MATERIALS AND METHODS

MATERIALS AND METHODS

The laboratory studies were conducted at Department of plant pathology ,Agriculture Collage and Research Institute, killikulam.

Spawn preparation :To prepare spawn we first took paddy or sorghum grains. It should be washed with water and remove the chaffy and damaged grains. after that the grain were boiled in vessel for 1 hours to soften the grains, excess water are removed from the boiled grains and spered uniformly over the hession cloth. Calcium carbonate was mixed with grains at 50% of moisture contant @20g|kg of grains.

after that use polythene bag size of 3/4th height and grains to be filled. Pvc using was placed on the edges of the bag down, the mouth of the bag was plugged tightly using non-absorbent cotton. Finally the bag was covered with a price of waste paper and tied tightly around the neck using rubber band. The bags were placed inside an autoclave and sterilized at 20lbs for 2 hours. After sterilization the bags were taken and placed inside the laminar air flow chamber fo inoculation .

The mycelial culture (8-10 mm dm disc) of plerotus sp. were cut and transformed to the bag. After finishing the innoculation the bag were incubated in clean room under temperature of 28 for 10 days. The rate of mycelial growth on the substrate was measured in days.

Bed preparation:

Paddy straw is good substrate for bed preparation. The substrate was act into 4-5 cm long bits. Soaked in cold water foe 4 hours and pasturied in hot water for 30 mins at 80oc using a transparent polythene bag as 30×60 cm length and thickness of gauge 80 for cultivation of oyster mushroom initially hand should be washed thoroughly using 70 % ethanol.

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The bottom of the bag was tied with thread and turn inside out. Maintain uniform moisture level of dried straw. Then the bed spawn was taken out and finlly pressed with hand and divided into 2 halves. From single spawn we make to beds. Small chopped straw (2-5cm long) were placed on bottom of the bag and make a layer up to 10 cm height, on which 40 g of spawn were placed. Second layer make up to 5 cm and again placed the spawn 40g. likewise five layer of straw and 4 layers of spawn were kept in polythene bag and finally tied the bag using rubber band. Requied number of ventilation holes of 1 cm depth were made randomly. Finally the beds were placed in spawn running room and maintained the temp at 28+-2 o c and RH 80 % for initiation of basidiocarp.

Some procedure to be followed for all bed substrate for standard bed substrate paddy straw is good. For this study. Paddy grain straw of pleurotus sp. were used. Four replications were maintained. Observation on days recquied for spawn running, sporocarp formation and weight of sporocarps to be recorded finally biological efficacy was calculated.

3.1. morphological Characterization of Pleurotus djamor isolates KKM 1 and Pleurotus florida Pleurotus djamor However, Pleurotus isolate Woody-1 is more leathery, contains less plectenchyma tissue in the pileus.

On the PDA medium. Mycelial characters of woody is thread like and Less fluffy. Stripe of the woody is Absent or undeveloped. wavy margin is preasnt the pileus. Plectenchymatou tissue is very less in the pileus of woody. Hence it is rubbery or fibric. More number of gillus present in woody. However, Pleurotus isolate Woody-1 is more leathery, contains less plectenchyma tissue in the pileus.

Pleurotus florida Mycelial characters is more fluffy on the medium. PF Pileus margin is wavy smooth margin with typical stripe. Plectenchymatou tissue in the pileus is more. That is why it is soft. More number of gillus present in PF.

Developmental stages of basidiocarps of Pleurotus spp.

Various isolates viz, Pleurotus sp. Isolate KKM1, Pleurotus florida were inoculated in baddy straw based beds for basidiocarps development observed from the time of inoculation to maturity. To observe the number of the days taken for the development of basidiocarps various stages such as primordial stage, mature stage and over matured stages were noted.

3.2.Chemical mutation:

Collection of basidiospores:

The Woody 1 matured pileus was collected aseptically. And cut pileus by using sterile blade. The pileus were kept in a petriplate, the gills are facing the down the lower plate. basidiospores were allowed to disperse. For the petri dish were incubated at 28oc for 2-4 hours. Add 10 ml of sterile water to the petridish to harvest the basidiospores.

Determination of number of basidiospores (permananent stock basidiospore):

Count the number of basidiospore in the harvested solution using haemocytometer and record the the basidospore count.

Dilution of basidiospore (working stock basidiospore)The harvested basidiospores were diluted with sterile water to to get 10-5 to 10-6 basidiospores /ml

Chemical mutation done by EMS

Take 500 µl of basidiospore suspension containing 105-106 spore count per ml. for different Ethyl methane sulfonate (EMS) concentration take 5 centrifuge tubes. Centrifuge the basidiospore solution to get pellet. Discard the supernant from the centrifuge tubes. finally leave upto 20 µl of basidiospore in centrifuge tube at the bottom. Add different concentration of EMS solution 40mM, 80mM, 120Mm, 160mM, 200mM. incubate for 1 hours in shaker at 150 rpm. after the incubation period basidiospores washing by using sterile water for 2-3 times. Count the number of basidiospore present in centrifuge tube by using haemocytometer. spread the basidiospore of 200 count on PDA plates. Incubate for 8 days. Look for basidiospore growing very fastly and thickly. breed mutated spores 1 in 10 unmutated Pleurotus florida. basidiospore that are opposite mating type.

Collection and storage of basidiospores:

The fruiting body of mature pileus of pleurotus sp. was taken. the isolate KKM1 and p. florida was cut into 3×3 sized pieces using a sterilized knife, a piece of pileus was taken and fixed in the sterile upper plate facing the inner side having petri dish with glue and in same way the gills are facing down on the lower plates. Incubate the petri dishes at 28 o c for 4-6 hours thus allowing the basidiospores to be dispersed. Collect the basidiospore by adding 5-10 ml sterilized water in the petri dish. Then the spores are taken in 50 ml falcom tube and it can be stored as a stock suspension. Using a haemocytometer, the number of basidiospore was counted. Then the basidiospore were diluted to 500 basidiospore per milli liter and it can be kept for working basidiospore stock.

Culturing the monokaryons:

The basidiospore stock, in which the work is going on was spread on the PDA medium in plates of about one hundred microlitre of basidiospore suspension which contains approximately 50 basidiospore using L-rod spreader. Incubation of the plate is done @ 28oc for 5-7 days or until the colony develops around each basidiospore giving monokaryotic mycelium with the diameter of 3-5 mm. different colonies of mycelium about 15 to20 number was picked from the plate culture and placed in a grid form in another PDA plate at equal distance.

Incubation was carried at 28oc for master plates for 2-3 days. Around the inoculated points, visible colonies of monokaryotic mycelia were soon. Further development of monokaryotic was stopped by keeping the grid plate @ 4 oc in refrigerator, where merging of mycelial colonies can be avoided.

Hybridization cross breeding of monokaryon:

A well grown monokaryotic isolate of Plerotus isolate KKM 1 and P.florida of about 13 and 30 number were selectively or respectively. Each of the two monokaryotic isolates are mated by inoculation them in a PDA medium at the distance of 2 cm apart from centre. Inoculation were carried till the isolate approach together and fuse with one another.

Analysing of successful crosseshybrid:

The putative dikaryotic mycelium was taken from the point of junction formed after the fusion of two monokaryon and changed into new PDA plate and inoculated for five days for growth of mycelium. during the incubation period , the phenotypic character of the putative dikaryotic mycelium were observed. The phenotypic character include the rate of growth, culture sectoring, growth defect and clam connection were present in the mycelium, the crosses were considered to be positive when viewed under the light microscope.

Spawn production:

Mother spawn was prepared by using different hybrid strains inoculated paddy grains in a polythene bag. Maintained at 3 replication and incubated at room temperature (28oc). The number of days taken for complete spawn development and better of growth were observed and noted.

Bed preparation:

The mushroom beds were prepared by using a paddy straw inoculated with different hybrid stains. Formation of basidiocarps, requirement of days for sporophore formation (in days) and their weight was observed and noted. And the biological efficiency was calculated.

Growth rate of hybrid strains on PDA medium:

To study the mycelial growth, 5 mm culture discs of both hybrid strain and the parent culture were observed from actively growing margins of fungus colony with sterilized cork borer and inoculate on PDA plates and incubated at 28oc and maintained at 3 replication. When any one of isolate was completely grown with the mycelium, the growth of the mycelium was observed and recorded.

Growth rate of hybrid strains on spawn substrate:

Mother spawn was prepared in flask by using paddy grains and inoculated with 5 discs of different hybrid strains per packet and maintained at 3 replication. The spawn were incubated at room temperature. The number of days taken for complete development of spawn and pattern of growth were observed and noted.

Study of morphological character of basidiocarps of selected hybrids:

Evaluation of biological efficiency of selected hybrid:

To evaluate the biological efficiency of selected hybrid strains, mushroom beds were placed along with parent stain for comparation. Days for formation of basidiocarps, and biological efficiency were recorded for each isolates 3 replication were maintained.

Statistical analysis:

The data were statistically analysed with the help of SPSS version 160.data were subjected the ANOVA at a significant level (p 0.05) and by using DMRT, the means were compared.