FABRICATION OF NANOCOMPOSITE-Trichoderma asperellum-CHITOSAN-

CELLULOSE ACETATE-ENHANCED FOLIAR ORGANIC FERTILIZER

AND ITS AGRICULTURAL APPLICATION

PHYSICAL SCIENCE – TEAM

Ken M. Suarez
Romeo Paul F. Bactol
Paul P. Manuel
Investigators

Eriza G. Mizona
Research Adviser

Dr. Juvy J. Monserate

Research Consultant

Juan R. Liwag Memorial High School

Bayanihan, City of Gapan, Nueva Ecija

Physical, Inorganic and Material Science (PIMS) Nanoworks Laboratory

Department of Chemistry, Central Luzon State University
 Fabrication of Nanocomposite-Trichoderma asperellum-Chitosan-
Cellulose Acetate-Enhanced Foliar Organic Fertilizer
and Its Agricultural Application

Ken M. Suarez, Romeo Paulo F. Bactol and Paul P. Manuel

Juan R. Liwag Memorial High School, Gapan City, Nueva Ecija, Philippines

ABSTRACT

Agriculture had been one of the fundamental aspects of Philippine economy and livelihood
however, agricultural productivity significantly reduced due to the inevitable problems faced by
farmers. This study aimed to enhance Fish Amino Acid (FAA) by incorporating chitosan, nano-
SiO2, ala-MMT and Trichoderma asperellum to increase its Nitrogen, Phosphorus and Potassium
content. Specifically, this study sought to determine the effects of the nanomaterials added to the
NanoBiofertilizer (NBF) to the spore count, antagonistic effect against Fusarium moniliforme and
resulting growth and development (plant height, number of leaves and fruits) in Tomato (Solanum
lycopersicum) plants. Treatments with different composition which includes the positive control
TaPdb, TaPdbCa, TaPdbCaAm and TaPdbCaNs were prepared. Treatments were added to 300ml
of FAA with 10ml chitosan solution a week after of the formulation of NBF treatments. Fifteen
milliliters of each NBF treatments were diluted in 200ml distilled water and were applied to
Tomato plants. Addition of nanomaterials slightly reduced the spore count of T. asperellum. NBF
treatments with T. asperellum showed significant difference to control group on eradicating F.
moniliforme. On the NPK analysis, FTCCAM has the highest NPK total with 4.52% followed by
FTCCNS with 4.35%. FTCCAM adhered the highest growth and leaf rate per day which also
showed significant difference compared to the control group. During the first week of fruiting, the
experimental group didn’t establish significant difference to the control group although there was
an increase in the number of fruits. Results revealed that the addition of nanomaterials could
provide improvisation in organic farming.

Keywords:
 TABLE OF CONTENTS
ABSTRACT..............................................................................................................................................
INTRODUCTION ................................................................................................................................... 1
METHODOLOGY .................................................................................................................................. 4
I. Materials ...................................................................................................................................... 5
II. Preparation and Synthesis of Organo-Modified Montmorillonite Nanocomposite ..................... 5
III. Preparation of Potato Dextrose Broth and Inoculation of T. asperellum .................................... 6
IV. Preparation of Treatments ........................................................................................................ 6
a) Preparation of Preliminary NBF Treatments ............................................................................. 6
b) Preparation of Chitosan Solution .............................................................................................. 7
c) Preparation of Nano-Bio-Fertilizer Treatments ......................................................................... 7
V. Application of Nano-Bio-Fertilizer Treatments to Solanum lycopersicum ..................................... 8
VI. Data Gathering and Analysis.......................................................................................................... 9
a) Spore Counting ............................................................................................................................ 9
b) Dual Culture Method ................................................................................................................. 10
c) NPK Analysis ........................................................................................................................ 11
d) Growth Performance .............................................................................................................. 11
Statistical Analysis ............................................................................................................................ 12
RESULTS AND DISCUSSIONS .......................................................................................................... 13
Spore Count ...................................................................................................................................... 13
Antagonistic Activity ......................................................................................................................... 14
Nitrogen-Phosphorus-Potassium Content ........................................................................................... 16
Absolute Growth Rate ....................................................................................................................... 18
Absolute Leaf Rate ............................................................................................................................ 20
Number of Fruits ............................................................................................................................... 22
CONCLUSION ..................................................................................................................................... 24
RECOMMENDATIONS ....................................................................................................................... 24
ACKNOWLEDGEMENT ..................................................................................................................... 25
BIBLIOGRAPHY ................................................................................................................................. 26
APPENDICES ...................................................................................................................................... 30
 1

INTRODUCTION Commented [MLL1]: Pakibaba pa yun page number

Agriculture had been one of the fundamental aspects of the Philippine economy and

livelihood since it contributes to the one-third of the labor force (Food and Agriculture

Organization of United Nations, 2016) however, ironically, the share of the agricultural sector in

the economy is only 10% (Philippine Statistics Authority, 2016). Moreover, the Philippines was

considered as a “mega biodiversity” due to its abundant natural resources and fertile soil which

signified its self-sufficiency and advantage in the field of agriculture (United Nations Development

Programme, 2014) still, paradoxically, reports from FAO (2010-2012) revealed that over the years,

the Philippines had been among the countries with the highest food inadequacy, import

expenditures and poverty incidence in its major food providers, the farmers and fishermen

(National Statistical Coordination Board).

Half of the agricultural sector involves crop production thus, farmers are challenged with

improving agricultural productivity (Philippine Statistics Authority, 2016) despite of the high cost

production and low income earning capacity in this sector (Department of Agriculture, 2016)

mainly caused by inevitable problems including natural calamities, pests, crop diseases and soil

infertility (PSA, 2016). Furthermore, farmers are expected to produce sufficient and affordable

food source for 105 million Filipinos that are increasing by 1.9% each year (Department of

Agriculture, 2016) thus, farming adaptations must be applied to effectively manage these

challenges on agriculture and food security.

The current farming system utilizes different strategies and technologies to modernize

agricultural techniques to yield better crops which mainly depends on the utilization of synthetic

agrochemicals. The application of both synthetic pesticides and fertilizers expanded rapidly

however, it also results to unfavorable consequences on human health and vast environmental
 2

effects such as eutrophication, contamination of groundwater and soil chemical residues (Net

Industries, 2016) resulting to soil degradation that reduces soil productivity of hectares of

agricultural land in the country (Asio et al. 2009). Thus, organic farming had been visualized as a

solution on restricting the adverse impact of synthetic agrochemicals to the environment (FAO,

2016). Utilization of microorganisms such as bacteria and fungi on organic farming had been an

effective way to improve the soil quality and organic material content of soil. Studies showed that

Trichoderma asperellum has no pre-harvest-interval and no issues on pesticide residue making it

very suitable for application as a bio-fertilizer. T. asperellum secretes natural substances which

were responsible for the solubilization of phospates in soil making it available for the usage of

plants (Real Impact, 2016). According to the work of Tuomisto et al. (2012), organic farming

practices compared to conventional farming had lower environmental impacts per unit of area but

lower yields per product unit.

Nanobiotechnology can enhance the understanding of the biology of various crops to

enhance different nanoscale properties and thus can potentially improve the yields as well as

develop improved systems to increase agricultural food production (Tarafdar et al. 2013).

Nanotechnology reduces costs of environmental protection and promotes the nutrient use

efficiency. Various nano-composite consists of N, P, K, micronutrients, mannose and amino acids

has the capability to increase the uptake and utilization of nutrients by grain crops (Guru et al.

2015). Nanoparticles such as Nano-SiO2 commonly known as Montmorillonite (MMT) provides

promising application in different sectors of agricultural biotechnology. Nano-SiO2 treated soil

exhibited elongated roots which enables plant to survive in conditions like drought and improved

silica uptake (Kannan, 2013). On the other hand, incorporation of chitosan on fertilizers can

possibly give off a slow-release effect on nutrients of fertilizers making them more desirable
 3

because of their longer effect on soil and plant when applied (Abdel-Aziz, Hasaneen and Omer,

2016).

This study is sought to fabricate an innovated organic fertilizer that can be appropriately

utilized in organic farming by incorporating T. asperellum together with its growth medium,

chitosan, Nano-SiO2 and organo-modified Nano-SiO2 to improve the NPK micronutrient of fish

amino acid. Also, the study is concerned on investigating and characterizing the Nano-Bio-

Fertilizer. Furthermore, this study can add up to the existing knowledge on organic farming

practices and techniques and aid the farmers to have improved systems of farming and to improve

their crop yield during harvest season to secure the food supply and help boost the economy of the

country.
 4

METHODOLOGY

Materials

Preparation and Synthesis of

Organo-Modified
Montmorillonite
Nanocomposite

Preparation of Potato
Dextrose Broth and
Inoculation of Trichoderma
asperellum

Preparation of Treatments

Spore Counting Antagonistic Test

NPK Analysis Data Gathering

Commented [MLL2]: Figure name?

 5

Materials and Methods

Experimental procedures for the Preparation and Synthesis of Organo-Modified

Montmorillonite Nanocomposite was performed in the Physical Inorganic and Material Sciences

Nanoworks Laboratory, Central Luzon State University, Science City of Muñoz, Nueva Ecija.

Preparation of treatments, inoculation and culturing of Trichoderma asperellum and counting were

conducted at Microbial Laboratory in Ramon Magsaysay Center for Agricultural Resources and

Environment Studies (RM-CARES) Organic Farming Training Center, Central Luzon State

University, Science City of Muñoz, Nueva Ecija. Nitrogen, Phosphorus and Potassium (NPK)

Analysis was done at to Regional Soils Laboratory, Integrated Laboratories Division, Regional

Field Office III, Department of Agriculture in San Fernando, Pampanga.

I. Materials

Nano-SiO2, Chitosan and Cellulose acetate (CA) from Sigma-Aldrich that was utilized in this

study were obtained from Physical Inorganic and Material Sciences Nanoworks Laboratory. T.

asperellum and Fusarium moniliforme were acquired from RM Cares Organic Farming Training

Center.

II. Preparation and Synthesis of Organo-Modified Montmorillonite Nanocomposite

Ala-MMT water suspension was prepared by dispersing 28.26 grams of nanofil

Montmorillonite (MMT) into 600 ml distilled water at 70 °C. The 3.3 g 12-aminolauric acid (ala)

and 21 ml concentrated hydrochloric acid were dissolved in 94.2 ml of hot distilled water at 70°C.

The prepared mixture was then poured into the previously prepared MMT-water suspension. The
 6

mixture was stirred for 24 hours using a magnetic stirrer, then vacuum-filtered and finally washed

with distilled water three times (De and Mukhopadhyay, 2014).

III. Preparation of Potato Dextrose Broth and Inoculation of T. asperellum

Two hundred (200) grams of sliced unpeeled potatoes were boiled in one liter distilled

water for 30 minutes and then filtered through cheesecloth saving effluent. Twenty (20) grams of

dextrose and 20 g of agar were boiled to dissolve. The prepared medium was autoclaved for 15

minutes at a temperature of 121°C at 15 pounds per square inch (psi). (U.S. Food and Drug

Administration, 2015). Medium were dispensed in 20-25 ml portions into sterile petri dishes and

10 mm of T. asperellum will be inoculated. The previously inoculated T. asperellum were

incubated for 14 days until surface area of the petri dishes are covered by the fungus.

IV. Preparation of Treatments

a) Preparation of Preliminary NBF Treatments

For the spore counting, four treatments with different compositions were prepared (See Table

1). Cellulose acetate was added to the potato dextrose broth with T. asperellum after a week. Nano-

SiO2 and ala-MMT nanocomposites were added after another one week then the treatment. The

prepared treatments were said to be finished a week after the addition of nanocomposites. All of

the treatments were observed and monitored per week.

 7

Table 1. Composition of Preliminary NBF Treatments

Potato Cellulose ala – Nano-Silicon

Trichoderma
Treatments Dextrose Acetate Montmorillonite Dioxide
asperellum
Broth (1.5g) (1.5g) (0.1g)
1 (Negative
+ + - - -
Control)
2 (TC) + + + - -
3 (TCAM) + + + + -
4 (TCNS) + + + - +
Legend: + presence; - absence

b) Preparation of Chitosan Solution

Chitosan solution was prepared by pouring 20 ml glacial acetic acid (GAA) into

180 ml of distilled water to acquire 10% glacial acetic acid solution. Fifty (50) ml of the

10% GAA solution was mixed with 0.5 grams of chitosan using magnetic stirrer for 30

minutes until dispersed thoroughly. Ten (10) percent glacial acetic acid solution and higher

concentration of acetic acid solution favor the bimodal demixing, leading to the formation Commented [MLL3]: Wala bang exact percentage?
Commented [MLL4]: Ano to? Pakisimplify
of a cellular structure and cause the chemical composition of chitosan equalize in different

phases (Cardea et al. 2013). Commented [MLL5]: check this

c) Preparation of Nano-Bio-Fertilizer Treatments

Nano-Bio-Fertilizer (NBF) with four treatments was prepared by adding 200 ml of

Fish amino acid (FAA) and 10 ml of chitosan solution to the previously prepared treatments

excluding the control group. Composition of treatments can be seen in Table 2.

 8

Table 2. Composition of NBF Treatments

Fish
Chitosan Preliminary NBF
Treatments Amino
Solution (10ml) Treatments
Acid (200ml)
1(Positive Control) + - None
2 (FTCC) + + Treatment 2
3 (FTCCAM) + + Treatment 3
4 (FTCCNS) + + Treatment 4
whereas: FTCC- Commented [MLL6]: Pakilagyan ng legend

V. Application of Nano-Bio-Fertilizer Treatments to Solanum lycopersicum

Fifteen (15) day old tomato seedlings were transplanted in propylene bags measuring 0. 25 Commented [MLL7]: Pakiverify to kasi 42 yun nakalagay
sa certification nyo
m X 0. 25 m X 0. 25 m, each filled with eight kilograms of autoclaved garden soil and with a hole

depth of 12 inches (Albert, n.d.). Tomato (Solanum lycopersicum) plants were then treated with

treatments prepared (See Table 3). A diameter of 10 milliliter mycelia discs from each treatment

culture plate was cut-out and, together with seven milliliters of potato dextrose from plate, was

poured into their corresponding bottles each containing 200 ml of FAA that was provided by RM-

CARES. Two amber bottles prepared for each treatment to avoid light exposure while on storage.

After 14 days one bottle from each of the treatment was sent to analytical laboratory for

Nitrogen, Phosphorus and Potassium (NPK) content analysis; the other bottles were used in

treating the S. lycopersicum plants. Prior to application, 15 ml treatments were diluted with 300

ml of water and then an amount of 105 ml diluted treatments (35 ml each) were sprinkled to the

plants seven days after the transferring of S. lycopersicum seedlings to pots. Ten (10) percent and Commented [MLL8]: particularly in what part?

higher concentration of fertilizer solution through foliar application could lead to the higher intake

of nutrients in the leaves of the plant (Khan, 2009). This latter procedure was repeated once more

after another seven days.

 9

Table 3. NBF Treatments on Plant Treatments

Plant Treatment Fertilizer Applied Total Composition of Fertilizer

1 (Negative Control) Untreated None
2 (Positive Control) FAA FAA
3 FTCC FAA+TA+CA+CHI
4 FTCCAM FAA+TA+CA+CHI+ALA-MMT
5 FTCCNS FAA+TA+CA+CHI+Nano-SiO2
whereas: FAA- Commented [MLL9]: Pakilagyan to ng legend para di
maliligaw yun judge

VI. Data Gathering and Analysis

a) Spore Counting

The spore count of each treatment was monitored per week. Using an autopipette,

20 microliters (μL) of Potato Dextrose Broth (PBD) with Trichoderma asperellum colonies Commented [MLL10]: Please do use first the spelled out
version of units of measurements before using its shortened
form
was transferred into a test tube containing 20 μL normal saline solution (NSS) to prepare a

1:3 suspension of T. asperellum spore. A five-microliter suspension was transferred into Commented [MLL11]: Numbers 0-9 must be in word
form while numbers higher than it must be in numerical
form.
the haemocytometer for visual counting under the microscope. The T. asperellum spore

count was calculated using the formula: (Abcam plc., 2016)

Average number of spore x volume factor x dilution factor

𝑇𝑟𝑖𝑐ℎ𝑜𝑑𝑒𝑟𝑚𝑎 𝑠𝑝𝑜𝑟𝑒 𝑐𝑜𝑢𝑛𝑡 𝑝𝑒𝑟 𝑚𝑙 = 𝑚𝑙

Where:

Trichoderma spore per mL = concentration of trichoderma spore per mL of Potato Dextrose

broth suspension

Average number of spore counted = mean number of trichoderma spores counted in one

large corner square of haemocytometer

 10

Volume factor = This is the inverse value of volume of one large corner square which is

equal to (1 / 0.1 mm3) 10 / mm3 or 104 / mL. Since 1 mm3 = 0.001 mL.

Dilution factor = The dilution of PDB (grown with trichoderma colonies) suspension with

NSS; if the dilution used in preparing suspension is 1 : 3 , then the dilution

factor is 3.

b) Dual Culture Method

The assay for antagonism was performed on PDA on petri dishes by the dual culture

method (Fokkema, 1978). Isolate of Fusarium moniliforme, a common fungal plant

pathogen was obtained from RM-CARES, Central Luzon State University, Science City of

Munoz, Nueva Ecija. The isolate was cultivated on PDA. To evaluate the in vitro

antagonistic activity of Trichoderma spp. and the nanomaterials against F. moniliforme,

five millimeter size discs of each culture of F. moniliforme and T. asperellum from the four

treatments (See Table 1) were placed opposite to each other and close to the periphery of

90 mm petri plates containing PDA. For the control, F. moniliforme was placed in a similar

manner on PDA plate. All pairings were carried out in three replications and incubated at

25 ᴼC. The antagonistic activity was assessed after 7 days of incubation by measuring the

radial growth of the F. moniliforme colony. Mycelial growth (MG) interactions were

assessed by cutting a five-millimeter size colony interaction on the PDA, then mounted it

on the glass slides with a coverslip and counted under a microscope. The two readings were

calculated into the percentage of mycelial inhibition of growth (PIMG) using the formula

(Skidmore and Dichinson);

(A1 – A2)
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑚𝑦𝑐𝑒𝑙𝑖𝑎𝑙 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑜𝑓 𝑔𝑟𝑜𝑤𝑡ℎ = 𝑥 100
2
 11

Where:

A1: The diameter of mycelium growth of pathogenic fungus in control

A2: The diameter of mycelium growth of pathogenic fungus with T. asperellum.

c) NPK Analysis

d) Amber bottles containing 100 ml of Nano-Bio-Fertilizer treatments were brought to

Regional Soils Laboratory in the Department of Agriculture Regional Field Office III

at San Fernando, Pampanga to quantify the total NPK content of each treatment by

means of Kjeldahl Method for total Nitrogen (N), Vanadomohybdate Method for total

Phosphorus (P) and Flame Atomic Emission Spectroscopy Method for total Potassium

(K).

e) Growth Performance Commented [MLL12]: Pakisingit na yun fruits

Height and number of leaves of S. lycopersicum were measured every seven days

while number of fruits were counted after 6 weeks to assess and monitor the effect of the

NBF treatments applied on plant treatments. Height were measured using vernier caliper

for the first week and tape measure for the following weeks. The length of the stem from

starting from soil to the highest point of the stem were recorded. Number of leaves were

counted using click-counter three times to ensure the accuracy of the counting. S.

lycopersicum plants were monitored for 4 weeks. Absolute growth and leaf rate was

computed using the formula (Benjamin, 2003):

H2 − H1
Absolute growth rate =
𝑇2 − 𝑇1
 12

Where:

H = Plant Height (millimeters)

T = Time (Day)

L2 − L1
Absolute growth rate =
𝑇2 − 𝑇1

Where:

L = Number of Leaves (millimeters)

T = Time (Day)

Statistical Analysis

Data gathered were analyzed using Complete Randomized Design (CRD) for Spore

Counting and NPK Analysis and Randomized Complete Block Design (RCBD) for

Antagonistic effect, Absolute growth rate and absolute leaf rate. Comparison between

groups was made by means of One-Way Analysis of Variance with Duncan Multiple Range

Test with the aid of QI Macros in MS Excel. Differences with p<0.05 between

experimental and control groups were considered.

 13

RESULTS AND DISCUSSIONS

Spore Count

2,500,000 2,290,000
2,070,000
2,000,000 1,850,000
Spore Count per ml

1,500,000
1,180,000

1,000,000
606,500 674,125
595,750
500,000 400,750

40,909 40,909 40,909 40,909

0
Negative Control TC TCAM TCNS

After 7 days After 14 days After 21 days

Figure 1. The effect of treatments in the spore production of Trichoderma asperellum

every 7 days for 3 weeks.
Legend: TC-Trichoderma Cellulose Acetate; TCAM-

Spore count of each NBF treatments was monitored and quantified to determine if the Commented [MLL13]: Pakicontinue yun legend sa itaas.
Then, paki-Arial yun lahat ng fonts don sa table para mas
formal. The same with sa lahat ng tables. Paki-green narin
nanomaterials could affect the growth of Trichoderma asperellum in different NBF treatments.
yun table sa Metho para isang theme nalang.

Graph 1 shows the mean spore count per milliliter of different preliminary NBF treatments.

It can be inferred that a week after the addition of cellulose acetate (CA), there is a gradual increase

in the spore count of each treatments. It can be also seen in the second week that TCNS (untreated

with Nano silica) exhibited the highest spore count at 6.7x105 spores/ml followed by TCAM

(untreated with ALA-MMT) then TC with 6.1x105 and 5.9x105 spores/ml respectively. On the

third week, which was a week after the addition of ALA-MMT on TCAM and Nano-SiO2 on

TCNS, it can be noted that TC with CA only showed the highest frequency in the spore count. The

results implied that the addition of nanomaterials in the treatments such as TCAM and TCNS

affected the production of T. asperellum spores that resulted to a slight decrease of 10-19% in
 14

spore concentration compared to TC which only has CA though all formulated NBFs have higher

frequency of spores by 40-48% than the negative control.

In the work of Prabu et al. (2013), they discussed that Nano-SiO2 showed minimal decrease

in pH. Since SiO2 is soluble in water, it bonds with hydrogen molecules converting it to silicon

colloids making them biologically active molecules (silicic acid). Hydrated (silicic acid) silica was

formed because of Nano-SiO2 through binding of Si–O to water molecules and hence reduces pH

that caused the effect in the reduction of spore count in the potato dextrose broth which was also

shown in the study of Singh et al. (2014) wherein a shift of pH from 7.0 to 5.5 slowly decrease the

biomass production of T. asperellum. Commented [MLL14]: Singh A, Shahid

M, Srivastava M, Pandey
S, Sharma
A, et al.
(2014) Optimal Physical Parameters for Growth of
Antagonistic Activity Trichoderma
Species at
Varying pH, Temperature and Agitation. Virol Mycol 3:
127.
45
41c
40
Radial Growth of Colony (mm)

35

30

25

20

15 13.33b 12.67ab
9.33a 9.33a
10

0
Potato dextrose Trichoderma TC TCAM TCNS
Agar asperellum Treatments
Figure 2. Radial Growth of Fusarium moniliforme Colonies under Preliminary NBF
Treatments *Means having the same lower case letter data labels are not significantly
different. Statistical differences were determined by one-way ANOVA (p<0.05)
followed by Duncan’s Multiple Range Test.
 15

The in vitro antagonistic potential of T. asperellum was investigated to determine if NBF

treatments can eradicate plant pathogens such as Fusarium moniliforme via dual culture method.

Fusarium species are one of the most common genus of soil-borne fungi that are affecting at least

81 out of the 101 economically important food crops such as tomato, potato, sugarcane, mango,

corn and rice. Thus, inhibiting its production is a vital process in improving the growth and
development of agricultural crops.
Commented [MLL15]: https://www.britannica.com/scien
ce/fusarium-wilt
http://www.fao.org/fileadmin/templates/agphome/docum
ents/Pests_Pesticides/caribbeantr4/06Evolution.pdf

Figure 2 exhibits radial growth of F. moniliforme colonies under Preliminary NBF

Treatments. It could be notably seen in the graph that TC and TCAM have no significant difference

with each other and showed the best antagonistic activity against F. moniliforme followed by

TCNS. It can be also inferred that all formulated NBF treatments showed significant difference

compared to the normal growth of the fungus in the control group exhibiting its potential in

inhibiting the growth of the plant pathogen. Based on the investigation of Komy et al. (2015), T.

asperellum isolates could significantly lower the mycelial growth of Fusarium strains. Secretion

of extracellular cell wall degrading enzymes namely protease, chitinase and β-glucanases that

penetrates on the cell wall of other fungi, secondary metabolites and peptaibols (Li et al. 2016).

However, the antagonistic effect of the NBF treatments could not be solely contributed by T.

asperellum since the result of the bioassays revealed that the addition of cellulose acetate and

Nano-silica increased the selective antifungal activity of the two NBF treatments by 30%.
 16

Nitrogen-Phosphorus-Potassium Content

3
2.43
2.5
2.1 2.1
1.96 1.94
Percentage (%)

2 1.83 1.78
1.46
1.5

0.5 0.29 0.31 0.31 0.31

0
FAA FTCC FTCCAM FTCCNS
Treatments

Nitrogen Phosporus Potassium

Figure 3. Total amount of Nitrogen, Phosphorus and Potassium in Treatments

Figure 3 exhibits the result of the analysis on the total amount (%) of Nitrogen, Phosphorus

and Potassium present in each treatments. It can be inferred that FTCCAM has the highest NPK

total with 4.52% followed by FTCCNS with 4.35% then FTCC with 4.24% and FAA (control)

with 3.71%.

FTCCAM has the highest percentage of Nitrogen with 2.43%. Organic fertilizers with an

adequate or high amount of nitrogen would be favorable since it is an essential nutrient that

promotes successful plant growth and associated with leafy, vegetative growth that stimulate well

development of foliage and fruit. Also, it is one of the part of the chlorophyll molecule that is also

include creating of food for the plants through photosynthesis (Meredith, 2016). Moreover, plant

having sufficient nitrogen will probably exhibit vigorous plant growth where leaves will also

develop a dark green color (The University of Hawaii, 2016).

Furthermore, all treatments excluding FAA showed the greatest percentage in Phosphorus

content. Organic fertilizers with rich of phosphorus content would be beneficial since it is the
 17

nutrient involving a metabolic process responsible for the transfer of energy from one point to

another in the plant. These mineral element is vital in root and flowering development (Meredith,

2016). Hence, different growth and development effects of phosphorus that are essential for the

plants include stimulated root development, increased stalk and stem strength, improved flower

formation and seed production, more uniform and earlier crop maturity, increased resistance to

plant diseases, and supports development throughout the cycle (The Mosaic Company, 2016).

FTCC and FTCCNS adhered the highest presence of Potassium in all the treatments with

2.1%. Potassium is one of the major mineral element responsible for plant stress tolerance that also

helps regulate the plant metabolism, root development and water pressure regulation inside and

outside of plant cells (Meredith, 2016). Furthermore, potassium plays a vital role in root growth

and promotes drought tolerance via formation of cellulose and reduces lodging, increases protein

content of plants, reduces water loss and wilting, and helps retard the spread of crop diseases and

nematodes (The Mosaic Company, 2016). Hence, fertilizer with appropriate amount of potassium

would be beneficial.

According to Department of Agriculture (2012), the minimum required total NPK content

for plant growth regulators which has fish amino acid as the main component was about 0.5%-

2.5%. Hence, it can be derived from the results of the analysis in Figure 2 that all the formulated

treatments surpassed the minimum requirement implied by DA in organic fertilizers. FTCCAM

has the highest total NPK content with 4.52% followed by FTCCNS with 4.35% then FTCC with

4.24% and FAA with 3.71%. However, none of the treatments reached or exceeded the

specifications on the total NPK content for foliar organic fertilizer which was about 5%-10%

(Department of Agriculture, 2016) though, treatment 3 established close value on it with 4.52% of

total NPK.
 18

Absolute Growth Rate

Absolute Growth Rate

50
45 42.22a
40 36.39b 37.22ab
33.89bc 33.61a
35 30.56c 31.22a 30.78a 30.78a
Height (mm)

30
25 22.94b
18.50a 16.94ab
20 15.44ac 14.33bc
15 12.94c
10
5
0
Negative FAA FTCC FTCCAM FTCCNS
Control

1st-2nd 2nd-3rd 3rd-4th

Figure 4. Absolute Growth Rate per Day of Plant Treatments from 1st week-4th week
*Means having the same lower case letter data labels are not significantly different.
Statistical differences were determined by one-way ANOVA (p<0.05) followed by
Duncan’s Multiple Range Test.

Figure 4 shows the absolute growth rate per day in 7 days interval in 4 weeks of applying

the plants with different NBF treatments. Growth rate was observed on plant treatments to know

how fast plants increase their height and develop stems after application of different NBF

treatments.

During the first week to second week, it can be noted in the experimental group that growth

rate of plants treated with FTCCAM showed the greatest growth with 18.50 mm growth per day

followed by plant FTCCNS then plant treatment FTCC. Also, it visible in the experimental group

that both plant FTCCAM and FTCCNS showed significant difference with FTCC. In comparison

to the experimental and the control group, the negative control has no significant differences with

FTCCAM and FTCCNS while FAA didn’t show significant differences to FTCCNS. It is

illustrated in the graph that FTCCAM and FTCCNS were two of the NBF treatments with
 19

promising performance in the first week. Furthermore, the similarities statistically on mean of the

control and experimental group was triggered due to the inclusion of chitosan on the experimental

treatments since it has the capability that adds up slow or controlled-release delivery on the

nutrients of fertilizers (Abdel-Aziz, Ahasteen and Omer, 2016).

From the second week to the third week, it is noticeable all plant treatment in the

experimental group showed efficient growth performance which were statistically the same.

Moreover, during this developmental phase of plants, it can be inferred that the effects of all

treatments in the experimental group were comparable to FAA.

On the third week to fourth week after NBF application, it is observable that FTCCAM

was notable in the experimental group in terms of the height performance though it has no

significant difference to the plants treated with FTCCNS. Similarly, means of FTCCNS and FTCC

didn’t showed significant difference. In contrast to results of the experimental and control group,

means of the experimental group exhibited significant difference to the means of the control group.

This indicates that experimental treatments particularly FTCCAM were highly comparable and

effective based on the statistical result.

Based on the results of the investigation, FTCCAM which was composed of

FAA+T.asperellum+CA+CHI+ALA-MMT performed the best in terms of growth rate per day all

throughout 4 weeks of observation and monitoring. Consistency on the promising performance

each week by FTCCAM was caused by the controlled-release effect brought by the chitosan on

the fertilizer applied. (Abdel-Aziz, Ahasteen and Omer, 2016). Moreover, this could be explained

by the nitrogen content of NBF treatments since this mineral element was one of the major element

responsible for the development and plant growth (De Pascale et al., 2006). Among all the
 20

treatments as seen in Figure 2, FTCCAM has the highest nitrogen content total implying that it has

the greatest capability to improve the growth rate in terms of height of plants.

Absolute Leaf Rate

Absolute Leaf Rate

45 40.33a
40 35.00b
34.67b 33.33bc
35
28.78c
30
Frequency

25
20 17.83a 17.89a
14.44b 14.50b 15.67b
15
10 6.11a
4.28b 4.28b 2.83b 4.00b
5
0
Negative FAA FTCC FTCCAM FTCCNS
Control

1st-2nd 2nd-3rd 3rd-4th

Figure 5. Absolute Leaf Rate per Day of Plant Treatments from 1st week-4th week
*Means having the same lower case letter data labels are not significantly different.
Statistical differences were determined by one-way ANOVA (p<0.05) followed by
Duncan’s Multiple Range Test.

Figure 5 exhibits the absolute leaf rate per day in 7 days interval in 4 weeks of plant

treatments treated with different NBF treatments. Leaf rate was observed on plant treatments to

distinguish how fast the tomato plants develop leaves after application of different NBF treatments.

From the first week to second week, it was noticeable in the experimental group that only

FTCCAM adhered significant difference with plants treated with FTCC and FTCCNS.

Furthermore, it can be inferred in the figure that the leaf rate of the experimental group was

comparable to the control group. Statistically, all plant treatments during the first week of

observation have same means excluding FTCCAM which has a higher mean.
 21

On the second week to third week, it can be seen in the experimental group that FTCCAM

was recorded with the highest leaf rate per day with 6.11 leaves and established significant

difference between FTCC and FTCCNS. Moreover, in the control group, the FAA exhibited

difference statistically comparable with the negative control. In comparison between the control

and the experimental group, it can be derived that FTCCAM was comparable to FAA while FTCC

and FTCCAM was comparable to the negative control.

For the last week of observation, it was distinguishable in the experimental group that

plants treated with FTCCAM was documented with the highest frequency of leaf rate with 40.33

leaves per day. Also, FTCCAM established significant difference with FTCC and FTCCNS.

Moreover, the FAA has a significant difference compared to the negative control. Furthermore, it

can be inferred in the figure that experimental group was comparable to the control group and

FTCCAM yielded the best performance among all the treatments.

With respect to the results of the investigation, plant treatment 4 which was applied with

FTCCAM of the NBF treatments composed of FAA+T.asperellum+CA+CHI+ALA-MMT

performed the best in terms of leaf rate per day all throughout 4 weeks of observation and

monitoring. Plant manufactures proteins that is essential to new cell growth and is mainly

responsible for leaf and stem growth by converting nitrogen (OK Plant Diet, 2014) thus, suggesting

that plant treatment applied with NBF treatment that has the highest nitrogen content can exhibit

the highest leaf rate per day. The result from the NPK Analysis and the application of NBF

treatments were parallel since the treatment with the highest nitrogen content which was FTCCAM

from the NPK analysis also showed the best leaf rate during application in Plant treatment 4.
 22

Number of Fruits

16

14 13.33a
11.67a
12
Number of Fruits

9.67a 10a
10
7.67a
8

0
Negative FAA FTCC FTCCAM FTCCNS
Control
Treatments

Figure 6. Average Number of Fruits per Plant after 42 days

*Means having the same lower case letter data labels are not significantly different.
Statistical differences were determined by one-way ANOVA (p<0.05) followed by
Duncan’s Multiple Range Test.

Figure 6 exhibits the average number of fruits per plant after 42 days treated with different

NBF treatments. It was noticeable that FTCC resulted to the highest average number of fruits with

13.33 followed by FTCCNS with 11.67 then FTCCAM with 10 next was FAA with 9.67 and lastly

the negative control with 7.67. Apparently, all treatments didn’t established significant difference

with each other. This was because it was the only first week of observation for the fruiting of

Solanum lycopersicum. However, it can be seen that the NBF treatments have greater means

compared to the control group which indicates that treatments can potentially increase the yield of

fruits by 4-27% and establish significant difference after more weeks or during harvesting.

Medium-sized tomato plant takes approximately 65 days or more to reach harvest time while main-

crop tomato plant take 75 days or more to reach harvest time (Rose, n.d). Also, the results of the

fruiting was parallel to the NPK analysis in Figure 3 since the experimental treatments have greater

phosphorus content which were responsible for promoted fruiting on plants (Carlson and Le
 23

Capitaine,2015) compared to the control group although the difference were just 0.02% which

triggered the similarities in the means statistically.

 24

CONCLUSION

The investigation revealed that addition of nanomaterials slightly reduced the spore count

of Trichoderma asperellum in the NBF treatments due to the formation of silicon colloids which

reduces the pH level of the growth medium that affected the growth of T. asperellum. Based on

the dual culture method, treatments with T. asperellum with nanomaterials established significant

difference to control group implying that the NBF formulations have antagonistic potential to

eradicate Fusarium moniliforme. Based on the NPK analysis, FTCCAM yielded the highest total

NPK content with 4.52% followed by FTCCNS with 4.35% then FTCC with 4.24% and FAA with

3.71%. All throughout the four-week observation on the growth rate of plant treatments, plant

treatment 4 which was treated with FTCCAM adhered the highest growth rate and established

significant difference with the control group. Also, plant treatment 4 exhibited the greatest leaf

rate per day within the observation period and showed significant difference compared with the

control group. Moreover, during the first week of observation for the fruiting of Solanum

lycopersicum, the experimental group didn’t establish significant difference to the control group

although results revealed that the experimental group promoted and increased the fruiting of S.

lycopersicum plant by 4-27%.

RECOMMENDATIONS

Based on the study conducted, the following recommendations are hereby drawn:

1. Further study should be conducted investigating other qualities and characteristics of

plant to precisely evaluate the total effect of the NBF treatments on the growth and

development of plants.
 25

2. Seek for the appropriate plant that will properly suit up the properties and NPK content

of the developed Nano-bio-fertilizer.

3. Explore the different additives or inoculants that can be applied or added in the NBF to

further increase the total NPK content.

4. Investigate and compare the developed Nano-Bio-Fertilizer and commercial fertilizer

in terms of effect to plant growth and soil.

5. Observe for the longer term effect such as for the fruiting and fruit quality of NBF

Treatments on Solanum lycopersicum and other crops

ACKNOWLEDGEMENT

The researchers would like to express their sincerest thanks and gratitude to Dr. Estrelita

Cunanan, Mrs. Elvira Pascua, Ms. Nenita Manalastas, Mr. Jeffrey Sta. Ines, Mr John Esteban and

their parents for the unending support, encouragement and guidance throughout this pursuit of

knowledge. Mostly, this piece of work is highly dedicated and offered unto the Lord. To God be

the Glory!
 26

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 30

APPENDICES

Image 1. Cellulose Acetate

Image 2. Organo-Modified Montmorillonite

 31

Image 3. Nano-Silica

Image 4. Preparation of ALA-MMT Mixture

 32

Image 5. Autoclaving of the prepared medium

Image 6. Transferring Potato Dextrose Broth to Microwaveable Container

 33

Image 7. Inoculation of Trichoderma asperellum

Image 8. Charging the Hemacytometer with PD Broth Suspension using Auto-Pippete

 34

Image 9. Counting of Trichoderma asperellum using Hemacytometer

Image 10. Preparation of Chitosan Solution

 35

Image 11. Preparation of NBF treatments

Image 12. Planting and application of NBF treatements

 36

Image 13. Gathering of data

Image. 14. Department of Agriculture: Regional Field Office II

 37

Image 15. Regional Soils Laboratory

Image 16. One-way ANOVA of Absolute Growth Rate per Day of Plant Treatments from 1st week to 2nd
week
 38

Image 17. One-way ANOVA of Absolute Leaf Rate per Day of Plant Treatments from 1st week to 2nd
week

Image 18. One-way ANOVA of Absolute Growth Rate per Day of Plant Treatments from 2nd week to 3rd
week
 39

Image 19. One-way ANOVA of Absolute Leaf Rate per Day of Plant Treatments from 2nd week to 3rd
week

Image 20. One-way ANOVA of Absolute Growth Rate per Day of Plant Treatments from 3rd week to 4th
week
 40

Image 21. One-way ANOVA of Absolute Leaf Rate per Day of Plant Treatments from 3rd week to 4th
week

Image 22. Certification on the analysis of trichoderma asperellum

 41

Image 23. NPK analysis results