Estimating the chemical composition of secondary compounds of Iraqi wild Agaricus bellaniae characterized morphologically and genetically

: This study, which is considered the first of its kind in the world and the Arab homeland, was carried out in the laboratory of mushroom production belonging to the Medicinal Plant Unit/ College Of Agricultural Engineering Sciences/ University of Baghdad during the period from July 21, 2016


Introduction:
The mushroom Agaricus bellaniae belongs to the Basidiomycota group, order Agaricales, and family Agaricaceae which is listed among edible mushrooms.It was classified as edible mushroom that grows during late summer to early fall season within weeds in the form of cuts, arches, or circles distributed in the huge eastern plains of Illinois in the USA 1 .This type of mushroom is characterized by the short, convex, and bell-shaped cap in the early stages and then, at the maturity, it expanded to become flat with inverted convex from the middle to the inside.The cap diameter is 3.5-8 cm containing yellowish-brown gills in contrary to the color of the cap, which is pale brown in color at the edges and dark brown in the middle that grades in color during maturity.The cap edge is soft and bump-free while the gills are free at the stem region.They are short and compact at initially with a white color turns to pink in the center of the cap at the aging ending in brown color at the button stage when the brittle, white ring appears on the stem.The stem height is 3.5 -7cm and its diameter 5-12 cm either uniform or widening at the base.Stem color turns to brown during aging, and sometimes pink bumps appear at the base.The flesh is white unchanging color after cutting.The spore print color is dark brown 2 .
The importance of edible genus Agaricus collected from the wild and consumed by humans is due to the high nutritional value, medicinal compound content, taste, and flavor.This mushroom genera included A. augustus Fr., A. campestris L.: Fr, A. arvensis schaeff, and A subrufescens 3 however, A.bisporus is regarded the commonest species among them for the nutritional value it has as it is characterized by the high content of protein in the fruit bodies that is around 11.01-29.14% varying according to the growth culture media 4 .Total carbohydrates content was 51.05% involving the digestible carbohydrates such as mannitol and glucose, usually forming less than 1%, in addition to glycogen forming 5-10% of the dried fruit bodies while the indigestible carbohydrates, including oligosaccharides such as trehalose and non starch as well as the polysaccharides such as lignin, betaglucan, and mannans, formed the major part of the carbohydrates found within the cultivated mushroom 5 .Mannitol and trehalose are among the saccharides abundantly found in the cultivated mushroom of both types, the white and the brown.The dried fruit bodies of A.bisporus (cultivated mushroom) also contain chitin, hemicellulose, mannans, and Beta-glucan which have beneficial characteristics for human health 6 .Rukaibaa 7 mentioned that the fruit bodies of the cultivated mushroom contain k, Fe, Zn, Cu, Na, Se, Mn, Cad, and Co where the amount of phosphor, calcium, magnesium, sodium, iron, and zinc is usually high in the dried fruit bodies .Ekhlas et al 6 found that the polysaccharides content in the dried fruit bodies of the cultivated white mushroom of the brown series enhances the immunity system in a human body and acts as an anti-tumors in-vitro as well as in-vivo., furthermore, the dried bodies of them contain anti-filamentary and anti-oxidants such as phenols and chitosan that prevent the fatty liver disease 8 as well as they contain several antibacterial compounds 9 .This mushroom has been recognized in North America through a study prior to this and symbolized at the National Center for Biotechnology Information (NCBI), with the symbols NR_145001.1,KJ877783.1 10 , and KJ877782.1 .Therefore, conducted for the first time in Iraq, this research aims to determine the nutritional and medicinal compounds in the mushroom Agaricus bellaniae which grows wildly in the middle region of Iraq at the high temperature weather.

Material and methods:
Samples collection: The samples of Agaricus bellaniae were obtained from the Salhia region, Baghdad where the temperature was about 50-55 °Con July 21, 2016 (Fig. 1).They were propagated and purified on Potato Dextrose Agar (PDA) media 11

Electrophoresis of PCR products
Preparation of agarose gel: The gel was prepared at a concentration of 1% by dissolving 0.5 g of acarose in 50 ml of 1 x TBE solution, heated using a microwave oven for 2 minutes, cooled to approximately 55 °C and adding 2 μl of ethidium bromide dye to it.

Preparation of gel mold and sample
Pour the gel at 55-50°C into the electric transfer mould.Place the comb at the end of the gel mould, after the end of the mold is blocked, and leave for half an hour to solidify.Then, remove the comb and add 1× TBE buffer to cover the surface of the gel.After PCR reaction, the samples results were electrophoresis on agarose gel 1.5% and sent to Macrogene Co. thatgave us nucleotides sequence for each isolate , this sequence enteredthe blast program to find the similarity between sequences of the isolates, while all isolates are recorded in NCBI.investigating the best extent of mycelium growth of the mushroom Agaricus bellaniae from the mother culture in vitro.
The mycelium growth in PDA was studied within various temperature extents including the following seven treatments (25,30,35,40,45,50,55,60) °C The parameters included are:  Number of days for completing the colony diameter.
 Number of days required for mycelium growth commence.The experiment involved culturing 5 dishes where the results were analyzed with Genstat software according to the Completely Randomized Design (CRD).investigating the chemical composition and the content of the medicinal secondary compounds of the mushroom.The remained part of the fruit body samples were dried and ground with a special grinder.Next, the samples were placed in tightly closed containers prepared for this purpose, and after that, they were sent to the Arab Republic of Egypt, Agricultural Research Center, Regional Food and Feed Center, to analyze the chemical elements and compositions and probe the percentage of medicinal compounds with GC-mass device where the following estimates have been involved.1.The medicinal compounds content in the fruit bodies estimated with the GC-mass device.2. The total antioxidants content, total phenols content and total flavonoids content in the dried fruit bodies calculated according to the procedure described by Albaldawi et al 13 .3. The content of the chemicals in the dried fruit bodies calculated according to the procedure described by Cho et al 14 .

Results and discussion:
The This diagnosis relied on the gene ITS4 gene.Firstly, the fungus' genetic material was extracted and checked for purity, and then it was amplified by PCR using the gene mentioned above.One package appeared, representing the whole genome extracted from the fungus.ITS4 gene amplification was done using the mentioned primers and the prepared program for this purpose.Table 4, demonstrates that Linoleic acid content in the dried fruit bodies was as the highest percentage that recorded 47.77 %.Linoleic acid is defined as a polyunsaturated fatty acid used in the biosynthesis of arachidonic acid (AA) as well as some other substances such as Prostaglandin, Leukotrienes (LTA, LTB, and LTC), and Thromboxane (TXA).All these linoleic acid products have a biological activity associated with human physiology and pathology.Linoleic acid is an essential fatty acid for humans, which must be obtained through diet forgood health.During an experiment on mice, lacking linoeate in the feed resulted in slight expansion in the skin, hair loss ,and poor wound healing , furthermore, it is considered an anti-cancer 16 .The percentage of sinaply alcohol was 0.99 7%.
It is an organic compound related to cinnamic acid, one of the mono lignin that is considered the initiator for the biosynthesis of different stilbenoids and coumarins, while the percentage of Melezitose was 0.51 %.This compound is molecularly decomposed into glucose and turanse, sucrose isomer, 17 .The percentage of the flavone complex Scutellarein was19.3% which importance lies as an anti-ischemic and anti-heart disease in humans 18 .The compound 4',6-Dimethoxyisoflavone-7-O-B-D was at the percentage of 1.15% which importance lies as a secondary compound regulates the fat metabolism in the liver and reduces the insulin allergy 19 .The percentage of Zearalenone was 0.77% that is one of the mycotoxins acting as an anti-oxidant leading to the programmed death of cancer cells 20 .Glycitin recorded 0.67 %, which is a glycosylisoflavone compound characterized by its activity inhibiting the cancer cell growth 21 .The percentage of Cholesta-4, 6-dien-3-one reached 0.87%.It is an important compound, as this was proven through laboratory experiments in mice, it reduces cholesterol in the body 22 .The Ethyl linoleate percentage reached 11.42%.It acts as an anti-fungal and anti-oxidant compound 23 .The percentage of Quercetin 3',4'-7-trimethyl ether was 0.92%.It is a flavone belonging to the polyphenols helping to prevent neurodegenerative diseases and inhibit six types of cancer.The percentage of γ-Sitrosterol was 0.89%.It is characterized by the coeffect on the human immune system and the protection against many diseases 24 .The percentage of oleic acid was 1.1 %.This fatty acid is considered as anti-inflammatory compound.Luteolin6, 8-C-diglucoside recorded the percentage of 1.1 %.It is one of the monounsaturated fatty acids known as omega 9 that is considered an antioxidant helping to decrease hypertension in addition to enhancing the cell membrane construction and brain development 25 .The percentage of vitamin E was 1.67%.This vitamin is known as antiinflammatory and anti-oxidant which is related to many diseases and cancer types such as prostate and skin cancer, moreover the skin diseases such as eczema 26 .The phytanic acid showed a percentage of 1.16%.It is one of the saturated fatty acids reducing prostate cancer occurrence 27 .The percentage of Trans Geranyl geraniol was 0.82%.This compound prevents the poisonous effect of cholesterol on cells without reducing the beneficial effect 28 .Curcumol which reached 1.02% reduces the incidence of breast cancer 29 .The percentage of Stigmasterol was 1.02%.It decreases cholesterol as a result of reducing its absorption since it is considered an anti-cancer of colon, ovary, prostate, and breast 30 . .
Though the oil percentage in the studied mushroom was high, most of the fatty acids were beneficial in the sense of health for humans that was confirmed by the results illustrated in  The results in Table 8, demonstrate the amino acid contents in the dried fruit bodies in the wild mushroom where the highest percentage was recorded by Glutamic acid 4.02% followed by Aspartic acid 2.26%.The data in the same table show that the powder of the dried fruit bodies contained 16 amino acids beneficial for human nutrition.Glutamic acid is necessary as it enhances the immunity system of the human body and maintains the health of newborns, adults, and pregnant women as well as it maintains the integrity of the membrane surrounding the digestive system 34,35 .Aspartic acid is distinguished by being involved in synthesizing and secreting hormone testosterone, which is responsible for fertility in men and important for athletes, as it participates in building muscles and increasing their strength 36,37 .

Conclusion:
According to the high medicinal and nutritional value of Agaricus bellaniae represented by this study, the results show that the dried fruit bodies are rich in secondary compounds such as 47.77% Linoleic acid and 0.369 ppm of selenium which are considered as anti-cancer compounds in addition to sodium and potassium which play an essential role in decreasing the hypertension of human.The dried fruit bodies of this mushroom are also distinguished by the high percentage of glutamic acid 4.02% and aspartic acid 2.26% which are important for human health.Therefore, according to the findings obtained, we recommend producing the mushroom on a commercial scale, taking into account introducing the treatments that increase the valuable medicinal and nutritional materials of the medicinal importance for the human body.
-We hereby confirm that all the Figures and Tables in the manuscript are mine ours.Besides, the Figures and images, which are not mine ours, have been given the permission for republication attached with the manuscript.-Ethical Clearance: The project was approved by the local ethical committee in University of Baghdad, Iraq.

Authors'contributions statement:
The project has been done in College of Agricultural Engineering Sciences,University of Baghdad.Z M.A-Q and RA.C wrote the manuscript .ZM.A-Q performed the statistical analysis .Z M.Al-Q and R A.C discussed the results and contributed the final manuscript.S k.M.A-T participated in the isolation coding of the fungi in this research in NCBI American organization .
in the Mushroom Laboratory of the Medicinal and Aromatic Plants Research Unit at the University of Baghdad, College of Agricultural Engineering Sciences where the isolation purification period lasted virtually for a year.

Fig. 2 ,Figure 2 .
Shows gel electrophoresis of DNA extracted from fungal isolates; a single bind appeared at the site 600 -700 bases and in two replicates 15M F1 Electrophoresis of the amplification product of the ITS4 gene showing the location of the segment between the molecular weight 600-700 nitrogenous basesThis bind was sent to the Korean company (Macrogene) to show the sequence of nitrogenous bases.Upon analyzing these results in the BLAST program, it was found that the isolate under study was related to Agaricus bellanniae, and the isolate matched with an isolate that had been registered in the genebank with the code NR145001.1.The strain under-study was recorded in the genebank with the searchers' names as a new isolate of Agaricus bellanniae and was given the international code MF987843.It is to be noted that this strain of fungus is being diagnosed for the first time in Iraq and the Middle East.

Table 2 . Morphological identification the fresh bodies of the wild mushroom Agaricus bellaniae
10mple of Agaricus bellaniae was obtained from Salhia region, Baghdad where the temperature was about 50-55 °Con July 21, 2016.They were propagated and purified on Potato Dextrose Agar (PDA) media10in the Mushroom Laboratory of the Medicinal and Aromatic Plants Research Unit at the University of Baghdad, College of Agricultural Engineering Sciences where the isolation purification period lasted virtually for a year.The morphological traits of the wild mushroom bodies were identified as illustrated in Table2.

and bell-shaped in the early stages and then, at the maturity, it expanded to become flat with inverted convex from the middle to the inside.
Cap diameter About 4-8.7 cm Cap colorOuter surface is dark brown in the middle, graduates to pale at the cap edges, while the inner part is characterized by its white flesh Gills Free at the stem region with white color at the edges and pink in the center of the cap at the commencing and then turn to dark brown at aging.Ring Brittle, white in color appears on the stem beyond the button stage completion Stem Heighted about 3.1-7.8cm with 4.6-11.8mm in diameter, widening at the base, browning in color during aging, and has pink bumps near the base.
The fungi molecular diagnosis was made by 18S with ITS1 and ITS4 primers usage .After PCR reaction samples results were electrophoresis on agarose gel 1.5% and sent to Macrogene Co.While Macrogene Co repeated a nucleotides sequence for each isolate, this sequence enteredthe blast program to find the similarity between sequences of the isolates.

Table 5 ,
22ustrates the content of total antioxidants, total phenols, and total flavonoids, in the fruit bodies of Agaricus bellaniae, to which the medicinal importance of the mushroom is due unlike what is found in the common species, A. bisporus22