In Vitro Inhibitory Potential of Water Hyacinth (Eichhornia crassipes) as an Antifungal

Agent against Early Blight (Alternaria solani)

Researchers:

Agarcio, Ralph Vinze D.C

Malones, Niña Carmela A.

Painandos, Egielyn E.

Robles, John Bernoulli R.

12-STEM ATAldaba

Capstone Project

Marcelo H. Del Pilar National High School

Senior High School Department

A.Y 2018-2019
Chapter 1

Problem and Its Background

1.1 Background of the Study

Reducing the number of pests, insects, blights that destroy crops is one of the major

problems of farmers in the Philippines. Aside from bacterial infections caused by bacterial spot

(Xanthomonas campestris) and wilt spot (Pseudomonas spp.), fungal diseases such as black early

late blight (Pseudocercospora sp.), powdery mildew (Liveilula taurica), and Early blight

(Alternaria Solani) infesting plants such as tomato, potato, and eggplant. (Philippine Star, 2009).

[1]

Early blight (Alternaria solani) is widely distributed across the globe and typically grows

where potato and tomato were largely produced including the United States and some Southeast

Asian countries like the Philippines (Kemmitt, 2013). [2] Dark pigmentation on the vegetative

part of the fungi or mycelium develops resistance to lysis that enable them to survive also in the

soil during wet periods or season including an alternating wet and dry periods that last longer

which allows the fungi’s sporulation.

Fungicides and pesticides such as penthiophyrad, boscalid, and pyraclostrobin were

actively used by farmers to eliminate this pest regardless of its harmful effects to humans and

land animals when in- contact especially when swallowed by marine animals like fish (“Early

blight of tomato”, 2018). [3] An article, “Potential Health Effects of Pesticides” (2014)

mentioned that pesticides also bring chronic diseases such as blood and nerve diseases, genetic

changes and eye irritations. [4]

Phytochemical examinations of potentially active plants that can inhibit the growth of

fungi such as A. Solani were observed in the past years. With the phytochemical constituents of

examined herbal plants such as garlic (Allium sativum), neem (Azadirachta indica) and

eucalyptus (Eucalyptus chamadulonsis), water hyacinth (Eichhornia crassipes) has been

indentified as one of the resolutions for this problem.

 With the given problems from the infestations, the researchers investigated the inhibitory

potential of water hyacinth (E. crassipes) against the fungi, A. solani causing early blight in

plants such as potato and tomato through developing a non-toxic, natural and alternative

antifungal extract This experimental research design will be beneficial for agriculturists and

farmers in the Philippines because aside from eradicating the early blight, the main cause of

blockage of water, brought by water hyacinth (E. crassipes) could be reduced.

1.2 Statement of the Problem

This study aims to investigate the inhibitory potential of water hyacinth (E. crassipes) extract

against the fungi A. solani that promotes early blight in plants. Furthermore, the researchers

sought to find answers in the following questions:

1. How will the different concentrations of water hyacinth (Eichhornia crassipes) extract

affect the efficiency of inhibiting Alternaria solani?

2. What are the possible implications of the study in the field of agricultural and

environmental science?

Hypotheses

Hypotheses

1. The different concentrations of water hyacinth (Eichhornia crassipes) extract will not

affect the efficiency of inhibiting Alternaria solani.

1.3 General Objectives:

Producing an alternative constituent of fungicide in reducing the growth of early blight

(A. solani) through preparation of extract obtained from water hyacinth (E. crassipes)

1.4 Specific objectives:

• To create a cost-efficient and harmless fungicides for tomato, potato and eggplant
• To identify the potential antifungal phytochemical constituents of water hyacinth (E.

crassipes) against A. solani

• To identify the zone of inhibition of the fungi in varying concentration of water hyacinth (E.

crassipes) extract

• To reduce the number of water hyacinth (E. crassipes) through an innovative development of

agricultural product

• To identify the possible advantages and beneficiaries of the research study especially in the

agricultural field

1.5 Scope and Delimitations

1.5.1 Scope

1. This study will be focusing on the effectiveness of the water hyacinth (E. crassipes) extract

to inhibit the fungi, A. solani which is responsible for early blight in tomatoes, potatoes and

eggplants.

2. The study shall be performed in vitro which means no organism will be subjected to testing

3. The study shall consider the identification of active phytochemical constituents of water

hyacinth (E. crassipes) as potential antifungal agent in plants

1.5.2 Delimitations

1. The main source of extract which will be used in the plant are the leaves of the plant, the

roots, stems and flowers will not be used and can be disregarded from the experimentation.

2. The study will no longer use fungicides or pesticides in A. solani, the extract itself shall

only be used in the set-ups.

3. The study shall not consider the use of any antifungal plants in inhibiting A. solani in plant

leaf, water hyacinth (E. crassipes) alone will be the main ingredient together with ethanol.
1.6 Significance of the Study

The findings of this study will be of a great benefit for the following:

Agriculturists/ Farmers. As an alternative antifungal solution, the extract of water hyacinth (E.

crassipes) could reduce the risk of using pesticides and fungicides that may harm the plant itself,

contaminate the soil and bring diseases to humans. This could be an alternative constituent for

antifungal solutions used because water hyacinth’s location is not difficult to find.

People living near Rivers. Since water hyacinth (E. crassipes) is constantly present in all rivers

in the Philippines, this study could reduce the number of the plant which causes blockage of

water during heavy rains, typhoon and severe flooding. It could help them to maintain the

cleanliness of the river and use the plant as an important constituent of antifungal activity aside

from using it as handicrafts.

Marine Animals. Rivers are primarily the favoured environment for the growth of water

hyacinth (E. crassipes). Together with water lilies (Nymphaeaceae), the sunlight and dissolving

of oxygen are interrupted and they are not easily permitted that causes dying of fishes and other

marine animals.

Students. Through this study, students who are interested in the Science field particularly in

agriculture, the results could help them to participate into an innovative idea of reducing

microbes in plants, by providing an alternative solution that also reduces another environmental

problem that the country faces.

Future researchers. The results of this study could help them to identify other plants or

organisms that can also inhibit the growth of the fungi A. solani and other pathogenic bacteria

that destroy the crops and other sectors of agriculture such as livestock production.
Notes in Chapter 1

1. Philippine Star. (2009). Tomato Planting Guide. Retrieved November 28, 2018, from

https://www.philstar.com/other-sections/gardening/2009/07/04/483137/tomato-planting- guide

2. Kemmit, G. (2002). Early blight of potato and tomato. Retrieved November 28, 2018, from

https://www.apsnet.org/edcenter/intropp/lessons/fungi/ascomycetes/Pages/PotatoTomato. Aspx

3. Early blight of tomato. (2018). Retrieved November 28, 2018, from

https://extension.umn.edu/diseases/early-blight-tomato

4. Potential Health Effects of Pesticides. (2018). Retrieved November 28, 2018, from

https://extension.psu.edu/potential-health-effects-of-pesticides
Chapter II

2.1 Review of Related Literature

2.1.1 Alternaria solani

Alternaria solani is a fungus that causes early blight in tomatoes, potatoes, and other

members in the Solanum family. [1, 2] It is considered to be the most impactful pathogen in the

decrease of tomato crop yields. [3, 4] The fungal disease is called “Early” Blight, but can occur

on any stage of plant growth, although older plants and older plant parts are more susceptible

than younger parts. [2, 5] Plants are also more susceptible to the fungi during the fruiting stage.

[6]. Leaf spots are circular and shape and can reach up to ½ inch in diameter. Dark or light

brown spots may appear individually or in groups all throughout the leaf, which in time can

cause the whole leaf to turn yellow, then brown, then fall off. If the disease persists on the plant

and spreads, it may cause complete defoliation of leaves; [7] when there are no more leaves to

support the plant, the plant dies subsequently. According to Central Statistics Authority, 2 per 9

tons of tomato or more than 20 % of supposed harvest in Ethiopia is wasted because of

Alternaria solani. [8] 35-79% yield lost were also reported on other countries. [9] Alternaria

solani thrives the most on warm and humid climates, when its plant host is moist or is always

wet and in contact with water. [2, 10] It can also grow and and undergo sporulation at alternating

wet and dry situations, as it can stay dormant, but still alive in dry environments [2] As it’s is a

type of fungi, Alternaria solani produces spores and can spread to other plants either by physical

contact or through airborne and water means. [11]

2.1.2 Fungicides for Alternaria solani

As Alternaria solani causes a huge impact on the tomato agricultural economy,

fungicides are being used to inhibit the growth of the said fungi. Fungicides such as ridomil gold,

sulphur, copper oxychloride, carbendzim, penthiopyrad, boscalid, pyraclostrobin and mancozeb

are being actively applied to decrease yield losses, [12] although these do have side effects.

Ridomil Gold, according to its production company itself, is “very toxic to aquatic life with long

lasting effects”. [13] Penthiopyrad has effects to pregnant and nursing mammals, exhibiting
minor up to serious complications on the child. [14] Mancozeb, on the other hand, also causes a

lot of health complications in the thyroid hormone system and nervous system [15] Although

effective, the long-term negative effects don’t make these fungicides efficient and worth it.

2.1.3 Eichhornia crassipes

According to Shanab et al [16], Eichhornia crassipes is an invasive aquatic weed known

to out-compete native plants and negatively affect microbes including phytoplankton. It is

originally from the amazon water basin but was then spread by humans to other warm water

locations. Since then, extreme its reproductive and propagative capabilities long caused

disruptions in the natural environment: threaten local native species diversity and disturb the

food chain and nutrient cycle and is expected to flourish even further with global warming. [17]

Eichhornia crassipes can grow, reproduce, and occupy a very large area, even if it only

starts with a small patch. Its height can grow up to 0.5 meter, but in terms of horizontal growth, it

can spread indefinitely. The leaves are densely veined, thick, glossy, and waxy, with

measurements 2 to 15 cm long and 2 to 10 cm wide. [18] The characteristics mentioned greatly

hinders human activities that are associated with bodies of water, such as boating and fishing.

[19]

2.1.4 Eichhornia crassipes Constituents

Although invasive and destructive, its activity of absorbing a lot of nutrients made it rich

with phytochemicals that have medicinal properties [20] Since Eichhornia crassipes is seen as a

useless weed, it would be ideal to be used as a fungicide or as substitute to inorganic and harmful

commercial fungicides. [21] A lot of photochemical analyses in studies have shown that

Eichhornia crassipes contain alkaloids, phthalate derivatives, reducing compounds, polyoses,

saponins, flavonoids, and terpenoids. [16, 19, 23]

Alkaloids, phthalate, saponins, flavonoids and terpenoids have all been verified to have

antibacterial/antimicrobial/antifungal properties by various studies. [24, 25, 26, 27, 28] On the

other hand, according to Shanab et al. (2012), “[the] extracts and fractions [in order to be] used

pharmaceutically could require the harvest of millions of tons/year”, [22] so even though it is
abundant and is proven to have medicinal properties, the sheer amount that will be needed to be

harvested and the method of harvesting may be difficult.

2.1.5 Alkaloids

Alkaloids are naturally occurring molecules that contain both carbon and nitrogen atoms.

Alkaloids are derived from plants and have thousands of different forms. Most alkaloids obtained

from plants are currently being used as medicinal drugs. These medicinal drugs have different

properties depending on which plant the alkaloid was extracted from but the unique and vast

structure of alkaloids are vital on the production of substances with anti-bacterial properties. [29]

2.1.6 Flavonoids

One of the phytochemical compounds of water hyacinth (E. crassipes) responsible for its

curative activity against pathogens and its potential as a remedy for illneses is the flavonoid.

Flavonoids are known as an antibacterial agent. Flavonoids have been considered as potential

substitute for antibiotics. According to Chen, et al., (2015), flavonoids contain antibacterial

mechanisms such as: inhibition of nucleic acid synthesis, inhibition of attachment and biofilm

formation, inhibition of porin in cell membrane, alteration of membrane permeability, and

reduction of pathogenicity. Flavonoids, together with tannin, are thought to be used for

antidiarrheal activity. Flavonoids are also as antioxidants and eradicate free radicals. Therefore,

it also has anticancer activity that protects the cell against all stages of carcinogenesis. [30]

2.1.7 Phthalates

Phthalates are esters of phthalic acids and are applied as plasticizers. It is most commonly

used for the flexibility of plastic and vinyl. Although it can cause several human and

environmental hazards, it is still considered to manifest moderate antialgal and antimicrobial

activities. [31]

2.1.8 Saponins

Saponins are natural glycosides responsible for many pharmacological uses. These

phytochemicals are commonly present in most vegetables and herbs. It is best known for its
foaming characteristics. Studies show that saponins from a specific plant extract is different from

saponins from other sources. Saponins have many medical benefits such as; cholesterol

reduction, antioxidant, immunity booster and bone loss reduction. In water hyacinth, saponins

and steroids are responsible for central nervous system activity. [32]

2.1.9 Terpenoids

Terpenoids are usually present in small amounts in living organisms. It represents the

largest and most diverse of beneficial chemicals. More than 40,000 terpenoids currently exist.

Plant terpenoids are beneficial especially for metabolic disorders and are also considered cancer-

fighting antioxidants. Terpenoids also contain analgesic and anti-inflammatory properties. [33]

2.2 Review of Related Studies

2.2.1 Research Investigation on Alternaria Solani in the Philippines

According to the Department of Science and Technology (DOST) (2018), Alternaria solani

results in serious crop losses in tomatoes and eggplants, most especially on wet seasons. [34] A

past study by Ona & Zaag(1998) conducted in Laguna, Philippines confirms the trend that

Alternaria solani is a serious production constraint in the warm tropics. [35] Desta et al. (2015)

and Koley & Mahapatra (2015) also describes Alternaria solani as one of the most pressing and

devastating problem in terms of tomato production on locations near the equator. [3, 36]

2.2.2 Eichhornia Crassipes as an antifungal agent

Jayanthi & Lalitha (2013) stated that Eichhornia crassipes’ different extracts also exhibit

significant anti-inflammatory effects. This anti-inflammatory characteristic has been tested on

mice and horses. [21] Haggag et al. (2017) supports Eichhornia crassipes medicinal properties

by proving its anti-fungal properties against various cultured samples [20].

2.2.3 Eichhornia Crassipes possessing antibacterial properties

In a study by Shanab et al. (2012), the Eichhornia crassipes were collected wholly from an

accessible source, then cleaned and air dried. The process of extraction used methanol, repeated

three times and then fractionated by thin layer chromatography (TLC). The crude methanolic
extract and the five produced fractions from TLC were tested using diffusion bioassay. The

crude extract and five fractions were proven to have antibacterial effects on both gram-positive

and gram-negative bacteria. Additionally, it has varying antifungal effects and slight antialgal

effects. [22]

2.2.4 Eichhornia Crassipes’s methanol extract containing anti-microbial properties

In another study conducted by Baral & Vaidya (2015), the Eichhornia crassipes were

collected from an accessible source, then cleaned and air dried. The already-prepared Eichhornia

crassipes were powdered by using a grinder. The powder was then subjected to different

extraction solvents: Soxhlet (hot method) extraction in methanol and water & Cold methanolic

and aqueous extraction. Upon testing and phytochemical analysis, the hot extraction method has

shown significant anti-microbial properties. [23]

2.2.5 Eichhornia Crassipes’s methanol extract containing anti-microbial properties

Alternaria solani is an economically important phytopathogenic and saprophytic fungi. It

is the causal agent for early light in tomato and potato plants. The growth of Alternaria solani is

influenced by different environmental factors such as temperature range, pH levels, light

intensity and growth media. In a pathogenicity study conducted by Chohan, B. et al., (2015),

Alternaria solani grew the maximum with a temperature of 25°C at 6.5 pH level and under

continuous light condition. Additionally, among six tomato varieties tested which includes roma,

sahal, salar, packit, reograndi and nagina, no variety was found resistant to the pathogen. [37]

2.2.6 Genome of A. solani as a molecular basis for pathogenicity test

In another study by Evenhuis, et al., (2018), they sequenced the Alternaria solani genome

in which they discovered that the gene effector proteins responsible for the development of the

disease occur in fast evolving and repeat-rich part of the genome. This genome sequencing of

Alternaria solani was the first among all the Alternaria species and provides a molecular basis

for the pathogenicity of Alternaria solani. [38]

2.2.7 Resistance of tomato against early blight

 Moreover, the growth of Alternaria solani can be prevented with the screening of tomato

parental lines. Anjanappa, et al., (2017), tested nine parental lines of tomatoes and eighteen

hybrids in which they employed the detached leaf method of screening. The researchers

concluded that in each season, the resistance of tomatoes against the early blight continuously

decreases. [39]

2.2.8 Silver and Selenium against A. solani

The antifungal activity of silver and selenium nanoparticles against Alternari solani was

examined in a study by Arafa, et al., (2016). Diagnosis of the disease in an early stage is

significant in optimizing the control of early blight. Silver nanoparticles exhibited significant

effect on the inhibition of the fungi at low concentration. It can also be a potential substitute for

chemical fungicides and can also boost plant immunity. [40]

2.2.9 Fungicide treatments against A. solani

A field experiment timing the application of fungicides against Alternaria solani was

conducted by Abuley, et al., (2017). Due to the prevalence of fungicide resistant mutants, the

study showed that spraying fungicide from the onset of the early blight symptom shows a

significant difference in minimizing the damage of the disease brought by Alternaria solani

compared to standard fungicide treatments. [41]

Definition of Terms

In Vitro

An experiment conducted in the laboratory that uses a test tube or laboratory dish that allows the

scientists to isolate specific cells, bacteria, and viruses and study them without the distractions of

having to look at a whole organism since this is a medical test wherein there are no direct contact

with human beings or animals, that is the reason why it considered safe when it comes in

studying a substance. It helps researchers to study the potential effects and risks of a drug before

exposing it to humans or animals. [42]

Ethanolic Extraction
This method is used to convert a substance from any matrix to ethanol. The process may be

under warm or cold conditions. It may also be conducted where the plant material mixture is

agitated by hand or any automated extractors that will control temperature, then inject the

ethanol and undertake clarification. [43]

Potato Dextrose Agar (PDA)

Basically, it used to detect yeasts and molds in dairy products and prepared foods. As well as for

the cultivation of yeasts and molds from clinical specimens that can be supplemented with acid

or antibiotics to inhibit bacterial growth. PDA can be used for growing clinically significant

yeast and molds. Encourages mold sporulation and pigment production in some dermatophytes

of nutritionally rich base (potato infusion). [44]

Zone of Inhibition Test

A test where it measure antibiotic resistance and the ability of solids and textiles to inhibit

microbial growth. There is a relation between the size of the zone of inhibition to the level of

antimicrobial activity present in the sample or product - the antimicrobial is more potent the

larger the zone of inhibition is. [45]

Fungal Pathogen

An intracellular pathogens, it indicates that at some point during the interaction between the host

and the invading species the pathogen lives inside the host cell. There are direct and indirect

effects of pathogenic fungi to their environments. The growth rate decreases as the susceptibility

of a plant to pests increases since the fungal pathogen affect its ability to compete for limited

resources, such as light and space. Another effect, is it infect inflorescences, fruits and seeds of a

plant that have a great impact when it comes to the plant's reproduction [46]
Notes in Chapter 2

1. Cox, R.J. & Simpson, T.J.. (2016). Comprehensive Natural Products II.

2. Kemmit, G.. (2013). Early blight of potato and tomato.

3. Koley, S. & Mahapatra, S.S.. (2016). In Vitro Efficacy of Systemic and Non-Systemic

Chemicals on The Growth Inhibition of Alternaria Solani Causing Early Leaf Blight of Tomato.

4. Jaggal, S., Srivastava, K., Sarma, B.K., Pal, C. & Kumar, R.. (2013). Studies on Growth

Conditions of the Tomato Alternaria Leaf Spot Causing Alternaria Solani L Materials and

Methods.

5. Kumar, S. & Srivastava, K.. (2013). Screening of Tomato Genotypes against Early Blight

(Alternaria solani) Under Field Condition.

6. Chohan, S., Perveen, R., Mehmood, M.A., Naz, S. & Akram, N.. (2015). Morpho-

physiological studies, management and screening of tomato germplasm against Alternaria

solani, the causal agent of tomato early blight.

7. UMassAmherst. (2013). Solanaceous, Early Blight.

8. Central Statistics Authority. (2009). Agricultural sample survey 2008/2007. Report on area

and production of crops (Private peasant holdings, main season).

9. Abhinandan, D., Randhawa, H.S. & Sharma, R.C.. (2004). Incidence of Alternaria leaf blight

in tomato and efficacy of commercial fungicides for its control. Annual Biological Research.

10. Sahu, D.K., Khare, C.P. & Patel, R.. (2014). Eco Friendly Management of Early Blight of

Tomato Using Botanical Plant Extracts.

11. Centers for Disease Control and Prevention. (2012). Principles of Epidemiology in Public

Health Practice, Third Edition. An Introduction to Applied Epidemiology and Biostatistics.

12. University of Minnesota Extension. (2018). Early blight of tomato.

13. Syngenta Agricultural Company. (2015). RIDOMIL GOLD MZ 68 WG.

14. Canada Pest Management Regulatory Agency. (2014). Registration Decision RD2014-17,

Penthiopyrad.

15. Axelstad, M., Boberg, J., Nellemann, C., Kiersgaard, M., Jacobsen, P.R., Christiansen, S.,

Hougaard, K.S., & Hass, U.. (2011). Exposure to the widely used fungicide mancozeb causes

thyroid hormone disruption in rat dams but no behavioral effects in the offspring.

16. Shanab, S., Shalaby, E., Lightfoot, D. & El-Shemy, H.. (2010). Allelopathic Effects of Water

Hyacinth [Eichhornia crassipes].

17. Aboul-Enein, A., Al-Abd, A., Shalaby, E., Abul-Ela, F., Nasr-Allah, A., Mahmoud, A. & El-

Shemy, H.. (2011). Eichhornia crassipes (Mart) solms, Plant Signaling & Behavior.

18. Southeast Exotic Pest Plant Council. (n.d.). Southeast Exotic Pest Plant Council Invasive

Plant Manual.

19. Kisebe, T.I.. (2016). Phytochemical Composition And Antibacterial Activity of Eichhornia

crassipes In Lake Victoria, Kisumu.

20. Haggag, M., Abou El Ella, S. & Abouziena, H.. (2016). Phytochemical Analysis, Antifungal,

Antimicrobial Activities and Application of Eichhornia Crassipes against Some Plant Pathogens.

21. Jayanthi, P. & Lalitha, P.. (2013). Antimicrobial activity of solvent extracts of Eichhornia

crassipes (Mart.) Solms.

22. Shanab, S. & Shalaby, E.. (2012). Biological activities and anticorrosion efficiency of water

hyacinth (Eichhornia crassipes)

23. Baral, B. & Vaidaya, G.S.. (2011). Biological and chemical assessment of water hyacinth

(Eichhornia crassipes) (mart.) Solms.) of Phewa Lake, Nepal.

24. Cushnie, T., Cushnie, B. & Lamb, A.. (2014). Alkaloids: an overview of their antibacterial,

antibiotic-enhancing and antivirulence activities.

25. Actega Coatings & Sealants. (n.d.). Phthalates.

26. Arabski, M., Ciuk, A., Czerwonka, G., Lankoff, A. & Kaca, W.. (2012). Effects of saponins

against clinical E. coli strains and eukaryotic cell line.

27. Alka, J., Padma, K. & Chitra, J.. (2012). Antifungal activity of flavonoids of Sida acuta Burm

f. against Candida albicans.

28. Joshi, S., Cjanotiya, C., Agarwal, G., Prakash, O., Pant, A. & Mathela, C.. (2008).

Terpenoid compositions, and antioxidant and antimicrobial properties of the rhizome essential

oils of different Hedychium species.

29. Amanda Robb. (n.d.). What Is an Alkaloid? - Definition & Examples.

30. Lata, N. & Dubey, V.. (2010). Preliminary phytochemical screening of Eichhornia

crassipes: the world’s worst aquatic Weed.

31. Manayi, A., Kurepaz-mahmoodabadi, M., Gohari, A., Ajani, Y. & Saeidnia, S.. (2014).

Presence of phthalate derivatives in the essential oils of a medicinal plant Achillea tenuifolia.

32. Top Culture. (n.d.). Saponins. Phytochemicals.

33. Mercola, J.. (2017). What are Terpenoids?

34. Department of Science and Technology. (2018). Development of Biofungicide for the

Control of Alternaria solani and Other Fungal Pathogens of Tomato and Eggplant.

35. Ona, I. & Vander Zaag, P.. (1988). Early blight (Alternaria solani) of lowland potato

(Solanum spp.): germplasm evaluation [Philippines] [1988]

36. Desta, M. & Yesuf, M.. (2014) Efficacy and Economics of Fungicides and their Application

Schedule for Early Blight (Alternaria solani) Management and Yield of Tomato at South Tigray,

Ethiopia.

37. Chohan, B., Mehmood, M., & Naz, S. (2015). Morpho-pysiological Studies, Management

and Screening of Tomato Genoplasms Against Alternaria Solani, the Causal Agent of Early

Blight. Multan : Bahauddin Zakariya University.

38. Evenhuis, B., et al. (2018). Gapless Genome Assembly of the Potato and Tomato Early

Blight Pathogen Alternaria Solani. Lelystad: Wageningen University and Research.

39. Anjanappa M., et al. (2017). Screening a set of tomato parental lines and their hybrids for

resistance to early blight (Alternaria solani) by detached leaf method. Karnataka: University of

Horticultural Sciences.

40. Arafa, R. A., et al., (2016). Evaluation of in vitro antifungal activity of silver and selenium

nanoparticles against Alternaria Solani caused Early Blight Disease on Potato. Cairo: Al Azhar

University.

41. Abuley, I. K., et al.,(2017). Timing the application of fungicides to control potato early

blight (Alternaria solani) in multi-location field trials in Denmark. Denmark: Department of

Agroecology.

42. Eldridge, L. (n.d.). What do the Terms In Vivo and In Vitro Mean? Retrieved December 2,

2018, from https://www.verywellhealth.com/what-does-in-vivo-and-in-vitro-mean-2249118\

43. June-Wells, M. (2018, July 11). Your Guide to Ethanol Extraction. Retrieved December 2,

2018, from https://www.cannabisbusinesstimes.com/article/your-guide-to-ethanol-extraction/

44. Aryal, S., Diane, Janine, Agnes, Rosa, Greene, R., & Singh, A. (2018, October 26). Potato

Dextrose Agar (PDA)- Principle, Uses, Composition, Procedure and Colony Characteristics.

Retrieved November 29, 2018, from https://microbiologyinfo.com/potato-dextrose-agar-pda-

principle-uses-composition-procedure-and-colony-characteristics/?

fbclid=IwAR26rjhlxH5wWxmNu3q0VayWnRWRV8j-kHBJxOOfkJrntG_L85qHva0w3vo

45. Microchem Laboratory. (n.d.). Retrieved December 2, 2018, from

https://microchemlab.com/test/zone-inhibition-test-antimicrobial-activity?

fbclid=IwAR3N8tHgHafp4WNfPra0wSDzkXgaMgvoq6xKQ6jMM1J7hUJwJTTzwsM3Xt8

46. Pathogenic Fungi. (n.d.). Retrieved November 22, 2018, from

https://www.sciencedirect.com/topics/immunology-and-microbiology/pathogenic-fungi?

fbclid=IwAR2DcfQblxUiFUyfVaouDBqL6qRJXvPvL41f331P5kCU8JeL_ej0LEXCahs
Chapter 3

Development of Project
 3.1 Project Research Design

The researchers will utilize experimental research design in the conduct of the study to

show the feasibility of water hyacinth ethanolic extract in the inhibition of fungal pathogen

Alternaria solani and also to assess the water hyacinth’s antifungal activity. A study conducted

by Shanab and Shalaby (2012), first showed the inhibitory capability of water hyacinth extract

against three pathogenic fungal species Asparagillus flavus, Asparagillus niger, and Candida

albicans. Thus, the researchers considered using similar inhibitor against the fung. For the

experimental setup, the researchers will have four tables with two replicates each. The water

hyacinth leaf extract will be soaked in ethanol in different concentrations (twenty five per cent,

fifty per cent, seventy-five per cent and one hundred per cent). [42]

For the controlled setup, the researchers will have two tables with two replicates each

with the leaf extract soaked at different concentrations. The researchers will use 100% water

hyacinth leaf extract for the positive and negative control. There will be 12 replicates in total.

[43]

3.1.1 Project Description

This is an experimental study aimed to show the inhibitory potential of water hyacinth

(E. crassipes) against Alternaria solani. With the fungal pathogen Alternaria solani as the

independent variable, the researchers will prove the feasibility of water hyacinth’s antifungal

activity by using different ethanol and leaf extract concentration against the fungal pathogen [44]

3.1.2. Conceptual Framework

1. Preparation of project
The researchers used an IPO model to illustrate
proposalthe conduct of the experimental study. In the
 Infoware 2. Gathering of related
input
- Related
box, the
literature
contents were categorizedstudies
into infoware, technoware and humanware. The
In vitro inhibition of
- Related studies 3. Gathering of materials Alternaria solani using
 methods
Technowareto be followed for the experimentation
4. Isolationwere
and put in the process box. And lastly, the leaf
water hyacinth
identification of extracts
- petri dishes
output of this study is the in vitro inhibition of Alternaria solani using water hyacinth leaf
pathogen
- Air drying oven 5. Maintenance of
- B.O.D incubator
extracts. Alternaria solani culture
 Humanware 6. Treatment of fungi with
- Consultants different concentrations
of leaf extract
INPUT 7. Inhibition period
PROCESS OUTPUT
8. Gathering and Analysis
of Data
3.1.3 Flowchart

With the help of the locals, the resarchers will collect leaf extract from Brgy.
Collection and
extraction of Matimbo, City of Malolos, Bulacan.
water hyacinth
from local areas

The collected leaf samples will be brought at the National Museum for verification
Verification of
the water and certification.
hyacinth leaf
samples

Water hyacinth leaf extraction will be done by putting it at an oven with a

temperature of 160° C for an hour. The air dried leaves will then be ground through
Water hyacinth mortar and pestle. The powdered leaves will be soaked in ethanol for 24 hours .
leaf extraction

The in vitro inhibition will be done using the Kirby Bauer method or disc diffusion
In vitro inhibition test. The potato dextrose agar (PDA) will be used as the nutrient medium for the
of Alternaria
solani using water experimental set-up.
hyacinth leaf
extracts

Twenty millimetres of the cultured Alternaria solani will be swabbed in the six
petri dishes with agar and different concentrations of water hyacinth ethanolic
Treatment of extracts will be applied.
Fungi
 Diseased tomato plants from the Malolos local market showing symptoms of early
Collection of blight will be used as samples for the isolation and culture of the pathogen
diseased Alternaria solani.
sample
The infected leaves will be brought in a laboratory and cut into small bits measuring
about 2mm. Twenty four petri dishes will be prepared for growing the Alternaria
Isolation and
solani and a piece of the specimen will be transferred on Potato Dextrose Agar
identification of (PDA) medium in the center of petri dish. These petri dishes will be incubated under
pathogen B.O.D incubator at 25 ±2°C.

The ethanolic leaf extracts will be put with the PDA medium consisting of fungi.
In vitro Set-up A with twenty- five percent extract, B with fifty per cent extract, C with
inhibition for
the seventy-five per cent extract and D with one hundred per cent extract.
experimental
set-ups

For the two controlled set-ups, one will not contain extract while the other one will
In vitro
inhibition for
contain one hundred per cent extract
the controlled
set-ups

Once the leaf extracts and alternaria solani have been put together, the inhibition
Inhibition period will then start. This wil last for 24 hours.
period

After the in vitro inhibition, the inhibition zone will be measured and the
Gathering and researchers will record the data based from the observation.
Analysis of
Data

3.1.4 Project Specifications

Three months will be allotted in the conduct of the study. The research will be conducted

in several locations. Brgy. Matimbo located in City of Malolos, Bulacan will be one of the

locales since there are sightings of water hyacinth needed for the study. Also, we will be using a

laboratory at the Department of Science and Technology in Taguig for the in vitro inhibition of

Alternaria solani and for the phytochemical testing of water hyacinth (Eichhornia crassipes).

For the analysis of data, the researchers will use different statistical treatment such as the

ANOVA (Analysis of Variance) test, to examine the difference in the outcome of the

experimental and controlled set-ups. The researchers will also utilize F- test to analyze the

variation between the set-ups. The data that will be obtained from the final measuring will be

analyzed using the IBM- Statistical Package for the Social Sciences (SPSS).

3.2 Project Development

3.2.1 Fabrication Process

3.2.1.1 Plant Verification

Sample of water hyacinth (E. crassipes) will be collected from the river part of Brgy.

Matimbo, City of Malolos, Bulacan that has been partially covered by the plant. The entire part

of water hyacinth will then be subjected to verification and identification in National Museum.

The taxonomical characteristics of the plant will then be determined before the treatment in

laboratory.

3.2.2 Testing Procedure

3.2.2.1 Phytochemical Analysis

Test for phlobatannins

For a positive result, boil a 10ml of the aqueous plant in 1% aqueous hydrochloric acid in a

conical flask or test tube, then there will be a quick formation of colour red.

Test for terpenoids

In a test tube, a fraction of 0.8 g of the plant sample, together with a 10 ml of methanol, shaked

and filtered, and be able to acquire an amount of 5ml plant extract sample. In the extracted plant

sample, a 2ml of chloroform was mixed and added a 3ml of sulphuric acid. The indication that

there is a presence of terpenoids is the formation of reddish brown colour in the selected plants.

Test for flavonoids

To confirm the presence of flavonoid in the selected plants, in a test tube 0.5 g of plant extract

was mixed with 10 ml of distill water, a solution of 5 ml of dilute ammonia was added to the

aqueous filtrate of each plant extract followed by putting a 1ml concentrated H₂S0₄. A

formation of a yellow orange color, indicates the presence of flavonoid.

Test for alkaloids

A mixture of 0.2 g of the plant samples and 3ml of hexane was palced in each test tube, shaked

and filtered. A portion of 5ml of 2% HCl was poured to the mixture. After being heated and
filtered, a few drops of picric acid was added. Formation of yellow color precipitate indicates the

presence of alkaloid.

Test for tannins using Braymer’s test

A 100ml portion of the plant extract were treated with 10% alcoholic ferric chloride solution.

Then a formation of color blue or greenish solution will be observed.

Test for rerpenoids using liebermann – Burchard test

A 1ml plant extract was treated with chloroform, acetic anhydride and drops of H2SO4. Then a

formation of a dark green color will be observed.

Test for Phenols using the Ferric chloride test

With a treatment of 5% ferric chloride to the portion of the plant extract, a formation of a deep

blue or black color will be observed.

3.2.2.2 Collection and extraction of water hyacinth

With the help of local residents, the researchers will be collecting water hyacinth plants at

Brgy. Matimbo, City of Malolos, Bulacan to be brought to the National Museum for

identification and certification. The root portion will be disregarded and the plant leaves will be

washed to free from debris. Water hyacinth leaf extraction will be done by putting it at an oven

with a temperature of 160° C for an hour. The air dried leaves will then be ground through

mortar and pestle. The powdered leaves will be soaked in distilled water with for 24 hours.

3.2.2.3 Collection of diseased sample

Diseased tomato plants from the Malolos local market showing symptoms of early blight

will be used as samples for the isolation and culture of the pathogen Alternaria solani. The

extraction of water hyacinth (Eichhornia crassipes) will be conducted at University of Sto.

Tomas and the maintenance of the culture and in vitro inhibition of the pathogen will be

conducted at Department of Science and Technology- Taguig.

3.2.2.4 Isolation and identification of pathogen

 1. The infected leaves will be brought in a laboratory and cut into small bits measuring

about 2mm.

2. Twenty four petri dishes will be prepared for growing the Alternaria solani and a piece of

the specimen will be transferred on Potato Dextrose Agar (PDA) medium in the center of

petri dish.

3. These petri dishes will be incubated under B.O.D incubator at 25 ±2°C for seven days

before the inhibition.

3.2.2.5 In Vitro Inhibition of Alternaria solani using water hyacinth (Eichhornia crassipes)

leaf extracts

After the fungus has been cultured, the researchers will start the in vitro inhibition by

using the Kirby Bauer method or disc diffusion test. Potato Dextrose Agar (PDA) will be used as

as the nutrient medium of the experimental set-up. The obtained extract from water hyacinth

(Eicchornia Crassipes) will be mixed with the PDA medium and will be sterilized for 120°C for

20 minutes. The PDA medium with the plant extract will be put in set-ups A, B, C, D the

experimental set-up and E, the controlled set up, treatment of the fungi will then follow. Twenty

millimetres of the cultured Alternaria solani will be swabbed in the six petri dishes with agar and

different concentrations of water hyacinth leaf extracts will be applied. This marks the start of

the inhibition period that will last for 48 hours to determine the efficacy of the applied

concentration with the given time.

3.2.3 Instruments, Tools, and Equipment Used

3.2.3.1 Potato Dextrose Agar (PDA)

Basically, it used to detect yeasts and molds in dairy products and prepared foods. As well as for

the cultivation of yeasts and molds from clinical specimens that can be supplemented with acid

or antibiotics to inhibit bacterial growth. PDA can be used for growing clinically significant

yeast and molds. Encourages mold sporulation and pigment production in some dermatophytes

of nutritionally rich base (potato infusion). [3]

3.2.3.2 Ethanol
Ethanol is formed during the hydration of ethane during fermentation of the sugar which is

commonly found in distilleries. It is commonly used in paints and varnishes. It plays an

important part in the medical field when developing medicines and instruments like

thermometers. Ethanol is widely used in veterinary medicine in the treatment of ethylene glycol

antifreeze poisoning. [4]

3.2.3.3 Petri Dish

Petri dish also known as a petri plate is a shallow cylindrical glass lidded dish that is typically

used to culture microorganisms. There are glass and plastic Petri dishes, and both can be

sterilized (using an autoclave) and re-used. Before being using the instrument, it is important to

ensure that the Petri dish is sterilized enough using an autoclave. This helps prevent the

contamination of the new culture. [5]

3.2.3.4 Air Drying Oven

Air Drying Oven has proven useful for drying soil, aggregate and fire-proofing samples as well

as other procedures calling for air drying at room temperature. Drying is still rapid and efficient

even if the 105°F (41°C) maximum temperature is below 140°F (60°C) is allowed for some

procedures because of the high air flow of 1—4 air changes per minute. [6]

3.2.3.5 BOD Incubator

BOD incubator is the most versatile and reliable low temperature incubator which is designed to

maintain at 20°C, necessary for Biological Oxygen Demand/Biochemical Oxygen Demand

(BOD) determination. BOD incubators provide controlled temperature conditions for accelerated

tests and exposures. [7]

3.2.4 Project Development Cost

The researchers considered the following cost for the development of the research project

Task Description Cost

Identification Of Plant And Animal Php 200.00

Specimens Botany And Zoology Divisions

Extraction And Phytochemical Analysis Of Php 1,804.00

Plant Material
Antifungal Activity Testing Php 1,650.00
Travel Expenses Php 500.00
Miscellaneous Fees Php 700.00
TOTAL COST Php

3.2.5 Timetable of Activities

Task Description Expected Date

Brainstorming of Topic November 14, 2018
Making of Chapters 1-3 November 15-22, 2018
Research Proposal November 23, 2018
Revising Chapters 1-3 November 24- December 6, 2018
Submission of the Edited Paper December 7, 2018
Finding of Water Hyacinth Location December 10-13, 2018
Finding of Possible Location of Infected Leaf December 16-19, 2018
Consultation at DOST-Taguig December 21, 2018
Collection of Infected Leaf and Water December 26, 2018

Hyacinth
Plant Identification and Validation December 27, 2018
Plant Extraction and Phytochemical Analysis/ December 28, 2018

Updating the result of analysis

Collecting Data and Start of Chapter 4
Start of Chapter 5
Preparation of Papers (ISEF Forms)
Final Revision of Paper
Preparation for Presentation
Presentation of Research Capstone Project
First week to second week of January
Second week to third week of January
First week of February
Second week of February
Second to fifth week of February
First week of March

3.3 Evaluation Procedure

The study aims to evaluate the inhibitory potential of water hyacinth (E. crassipes)

extract through an ethanol against the pathogenic fungi, A. solani. Through Kirby Bauer Method

or Disc Diffusion Test which primarily refers to the procedure done to measure the susceptibility

of an organism in antibiotics, particularly bacteria (Boundless Microbiology, n.d) [8], the

inhibition period will be done in vitro with the aid of PDA (Potato Dextrose Agar), a base for
culturing fungi. After the treatment, the zone of inhibition shall be measured in varying level of

concentration.

Zone of Inhibition of A. Solani after 24 hours (in mm)

Set-up Sample
1 2 3 4 5 6
A. 25% concentration water
hyacinth extract
B. 50% concentration water
hyacinth extract
C. 75% concentration water
hyacinth extract
D. 100% concentration
water hyacinth extract
E. Controlled Set-up

3.3 Evaluation Criteria

Notes in Chapter 3

1. Haggag, M. W., Abou El Ella, S., & Abouziena, H. (2017). Phytochemical Analysis,

Antifungal, Antimicrobial Activities and Application of Eichhornia crassipes Against Some Plant

Pathogens. Planta Daninha, 35, 3-4.

2. Isebe, T. I. (2016). Phytochemical Composition And Antibacterial Activity Of Eichhornia

Crassipes In Lake Victoria, Kisumu. International Journal Of Scientific & Technology, 5(9), 4-5.

3. Aryal, S., Diane, Janine, Agnes, Rosa, Greene, R., & Singh, A. (2018, October 26). Potato

Dextrose Agar (PDA)- Principle, Uses, Composition, Procedure and Colony Characteristics.

Retrieved November 29, 2018, from https://microbiologyinfo.com/potato-dextrose-agar-pda-

principle-uses-composition-procedure-and-colony-characteristics/?

fbclid=IwAR26rjhlxH5wWxmNu3q0VayWnRWRV8j-kHBJxOOfkJrntG_L85qHva0w3vo

4. Richardson, J. A. (2013). Ethanol. Retrieved December 19, 2018, from

https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-

science/ethanol

5. Chemistry Glossary. (n.d.). Retrieved December 19, 2018, from

https://glossary.periodni.com/glossary.php?en=Petri dish
 6. Air Drying Oven. (n.d.). Retrieved December 19, 2018, from

https://www.globalgilson.com/air-drying-oven

7. Acmas. (2014, April 19). Retrieved December 19, 2018, from

http://www.acmasindia.com/blog/bod-incubator/

8. Boundless. (n.d.). Boundless Microbiology. Retrieved from

https://courses.lumenlearning.com/boundless-microbiology/chapter/measuring-drug-

susceptibility/

Gonzalez, M., A. Zamilpa, S. Marquina, V. Navarro and L. Alvarez. 2004. J. Natural Products.

67, 938

Chuang, P. H., C. W. Lee, J. Y. Chou, M. Murugan, B. J. Shieh and H. M. Chen. 2007. Anti-

fungal activity of crude extracts and essential oil of Moringa oleifera Lam. Bioresource

Technology 98 232-236. Doi:10.1016/j.biortech.2005.11.003

Gupta, A. K. 1995. Gliricidia sepium. 270 Plantas Medicinales Iberoamericanas 1st edition.

Bogota: Presencia Ltda; 388-389

Rastrelli, L., A. Caceres, F. De Simone and R. Aquino. 1999. Studies on the constituents

of Gliricidia sepium (Leguminosae) leaves and roots: Isolation and structure elucidation of new

triterpenoid and aromatic compounds. J. Agric. Food Chem. 47:1537-1540

Baral, B., Vaidya, G. S., & Bhattarai, N. (n.d.). Bioactivity and biochemical analysis of water

hyacinth (Eichhornia crassipes). Retrieved January 17, 2019, from

https://www.nepjol.info/index.php/BOTOR/article/view/5556

Dermatophyte. (n.d.). Retrieved January 17, 2019, from

https://www.sciencedirect.com/topics/medicine-and-dentistry/dermatophyte

Dermatophytoses - Skin Disorders. (n.d.). Retrieved January 17, 2019, from

https://www.msdmanuals.com/home/skin-disorders/fungal-skin-infections/overview-of-

dermatophytoses-ringworm,-tinea
Hainer, B. L. (2003, January 01). Dermatophyte Infections. Retrieved January 17, 2019, from

https://www.aafp.org/afp/2003/0101/p101.html

Jr, J. S. (n.d.). Hidden fungi. Retrieved January 17, 2019, from

http://www.pchrd.dost.gov.ph/index.php/news/library-health-news/5543-hidden-fungi

Nordqvist, C. (2017, February 27). Athlete's foot: Symptoms, causes, and treatments. Retrieved

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