The Diversity of Endophytic Aspergillus

In plants, endophytic fungi and plant are closely related, endophytic fungi can use substances in plants as nutrients to survive. In return, they bring many benefits to the plant, playing an important role in protecting the host plant against the harmful effects of insects, harmful microorganisms or environmental dis-advantages. Recently, secondary fungi metabolites, especially endophytic fungi, are gaining interest because they can produce many bioactive metabolites with antibacterial, anticancer and antioxidant properties. Some endophytic fungi are noted as Aspergillus, Penicllium, Fusarium due to the production of many metabolites for biological effects such as antibacterial, antiviral, anticancer, etc. in which Aspergillus species product many compounds have properties antibacterial such as terrequinon A, terrefuranon, Na-acetyl aszonalemin, etc.


Endophytic microorganisms
Endophytic microorganisms are microorganisms that live in the plant tissue beneath the epidermal cell layers without harming or infecting the host plant, endophytic microorganisms that live in the intercellular space of tissues and thereby they can invade living cells [1].

Endophytic fungi
Endophytic fungi account for a high percentage of the current group of endophytic plant microorganisms. They are considered a source of many new substances, including many active substances with interesting biological effects. These fungal forms can be detected incidentally in the deep tissues of normally growing host plants. They are endogenous to the host plant and, thanks to their strong biosynthetic capacity, are able to produce a large number of metabolites. This may lead to the emergence of new bioactive substances and promises to develop production on an industrial scale. In addition, the substances produced by endophytic fungi are considered as an agent to help balance the microflora on the host plant to prevent pathogens [2,3].

Relationship between endophytic fungi and plants
Endophytic fungi can be easily isolated from a surface-sterilized piece of plant tissue. The number of endophytic fungi found was also very variable when examining different plant samples, this number can range from one to several hundred strains.
The presence of endophytic fungi in plant tissues can be explained in many different ways. But perhaps the most plausible is the hypothesis that endophytic fungi arose from some plant pathology in the evolution of plants. The tree also has a microflora, in which there are strains that exist dormant and cause disease only when the tree is old and weak or facing adverse living conditions. The interaction between the host plant and the pathogenic microorganism during long-term development has resulted in genetic mutations from the pathogenic microorganisms to yield useful strains of endophytic fungi without causing disease [1].
Between endophytic fungi and host plants there is a symbiotic relationship, mutualism or mutualism, etc. The symbiotic or mutualistic relationship between endophytic fungi and plants is shown quite closely. At times they are closely linked as a single individual and contribute to the distinctive character of the tree.
Endophytic fungi promote ecological adaptation of host plants. In some plant species with endophytic fungi, it has been found that they have increased drought tolerance or tolerance of aluminum toxicity in water sources, in habitats, etc. In addition to protecting plants against a number of factors detrimental to the host such as herbivores or insects, many natural products produced by endophytic fungi have also been observed, monitored and concluded on the ability to prevent, inhibit or kill many different pathogens that invade plant tissues. That is also the reason why some endogenous fungal strains can produce phytochemicals that give the host plant a unique and distinctive character [3,4].
For example: in the early 1990s, a novel taxol-producing endophytic fungus, Taxomyces andreanae, was isolated from Taxus brevifolia. This set the stage for a more comprehensive examination of the ability of other Taxus species and other plants to yield endophytes producing taxol. There is an endophytic fungi strain that produces taxol, an important active substance of great significance in the field of medicine.

Basic principles for selecting plants to isolate endophytic fungi
Young plant tissues are more suitable for isolating endophytic fungi than mature tissues because adult tissues often contain many different types of fungi, making isolation difficult. Collected plant samples should be stored at 4°C until endophytic fungi are isolated, and isolation should be carried out as soon as possible to avoid airborne bacterial contamination.
To obtain endophytic fungi with biological activity, it is necessary to select plant species that are outstanding in terms of biology, age, endemism, botanical history, and habitat of the host plant. Many studies have shown that medicinal plants and plants living in special environments are frequently studied to screen for endophytic fungi that produce antibiotic substances [1].

Plants live in unusual biological environments
With these plants, the unusual environment and harsh natural conditions force the tree to survive, a special element is needed to make the plant highly resistant. And one would expect that factor to be beneficial endophytic fungi [4].
For example: showed that an aquatic plant, Rhyncholacis penicillata, which lives in harsh aquatic environment which may be constantly wounded by passing rocks and 3 The Diversity of Endophytic Aspergillus DOI: http://dx.doi.org /10.5772/intechopen.99717 other debris, resists infection by common oomyceteous fungi that cause disease. The possibility that endophytes associated with this aquatic plant may produce antifungal agents that protect the plant from attack by pathogenic fungi is feasible. A novel antioomycetous compound, oocydin A was discovered from the endophytic strain Serratia marcescens from this plant.

Plants are used as folk medicine
A number of plants have been used according to folk experience, from generation to generation for wound healing, antifungal, antibacterial, etc.
For example: A study of endophytic fungi producing novel bioactive substances in Brazil. It is a plant named "Mexican Sunflower" -Tithonia diversifolia (Asteraceae) that is often mentioned with many interesting points. For a long time, based on oral experience, people have used this plant to cure a number of diseases such as malaria, diarrhea, viral fever, hepatitis and to heal open wounds. In addition, extracts from T.diversifolia with anti-inflammatory, amoebic, antispasmodic, antifungal, antibacterial, and antiviral activities were also mentioned. Based on that information, scientists have isolated and isolated Phoma sorghina, an endogenous fungus from the leaves of this "Mexican sunflower". From this, six anthraquinone derivatives were obtained from fungal metabolites, half of which are new known substances [1].

Ecologically specific plants
Plants with unusually long lifespans, growing in areas of great biological change, or living in ancient soils are also ideal research subjects to provide endogenous fungi new. Plants surrounded by plants infected with the pathogen, but not infected, are more likely to harbor endophytic fungi with antimicrobial activity than other host plants.

Endemic plants
Endemic plant species that have a normal lifespan, or occupy a certain area of land in the wild. Chaetomium globosum isolated from leaves of endemic plant Maytenus hookeri distributed only in Yunnan regions, China produces chaetoglobosin B which inhibits tuberculosis bacteria [5].

Diversity of endophytic fungi
Endophytic fungi are very abundant, according to a study by Matsushima in 1971 conducted on some angiosperms and conifers in North America and Panama, which suggested that most of the endophytic fungi belong to the Ascomycetes class [4].
According to the statistics of scientists studying three plant families in Southeast Arizona -USA, forests in North Carolina and Northern forests shows that: • Surveying the host plant representing the Fagaceae family obtained 44 endogenous fungal strains in which the genus Sordariomycetes predominated.
• Surveying Pinus ponderosa trees representing the Pinaceae family obtained 111 endogenous fungal strains, in which the genus Leotiomycetes predominated.

Endogenous fungi produce biologically active substances
Many endogenous fungi in plants have been isolated, they have the ability to produce biologically active substances such as antibiotic, antibacterial, antifungal, tumor suppressor, antioxidant and other biological activities.

Endogenous fungi with antibacterial and antifungal properties
Many studies on antibacterial and antifungal activities are produced from endogenous fungi, mainly belonging to the following groups: alkaloids, peptides, steroids, terpenoids, phenols, quinines and flavonoids, etc. These compounds account for only a part small in the total number of active substances produced by endophytic fungal species, they are clearly an excellent and novel potential source for the production of new antibiotics. This holds great promise for solving the problem of drug resistance in bacteria because these antibiotics are novel and highly active compounds Table 1.
Besides, Phomopsis is an endogenous fungus that produces phomopsichalasin, representing the first group of compounds of cytochalasin type.

Endogenous fungal tumor suppressor
Since 1990, Taxomyces andreanae was first isolated from T. brevifolia, this fungus produces paclitaxel -an inhibitor of achromatic spindles during cell division, with a mass spectrum similar to paclitaxel extracted from Taxus. Subsequently, several paclitaxel-producing fungi have been isolated from many plants and other Taxus species Table 2.

Endophytic fungi that are active against insects
Many studies have demonstrated the importance of endophytic fungi in the production of insect repellents, which have many implications for crop protection and increase in agricultural yields Table 3.

Endophytic fungi
Isolation source Antibiotic Antimicrobial effects Ref.

Endogenous fungi with antioxidant activity
Antioxidants are substances that react with free radicals generated during oxidation, thus preventing or slowing down this process. Antioxidants prevent and treat diseases such as cancer, cardiovascular (atherosclerosis, hypertension, ischemia), diabetes, neurodegenerative diseases (Parkinson's disease), arthritis and aging, etc. Endophytic fungi in higher plants are a source of many new antioxidant active substances Table 4 [10,11].

Endogenous fungi produce other biologically active substances
Endogenous fungi are also known as a source of many other biological metabolites, such as anti-inflammatory, antidiabetic, hypoglycemic, immunosuppressive, etc. used to prevent rejection in organ transplants and can be used to treat autoimmune diseases such as rheumatoid arthritis, insulin dependent diabetes also known from endogenous fungi.
Endophytic fungi of the genus Chaetomium produce chaetoglobosin, which is a cytochalasin analog that inhibits actin polymerization. The genus Chaetomium is able to produce cytostatic metabolites including chaetomin, chaetoglobosin A, C, D and G, chaetoquadrin, oxaspirodion, chaetospiron, orsellide and chaetocyclinon

Introduction of the endophytic Aspergillus
Aspergillus is a large genus of fungi and one of the most studied, according to Thom and Church in 1918, the genus is divided into 18 groups, 132 species and 18 orders. They account for a large proportion in the natural environment and are easy to culture in the laboratory. The economic importance of several species has attracted much research on Aspergillus. Furthermore, this common genus of fungi is involved in many industrial processes including production of enzymes (such as amylase), chemicals (such as citric acid) and food (such as soy sauce) are one of the plant endogenous fungal genera known for its ability to produce many biologically active substances with many practical applications [14].

Classify
Aspergillus was first classified in 1809 by the Italian priest and biologist Micheli by observation under a microscope. Nowaday, "Aspergillum" is also the name for asexual spores that form the common structure for all species of the genus Aspergillus; about a third of species in the genus have a sexual reproduction stage. According to the classification: Kingdom: Fungi Division: Ascomycota Class: Eurotiomycetes Order: Eurotiales Family: Trichocomaceae Genus: Aspergillus

Characteristics of Aspergillus
The characteristics of color (black, brown, yellow, red, white, blue, etc), growth rate, edge of mushroom cluster and surface texture of mushroom cluster vary depending on species and growing conditions. The mycelium of Aspergillus belongs to the group of colorless, segmented, branched hyphae that can produce many enzymes and some toxins. Aspergillus mycelium is vigorous and produces many spores attached to a long vertical spore stalk, which grows from a special cell located in the trophic fiber called a foot cell [9,15].
The spore-bearing head includes the spores: spore, flask, vesicle and spore stalk. The properties of each component vary from species to species and are characteristics that help identify species. Most species have the same shape, size, and color of spore-bearing heads as the cluster Figure 2. Some

Ecological characteristics and distribution
Aspergillus is very aerobic, found in most oxygen-rich environments, usually growing on the surface of a substrate. Normally, fungi grow on carbon-rich substrates such as monosaccharides (glucose) and polysaccharides (amylase).
Aspergillus is widely distributed in the environment and can grow almost anywhere, especially in places with high humidity. Aspergillus grows by saprophyte on decaying plants, compost, and humus. Most Aspergillus species can live on a variety of substrates such as feces, human tissues, and ancient parchment. Aspergillus species grow well and produce many spores at a temperature of about 23-26°C. However, there are some species such as A. terreus, Acronurus carneus, A. jcheri which thrives at 35°C or A. fumigatus which grows well at 45°C even up to 50°C. Temperature also affects the shape of the attachment spore tip. A. janus species produces two different types of attached spore heads, the ratio of these two forms is affected by temperature, at 18-20°C, most of the spore heads are white, clubheadshaped, and long spore stalks; but at 30°C, most of the spore heads are dark green, spherical, and short spore stalks.

Some biologically active substances produced by endophytic Aspergillus
Apergillus sp. has the ability to produce many biological active substances such as antibacterial, antiviral, cytotoxic and antioxidant activities, etc.

Isolation of biologically active endophytic fungus Aspergillus
In many plants, the microflora is entirely endophytic fungal. This suggests that endophytic fungi may have a more favorable biological interaction than endophytic bacteria with respect to host plants. The strains of endogenous fungi with active substances are very diverse in both morphology and reproduction, and some strains have very special forms of reproduction.
Identification of Aspergillus strains by ITS gene sequencing method and searching on BLAST SEARCH gave similar results as the morphological method, contributing to the confirmation of strains with high biological activity which the subject isolated was Aspergillus [16].
Aspergillus spiecies isolated from plants have been shown to be able to produce many secondary metabolites with valuable biological effects such as anticancer, antiviral, antimicrobial compounds. Strains of Aspergillus isolated from galangal, turmeric, tangerine, and kumquat plants could produce metabolites with high activity against S. aureus and MRSA.  Conditions affecting the biological activity of the endophytic fungus Aspergillus • pH: Importantly affects the growth, metabolism of fungi, enzyme activity, intermediate products, dissociation, dissolution, etc., thus affecting the biosynthesis of active ingredients antibacterial of fungi.
• Temperature: Like other microorganisms, the temperature of the environment also greatly affects the growth and development of fungi.
• Oxygen concentration: Oxygen concentration is very important and necessary for the survival and growth of aerobic microorganisms.
• Carbon source-nitrogen source: The choice of carbon and nitrogen sources greatly affects the activity of secondary substances. Different carbon sources such as dextrose, lactose, sucrose, fructose, starch, molasses and glycerol are believed to be suitable carbon sources for metabolism in various fungi. Organic and inorganic nitrogen sources such as NaNO 3 , yeast extract, meat extract and soybean meal, NH 4 NO 3 , (NH 4 ) 2 SO 4 , etc. can help increase biological activity in fungi [2,41,42].
All the optimization was performed based on % inhibition of bacterial growth when challenged with 10 μg/μl metabolite concentration. Among different media used, potato dextrose broth (PDB) and sabouraud's dextrose broth (SDB) proven to be better media for growth of fungus as well as metabolites production 1% yeast extract and 4% dextrose resulted in higher cell inhibition. Ethyl acetate served as good extracting solvent [19].
• Addition of vegetable oil to the environment: Vegetable oil can be used to supplement the carbon source during lovastatin production in Aspergillus. Palm oil and soybean oil significantly increased the biomass and lovastatin production of A. terreus [43,44].
• Trace elements: Fe and Zn are necessary for the biosynthesis of some antibiotics. It is possible that these two minerals have a positive effect on the antibiotic biosynthesis of Aspergillus.
• Salt concentration: Salt concentration affects the antibacterial activity of Aspergillus. For example, strain A. terreus has strong antibiotic activity in the range of NaCl salt concentrations from 0 to 1%. When the salt concentration is above 1%, the antibiotic activity of this strain decreases and at a NaCl concentration of 6%, A. terreus strain is no longer capable of biologically active substances. A. terreus is not only of research interest in terms of antimicrobial activity, but it is well known for its ability to produce lovastatin. According to the study of Pawlak et al. in 2012 on optimal conditions for lovastatin production of A. terreus, the authors determined that ventilation is essential for aerobic biological response [45].
Endophytic fungal populations of the genus Aspergillus have been isolated from many plants and have been shown to produce a wide range of biologically active substances including antifungal, antibacterial, anticancer, etc. In addition to the characteristics of resistance to MRSA and S. aureus as published by many studies, some strains of A. terreus isolated from soil or from plants have a spectrum of effects on a number of other bacteria such as E. coli, P. aeruginosa, Streptococcus faecalis. This shows the potential to study antibacterial compounds of Aspergillus.

Determination of biological activity of secondary compounds
In the world, there are many studies on the role and application of biologically active substances produced by endogenous fungi. Some endophytic fungal strains have the ability to produce important antibiotics to prevent the invasion of pathogenic organisms to host plants, which are significant in the control of plant diseases and insect pests. Some endogenous fungi are able to synthesize biologically active substances used as anticancer drugs, produce tumor suppressor antibiotics, immunostimulants, and antioxidants, and have biological activities. These compounds mainly belong to the groups of alkaloids, steroids, flavonoids and terpenoid derivatives and other substances, etc. Endogenous fungi also perform a resistance mechanism against plant diseases by producing substance with antibacterial activity. Screening for antimicrobial compounds from endogenous fungi is a way to kill resistant bacteria in humans and plants.
In addition, the natural metabolites of endogenous fungi also help to protect natural resources and meet the requirements of pharmaceutical production from plant origin by fermentation. Many biologically active substances are produced by endogenous fungi during growth and development. Finding and discovering those active ingredients is the goal that biopharmaceutical researchers are constantly reaching for.

The interaction between the host plant and the endophytic fungus Aspergillus
There is a complex relationship between endophytic fungi and host plants, the interaction between host and endophytic fungi can be endogenous or symbiotic depending on genetic predisposition, developmental stage, nutritional status and environmental factors.
Commensalism helps the endogenous fungi to survive by being supplied with nutrients without affecting the host plant. Mutual beneficial relationships of endophytic fungi and host plants through the provision of energy, nutrients, shelter as well as protection under environmental stress. On the other hand, endophytic fungi indirectly benefit from host plant growth by producing secondary metabolites that help host plants adapt to abiotic factors such as light, drought and stress such as herbivores, insect and nematode attacks or pathogens.
Schulz and Boyle in 2005, the authors proposed that the endophysis of endophytic fungal is a balanced antagonism between host and endophytic fungi, and provided endogenous virulence and protective capacity of the balanced host plant showed no significant symptoms.
Once the host-endophytic interactions become imbalanced, or disease in the host plant or host defense tissues kills the pathogenic endophytic fungi. Whether the interaction is balanced or unbalanced depends on the host-endophytic condition, virulence of the fungi, host defenses, toxicity, environment, and nutritional status and growth stages of the host plant and endophytic fungi.
Therefore, commensal relationships require a balance between the defense responses of the host plant and the nutritional requirements of the endophytic fungi. In agreement with the 2006 study by Kogel et al., endophytic fungi share structural similarities with pathogens and both have many similar virulence factors, such as production of Metabolites and exoenzymes are required to infect and colonize the host plant, so endophytic fungi are subject to self-recognition, the host