ISOLATION OF PURE CULTURE OF MEDICINAL HERB MACROFUNGUS Inonotus Hispidus (Bull.) P.Karst. IN UZBEKISTAN

ABSTRACT

This article presents information about the pure culture of medicinal Inonotus hispidus (Bull.) P.Karst., macrofungus in Uzbekistan. I.hispidus was collected from the bark of a growing mulberry tree. Identification of the fungus is done by preparing temporary preparations in laboratory conditions and using them under a microscope, and by mycological analysis of the given samples, the composition of the species is determined. In order to separate the pure culture, it was planted in two different nutrient media. These nutrients were found to be effective as the macrofungus I. hispidus grew well in both nutrient media.

АННОТАЦИЯ

В данной статье представлены сведения о чистой культуре лекарственного Inonotus hispidus (Bull.) P.Karst., макрогриба в Узбекистане. I. hispidus собирали с коры растущего тутового дерева. Идентификацию гриба производили путем приготовления временных препаратов в лабораторных условиях и использования их под микроскопом, а путем микологического анализа полученных проб определяли видовой состав. Для выделения чистой культуры, гриб высевали на две разные питательные среды. Эти питательные среды оказались эффективными, так как макрогриб I.hispidus хорошо рос в обеих питательных средах.

Keywords: Inonotus hispidus, spores, macrofungus, mycelia

Ключевые слова: Inonotus hispidus, споры, макрогриб, мицелий

Introduction

Currently, world mycologists are thoroughly studying the natural reserves of macromycetes and are trying to find scientifically based ways to use some promising species for consumption and as medicinal products for folk medicine [6].

Information about the medicinal properties of macrofungi has been widely used since ancient times in the experience of many countries of the world, especially in the countries of South-East Asia. Over the past two decades, intensive research has been conducted on their biochemical composition and medicinal properties. Today, foreign companies produce various nutritional products, medicines and cosmetic products from them, which are sold for billions of US dollars and bring huge profits [4]. There is information about the strengthening of the immune system and lowering of cholesterol levels, anti-tumor, anti-viral and other beneficial properties of Doivor macrofungi [4]. 

In China, Inonotus hispidus (Bull.) P.Karst., is a famous medicinal macrofungus with a long history. Secondary metabolites from I.hispidus and their biological activity have been discovered and investigated by scientists in the last 60 years [9]. 

I.hispidus has been used as a traditional medicine to treat dyspepsia, cancer and diabetes. Many studies have confirmed the antimicrobial, antioxidant, anti-inflammatory and biological activity of extracts from this species [8]. I.hispidus fungus is a facultative saprophyte (brown rot Basidiomycete) on various broadleaf trees in China and Europe, including mulberry (Morus alba L.), (Fraxinus mandshurica Rupr.), Populus euphratica Oliv., Ulmus campestris L., Sorbus aucuparia L., and Acer saccharum Marshall [12,13].

It is known that a number of works are being carried out in our Republic regarding the study of macrofungi. In 2020-2022, in the course of our scientific research to study macromycetes of deserts, hills and mountain ranges in Kashkadarya region, the medicinal fungus I.hispidus (Bull.) was found in Shahrisabz and Yakkabog districts.

I.hispidus is a fungus belonging to the genus Inonotus of the family Hymenochaetaceae, class Agaricomycetes of macromycetes, order Hymenochaetales, which mainly grows on the trunks of growing trees and causes severe white rot disease in wood.

This fungus was first introduced in Uzbekistan by A.A. Petrova (1989) [7] in the Zomin mountain forest reserve, G.M. Baltaeva (1992) [1] studied macromycetes belonging to the Polyporaceae family in the conditions of Uzbekistan. Later, in 2005, during the study of macromycetes of the Fergana Valley, it was reported that Juglans regia, Malus domestica, and Cerasus avium are abundant in trees [6].

The purpose of the study is to obtain a pure culture of the fungus I. hispidus on the basis of morphological and cultural characteristics. Only the floristic distribution of this fungus has been studied.

Research source and methods

In this research, the samples taken from the bark of the mulberry tree, where the fruit bodies of the fungus I. hispidus were collected during the mycological research conducted in June 2020, May 2021 in Shahrisabz, and in June 2022 in the hilly zone of Yakkabog district, served as a source. The identification of the fungus was carried out by preparing temporary preparations under laboratory conditions and viewing them under a microscope (Roskin 1967) [10]. Experimental mycology and other methods were used to identify fungi and isolate a pure culture from the fruit body and substrate of the fungus [2-5].

Results of the study

In the course of our field research conducted along the territory of Kashkadarya region, the fungus I. hispidus was found on the trunk of a mulberry tree growing in the gardens of Yakkabog and Shakhrisabz districts. It was noted that this fungus grows on a thick trunk of a mulberry tree 1 meter above the ground. The fact that the external morphological signs of the fungus depend on the environment in which it grows was also clearly visible in the field. We set ourselves the goal of obtaining a pure culture of this medicinal mushroom and developing an optimal technology for growing it in laboratory conditions in the future.

The fruit body is annual, tuberous, semicircular, juicy at the beginning, soft, the surface of the fruit body is hairy, yellow or yellowish brown. The edge is blunt, sometimes it bears fruit from below, sometimes it is located in 2-3 lobes, and it widely adheres to the substrate. The size of the cover is 10 × 16 × 8 cm. The upper surface of young caps is red-orange, velvety, and the edge is the same color as the rest of the surface.

The lower part of the fruit is white, the holes are round, angular, and their width is 1-3 mm. Spores are almost spherical or broadly elliptical, smooth, 8.3–11 × 7–9 µm.

a                                                           b

Figure 1. Inonotus hispidus (Bull.) P.Karst., а- fruit body; b- spores (Оrigin. Kuziboev Kh)

Pure culture was obtained using experimental mycology methods.

In this case, two types of optimal nutrient media were selected and prepared for the good growth of the fungus.

Potato-glucose agar and Sabouraud glucose agar nutrient media were used.

Potato-glucose agar nutrient medium. For this, clean and crushed potato tubers - 200 gr., glucose - 10 gr., agar-agar - 15-20 gr., water - 1 l. was measured in quantity. Mashed potatoes were thoroughly boiled in water. We filtered the broth. Agar-agar was added to the prepared broth and stirred from time to time until it was completely dissolved. Then it was autoclaved and sterilized.

In order to isolate a pure culture of I.hispidus fungus from the fruit body, an undamaged fruit body was selected and washed in running water to remove various foreign substances. Using a sterilized scalpel, a small piece of 0.5-1.5 cm was cut from the first cleaned fruit body, dipped in 3% hydrogen peroxide liquid for a few seconds, and planted in Petri dishes and test tubes in both nutrient mediums near the alcohol lamp. The seeded Petri dishes were placed in a thermostat at 28℃.

It formed mycelia in 3-4 days on both media and covered the Petri dish within a week (Figure 2).

Figure 2. Growth of Inonotus hispidus fungus in two different nutrient media

The mycelium was observed to grow white in color in Petri dishes. The daily growth of mycelium planted in both nutrients was 0.6-0.7 cm. It was observed that the mycelium attached to the substrate and grew densely (Fig. 3a).

In conclusion, the type of fungus was determined by observing its spores under a microscope, and in order to isolate pure cultures from it, it was planted in two different nutrient media. As a result of the study, it was found that these nutrients are effective because both nutrients grow well in the media. The work was successfully carried out, and for the first time in Uzbekistan, a pure culture of the medicinal fungus I. hispidus was obtained.

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