New Era University

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CHAPTER I

PROBLEM AND ITS BACKGROUND

Antibacterial resistance among bacterial pathogens in recent time is a critical

area of public health concern. The usual causative agents of infectious diseases

(especially bacteria) are becoming increasingly resistant to some or most antibiotics.

The cost of drugs in use today is too expensive for the majority of the population in the

third world countries and therefore the search for some cheap sources of antimicrobial

substances in nature become inevitable. The use of plants as therapeutic agents in

addition to being used as food is age long. In developing countries, thousands of rural

communities still depend mainly on folklore medicine to cure diseases. Medicinal plants

are cheap and handy to most of the populations on the globe. As a result of proximity,

reliability and age long practice, people still depend largely on traditional medicine for

their health care delivery.

The study of fungal endophytes, and in particular their diversity and the

understanding of symbiotic interaction between fungal endophytes and their host, is

related to several aspects of biology such as to investigate the evolutionary origins of

these diverse symbiotic fungi, to determine the community structure and metabolic

activity of all fungal symbionts associated with plants across landscapes, to learn more
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about the contribution of endophytes to plant gene expression and several questions

concerning the biology of endophytes. (Rodriguez, White et al. 2009). The endophytes

are rich sources of bioactive metabolites, which have important potentials in medicine,

agriculture and industries.

The goal of the study was to isolate and characterize the endophytic fungi from

Corazon De Maria (Caladium bicolor), and then screen for antibacterial activities against

Staphylococcus aureus to guides as an organic medicine that matters in human, and

provide the decent knowledge and improvements in the upcoming generations.

Statement of the Problem

The study generally looks at the outcomes and effects of Endophytic fungi

isloated from Corazon de Maria (Caladium bicolor) plant extract.

Specifically, the study will attempt to answer the following questions:

1. Is there any endophytic fungus isolated from Corazon De Maria?

2. Is there any bioactive compound presents on the endophytic fungi?

3. Is there any antibacterial activity of endophytic fungi isolated from Corazon de

Maria pure plant extract?

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4. Is there any significant increase or decrease of the bacteria?

5. Is there a significant difference between the antibacterial activities of endophytic

fungi cultures against Staphylococcus aureus?

Statement of the Hypotheses

Based on the problem statement, the following null hypothesis (Ho) and

alternative hypothesis (Ha) are formulated:

HYPOTHESIS 1

Ha1: There is endophytic fungus isolated from Corazon De Maria.

Ho1: There is no endophytic fungus isolated from Corazon De Maria.

HYPOTHESIS 2

Ha2: There are bioactive compound presents on the endophytic fungi.

Ho2: There are no bioactive compound presents on the endophytic fungi.

HYPOTHESIS 3

Ha3: There is antibacterial activity of endophytic fungi from Corazon de Maria pure plant

extract.
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Ho3: There is no antibacterial activity of endophytic fungi from Corazon de Maria pure

plant extract.

HYPOTHESIS 4

Ha4: There is no significant increase or decrease of the bacteria.

Ho4: There is no significant increase or decrease of the bacteria.

HYPOTHESIS 5

Ha5: There is no significant difference between the antibacterial activities of endophytic

fungi cultures against Staphylococcus aureus.

Ho5: There is no significant difference between the antibacterial activities of endophytic

fungi cultures against Staphylococcus aureus.

Significance of the Study

The significance of the study was to give functional data about the isolation,

characterization and antibacterial activities of the endophytic fungi isolated from

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Corazon de Maria (Caladium bicolor) wherein it collaborate the process and its effects

on the field of biology research.

This study will serve as theoretical model for future studies of the same nature if

ever the existing problem is penetrated in this case.

Future researchers will benefit from this study, and it will provide them facts

needed to compare their study.

Scope and Limitation of the Study

The researchers shall only be focused on the analysis of Endophytic Fungi from

Corazon de Maria plant extract to be harvested in the area of Pasong Tamo, Quezon

City through isolating and identifying the endophytic fungi within the plant and screen for

antibacterial capacity against Staphylococcus aureus. The study involves knowing the

bioactive compounds present in the plant extract.

The researchers shall only be done the research in almost five months due to the

limited period of time.

Definition of Terms

Alkaloids is any of numerous usually colorless,complex and bitter organic bases

containing nitrogen and usually oxygen.

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Amylase is any of a group of enzymes that catalyze the hydrolysis of starch and

glycogen.

Antibacterial is anything that destroys bacteria or suppresses their growth or their

ability to reproduce.

Antibiotic is a drug that is used to kill harmful bacteria and to cure infections.

Antiseptic is preventing the growth of microorganism.

Aspergillus is any of a genus of ascomycetous fungi with branched radiate

sporophores including many common molds.

Artemisinin

Bioactive metabolites are having an effect on a living organism related to the product

of metabolism.

Biotope is a region uniform in environmental condition and in its populations of animals

and plants for which it is the habitat.

Corazon De Maria (Caladium bicolor) is a long-lived perennial herb arising from a

fleshy, roundish, underground stem.

Endophytes are an endosymbiont, often a bacterium or fungus that lives within

a plant for at least part of its life without causing apparent disease.

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Endophytic fungi are a group of fungi that colonize living, internal tissues of plants

without causing any immediate, overt negative effects (Hirsch and Braun, 1992)

Host is a living animal or plant on or in which a parasite lives.

Folkloric is a traditional customs preserved among a people.

Flavonoid is any of a group of oxygen-containing aromatic antioxidant compounds that

includes many common pigments.

Fungus is any one of the group relate plants that have no flowers and that live on dead

or decaying things.

Furunculosis is a highly infectious disease of various salmonoid fishes that is caused

by a bacterium.

Isocoumarin

Isolation is a segregation of a group of organisms from related forms in such a manner

as to prevent crossing.

Laxative is a medicine or food that makes it easier for solid waste to pass through the

body.

Microbe is an extremely small living thing that can only be seen through microscope.

Pathogen is a specific causative agent (as a bacterium or virus) of disease.

Phenols is a corrosive poisonous crystalline acidic compound present in tars of coal

and wood that in dilute solution is used as a disinfectant.

Phenolic acid

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Phytohormone is one produced by a plant and active elsewhere that at the site of

production.

Peptide is any of various amides that are derived from two or more amino acids by

combination of amino group of one acid with carboxyl group of another.

Protease is any of numerous enzyme that hydrolyze proteins.

Proximity is the state of being near.

Purgative is a medicine or food that causes the bowels to empty.

Quinones are biologically important as coenzymes, hydrogen acceptors or vitamins.

Staphylococcus aureus is gram positive spherical bacteria that occur singly, in pairs

or tetrads, or in irregular clusters and include causative agents of skin infections.

Symbiotic is the relationship between two different kinds of living things that live

together and depend on each other.

Steroid is a natural substance that is produced in the body.

Terpenoid is any of various isomeric hydrocarbons found present in essential oils.

Therapeutic is relating to treatment of disease by remedial agents.

Tyrosinase is a copper-containing enzyme that promotes oxidation of phenols.

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Chapter II

REVIEW OF RELATED LITERATURE AND STUDIES

Health is one of the most important parts in human life. There is no doubt that

modern medicine has been highly effective in the prevention and treatment of many

diseases. But due to its price tag, millions of people in the world cannot afford it. As a

result, mostly rely on traditional medicine to cure diseases. There are about 80% of the

world’s population depend on the traditional medicine according to World Health

Organization (Mohammad et al. 2013).

People still depend largely on traditional medicine for their health care delivery

because to most of the populations on the globe, medicinal plants are cheap and handy.

In almost of the world’s population, the search for some cheap sources of antimicrobial

substances in plants becomes popular because of too expensive drugs. Attention has

been directed towards medicinal plants as these plants have been traditionally used to

treat symptoms and multiple diseases. It is established that these plants comprise

microorganisms, collectively known as endophytes.

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Microorganisms that are present in living tissues of a variety of plants,

without causing any indication of diseases are the Endophytic fungi (Stone et al. 2000).

They are rich sources of bioactive metabolites and have important capabilities not only

in medicine but also in agriculture and industries. They are known to produce

metabolites such as alkaloids, terpenoids, steroids, quinones, isocoumarin derivatives,

flavanoids, phenols, phenolic acids, and peptides. The Species produce anti-cancer

compounds, antimicrobial agents, and compounds that can be utilized industrially. For

developing therapeutic drugs, seeking out of bioactive compound from isolated

endophytic fungi is considered. Therefore, isolation and synthesizing of antibiotics,

therapeutic agents and agrochemicals from endophytic fungi are necessary. They are

highly effective, low toxic and having minor environmental impact (Ananda et al., 2012).

Endophytes are synergistic to their host. At times they are known to prevent

the host from successfully attacking fungi and pests by producing special substances

such as secondary metabolites and in return demanding nutrition (Strobel and Daisy

2003). The array of metabolites and other chemicals synthesized by the endophytes

endow the plants with more resistance to nematodes, insects and livestock. Plants

inhabited with specific endophytes are often able to grow faster due to the production of

phytohormones and become so competitive that they dominate in a particular

environment.

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The benefit of symbiotic relationship for the endophyte is that the host plant

is able to supply the necessary nutrients and compounds required for the endophyte to

complete its life cycle. Unlike the host plant, many endophytes are able to survive under

quite extreme and inhospitable conditions (Bacon and White 2000).

Fungal endophytes have evolved two transmission modes. These are

vertical and horizontal transmissions, of which the former transmits the systemic fungus

from plant to offspring via host seeds, and the latter operates by sexual or asexual

spore transfer (Saikkonen et al. 2004).

In a co-evolutionary view, endophytic microbes improve the resistance of the host plant

to adversity by the secretion of the bioactive secondary metabolites (Strobel and Daisy

2003). The number of secondary metabolites produced by fungal endophyte is larger

than that of any other endophytic microorganism class (Zhang et al. 2006).

The search for novel secondary metabolites should be concentrated on

those organisms inhabiting unique and exceptional biotopes. Continual metabolic

interactions between fungus and plant may enhance the synthesis of secondary

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metabolites with novel properties (Schulz et al. 2002). This may of course be a

consequence of high frequency of isolation of endophytic fungi from plants.

Endophytes are the chemical synthesizers inside plants (Owen and

Hundley 2004). The secondary metabolites produced by endophytes associated with

medicinal plants can be exploited for curing many diseases (Tejesvi et al. 2007). The

various natural products produced by endophytic fungi possess unique structures and

bioactivities, thus representing a huge reservoir which offers an enormous potential for

exploitation in agricultural and industrial areas (Tan and Zou 2001).

Attempts have been made to isolate and identify various bioactive

metabolites from endophytic fungi. Fermentation of endophytic fungi with potential for

bioactive compound production has several advantages, like reproducible and

dependable productivity. It can be grown in fermenters to provide inexhaustible supply

of bioactive compound and thus can be exploited commercially. Direct changes in the

culture conditions can be explored as a method of optimizing various biosynthetic

pathways which lead to the production of derivatives and analogues of novel

compounds (Strobel et al. 2004).

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It is believed that the plant endophytic fungi as a novel mine of natural

bioactive compounds have their great potential applications in agriculture, medicine and

food industry (Strobet et al. 2004).

Meanwhile, a problem in the area of public health concern is the antibacterial

resistance among bacterial pathogens. The normal contributing agents of infectious

diseases such as bacteria are becoming increasingly resistant to antibiotics.

Antimicrobial resistance (AMR) is resistance of a microorganism to an antimicrobial

drug that was originally effective for treatment of infections caused by it. Resistant

microorganisms (including bacteria, fungi, viruses and parasites) are able to withstand

attack by antimicrobial drugs, such as antibacterial drugs (e.g., antibiotics), antifungals,

antivirals, and antimalarials, so that standard treatments become ineffective and

infections persist, increasing the risk of spread to others.

The evolution of resistant strains is a natural phenomenon that occurs when

microorganisms replicate themselves erroneously or when resistant traits are

exchanged between them. The use and misuse of antimicrobial drugs accelerates the

emergence of drug-resistant strains. Poor infection control practices, inadequate

sanitary conditions and inappropriate food-handling encourage the further spread of

antimicrobial resistance.
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According to World Health Organization (WHO), Antimicrobial resistance

(AMR) threatens the effective prevention and treatment of an ever-increasing range of

infections caused by bacteria, parasites, viruses and fungi. It is an increasingly serious

threat to global public health that requires action across all government sectors and

society. AMR is present in all parts of the world. New resistance mechanisms emerge

and spread globally. In 2012, there were about 450 000 new cases of multidrug-

resistant tuberculosis (MDR-TB). Extensively drug-resistant tuberculosis (XDR-TB) has

been identified in 92 countries. MDR-TB requires treatment courses that are much

longer and less effective than those for non-resistant TB. Resistance to earlier

generation antimalarial drugs is widespread in most malaria-endemic countries. Further

spread or emergence in other regions, of artemisinin-resistant strains of malaria could

jeopardize important recent gains in control of the disease.

So establishing a balanced correlation between chemical, biological and

therapeutic activities of medicinal plants which are precursors for most conventional

drugs is inevitable. Studies regarding the chemical analyses and the antibacterial

activities of selected medicinal plants proved that lot of them have been confirmed to

contain antibacterial activity which the minerals and phytochemical components of the

medicinal plants may have been responsible (Fagbohun et al., 2010).

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The knowledge on chemical, biological and therapeutic activities of medicinal

plants is essential to provide decent information and improvements of plants which used

as traditional medicine.

Different studies on the subject of antibacterial activity of endophytic fungi

isolated from diverse plants had claimed success in conducting their studies.

In a study conducted by Pavithra and colleagues, endophytic fungi was

isolated from Ocimum Species and assessed against Pseudomonas aeroginosa,

Mycobacterium smegmatis, Salmonella typhimurium, Candida albicans and Penicillium

chrysogenum and production of enzymes such as amylase, protease and tyrosinase.

The results of the study suggest that endophytic fungi associated with Tulsi are potential

agents for antimicrobial activity and production of enzyme.

Another study regarding isolation and characterization of endophytic fungi is

the study conducted by Anitha and colleagues. They had been successful in isolating

endophytes from endemic medicinal plants of Tiruluma hills in India wherein a total of

14 fungal species: Fusarium oxysporum, Colletotrichum falcatum, Pestalotiopsis

species , Aspergillus fumigatus, Aspergillus flavipes, Sterile mycelia,

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Penicilliumsenticosum, Gliocladium roseum,Phomopsis jacquiniana, Nigrospora

sphaerica, Leptosphaeriaspecies, Phomopsis archeri , Alternaria alternata, Aspergillus

niger were isolated from segments of Boswellia ovalifoliolata, Pterocarpus santalinus,

Shorea thumbuggaia, Syzygium alternifolium. The presence of endophytic fungi was

identified based on the morphology of the fungal culture and characteristics of the

spores.

The occurrence of endophytic microorganisms in diverse plant species is

widely studied (Anitha et al. 2013). Thus, the knowledge of their occurrence on plants

is needed to give functional data about the antibacterial activities of the endophytic fungi

and wherein it will provide further information on the field of biology research.

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