The Endophytic Fungus Penicillium Setosum

The endophytic fungus Penicillium setosum was isolated from a narrowly studied habitat, i.e from microbiome of Withania somnifera (L) Dunal. This plant has great importance in the Ayurveda system because of their noticeable pharmacological functions contributed by their secondary metabolites. So, the exploration of such astonishing medicinal plant for the isolation of endophytes have much significance. Environmental conditions and plant physiological conditions together favors the growth of certain spectacular micro-organisms. Majorly plant-based compound producing organisms have been getting much more attention among endophytes. The P. setosum is found as an effectual bio-synthesizer of plant derived compounds, comes in the group flavonoids and was also able to produce some characteristics compounds of Penicillium species. According to Hardoim et al. (2015), plant mutualists are largely enriched with diverse genes that are involved in the biosynthetic processes and functions of compounds that are related to plants. Since the identified secondary metabolite pattern of P. setosum is agreement with the plant-mutualistic relation.

This particular fungus-host interaction definitely cause alteration in the phytochemical profiling of a host plant. It may be achieved either by synthesizing or stimulating the production of plant secondary metabolites (Ludwig-Müller 2015). This absolutely enhances the quality and quantity of the crude drugs of the plant, as well as favors the adaptableness of both endophytic fungi and their host plants (Jia et al. 2016). Uniqueness of the P. setosum from other members of section Lanata-divaricata was chemotaxonaomically confirmed by analyzing the secondary metabolite pattern obtained using high resolution LC-MS. The presence of andrastin D and anthraquinone (quinalizarin) in the culture extract defines the relation of this isolate to the other members of Penicillium section Lanata-divaricata (Taniwaki et al. 2015, C M Visagie et al. 2016), while the discrepancy is showed by the presence of patulin, which is mostly produced by Penicillium spp. of other sections (Frisvad 2018). So far Penicillium brefeldianum, Eupenicillium javanicum (=Penicillium javanicum), Eupenicillium sp. 1 and sp.2, Penicillium simplicissimum are reported from section Lanata-divaricata for the patulin production (Okeke et al. 1993).

But these strains are not presently included in the revised list efficient producers of patulin, by reason of, afore mentioned strains are not available to scientific community (Frisvad 2018). Based on the phylogenetic analyses of our previous study, the P. setosum is most closely related to P. javanicum (CBS 341.48T), by literally which is not reported for the production of patulin. So this result characteristically distinct this isolate from other members of section Lanata-divaricata. Patulin is both a mycotoxin and an antibiotic. It can also act as a phytotoxin (Ismaiel et al. 2015), and is reported to active against plant pathogenic fungi and bacteria (Gilliver 1946).

According to Houbraken et al. (2016) the phylogenetic relationships of interspecies patulin-producing Penicillium want to be stated from further in-depth phylogenetic analysis. Many of the fungal secondary metabolic genes come in Type I polyketide synthases (PKS) superfamily, herewith patulin, citromycetin, are the identified compounds from PKS 1 family. While flavonoids are derived through phenylpropanoid pathway, their genes are involved in the Type III polyketide synthases (PKS) superfamily. Chalcone synthase (CHS), chalcone isomerase (CHIs), phenylalanine ammonia lyase (PAL) genes are majorly explained from this family. Yeast like Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the filamentous fungus Neurospora crassa (Gensheimer and Mushegian, 2004) were previously reported for the presence of CHIs genes. This provides a preliminary sign for the existence of flavonoid like pathway also in fungi. But it is not yet reported in Penicillium species. 3-deoxy-D-arabino-heptulosonate7-phosphate (DAHP), which is found in the shikimic acid pathway and derivatives of certain coumarin compounds (considered as phenylpropanoid) (Vogt 2010) and flavonoids, provides the sign of phenyl propanoid pathway in this Penicillium species.Some phenylpropanoid-derived metabolites are limited to a few plant species or family. So they are considered as chemotaxonomic markers, thereby plants are chemotaxonomically grouped on the basis of similar flavonoid pattern (Cuyckens & Claeys 2004). W. somnifera has been reported for the presence of several group of flavonoids e.g. quercetin, kaempferol, catechins (Kandil et al. 1994, Alam et al. 2011). The biosynthesizing ability of flavonoids is prominently restricted with the plants, with a few exceptions in contrast to animals and fungi (Cuyckens & Claeys 2004).

So the P. setosum capacity to synthesize flavonoid is highly astonishing and significantly increase the advent of this study. Liquid chromatography coupled with mass spectrometry offers the rapid study of flavonoids, their ion chemistry, and the elucidation of structure even at low concentration in the extract (Kumar 2017). The occurrence of potent biologically active compounds such as quercetin and dihydroqueretin in the culture extract is the major highlight of this study. Structurally the quercetin and dihydroquercetin differ only at the double bond between the C2 and C3 carbons on the center (C) ring, but significantly varies with the biological activities. Quercetin is a fat soluble found as the most conspicuous compound in the group of flavonoid. While taxifolin is a water-soluble compound, it is getting much more attentiveness in its biological function now, because it is less toxic than quercetin and non-mutagenic. It can be effectively used in cancer therapy to prevent the development and proliferation of cancer. It is also used as a combined drug with antibiotics of linolizide etc. Conversely in nature dihydroquercetin is less abundant than quercetin and yet no reports on their microbial origin. The role of flavonoids in endophytic fungi has not yet been recognized.

So studying on these compounds have much relevance, for tracking to resolve the role of these compounds in the mutualistic relation and their role in response with the environment. Reports have shown that some of the endophtytic fungi isolated from medicinal plants are able to produce phenolic and flavonoid compounds (Lunardelli Negreiros de Carvalho et al. 2016). The probable explanation for this is the internal compartments of plants are unfavourable niches for aerobic microorganisms, due to the presence abrupt burst of reactive oxygen species (ROS) and reactive nitrogen species (RNS). To stand against this situation, many of the plant mutualists metabolize plant stress related compounds, by producing active compounds (Hardoim et al. 2015). Thereby mutualistic and/or symbiotic relationships between the fungi and host plant alters the responsiveness of both partners.

Based on the secondary metabolite pattern obtained from colony agar plug and culture broth extract method, both found as sufficient to understand the extrolite profiling of Penicillium spp. (Frisvad et al. 2008). However, colony agar plug method can be deliberated as a preliminary screening method for determining the chemical nature of secondary metabolites. This is because it is inexpensive, less intensive and time consuming, easy to perform and versatile to discover many extrolites even from five agar plugs of fungal colony. Culture broth extraction method can be adopted for the large-scale production and functional characterization of metabolites.