Abstract: Background and Objective: In nature, plants incorporate a wide scope of restoratively esteemed bioactive compounds. The fungal endophytes are the organisms which grow inside the plant parts such as roots, stems, leaves and flowers and produce similar bioactive compounds of medicinal significance. The fungal endophytes from the plant Plectranthus amboinicus were not well explored in West Malaysia. Therefore, this study was designed with the objectives to harness novel endophytic bioactive compound producing fungal strains from this plant. Materials and Methods: Screening for fungal endophytes was performed by using surface sterilisation method. Sterile explants were placed on a potato dextrose agar (PDA) plates and incubated under 28°C. These fungal endophytes were identified microscopically by lactophenol-cotton blue staining protocol and molecular characterization was performed by amplification of randomly amplified polymorphic DNA tool. Results: A total of ten fungal endophytes were isolated and identified. The prevalent endophytic fungal strain found was of Penicillium chrysogenum in comparison with Aspergillus niger, Fusarium oxysporum and Alternaria solani. Therefore, these strains selected for assessment of genetic diversity. Conclusion: The most prevalent bioactive compound producing fungal strain found was of Penicillium chrysogenum associated endophytically with the plant Plectranthus amboinicus.
INTRODUCTION
Emergence of pathogenic strains is a growing concern all over the world. Drug resistant pathogens or super bugs are using new methods for survival. These pathogens are having drug resistance genes either on plasmids or integrated with chromosomes1. Recently it was found that these pathogens also shifted the cell wall synthesis precursor molecules to escape the target action from drugs acting on cell wall1. A large number of side effects were seen in patients due to overuse of synthetic drugs2. Therefore, the search for new bioactive compounds from natural resources is increasing year by year.
Large community of medicinal plants identified all over the world as potentialistic drug producer in natural manner. Herbal medicines are used all over the world for health care management. Plectranthus amboinicus is a perennial herb and possesses antimicrobial and antioxidant properties1. It is traditionally used to treat conditions like coughing, skin infections and fever. The leaves of this herb are used for flavoring and in preparation of traditional food2. The earlier research data discloses that this herb contains 70 volatile and 30 non-volatile compounds of phytochemicals with significant pharmacological properties3.
Endophytes are organisms that colonize in healthy tissues of plants and produce metabolites of pharmaceutical significance. During growth and development of medicinal plants, these endophytes colonize naturally in various plant parts and maintain healthy relationship. These endophytic organisms produce various bioactive compounds, which help the plant to metabolize certain nutrients, in return the plant facilitate the colonization and survival of these organisms. Pestalotiopsis sp., Taxomyces sp. and Penicillium species are some important genera associated endophytically with various medicinal plants and producing unique bioactive compounds of medicinal importance. Research studies have shown, that the endophytic Penicillium species produce unique bioactive compounds such as terminatone, ergosterol, 4-hydroxybenzaldehyde and 4-hydroxy-hexadec-6-6-enoic acid methyl ester4. These compounds possess antimicrobial and antioxidant activity. Toghueo and Boyom5, discussed in their comprehensive review about various potentialistic fungal endophytes isolated from Terminalia species.
Maheshwari et al.6 explored endophytic mycoflora and its biological activities from Terminalia arjuna medicinal plant of Indian region.
Similarly, Rajeswari et al.7 found out various significant endophytic fungi from Terminalia chebula plant of the Eastern Ghats, Tamil Nadu, India.
Kim et al.8 discovered two species of Penicillium from Korea in their research work. These Penicillium species were significant bioactive compound producers.
In India, diverse endophytic fungal communities explored for the discovery of novel bioactive compounds at molecular level by Verma et al.9 and Tiwari10. Therefore, in the present work, novel fungal endophytic strains from the herb Plectranthus amboinicus were isolated, identified and molecularly characterized in the search of novel unique bioactive compounds.
MATERIALS AND METHODS
Research duration: The research was conducted in Department of Microbiology, Management and Science University, Shah Alam, Malaysia for duration of 6 months (April-October, 2019).
Sample processing: The samples were collected from different regions of Selangor, Malaysia. This herb is grown easily in pods. The healthy plant parts (Leaves and roots) were collected and transferred in sterilized poly bags. The plant parts were processed in MSU microbiology laboratory by standard protocol.
Isolation and identification of fungal endophytes: Isolation of fungal endophytes was carried out by standard protocol given by Tiwari and Chittora11. The isolated fungal endophytes were identified on the basis of macroscopic and microscopic characteristics.
Colonization frequency: The colonization frequency was calculated by using the formula given below11:
Cultivation of P. chrysogenum strains: Initially numbering was given to all endophytic fungal strains isolated. The P. chrysogenum strains were named as P. chrysogenum-I, P. chrysogenum-II, P. chrysogenum-III, P. chrysogenum-IV, P. chrysogenum-V and P. chrysogenum-VI. All 6 novel endophytic fungal strains of P. chrysogenum (I-VI) were grown in potato dextrose agar medium, were transferred to czapek yeast autolysate (CYA) agar medium containing plates and slants by maintaining all aseptic conditions. After 7 days incubation at 25°C, the phialides, conidiophore and conidia of pure cultures were examined for the identification by lactophenol cotton blue staining protocol. For the cultivation of these strains, an agar cube of equal size was cut (Same size, Cork borer) from the pure culture PDA plate on laminar air flow by maintaining all aseptic conditions. This cube was transferred to Czapek Yeast Autolysate (CYA) broth for penicillin production and incubated in shaker incubator at 25°C, 100 rpm (rotation per minute) for 10 days in dark12. Shake flask culturing steps were performed in 500 mL Erlenmeyer flasks containing 100 mL growth medium protocol standardized by Chen et al.12.
Penicillin extraction: The 4 step extraction method was used for the extraction of penicillin from these novel strains of P. chrysogenum as described by Rafi13.
Sensitivity estimation: The penicillin extract poured in wells of Mueller-Hinton agar medium containing plates flooded with Staphylococcus aureus clinical isolate. After two days of incubation, the diameter of zone of inhibition was measured14.
Assessment of genetic diversity: Genomic DNA from six novel Penicillium chrysogenum strains was isolated15. Assessment of genetic diversity among six endophytic fungal strains of P. chrysogenum isolated from Plectranthus amboinicus were carried out using PCR based RAPD primers and 32 random decamer primers were used. Out of 32 primers screened, 8 primers amplified and produce results. Percentage of polymorphism was calculated on the basis of number of polymorphic bands/number of total bands. The clade and dendrogram constructed by unweighted pair group method based on Jaccard distance15.
Statistical analysis: The prevalence of fungal endophytes was calculated by colonization frequency (%) and the graph was constructed in Microsoft excel by number of identified endophytic fungal genera obtained during this research study.
RESULTS
Colonization of endophytic fungal genera in Plectranthus amboinicus: The healthy plant Plectranthus amboinicus collected for processing was shown in Fig. 1. After five days of incubation, the sporulating fungal endophytes started growing on potato dextrose agar medium from leafy and nodal explants, which were presented in Fig. 2a, b. These fungal endophytes were transferred carefully to another set of potato dextrose agar plates for the maintenance of pure culture and further investigation.
| Fig. 1: | Healthy Plectranthus amboinicus plant |
| Fig. 2(a-b): | Sporulating fungal endophytes from nodal and leafy explants of Plectranthus amboinicus L. |
| Fig. 3: | Pure culture of Penicillium chrysogenum strain after 2 days of incubation |
| Fig. 4: | Microscopic view of Penicillium chrysogenum strain at 40×magnification showing broom shape structure and conidia |
The pure culture of P. chrysogenum strain on potato dextrose agar medium after 2 days of incubation is shown in Fig. 3. The microscopic view of P. chrysogenum strain showing phialides and conidia (Broom shaped structure) by lactophenol cotton blue staining presented in Fig. 4.
| Fig. 5: | Aqueous extract (1 mg mL–1) of novel P. chrysogenum strains (I-VI). |
P. chrysogenum strain-VI has shown maximum zone of inhibition (1 cm) against methicillin resistant Staphylococcus aureus (MRSA) clinical isolate |
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Measurement of zone of inhibition and sensitivity test: The aqueous extract of Penicillium chrysogenum strains (I-VI) shown zone of inhibition against methicillin resistant Staphylococcus aureus (MRSA) clinical isolate. The maximum zone of inhibition found was of 1 cm with P. chrysogenum -VI strain (1 cm) in comparison with other strains (I, II, III, IV and V) of P. chrysogenum which has shown zone of inhibition of 0.2-1 cm (Fig. 5). Therefore, the VI strain of P. chrysogenum was found as maximum penicillin producer at batch level.
Prevalence: A total of ten novel endophytic fungal strains were isolated and identified from the plant Plectranthus amboinicus. The most prevalent endophytic fungal species found was Penicillium chrysogenum in comparison with Aspergillus niger, Fusarium oxysporum and Alternaria solani (Fig. 6).
Genetic diversity assessment: The genetic diversity assessment was performed by randomly amplified polymorphic DNA (RAPD) tool. A total of 39 loci were amplified. Out of 39 bands, 21 (53.84%) were found to be polymorphic and 18 (46.15%) were found to be monomorphic bands.
| Fig. 6: | Prevalence of novel endophytic fungal strains isolated from Plectranthus amboinicus |
| Fig. 7: | Dendrogram showing genetic diversity among 6 Penicillium chrysogenum strains |
Dendrogram: Dendrogram was constructed by unweighted pair group method based on Jaccard distance. Six novel strains of P. chrysogenum aliened into three large clades (I, II and III). Each clade contains two strains which are the closest to each other. Clade I contains VI and II, clade II contains IV and I and clade III contains V and III. These strains are the closest match with each other (Fig. 7).
DISCUSSION
In the present study, results confirmed that the P. chrysogenum (Fig. 6) is most prevalent endophytic fungal strain associated with the plants of Plectranthus amboinicus in West Malaysia. These P. chrysogenum strains are symbiotic and naturally selected by the plant during development. Surprisingly, these strains are producing penicillin in small scale.
The significant P. chrysogenum fungal strain was VI (Clade I) in comparison with other strains (I, II, III, IV and V). This strain significantly inhibited the growth of Staphylococcus aureus clinical isolate with zone of inhibition of 1 centimeter. These strains were assessed for genetic diversity by randomly amplified polymorphic DNA (RAPD) tool and shown closest match between VI and II strain in clade I. Various researchers used RAPD tool for the genetic diversity assessment of endophytic fungal strains and found it significant.
Similar results were found by Astuti et al.16 in which, potentialistic bioactive compound producing fungal endophytes isolated from this plant. These endophytes also shown antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) and other drug resistant pathogens.
Gaikwad et al.17 also studied diverse endophytes from this plant and found Aspergillus flavus most prevalent in comparison with other endophytes.
Rajendran et al.18 isolated novel fungal endophyte Pestalotiopsis microspora EF01 from the plant Plectranthus amboinicus. This fungus was producing taxol (an anticancerous drug) and shown significant anticancerous activity against human liver carcinoma cell lines.
From the present work, it was found that the endophytic P. chrysogenum strains were significant penicillin and other bioactive compound producer. Similar results were found by Korejo et al.19 in which the endophytic Penicillium species had shown antibacterial and antifungal activity.
In Philippines, Campos et al.20 reported myco-pharmacological properties of endophytic fungi from the Plectranthus amboinicus. The major fungal strains isolated in this research work, were A. tamarii, A. terreus and A. niger. These strains were producing saponins, tannins, phenols, quinones, sterols and terpenoids. Moreover, these strains were significantly inhibiting the growth of Staphylococcus aureus and Escherichia coli bacterial pathogens In vitro. Contradictory, in the present work, P. chrysogenum endophytic fungal strains were found prevalent and potentialistic penicillin producer at batch level.
In India, Sukrutha et al.21 carried out the research work and found that, isolated endophytic fungal strains were producing polyunsaturated fatty acids in significant manner. Although these strains were isolated from various medicinal plants including the plant P. amboinicus.
In Pakistan, Urooj et al.22 reported that the endophytic Penicillium species is involved and suppressing the growth of soil borne root rotting fungi M. phaseolina, R. solani, F. solani and F. oxysporum In vitro. It proved that the endophytic Penicillium species is producing distinguished secondary metabolites in association with medicinal plants.
Furthermore, in Brazil, leishmanicidal and fungicidal activity of lipases obtained from endophytic fungal extracts were demonstrated In vitro by Alves et al.23. This shows that a large number of secondary metabolites are produced by the fungal endophytes in association with medicinal plants of unique environmental niches.
Houbraken et al.24 discovered new penicillin producing Penicillium species. The present work also confirms that the Penicillium species has enormous potential for the production of penicillin and other related bioactive compounds.
CONCLUSION
This study concluded that Plectranthus amboinicus L. harbors a large number of fungal endophytes of medicinal importance. The novel Penicillium chrysogenum strains isolated from this plant are significant penicillin producer and RAPD tool is effective for the genetic diversity assessment of these novel strains. Further research work is in progress for the purification of penicillin and other related bioactive compounds from these novel P. chrysogenum strains.
SIGNIFICANCE STATEMENT
This study discovered that the P. chrysogenum strains were dominantly colonized in Plectranthus amboinicus L. plants of West Malaysia. This study will help the researchers to uncover the critical areas of plant and fungal endophyte interaction that many researchers were not able to explore. Thus, a new theory on unique endophytic interaction between P. chrysogenum and Plectranthus amboinicus L. plants may be arrived at.
ACKNOWLEDGMENT
This study has been carried out as a partial fulfillment of bachelor’s degree (Bachelor of Medical Sciences, International Medical School) in Management and Science University. Therefore, authors would like to thank microbiology laboratory, Management and Science University for providing laboratory facilities.