Where to Find? A Report for Some Terrestrial Fungal Isolates, and Selected Applications Using Fungal Secondary Metabolites

Biodiversity of mycobiota is getting more attention, since fungi and their metabolites represent the future alternatives of many currently used chemical compounds. In this review, the fungal mycobiota associated with leaves of some plants were discussed comparing the literatures focusing on some economically important plants such as wheat, sugarcane, and some medical plants. Furthermore, the role of some fungi as promising small factories that could be used for industrial applications such as myco-diesel oil production, and agricultural applications such as plant growth promoters


Introduction
Biodiversity describes the variability among living organisms within a given ecosystem, and it is often used to measure the health of biological systems. Documenting and describing fungal diversity is an extremely difficult mission that requires years of experience due to their ability to appear in various morphologies from a single individual, and their confusing similarity in appearance to non closley-related taxa [1]. There are many methods used from decades to analyze fungal biodiversity. The classical methods rely on direct observation of fungi. These methods are cheaper and need no specific equipment in comparison with molecular based methods which rely on sampling fungal DNA. Classical methods are commonly used specially in developing countries, which usually cannot afford applying molecular methods. Classical methods are hence accompanied by doing many investigations, and record fungi over a specific area and /or amount of substrate [2,3]. Putting also in consideration ecological variables, such as pH, soil nutrients composition, age of the plant, time of the year, and some other weather-related variables [4].
Fungi are ubiquitous, and new species can be isolated even from the most well studied areas [5]. The word phylloplane point to the surface of plant leaves which represents a complex terrestrial habitat and so studying phylloplane mycobiota is concerned with the fungal flora growing on the surface of plant leaves [3]. Many studies focused on investigating phylloplane fungal flora of several plants growing or cultivated in Egypt and other parts of the world [6][7][8][9][10][11][12][13]. There are two groups of phylloplane fungi: residents and casuals. Residents multiply on the surface of healthy leaves without harming the host plant or affecting it. While, casuals though existing on the surface of the leaf, they cannot grow on it. [14,15]. Studying biodiversity of mycobiota is very important for identifying and documenting changes and similarities between species. Moreover, various novel natural compounds can be isolated and identified from such mycobiota with promising potential biological, medical and industrial applications [16]. In this review, phylloplane mycobiota from some economically important plants and some medicinal plants were described. Moreover, using some fungal strains as small factories to produce valuable compounds involved in many applications was discussed.

Isolation of Phylloplane Mycobiota
A common method for isolation of phylloplane mycobiota, the agar plate method, was described by Elkhateeb [4], include washing leaves of the plant several times with distilled water, and dry them between sterile filter paper, followed by cutting the leaf into equal segments (about 1cm each). Five segments were placed in each plate on the surface of the agar medium supplemented with appropriate antibacterial. Then, the plates were incubated at 28 o C ± 1 for 7-10 days, and the developing fungi were counted, identified, and calculated as colony forming unit "cfu" per 100 segments of fresh leaves.
Another commonly used method is moist chamber plate's method [17], which doesn't require using any type of medium for the growth of Phylloplane fungi. In this method the leaf segments of 1 Cm square, both sterile an unsterile were placed separately on the moist chamber plates using a sterile forceps and pressed genteelly, followed by incubation at 25±3ºC in the incubation chamber for 7 to 21 days. But starting from the fourth day monitoring plates is very important in order to check the growth of fungi. The fungi on moist chamber were then counted.

Mycobiota from Economically Important Plants
Wheat is one of the most economically important plants worldwide. Many fungal genera were listed to be isolated from wheat. Abdel-Hafez [18] reported that the most common phylloplane genera isolated from wheat leaves cultivated in Saudi Arabia were Cladosporium, Aspergillus, and Alternaria, which came in accordance with Abdel-Hafez et al. [19] who reported the same genera Alternaria, Aspergillus, and Cladosporium, but he also added Cochliobolus, Gibberella, Nectria, and Penicillium. Solated to the list of common mycobiota isolated from wheat fields in El-Kharga Oasis, Western desert, Egypt. On the other hand, Mazen et al. [20] nominated the most common species isolated from phylloplane of wheat plants as Aspergillus niger, Penicillium corylophilum and Alternaria alternata.

Fungal Secondary Metabolites with Beneficial Properties
It is already known that fungi secretes a wide range of secondary metabolites which represent an excellent source of compounds with promising applications for industry, medicine, and agricultural fields. For example, Epicoccum Sp. is known to secrete many secondary metabolites such as epicorazines which has an antibacterial activity [27,28]. Trichoderma Sp. is widely used as biofertilizers and biocontrol agents in agriculture due to its secreted secondary metabolites that exert different biological activities towards plants and other microbes [29]. T. harzianum produces an important metabolite, harzianic acid (HA) which promotes plant growth [30]. Another metabolite was isolated from the culture filtrate of a T. harzianum strain a, termed isoharzianic acid (iso-HA), which has antifungal activities In vitro aganist Sclerotinia sclerotiorum and Rhizoctonia solani. [30][31][32][33][34][35][36][37][38][39].

Oil Production
The current energy crisis has highlighted the importance of searching for potent alternative oils and for synthesis of microbial lipids, which represent the raw material for biodiesel production [40]. Many fungi can accumulate high level of lipids and grow as pellets that can easily be harvest from cell broth [41]. One of the most potent fungus in oil production is Mucor circinelloides. Pellet formation in submerged fungal cultivation of M. circinelloides depends strongly on adjusting pH during cell growth [42]. Many agricultural wastes such as Sugar beet pulp (SBP) can be used as substrate for production of fungal oil from Mucor circinelloides and Aspergillus oryzae, which is an economical way to offer an alternative to oil to be used as an energy source. Another fungus, Cunninghamella japonica, was reported as a promising lipid producer [43][44][45].

Conclusion
As described in this review, even the well-studied area can yield new fungal isolates with metabolites to be investigated. Further studies on the isolation and identification of fungi as well as investigating and prospecting their secondary metabolites for biotechnological uses or biologically important application, represent a critical need nowadays. Fungi isolated from medicinal plants usually exerts novel and important biological activities. Documenting the species composition of fungal mycobiota existing over the world and comparing this information may contribute in understanding relation between presence of such genera and many other factors.