowth without initial deadlock; C A1 , partial replacement after initial deadlock with contact; C A2 , complete replacement after initial deadlock with contact; C B1 , partial replacement after initial deadlock at a distance; C B2 , complete replacement after initial deadlock at a distance; (* overgrowth of the mushroom by the phytopathogenic fungus).  Therefore five new species were subjected to identify on the basis of molecular indentification. The size of the DNA of five mushroom samples was around 600bp in (Figure 4). The rDNA-ITS (Ribosomal DNA Internal Transcribed Spacers) fragments of the genomic DNA were amplified using ITS1 and ITS4 primers. The PCR amplification products ( Figure 5) showed that 8/12 gave around (500 bp) amplified band, while 33/12 and 112/12 bands around 490 bp, 45/12 and 48/12 showed bands at 650 bp. These PCR products were gel purified, run in 1% agarose gel, and processed for nucleotide sequencing. The nucleotide sequences of five samples were obtained and then analyzed for Basic Local Alignment Search Tool (BLAST). The BLAST result were presented in (  (Table 5). Tested 112/12 collections are potential producers of antifungal metabolites which significantly suppressed the growth of test phytopathogenic fungi. Among tested species the highest activity was detected in 112/12.

Discussion:-
Antagonistic activity of mushrooms against fungi has been reported. Changes in colony colour were due to diffusion of metabolites of two species and this pigmentation occurred by mycelial phenoloxidase or peroxidase activity 18,19 .
The mushroom produce metabolites with antifungal properties. The most active mushrooms were 45 and 112. This study provides a simple strategy that used to answer relevant questions how mushroom can overpowered the growth of pathogenic fungi. The human getting infected indirectly through foods of plant origin, animal origin through fungal toxins 20 . As discussed by 21 that Fusarium spp. produce mycotoxins Deoxynivalenol (DON) affects animal and human health causing acute temporary nausea, vomiting, diarrhoea, abdominal pain, headache, dizziness, and fever. Therefore against Fusarium all five sample gave good results but 33/12 and 45/2 showed better results and had more tendency to suppress the Fusarium sp. Fungal pathogens cause disease in plant and animal hosts 22 . The 23 determined that mushrooms had more species with antifungal, (Pleurotus ostreatus, Pleurotaceae) has shown effectiveness against Aspergillus niger causing aspergillosis lung disease, a malady that can pose a serious threat to persons with compromised immune systems. Hericium erinaceus has also shown anti-fungal activity against the mold A. niger. L. edodes represents a promising medicinal mushroom to control the infection by A. parasiticus and the aflatoxin production. 24 cyanthus sp. inhibition of human pathogenic fungus (Aspergillus fumigatus) 25 .
The analysis of genomic DNA using PCR-based methods has proven to be a fast and reliable method to determined genetic relationships among basidiomycetes 26 . Nuclear rDNA, and particularly the internal transcribed spacer (ITS) regions are good targets for the Phylogenetic analysis in fungi because the ITS regions are often highly variable between isolates of the same species 27,28 . The genomic DNA of the mushroom was extracted, the rDNA -ITS fragment of the genomic DNA was amplified using ITS1 and ITS4 primers and subjected to nucleotide sequence determination.

Conclusion:-
Therefore Studies aiming at the isolation and identification of basidiomycetes because with increasing demand for edible and medicinal mushroom, it becomes a necessity to unravel the rich biodiversity of basidiomycetes. Identification of those high-quality fungal species is not only necessary but has great economic significance as it will help in detecting fraudulent products being marketed. The best of our knowledge, this study is the first to investigate the potential of basidiomycetes against the pathogenic fungi and can serve to stimulate the investigation.