International Journal of Farming and Allied Sciences Assessment Of Total RNA Extraction From Edible Mushroom (Agaricus bisporus) With Three Current Methods

ABSTRACT: Nucleic acid extraction is an attractive alternative to labor-intensive manual methods. In this report, a few methods of total RNA Extraction from Edible mushroom (Agaricus bisporus) compared. This methods consist AGPC(Acid Guanidinium-Phenol-Chloroform), The method according by Lithium chloride and RNx total RNA isolation Kit (CinnaGen). Best method selected for the preparation of high quality and intact total RNA from A. bisporus. Based on the Ethidium Bromide straining pattern of 28s and 18s rRNAs, either denaturing (formaldehyde/agarose) or nondenaturing (TAE/agarose) gel electrophoresis could be used to determine the general integrity of the RNA preparation. Analysis by either method revealed sharp rRNA bands indicative of high quality RNA. yield of RNA was acceptable in any three method but RNA quality in AGPC method similar with RNx Total RNA isolation kit (CinnaGen)

Screening of the White Button Mushroom (Agaricusbisporus) Homokaryons and Producing New Hybrid Strain by SSR Markers

Introduction: Edible white button mushroom (Agaricusbisporus) is the most common edible mushroom in Iran and the world. The yield of this mushroom is less than the average of yield in the world because of strain degeneration and using strains with low yield. Most of the current hybrids are either identical or very similar to the first hybrids. Ongoing breeding programs are exploiting the variability in Agaricus germplasm to produce new varieties with better traits including higher yield and resistance to biotic and abiotic stresses. One of the breeding programs is F1 production from parental homokaryons crossing. These homokaryonsis were isolated among germinated basidiospores on the culture media. During the last decades, various molecular markers based on nucleic acid polymorphisms (such as Restriction Fragment Length Polymorphism, Random Amplification of Polymorphic DNA, Amplified fragment of Length Polymorphism, Inter Simple Sequence Repeat, Simple Sequence Repeat markers) have been used to differentiate homokaryons and heterokaryons. Microsatellites consist of short tandem repeat motifs distributed throughout the genome. Microsatellites are usually highly polymorphic due to a high degree of variation in the number of repeats among individuals. Microsatellite markers are multiallelic and co-dominant and thus tend to be more informative than other marker systems. Microsatellite markers have been widely developed in animals and plants and more recently in fungal species. The presence of microsatellites in the genome of A. bisporus was previously reported. Materials and Methods: In this research, 160 germinated basidiospores were collected from commercially cultivated strain A15 and they were grown on compost extract agar (CEA). The mycelial growth rate of these160 isolates was evaluated at 25°C on CEA medium. 18 isolates with slow growing rate were selected from 160 isolates. In the next step, co-dominant SSR markers were used to homokaryons detection. Ten SSR primers showed polymorphism in parental control samples that were used to this experiment. The isolates were divided into two general homoallelic and heteroallelic groups and seven isolates from homoallellic group, which showed one-band pattern, characterized as putative homokaryon. Genetic similarity was calculated by NTSYSpc software version 2.02 e using UPGMA method. In the next step of experiment, the isolates (4 and 8) had minimum genetic similarity that was crossed to produce hybrid. In order to confirm the hybrid formation, PCR-SSR reaction with a primer (AbSSR 45) was performed. Results and Discussions: Basidiospores were collected and allowed to germinate on CEA medium. Putative homokaryons were different in colony morphology and growth rate compared to the original heterokaryons. Mycelium samples showed different colony morphology including tomentose, apprised and strandy mycelium. Different growth rate can be affected by genetic factors in nucleus and mitoconderia. After four weeks, mycelium browning was appeared in liquid compost extract medium and created a disturbance in DNA extraction. To solve this problem, DNA was extracted from three-week old mycelium. Mycelium browning may cause by phenolic compounds produced by mycelium and enzymes that catalyze melanin biosynthesis reactions. Ten primers were used to homokaryon isolation. These primers were situated on the 9 linkage groups of 13 haploid chromosomes. Seven isolates were distinguished as putative homokaryon that showed one-band in all primers on the gel electrophoresis. The results of genetic similarity calculation showed that this index was variable between 0.17 to 0.67in 7 homokaryon isolates and the minimum genetic similarity (0.17) was observed between isolates 4 and 8. These two isolates were crossed and the result of this crossing was N1 hybrid. Also, other homokaryon isolates were crossed and mating incompatibility was observed in some of them. According to these observations, it is suggested that in future studies, in addition to genetic similarity, sexual incompatibility should also be considered. Hybrid N1 produced aerial mycelium and had higher growth rate in comparison to parental homokaryons and similar to heterokaryon control, had two-bands pattern. This two bands pattern indicates the presence of two non-sister nucleuse in each cells. Finally, the results showed that SSR marker can result to accurate detection of homokaryons. Conclusions: The aim of the present study was screening homokaryon isolates of A.bisporus using SSR markers to obtain hybrid. Results showed that growth rate of homokaryon isolates were lower than the heterokaryons. Since, SSR markers were able to show high polymorphism in the isolates, thus it can be said that these markers are suitable to homokaryon screening. Final result of this study is N1 hybrid that can compare to commercially cultivated strains

Optimization of King Oyster Mushroom (Pleurotus eryngii) Substrate Using Lignocellulosic Affordable Wastes

Introduction: King oyster mushroom (Pleurotus eryngii) belongs to Basidiomycota division, Agaricomycetes class and Pleurotaceae family. This mushroom generally grows on wood wastes of Apiaceae family. The Pleurotus eryngii is found in pastures, meadows, gardens and seldom in grassy forest clearings and hilly areas. The Pleurotus of the Umbellifers occupy an area in the Northern hemisphere between the 30 and 50º N. These species are mainly found in the subtropical regions of the Mediterranean, Central Europe, Russia, Ukraine, Central Asia and Iran. The P. eryngii sensulato is the only taxon within the genus, which grows in association with plants. P. eryngii has distinguishable characteristics such as coherent texture, unique form, favorable taste and high durability. Mushroom cultivation represents the only current economically viable biotechnology process for the conversion of waste plant residues from forests and agriculture. The species of these genera show much diversity in their adaptation the varying agro-climatic condition which makes more cultivated species than other mushrooms. Special ability of Pleurotus family is growing in lingocellulosic plant or agricultural wastes without needing to prepared compost and casing soil. Pleurotus is an efficient lignin- degrading mushroom and can grow and yield well on different types of lignocellulolosic materials. Type of substrates for mushroom growing depends on available plant or agricultural wastes. In Europe, wheat straw is used for mushroom growing; whereas in Asian South-East countries sawdust is more popular. Different materials for cultivating of P. eryngii have been suggested in different regions of the world; but a few studies have been done on suitability of various lignocellulosic affordable wastes for P. eryngii production in Iran. Therefore, the current study aims to evaluate effects of various locally available agro wastes on the growth characteristics of King oyster mushroom (P. eryngii). Materials and Methods: Sawdust was utilized as the main substrate obtained from beech and populous trees (1:1). After being rinsed off in water and supplemented with calcium sulfate (3%) and calcium carbonate (3%), the substrate was filled in 20 × 40 cm polyethylene bags weighted to 800 grams. Sterilization was performed at 121 °C under pressure of 1.5 bars for two hours. A cultivated P. eryngii strain was then inoculated in the cooled material at a rate of 3% of dry/fresh substrate. The experiments were conducted based on a completely randomized design with five treatments and four replications, measuring mycelial growth (MG), number of fruiting bodies (NFB), mushroom weight, and biological efficiency (BE). AMG was measured in both test tubes and in petri plates in different pH levels (5.5, 7, and 8.5). Data were analyzed by JAMP 4.0, while graphs were drawn by Microsoft Excel 2007 and SigmaPlot 12.0 software. Results and Discussion: The pH of 7 was found to be the best for obtaining maximal MG under all treatments after seven days. The highest amount of MG was obtained with substrate No. 1, while the least was observed in the culture of substrate No. 5. The substrates No. 1 and No. 5 generated the highest and lowest NFBs (p≤0.05). However, there was no significant difference (p≥0.05) in NFB between substrates No. 1 and 3 or between substrates No. 2, 4 and 5. The BE percentages obtained from experimental treatments No. 1, 2, 3, 4, and 5 were 64.81, 49.74, 59.22, 28.72, and 19.8, respectively. The comparison of means of different growth characteristics revealed that there was no significant difference between substrates No. 1 and 3 or between substrates No. 4 and 5 (p≥0.05). Conclusion: In this time, only two species (Agaricus bisporus and P. ostreatus) are producing in Iran, whereas at least 10 species of edible mushrooms are cultivating in the world. King oyster mushroom has low cost of production and distinguishable characteristics. Therefore, this mushroom can be use as alternative for button mushroom (A. bisporus). Many kind of agricultural wastes are in use for mushroom cultivation. Understanding the effects of substrate materials on mushroom production will be very valuable. The average number of fruits and biological efficiency of treatment No.1 showed significant difference with other treatments. Hence, the treatment No. 1 could be used for commercial production of King oyster mushrooms in Iran. Growth rate of P. eryngii was very diverse, in respect to the determinate values of the environmental factors. On the basis of the average growth rate of the strains, we could conclude what are the optimum ecological values of the species, though these conclusions did not always coincide with the optimum values of the certain strains. However, more research needs to be done to obtain regular and homogeneous supply of this mushroom