A system for the isolation, identification and analysis of fungal endophytes from selected medicinal plants

Abstract

A system for the isolation, identification, and diversity and enzyme analysis of endophytic fungi from medicinal plants, consisting of: a plant tissue processing unit configured to process fresh and healthy tissue of medicinal plants; a sterilization device configured to sterilize the surfaces of the plant tissue; an isolation chamber equipped with culture media for isolating endophytic fungi from the sterilized plant tissues; an incubation unit configured to maintain optimal growth conditions for the endophytic fungi; a morphological identification unit comprising optical devices configured to examine cultural characteristics, fruiting bodies and spores of isolated fungi; a diversity analysis unit configured to calculate abundance indices, isolation rates, and colonization rates of the endophytic fungi; and an enzyme screening device with substrate-specific media plates configured to test extracellular enzyme activity.

Description

FIELD OF THE INVENTIONThe present disclosure relates to a system for isolating, identifying and analysing endophytic fungi from medicinal plants. More particularly, the present invention relates to a system for isolating, identifying and analyzing the diversity and enzymatic potential of fungal endophytes from selected medicinal plants for bioindustral applications.BACKGROUND OF THE INVENTIONEndophytic fungi enter into symbiotic relationships with plants without causing disease symptoms. These fungi, which colonize plant tissue internally, provide a rich source of novel bioactive compounds with important industrial and pharmaceutical applications, including agrochemicals, antibiotics, antiparasitics, antioxidants, biopesticides, and cancers. In addition to producing valuable metabolites, endophytes also enhance the resistance of the host plant to environmental stress.The variety of endophytic fungal community varies depending on the host plant species, geographical location, climates and plant age. Understanding this variety presents both a challenge and a chance to discover new fungal species that can produce novel compounds and enzymes.Endophytic fungi have become known due to their production of extracellular enzymes. These enzymes make use of them for nutrient recovery from host plants, for hydrolysis of foodstuffs and for defense against pathogens. Because of their diverse functions and stability, these enzymes offer potential industrial applications. This invention relates to the systematic isolation, identification and analysis of endophytic fungi from medicinal plants, in particular to their ability to produce enzymes.In view of the foregoing discussion, the present invention provides a system for isolating, identifying, diversity studies and enzyme analysis of endophytic fungi from medicinal plants.SUMMARY OF THE INVENTIONThe present disclosure relates to a system for isolating, identifying, diversity analysis and enzyme analysis of endophytic fungi from medicinal plants for bioindustrial applications. The invention provides an integrated system for isolation, identification and analysis of endophytic fungi from medicinal plants, with particular focus on their diversity and ability to produce extracellular enzyme. The system combines special equipment for processing plant tissue, for isolation of fungi, for carrying out diversity analyses and for screening for enzyme activity, thus offering a comprehensive platform for investigating the biotechnological potential of these fungi.The present disclosure aims to provide a system for isolation, identification as well as diversity and enzyme analysis of endophytic fungi from medicinal plants. The system comprises: a plant tissue processing unit for processing fresh and healthy tissue from medicinal plants; a sterilization device for surface sterilization of the plant tissue; an isolation chamber with nutrient media for isolation of endophytic fungi from the sterilized plant tissue; an incubation unit for maintaining optimal growth conditions for the endophytic fungi; a morphological identification unit with optical equipment for examining cultural characteristics, fruit bodies and spores of isolated fungi; a diversity analysis unit for calculating frequency indices, isolation rates and colonization rates of the endophytic fungi; and an enzyme screening device with substrate-specific media plates for assaying extracellular enzyme activity.It is an object of the present disclosure to provide a system for isolation, identification and analysis of endophytic fungi from medicinal plants for bioinustral applications.Another object of the present disclosure is to develop a systematic approach for isolating and identifying endophytic fungi from medicinal plants, including hazardous species.Another object of the present disclosure is to enable comprehensive diversity analysis of endophytic fungal community using multiple statistical indices.Another object of the present disclosure is to enable efficient screening of isolated fungi for their ability to produce extracellular enzymes.Another object of the present disclosure is to provide a system for comparing endophytic fungal community across different plant species and tissues.In order to further clarify the advantages and features of the present disclosure, the invention will be explained in more detail with reference to specific embodiments that are illustrated in the accompanying drawings. This drawing shows only typical embodiments of the invention and is therefore not to be considered as limiting its scope. The invention will be described and explained in more detail with reference to the accompanying drawings.BRIEF DESCRIPTION OF THE FIGUREThese and other features, aspects, and advantages of the present disclosure will become more fully understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. The following applies here: FIG. 1 is a block diagram of a system for isolation, identification, and diversity and enzyme analysis of endophytic fungi from medicinal plants according to an embodiment of the present disclosure.Those skilled in the art will also appreciate that the elements in the drawing are shown for simplicity and are not necessarily to scale. For example, the flowcharts illustrate the method using the key steps to improve understanding of aspects of the present disclosure. Also, as for the construction of the apparatus, individual or plural components of the apparatus may be represented by conventional symbols in the drawing. The drawing may only show the specific details relevant to understanding the embodiments of the present disclosure so as not to obscure the drawing with details readily apparent to those skilled in the art after the present description.DETAILED DESCRIPTION:In order to promote an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and will be described in an comprehensible manner. However, the scope of the invention is not limited thereby. Changes and further modifications of the illustrated system, as well as further applications of the principles of the invention, are possible, as would normally occur to a person skilled in the art.It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.References throughout this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the phrases "in one embodiment," "in another embodiment," and similar phrases in this specification may or may not refer to the same embodiment.The terms "comprises," "comprising," or other variations thereof are intended to cover a non-exclusive inclusion, such that a process or method comprising a list of steps may include not only those steps, but also other steps not expressly listed or inherent in that process or method. Likewise, the phrase "comprises... for" one or more devices, subsystems, elements, structures, or components does not exclude, without further limitations, the existence of other devices, subsystems, elements, structures, components, or additional devices, subsystems, elements, structures, or components.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. The systems, methods, and examples provided herein are for illustrative purposes only and are not to be considered limiting.Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.FIG. 1 shows a block diagram of a system ( 100) for isolation, identification and diversity and enzyme analysis of endophytic fungi from medicinal plants according to an embodiment of the present disclosure.Referring to Figure 1, the system (100) comprises: a plant tissue processing unit (102) for processing fresh and healthy tissue from medicinal plants; a sterilization device (104) for surface sterilization of the plant tissue; an isolation chamber (106) containing culture media for isolation of endophytic fungi from the sterilized plant tissue; an incubation unit (108) for maintaining optimal growth conditions for the endophytic fungi; a morphological identification unit (110) having optical equipment for examining cultural characteristics, fruit bodies and spores of isolated fungi; a diversity analysis unit (112) for calculating frequency indices, isolation rates and colonization rates of the endophytic fungi; and an enzyme screening device (114) having substrate specific media plates for assaying for extracellular enzyme activity.In one embodiment, the diversity analyzer (112) is also configured to calculate the Simpson diversity index, the Simpson dominance index, the artichum, the Shannon-Wiener index, and the uniformity.In one embodiment, the enzyme screening device (114) comprises: a starchy media plate configured to test amylase activity; a hexadecyl; a trimethylammonium bromide media plate configured to test pectinase activity; a carboxymethylcellulose media plate configured to test cellulase activity; a 1-naphthol media plate configured to test laccase activity; and an L-asparagine media plate configured to test asparaginase activity.In one embodiment, the system (100) further comprises a statistical analysis unit (116) configured to perform variance analyses and Duncans multi-range assays on data obtained from the enzyme screening device.In one embodiment, the plant tissue processing unit (102) is configured to process tissue from plants including tinospora. Cordifolia, Piper nigrum, Piper longum, Zingiber officinale, Hedychium Coronarium and Hedychium flavescens.In one embodiment, the system (100) further comprises a Herbaricium preservation unit (118) for depositing plant samples used for isolation of endophytic fungi.In one embodiment, the system (100) further comprises a fungal storage unit (120) configured to maintain viable cultures of isolated endophytic fungi at suitable temperatures.In one embodiment, the isolation chamber (106) includes separate compartments for processing various plant tissues, including leaves, stems, and roots.The present invention relates to a system for the isolation, identification and analysis of endophytic fungi from medicinal plants for bioinustral applications. The system comprises several specialized units, each of which performs a specific function in the study of endophytic fungi. The plant tissue processing unit is designed for processing fresh and healthy tissue from medicinal plants and ensures that the samples remain viable for further analysis. In order to remove surface contamination, the plant tissue is surface-sterilized with a sterilizing apparatus before further processing. After sterilization, the plant tissue is transferred to an isolation chamber equipped with nutrient media specially prepared for isolation of endophytic fungi. The isolated fungi are then incubated in an incubation unit which maintains optimum growth conditions for their development. For identification, a morphological identification unit integrated into the system is integrated. This includes optical instruments for investigating the cultural characteristics, fruiting bodies and spores of the isolated fungi. For evaluating fungus diversity, a diversity analysis unit is integrated which calculates frequency indices, isolation rates and colonization rates of the endophytic fungi. In addition, extended diversity metrics such as Simpsons diversity index, Simpsons dominance index, Artichum, Shannon-Wiener index, and uniformity may be determined. To investigate the enzymatic potential of the isolated fungi, the system has an enzyme screening device with substrate-specific media plates. The screening apparatus consists of a starchy media plate for amylase activity detection, a hexadecyl trimethylammonium bromide containing media plate for pectinase activity, carboxymethylcellulose containing media plate for cellulase activity, 1-naphthol containing media plate for laccase activity and L-asparagine containing media plate for asparaginase activity. The system also includes a statistical analysis unit that facilitates data analysis by analysis of variance (ANOVA) and Duncans Multiple Range (DMRT) tests of enzyme activity data. The plant tissue processing unit of the system is configured for processing tissues of medicinal plants including Tinospora. Cordifolia, Piper nigrum, Piper longum, Zingiber officinale, Hedychium Coronarium and Hedychium flavescens. In addition to the major components, the system has a Herbarium preservation unit for storing plant samples used to isolate endophytic fungi to ensure the availability of reference samples for future studies. Moreover, a fungal storage unit is integrated to store viable cultures of isolated endophytic fungi at suitable temperatures for further research and applications. The isolation chamber is provided with separate chambers for the processing of various plant tissue types including leaves, stems and roots. This minimizes cross-contamination and increases the specificity of fungal isolation.Endophytic fungi were obtained from fresh and healthy tissue from six wild medicinal plants collected in the region Bisle in the Westghet in Karnataka. These plants were deposited as Tinosspora cordifolia(Willd.) Hook. f. & Thomson, Piper nigrum L., Piper longum L., Zingiber officinale Roscoe, Hedychium Coronarium J. Koenig and Hedychium flavescens Carey ex Roscoe. The herbicides of these plant samples were deposited with the National Ayurveda Dietarys Research Institute of the Central Council for Research in Ayurveda and Sidha, Division AYUSH, Ministry of Health and Family Care of the Indian Government in Jayanagar, Bangalore, India. The medicinal use of these plants has been documented for reference purposes.Isolation and identification of endophytic fungi was performed according to standard protocols established in previous studies. Identification was made from culture characteristics, the morphology of the fruit bodies and the spore structures using standard identification manuals. The frequency of endophytic fungi was determined by the absolute frequency (f), which represents the total number of fungi isolated. The relative frequency (fr) was calculated to determine fungal species density, expressed as the number of isolates of each species divided by the total number of isolates, represented as a percentage. The isolation rate (IR) was calculated as the number of isolates obtained from tissue segments divided by the total number of tissue segments used in the study. In addition, the rate of endophytic fungus (CR) occupancy was calculated as a percentage of all isolates obtained from different tissue segments relative to the total number of isolates from all incubated tissue segments.CR = total number of isolates recovered from different tissue segments / total number of isolates recovered from all incubated tissue segmentsIR = Number of isolates recovered from tissue segments / Total number of tissue segmentsDiversity analysis was performed to evaluate the fungal population in the studied medicinal plants. Simpsons diversity index, simpsons dominance index (D), Artichum (S), Shannon-Wiener index (H) and uniformity (E) were calculated to evaluate fungal diversity and distribution patterns. The enzyme production potential of the isolated endophytic fungi was determined by preliminary screening by the plate assay method. Mycelium plugs (5 mm diameter) were placed on solid media with specific substrates for the targeted enzyme detection. Starch was tested for amylase activity, hexadecyl Pectinase activity was tested with trimethylammonium bromide, cellulase activity with carboxymethylcellulose, laccase activity with 1-naphthol and asparaginase activity with L-asparagine. After an incubation time of seven days at room temperature, enzyme activity was determined by the observable reactions.For statistical validation of the experimental results, the data were evaluated using the SPSS version 20 software. The results were subjected to analysis of variance (ANOVA) and significant differences between the mean values were determined with Duncans Multiple Range Test (DMRT). The triplicate experiments assured the reliability of the results and contributed to a well-established assessment of endophytic fungal diversity and its enzymatic potential.A total of 112 fungal isolates corresponding to 26 different fungal taxa were obtained from 480 tissue fragments. These taxa were classified by morphological characteristics into hyphomycetes (38.4%), ascomycetes (34.6%), coelomycetes (15.38%) and zygomycetes (7.69%). The extent of fungal colonization varied between different plant tissues, with leaves harboring a greater variety of endophytic fungi than other tissues. Among the plant species studied, T. cordifoliawas found to have the highest isolation rate (IR), while P. nigrumand Z. officinalewere found to have comparatively lower IR values. The fungal infestation rate (CR) also varied between the six plant species analyzed. Several fungal genera, including Aspergillus, Alternaria, Cladosporium, Colletotrichum, Fusarium, Mucor, Penicillium, Rhizopusand Myceliasterilia, showed a high relative frequency of occurrence. In contrast, acremonium, cyldrocphalum, dreshclera, lasiodiplocia, myrothecium, nigrospora, paecilomycesand torulawere more rarely found. Overall, all plant samples were associated with a wide range of endophytic fungi, with both isolation and colonization rates varying.The drawings and the foregoing description show examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be divided into multiple functional elements. Elements of one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Moreover, the actions of a flow chart need not be performed in the order shown; nor do all actions necessarily need to be performed. Also, actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is by no means limited by these specific examples. Numerous variations, whether or not explicitly stated in the specification, such as differences in structure, dimensions, and material use, are possible. The scope of the embodiments is at least as broad as recited in the following claims.Advantages, other advantages and solutions to problems have been described above with reference to specific embodiments. However, the advantages, merits, solutions to problems and any components that may result in an advantage, merit or solution being introduced or enhanced are not to be understood as critical, required or essential features or components of individual or all claims.REFERENCES100 A system for isolation, identification, diversity and enzyme analysis of endophytic fungi from medicinal plants 102 plant tissue processing unit 104 sterilization devices 106 isolation chamber 108 incubation unit 110 morphological identification unit 112 unit diversity analysis 114 enzyme screening devices 116 unit for statistical analysis 118 herbaricium conservation unit 120 fungal warehouse

Claims

A system for isolation, identification and diversity and enzyme analysis of endophytic fungi from medical plants, comprising: a plant tissue processing unit configured to process fresh and healthy tissue from medical plants; a sterilization device configured to sterilize the surfaces of the plant tissue; an isolation chamber equipped with culture media for isolating endophytic fungi from the sterilized plant tissues; an incubation unit configured to maintain optimal growth conditions for the endophytic fungi; a morphological identification unit comprising optical devices configured to examine cultural characteristics, fruit bodies and spores of isolated fungi; a diversity analyzer configured to calculate frequency indices, isolation rates and colonization rates of endophytic fungi; and an enzyme screening apparatus with substrate specific media plates configured to assay extracellular enzyme activity.The system of claim 1, wherein the diversity analyzer is further configured to calculate the Simpson diversity index, the Simpson dominance index, the Artichum, the Shannon-Wiener index, and the uniformity.The system of claim 1, wherein the enzyme screening device comprises: a starchy media plate configured to test amylase activity; a hexadecyl trimethylammonium bromide containing media plate configured to test pectinase activity; a carboxymethylcellulose containing media plate configured to test cellulase activity; a 1-naphthol containing media plate configured to test laccase activity; and an L-asparagine containing media plate configured to test asparaginase activity.The system of claim 1, further comprising a statistical analysis unit configured to perform variance analyses and Duncans multi-range assays on data obtained from the enzyme screening device.The system of claim 1, wherein the plant tissue processing unit is configured to process tissue from plants including tinospora. Cordifolia, Piper nigrum, Piper longum, Zingiber officinale, Hedychium Coronarium and Hedychium flavescens.The system of claim 1, further comprising a Herbarium preservation unit for depositing plant samples used for isolation of endophytic fungi.The system of claim 1, further comprising a fungal storage unit configured to maintain viable cultures of isolated endophytic fungi at suitable temperatures.The system of claim 1, wherein the isolation chamber comprises separate compartments for processing different plant tissues including leaves, stems and roots.

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