test for delaying the senescence of tomato
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TEST FOR DELAYING THE SENESCENCE OF TOMATO
(Lycopersicum esculentum) USING DIFFERENT LEVEL OFPOTASSIUM
ALUMINUM SULFATE (ALUM)
CHAPTER 1
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INTRODUCTION
1.1 Background of the Study
Tomato is one of the most important vegetables in Asia and Africa and these
countries account for more than 65% of global tomato production. Tomato is rich in
nutrients such as vitamins, minerals, and antioxidants, which are important to well
balanced human diets. Tomato is also an important dietary component because it
contains high level of lycopene, an antioxidant that reduces the risks associated with
several cancers and neurodegenerative diseases.
Tomato is susceptible to several insects and mite pests as well as plant
diseases. Chemical pesticides are being used indiscriminately to manage these pests in
South and Southeast Asia and part of Africa. In addition, chemical fertilizers and
insecticides and sometimes overused in tomato production, which may contaminate
groundwater. Intensive agrochemical use in tomato husbandry substantially increase
the production cost and may pose serious risks to producers, consumers, and the
overall health of the environment (Srinivasan R [Ed.] 2010).
Filipino tomato farmers are often challenged by numerous plant diseases that are
promoted by warm, and sometime moist climate. The conditions that promote tomato
diseases also favors the development of tomato rots, in field and even during handling,
storing and transporting.
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Tomato fruit rot are generally caused by microscopic opportunistic pathogens
that live on plant debris. These pathogens can, however, infect tissues that are
wounded and/or exposed to the environment. The opportunists are ubiquitous in the
natural environment, in part because they are saprophytes.
Rhizopus stolonifer, one of postharvest diseases in tomatoes, is mainly
responsible for significant losses of tomatoes both before and after harvesting
especially during storage]. Chemical fungicides such as Methyl (1-butylcarbamoyl)-2-
benzimidazole carbamate (benomyl) are normally utilized to control postharvest
diseases in tomatoes during storage. Unfortunately, such chemical fungicides may
induce fungicide-resistant strains. Furthermore, the growing consumer awareness
and/or demand for healthy foods have increased the quest for more efficient methods
with minimum health and environmental impact for the control of diseases. Therefore,
alternative methods to preserve fruits and vegetable have been explored by many
researchers (Turkhan, 2010).
Potassium Alum Sulfate (KAl(SO4)212H2O) or alum has shown a good fungicidal
effect against moulds. Powdered commercial alum, due to its eco-friendly and
biodegradable nature, has been used as deodorant for a long period of time. Application
of alum to control fungi in tomatoes has, however, never been examined. The objectives
of this study were to investigate the antifungal activity of alum against Rhizopus
stoloniferand to evaluate the potential application of alum to control postharvest
spoilage on tomatoes during storage (Turkhan, 2010)
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1.2 Statement of the Problem
This research was conducted to test the effectiveness of alum sulfate on inhibiting
the growth of fungi and determine which concentration (1.0mg/L, 2.0mg./L or 3.0mg./L)
is most effective.
1.3 General Objectives
This research is conducted with the end in view of qualifying the possibility of
using alum sulfate in inhibiting the growth and spread if pathogenic Rhizopus stolonifer
on commercial tomato. This may likewise help farmers whose products are viable to
Rhizopus stolonifer infestation to fully protect and preserve the market value of their
products for more income with less cost on post-harvest crop protection.
1.4 Specific Objective
This research will endeavor to come up with a less cost but effective medium in
controlling the infestation of Rhizopus stolonifer on tomatoes.
1.5 Hypotheses
Null Hypothesis: Alum cannot inhibit the growth ofRhizopus stolonifer
Alternative Hypothesis: Alum inhibit the growth ofRhizopus stolonifer
1.6 Significance of the Study
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The current antifungal problems in the society, the study about the antifungal
effectiveness of alum sulphate will be a very good contribution to the development of
antifungal medicines or drugs in the near future. It will also compare and determine the
effect of the different concentrations of alum to bread molds (Rhizopus Stolonifer). This
research study will also provide a cheaper and easier solution in treating fungal
infections or diseases especially that drugs for fungal infections are becoming more and
more expensive. Thus, it will be very beneficial for antifungal research and development
and also to the people suffering antifungal problems.
1.7 Scope and Limitations
This study was conducted on August 2013 under ambient room conditions.
Commercial alum sulphate was used as the only experimental chemical with water and
baking powder as negative and positive control. This research used Rhizopus Stolonifer
for the antifungal test. Rhizopus stolonifer was procured from the research laboratory of
Notre Dame Integrated Basic Education Department, General Santos City.
1.8 Operational Definition of Terms
In Vitro
In Vivo
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Potassium aluminum sulfate (alum), a chemically hydrated aluminum potassium sulfate
that possesses a specific crystal shape with a chemical formula
KAl(SO4)2.12H2O. It is no-toxic, has somewhat a sweet acidic taste that
dissolves easily in water and reacts with acid.
Tomato,
Tomato size,
Post harvest,
Growth inhibition,
Rhizopus stolonifer,
Antifungal
Radial Infestation
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Inoculum
Chapter 2
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Review of Related Literature
This chapter places the current study into the context of previous research. It
consists of both the theoretical and conceptual framework of the present study, the
critique of both related studies and literature that are related to the present study, as
well as the operational definition of terms that are based on observable characteristics
and how it is used in the study.
Figure 1. Theoretical Framework
This portion showed the conceptual framework of the research, the major steps
undertaken towards the desired result.
Related Literature
Alum sulfate
Rhizopus stolonifer
In Vitro
a. Growth Inhibition ofRhizopusstoloniferon Commercial Tomato
b. Color and c. Firmness
In Vivo
Anti-fungal Screening ofAlum
Sulfate
Recommending the Use of Potassium
Alum Sulfate Against Rhizopus
stolonifer
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Philippine is a tropical country whose climate favoured all sort of microbial
habitation. Philippine government through its lead agency the Department of Health
tried to suppress pathogenic microorganism in spreading and contaminating different
types of diseased be it human or animals. In like manner, the Department of Agriculture
also heightened their stand to safeguard and help protect Filipino farmers in their effort
to produce quality and abundant farm products for the end consumers and for their
livelihood (DA, 2010).
Seeing the potential of alum in controlling fungal infection in human body ignite
the researcher scientific thought of exploring the possible application of alum to the
commercial tomato in order to extend the shelf life to more than two (2) weeks prior to
its final deterioration caused by fungal infection.
Potassium Aluminum Sulfate (Alum) is a widely-used and versatile industrial
chemical, playing an important role in the production of many essentials seen and used
every day in the home and industry.
Most of the alum produced today is used in the pulp & paper industry as well as
water and wastewater treatment. It is inexpensive and effective for a broad range of
treatment problems because it can function as a coagulant, flocculant, precipitant and
emulsion breaker. As a coagulant and flocculant, alum removes turbidity, total organic
carbon (TOC) which can be disinfection byproduct precursors, suspended solids and
colloidal color, reduces biochemical oxygen demand (BOD) and clarifies potable,
processes and waste water.
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Alum is widely used for lake restoration, treatment and nutrient inactivation. It
also is used in the production of aluminum chemicals, fire extinguisher compounds, soil
additives and fertilizer, soaps, greases, drugs and cosmetics. It even enters into the
sports arena, as an additive to give major league baseball covers their hard, tough
hides. In short, alum touches the lives of just about everyone in many ways.
http://www.generalchemical.com/aluminum-sulfate.html
Rhizopus stolonifer (black bread mold) is a widely distributed Mucoralean mold.
Commonly found on bread surfaces, it takes food and nutrients from the bread and
causes damage to the surface where it lives.
Asexual spores are formed within sporangia, which break to release the spores mature.
Germination of these spores forms the haploid hyphae of a new mycelium. R. stolonifer
grows rapidly at temperatures between 15 and 30C.
Rhizopus stolonifer is a heterothallic species (Kwon, 2001), in that sexual
reproduction happens only when opposite mating types (designated + and -) come in
contact. Successful mating results in the formation of durable zygospores at the point of
contact. Subsequently, the zygospore germinates and forms a sporangiophore whose
sporangium contains both + and - haploid spores. There are three varieties: R.
stolonifervar. stoloniferproduces straight, erect sporangiophores, whereas those ofR.
stolonifer var. lyococcos are curved. A closely related species, Rhizopus sexualis,
differs primarily in being homothallic (self-compatible).
http://www.generalchemical.com/aluminum-sulfate.htmlhttp://en.wikipedia.org/wiki/Moldhttp://en.wikipedia.org/wiki/Sporangiumhttp://en.wikipedia.org/wiki/Heterothallichttp://en.wikipedia.org/wiki/Zygosporeshttp://en.wikipedia.org/w/index.php?title=Rhizopus_sexualis&action=edit&redlink=1http://en.wikipedia.org/wiki/Homothallichttp://www.generalchemical.com/aluminum-sulfate.htmlhttp://en.wikipedia.org/wiki/Moldhttp://en.wikipedia.org/wiki/Sporangiumhttp://en.wikipedia.org/wiki/Heterothallichttp://en.wikipedia.org/wiki/Zygosporeshttp://en.wikipedia.org/w/index.php?title=Rhizopus_sexualis&action=edit&redlink=1http://en.wikipedia.org/wiki/Homothallic -
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The Rhiopus stoloniferare opportunists and are ubiquitous (found everywhere) in
the natural environment, in part because they are good saprophytes. Mechanical
injuries (e.g., cuts, punctures) that occur during harvest and handling are a frequent site
for decay development beginning on the fruit surface. By contrast, internalized
pathogens (those that have entered tissues beneath the fruit surface) cause lesions that
begin inside the fruit. Internal bruises may occur during harvest, and certain fungi can
colonize the damaged tissues, producing an internal black rot.
Green tomatoes are normally resistant to sour rot caused by Geotrichum
candidum. However, if green fruit have been chill injured or are congested with water,
sour rot will develop and produce a watery decay often associated with wet boxes.
Once harvested, fruits and vegetables have a limited postharvest life. They no longer
receive water or nutrition from the plant. Naturally occurring senescence in produce
leads to a softening of the tissues and often a loss of preformed antimicrobial
substances. These changes in the fruit or vegetable also make it less desirable to
consumers. This correlation between senescence, susceptibility to decay and loss of
edible quality has a great impact on decay control methods. Therefore, handling
methods that preserve the freshly harvested quality of the crop, such as cooling, are
likely to minimize the development of decay. Pathogens are present in all production
areas and are most numerous when the weather becomes warm and wet. Movement of
weather fronts or tropical storms through production areas can also affect the
susceptibility of tomato fruit to decay (Kwon, 2001).
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The relative perishability and storage shelf life of fresh tomato produce is less
than two (2) weeks comparable to strawberry asparagus broccoli and lettuce (Kader,
1978).
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Chapter 3
Methodology and Research Design
This chapter deals with the experimental methods of research used. The techniques
used under experimental research method as well as the data gathering tools and
analytical tools used will be further explained in this chapter.
3.1 Research Design
The proponent used the experimental research method which involves: In vitro and
In Vivo Screening of the alum suflate against Rhizopus stolonifer. Data gathering,
organizing, tabulating, depicting and analyzing using the One Way Analysis of Variance
and Spearman Product Moment Correlation for homogeneity of data.
The In Vitro was carried out in five (5) treatments [T1
as negative control, water; T2
1.0 mg/L alum; T3 2.0mg/L alum; T4 3.0 mg/L alum and T5 baking powder] and each
treatment is replicated (3) three times.
The In Vivo was carried out using a selected tomatoes procured from the Public
market of General Santos City. The selection was made in order to experiment on similar
size, color, texture and age of tomatoes under investigation. The same level of
concentration of alum was employed in the In Vivo Screening.
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Table 1.0 Experimental Design
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg./L Alum2.0 mg./L Alum
3.0 mg./L Alum
Baking Power
Table 1.0 represents the distribution of petri disc containing the agar impregnated
with Rhizopus stolonifer for growth inhibition protocol by alum sulfate.
The same arrangement will be followed in the experimental design for the In -
Vivo investigation.
Data Gathering:
In Vitro Screening data will be gathered and recorded 24 hours from the conduct of
the experimentation. Data such as the minimum zone of inhibition will be measured using
the micrometer caliper.
The In Vivo Screening data will be gathered and collected on the span of nine (9)
days or until the tomatoes under study are decomposing due to Rhizopus stolonifer
invasion. Data to be collected included the following, such as color, texture, and water loss.
3.2 Materials and Equipments
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Aluminum sulfate
Commercially available aluminum sulfate is procured from the supermarket at a
lower price.
Tomatoes
Commercially grown tomatoes (Lycopersicon esculentum Mill.) at the red colour
stage used in this work were obtained from Public market of General Santos City at
P15.00 per kilo. Tomatoes were selected to have uniform size, color, and texture.
Tomatoes with apparent injuries were removed. Before treatments, tomatoes were
immersed in 70% ethanol for 1 min, washed with ionized water, and then air dried.
Cultures
Rhizopus stolonifer was procured from Notre Dame of Dadiangas University,
Integrated Basic Education Department. R. stolonifer was incubated on Malt Extract
Agar for 810 days at 25C.
Preparation of inoculum
Spores suspensions were obtained from mycelium grown on MEA medium at
25C for 14 days and were collected by flooding the surface of the plates with ~5 ml
sterile saline solution (NaCl, 8.5 g/l water) containing Tween 80 (0.1% v/v). After
counting the spores using a haemocytometer, the suspension was standardized to
concentrations of 107 spore/ml by dilution with sterile water before using. The viability of
all strains checked using quantitative colony counts were at 10 7 CFU/ml. (Burton, 2007)
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Preparation of the Alum concentration
Alum concentrate is prepared by mass/volume concentration ratio.
3.3 Anti-fungal Screening
In vitro test
The agar dilution method was employed. Each concentration of alum sulfate at
1.0mg/L, 2.0mg/L, and 3.0mg/L, with water and baking powder as negative and
positive control respectively. Three replicates were prepared for each treatment. The
impregnated plates were then incubated at 25 C for 3 days in an incubator. The zone
of inhibition is then gathered and recorded to statistical analysis.
In vivo test
Fruit were dipped for 20 min in the respective alum sulfate concentrations of 1.0mg/L,
2.0mg/L, 3.0mg/L in distilled water. After drying, they were evenly sprayed with a spore
suspension ofR. stoloniferand held at 25 C for 9 days. Each treatment was replicated
three times with 10 fruits per replicate. Following incubation, tomatoes were individually
rated for mould growth on a scale of 0 to 5, with 0 denoting clean specimens and 5
representing heavy mould growth (0=clean, 1=20%, 2=40%, 3=60%, 4=80%, 5=100%
of mould growth). (Matan, 2011)
2.6 Color Measurement
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Inset photo is taken from California Tomato Commission. This is the Standard for
measuring the acceptability of importing tomatoes in California.
Skin colour values of each of tomatoes at 0th and 9th day were measured
according to the Color Chart from California Tomato Commission. Corresponding color
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changed for nine (9) days will be recorded. Extended observation may occur until the
onset of Rhizopus stolonifer infestation become visible and tomatoes will undergo
deterioration. Analysis of Variance will be used to determine the significant difference in
the mean of tomato deterioration.
2.7 Textural analysis
Tomatoes firmness was assessed before and after about 9 days of storage at
25C using a nondestructive tests. The Modified penetrometer deformation apparatus is
constructed. A 500-g weight was applied (radially) for a constant time period and
deformation was measured (mm) for a single point.
Instron axial (vertical) deformation. Fruits were subjected to axial compression of
an initial pre-load of 0.1 kg and subsequently to a total load of 1.0 kg. Resultant delta
deformation was measured in millimeters. One data point per fruit was taken. (This
variation was included because historically most whole fruit firmness studies have been
in axial mode.)
A custom brace to gently hold tomatoes in a radial position was designed and the
Instron method employed a flat disk probe, 3 in. in diameter, and a 2 kg load cell. The
diameter at each position was measured and recorded prior to compression. The
crosshead moved at a constant speed of 20 mm/min. Spearmans Rho, which is a
measure of linear association between ranks of variables, similar to the linear
regression for parametric statistics, was utilized for analysis of the data. (Matan, 2007)
Twenty-five replicates were taken per sample. Firmness values were obtained
from the maximum peak of the first compression.
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Table ____. Relationship between Subjective Firmness Measurements for WholeTomato Fruit Firmness
2.8 Statistical analyses
All variables were tested for normality applying the Spearmans Rho for
homogeneity. Data transformation was done, where necessary. All results were
expressed as mean standard deviation SD. The data was statistically treated by one-
way ANOVA and Tukey post hoc test with p < 0.05 was considered to be statistically
significant.
Chapter IV
Results and Discussion
This chapter presents the data collected during the course of the investigation as
well as the appropriate statistical used in the analyses of the collected data. Data were
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presented in tabular form with corresponding graphical presentation for visual
comprehension.
There were two sets of data collected and analyzed. The In Vitro and the In vivo
data as observed during the investigation. Statistical tool used were One Way Analysis
of Variance and Spearman Rho for correlation of the nonparametric In Vivo observation.
4.1 In vitro Screening
Antifungal Screening of the growth inhibitory effect of Rhizopus stolonifer using
potassium aluminum sulfate in different concentration level. The test is carried out in
petri disc using agar diffusion method.
Table xx: Growth Inhibition of Rhizopus stolonifer by Potassium Aluminum Sulfate
TreatmentReplication
Total Mean1 2 3
Water
0.5 mg/L
1.0mg/L1.5mg/L
Baking Powder
Total
ANOVA
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In Vivo Screening Result
A. Mould Growth Inhibition of Rhizopus stolonifer
Table ____. Radius ofRhizopus stoloniferGrowth (mm)
Treatment
Replication
Total
MeanIndex
1 2 3
Water
0.5 mg/L
1.0mg/L
1.5mg/L
Baking Powder
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B. Color Change
Table _____ Tomato Fruit Color Index Day 1
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
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Table _____ Tomato Fruit Color Index Day 3
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
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Table _____ Tomato Fruit Color Index Day 5
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
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Table _____ Tomato Fruit Color Index Day 7
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
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Table _____ Tomato Fruit Color Index Day 9
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
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Table _____ Tomato Fruit Color Index Day 11
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
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C. Firmness Index Using Modified Penetrometer Deformation Apparatus
Table _____ Tomato Fruit Color Index Day 1
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
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Table _____ Tomato Fruit Color Index Day 11
TreatmentsReplication
Total Mean1 2 3
Water
1.0 mg/L
2.0 mg/L
3.0 mg/L
BakingSoda
Spearmans Rho
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Fungal growth inhibition of citronella oil against Rhizopus stoloniferare shown in
Table 1. The MIC of citronella oil against R. stoloniferon agar was 5L/mL. Fungal
resistance of tomatoes dip-treated with various concentrations of citronella oil against
R. stoloniferis shown in Figures 1 and 2. The results are presented as the average
ratings of 25 five specimens. It is clear that citronella oil was not active against R.
stoloniferon tomatoes at the concentration less than 5 L/mL. Above that concentration,
antifungal activity of citronella oil gradually increased with increasing concentration of
the oil. A complete protection of tomatoes from R. stoloniferfor up to 9 days was
achieved at the concentration of citronella oil at 20 L/mL. It should be noted that
antifungal activity of citronella oil should be inherently arisen from components within
the oil rather than from the moisture exclusion effect since the control stakes dip-treated
with vegetable oil showed 100% mold coverage.
Chapter 5
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SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATION
This chapter presents the summary of findings and conclusions made from the study and
recommendations given by the researcher.
5.1 Summary of Findings
The water reservoirs we have these days are being contaminated with many different
chemicals from either industrial wastes and it may be naturally occurring. These chemicals can
therefore be harmful to living things. This study was conducted to test if Sphagnum moss
(Sphagnum flexuosum) can absorb the heavy metals used in this project.
In conducting this study, the proponents collected some Sphagnum moss and placed it
equally upon containers with contaminated waters with Copper and Lead with 3 replications
each. After an observation of 7 days, the water in the contaminated water changed and can be
concluded that there was an effect. To prove this, water in the containers was removed and
placed separate containers to be studied. It was discovered and confirmed that the moss had an
effect and greatly reduced the amount of contaminants that was originally present in the water.
Though no study was conducted to test if the heavy metals were transferred to the
moss, it can be already directly concluded that the moss absorbed it because there could not be
another explanation to how the heavy metals were removed from water.
5.2 Conclusions
Based on the experimentation performed, results and information drawn together, the
researcher was able to formulate the following conclusions:
http://en.wikipedia.org/w/index.php?title=Sphagnum_flexuosum&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Sphagnum_flexuosum&action=edit&redlink=1 -
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5.2.1. Sphagnum moss was proven to remove copper and lead in water.
5.2.2. Sphagnum moss was effective in adsorbing copper and lead content in water
5.2.3. The proponents determined the change in amount of copper and lead when using
Sphagnum moss from the color change of the contaminated water in all replications.
5.3 Recommendations
From the findings of the study, it can be observed that there were research gaps. Results
can be improved and further benefits can be disclosed with the following recommendations:
5.3.1. People shall cultivate Sphagnum moss as a plant in the surroundings to ensure that
water surrounding them shall be free from chemicals such as copper, and lead.
5.3.2. Further research will be conducted to determine the amount of the said chemicals
absorbed day by day in the experimentation.
5.3.3. Another study shall be conducted using another kind of moss to test the effects on
the said chemicals.
5.3.4. Another experimentation shall be conducted with other contaminants.
5.3.5. The government will conduct the study to improve the research.
5.3.6. A further study shall be conducted to prove that the moss absorbed the
Heavy metals
BIBLIOGRAPHY
Books
Burton and Engelkirk,. 2007. Burtons Microbiology for the Health Science
Siegel, S. 1998. Nonparametric Statistics for Behavioral Sciences
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Journals
Bartz, J.A, Steven, R.A and Mahovic, M. Guide t0 Identifying and ControllingPostharvest Tomato Diseases in Florida, J, p.23
Barrett, D. M.et all. 1998.Textural Modification of Processing Tomatoes, 185 190
Kader, A. A. 1978, American Society of Horticultural Science Vol. 103, p.101
Kwon, J.H, et all. 2001. Rhizopus Soft Rot on Cherry Tomato by Rhizopus stolonifer inKorea, Mycobiology 176-178.
Matan, N, W., 2011. Postharvest Control of Rhizopus stolonifer on Tomato by CitronellaOil, Thailand. p 23
Radzevicius, A. et all. 2012. Tomato Ripeness Influence on Fruit Quality, World
Academy of Science and Technology Vol. 64 J, 653
Turkhan, H.A et all. 2010. The Effect of Head Rot Disease (Rhizopus stolonifer)On Sunflower Genotype at Two Different Growth Stages, Turkish Journal offiled Crops Vol. 15(1), p. 94
www.lagorio.com, California Tomato Commission
http://www.lagorio.com/http://www.lagorio.com/ -
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