Effectiveness of Wood Vinegar (Pyroligneous Acid) Against Fusarium Proliferatum
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INTRODUCTION
Rationale of the Study
Fusarium proliferatum is known as the conidial stage of Giberellafujikuroi, which is an ascomycetous seed-borne fungus that causes bakanae, a disease that influences rice (Nelson, 1994). There are over 100 species of the genus, Fusarium, and they are most common in tropical and subtropical areas. The presence of pathogenic conidia may cause the discoloration in kernels.
F. proliferatum is a filamentous fungus or mould found in aerobiologic surveys worldwide. It is a major parasite of rice, sugar cane, sorghum, bean, soybean, and is especially common on maize grains (Pitt, et al. 1994).
According to (De Leon and Pandey 1989; King and Scott 1981; Ochor et al. 1987), F. proliferatum causes a disease called Fusarium kernel rot. In parts of the USA and lowlands tropics, this is one of the most important ear diseases and is associated with warm, dry years and insect damaged fields.
Czembor, et al. (2015) stated that maize is one of the most important crops in Poland. Diseases caused by Fusarium spp. can affect the yield and grain quality of maize because of contamination with numerous mycotoxins produced by these fungi. Similar results were obtained from 148 samples of maize from the Philippines, and 82 samples of maize from Indonesia. F. proliferatum persisted in high numbers in stored maize from the both countries (Pitt, et al.,1993).
(Zainudinet al., 2008b; NurIzzati and Salleh, 2009), stated that F. proliferatum have been isolated and found to be associated with bakanae disease in Malaysian rice varieties.
Measures were taken to control the disease such as the chlorothalonil which is a commonly used fungicide in rural and urban environments that can be applied through direct overspray and drift from aerial applications especially on those plants that are affected by fungal diseases ( Sherrard et al. 2003). Yet, chlorothalonil is a little bit toxic to mammals, because it can cause eye and skin irritation in certain formulations. (Walker, M. M. and L. H. Keith. 1992).
On the other hand, wood vinegar, a liquid, which is a by-product of burning fresh wood in an airless container, contains methanol, acetic acid, acetone, fractural and related products miscible with water and alcohol. It is widely used in agricultural crop production toward plant growth stimulation, germination, soil disinfection and the control of weed, disease and pest (Kim et al. 2011; Agricultural Chemistry Group, Agricultural Production Sciences Research and Development Office
Department of Agriculture, 1998;; Hawley, 1981).
Anchored on the mentioned studies, the researchers are compelled to investigate the effectiveness of Pyroligneous Acid (wood vinegar) against Fusarium proliferatum.
Conceptual Framework
Dependent variable
* Fungal Growth Inhibition in Fusarium proliferatum treated with Commercial fungicide, Distilled water , 10%, 50%, 100% concentrations of Wood Vinegar Extracts * Change in mm of the zone of inhibition
Independent variable
* Different Concentrations of Wood Vinegar Extracts (10%, 50% and 100%) * Types of Treatments (Commercial fungicide, Distilled water , 10%, 50%, 100% concentrations of Wood Vinegar Extracts) * Varying Observation Time (6th, 12th, 18th, and 24th hour)
Fig 1.Paradigm on the effect of the different concentrations of Wood Vinegar Extracts, Types of treatments and the Varying Timezones in the growth of Fusarium proliferatum
Figure 1 exhibits the possible effect of the different concentrations of Wood Vinegar Extracts, types of treatments and the varying time zones in the growth of Fusarium proliferatum. The inputs in the study or the independent variables are the different concentrations of Wood Vinegar Extracts (10%, 50% and 100%), Types of Treatments (Positive Control, Negative Control, 10%, 50%, 100% concentrations of Wood Vinegar Extracts) and Varying Observation Timezones (6th, 12th, 18th and 24th hour). The independent variables mentioned above were tested to Fusarium proliferatum to determine their effect in terms of the inhibition of the fungal growth as observed in the change of the size in mm of the mentioned pathogen.
Statement of the Problem
This study aimed to investigate the effectiveness of wood vinegar against
Fusarium proliferatum.
It specifically tried to answer the following questions:
1. Which of the following treatments has the longest diameter in the zone of inhibition:
1.1 10% of wood vinegar only
1.2 50% of wood vinegar only
1.3 100% of wood vinegar only
1.4 Commercial Fungicide (positive control)
1.5 Distilled Water (negative control)
2. What are the effects of the various treatments, (10% of wood vinegar, 50% of wood vinegar, 100% of wood vinegar, Commercial Fungicide, and distilled water) in the diameter of the zone of inhibition?
3. How effective is the wood vinegar in inhibiting the Fusarium proliferatum in:
3.1 6 hours
3.2 12 hours
3.3 18 hours
3.4 24 hours
4. Which experimental treatment is comparable to the commercial fungicide in terms of size of the diameter of the zone of inhibition?
Statement of Hypotheses
The following hypotheses were tested at α= 0.01 level of significance in this study.
Ho1: The various treatments have no significant difference in the diameter of the zone of inhibition of Fusarium proliferatum.
Ho2: The diverse treatments have no significant effect in the diameter of the zone of inhibition.
Ho3: The hours of exposure has no significant difference in inhibiting the growth of Fusarium proliferatum.
Ho4: The experimental treatments with the highest zone of inhibition have no significant difference to the commercial fungicide in the size of the diameter of the zone of inhibition.
Significance of the Study
Due to the spread of rice problems taken by these fungal infections (Fusarium proliferatum), the rapid multiplication of these fungi and the lack of information about alternative prevention, the upshot of this work shall bring countless benefits to the:
DEPARTMENT OF AGRICULTURE. The said government agency can recommend and endorse the product to the farmers to use the wood vinegar as a fungicide.
SUPERMARKET. The study would help the supermarket to ensure a good sell of rice which is free from P. proliferatum.
FARMER. The result of this study can help to farmers to use the alternative fungicide to fight the epidemic of rice blast that could cause the destruction of crops which eventually can decrease their harvest.
CONSUMER. The consumers would be benefitted because they are eating rice which are safe.
RESEARCHERS. The study is essential to the researchers for it gives them a chance to undergo hands on learning. They are able to experience how the theories in science are put into practice. Moreover, it hones their mathematical abilities when they statistically treat the gathered data.
FUTURE RESEARCHERS-Future researchers can refer to this study on the proper handling of fungi.
Scope and Limitations of the Study
The investigation was directed to determine the effectiveness of Wood Vinegar as an antifungal against Fusarim proliferatum. The researchers looked into the ability of Wood Vinegar to inhibit the growth of fungi. The positive control used was limited to Commercial fungicide, while the negative control used was distilled water since it was the solvent used in diluting the wood vinegar at 10%, 50% and 100% concentration of wood vinegar. Potato Dextrose Agar was used as growth media for Fusarium proliferatum for better dispersion of the treatments utilized in the study. The observation time was limited only at 6th, 12th, 18th and 24th hours. This study was limited on the use of 0.5 McFarland Standard as a quality control program for adjusting densities of fungal suspensions that were used for identification and susceptibility testing.
Definition of Terms
For the purpose of this study and to facilitate the comprehension of this work, the terms hereafter mentioned are hereby defined.
Aerate. It refers to putting air or a gas into something, such as soil or a liquid.(http://www.merriam-webster.com/dictionary/aerate)
Advancing margin. It refers to the advanced edge or border of the rice grain. (http://www.oxforddictionaries.com/us/definition/american_english/advancingmargin)
Agar.A gelatinous material derived from certain marine algae. It is used as a base for bacterial culture media and as a stabilizer and thickener in many food products. (http://www.thefreedictionary.com/agar)
Concentration.The strength of a solution, especially the amount of dissolved substance in a given volume of solvent, usually expressed in percentages. (http://www.thefreedictionary.com/concentration)
Confounding Variables.An extraneous variable whose presence affects the variables being studied so that the results you get do not reflect the actual relationship between the variables under investigation. (http://www.alleydog.com/glossary/definition.php?term=Confounding%20Variable)
Conidia.It issometimes termed asexual chlamydospores, or chlamydoconidia are asexual, non-motile spores of a fungus, from the Greek word for dust, konis..
Diameter.is defined as the width of a circle or cylindrical object. In this study it refers to the change in the colony of the bacteria. (http://www.thefreedictionary.com/diameter)
Eradicate. It means to destroy completely or put an end to.(http://www.oxforddictionaries.com/us/definition/american_english/eradicate)
Filamentous. It is a long thin cellular structure characteristic of many fungi, usually having multiple nuclei and often divided by septa.(http://www.thefreedictionary.com/filamentous+body)
Fumonism.A fumonisin is a mycotoxin derived from Fusarium, Liseola section (Medical Subject Headings, Karolinska Institute, 13 April 2010).
FUM cluster. It refers to a group of genes exhibiting a common expression pattern during fumonisin biosynthesis (Stepien et al., 2010).
Inoculation.It is the act of inoculating or of being inoculated; vaccination (http://www.merriam-webster.com/dictionary/inoculation)
McFarland Standard. In this study, McFarland Equivalence Standards are intended to be part of a quality control program for adjusting densities of fungal suspensions instead of bacteria that are used for identification and susceptibility testing. (http://www.pro-lab.com/inserts/McFarland.pdf)
Morphology.It is a branch of biology dealing with the study of the form and structure of organisms and their specific structural features. (http://www.askoxford.com.)
Mycotoxin.It is a toxic secondary metabolite produced by organisms of the fungi kingdom, commonly known as molds. (Richard, JL 2007)
Pathogenicity.It refers to the ability of an organism to cause disease (i.e., harm the host). (http://www.tulane.edu/~wiser/protozoology/notes/Path.html)
Polymorphism.It refers to more than one form of an organism which might be due to mutation. (Ford E.B. 1965)
Potato Dextrose Agar. It is a general purpose medium made up of dehydrated potato infusion and dextrose that serves as a growth medium of fungi. The agar is added as a solidifying agent. (United StatesPharmacopeial Convention (2007)
Seed Rot. It refers to the disease caused by F. proliferatum. (http://www.ipm.ucdavis.edu/PMG/r682100111.html)
Teleomorph.It is the final, sexual reproductive form in the life cycle of any fungus of the Division Ascomycota and Basidiomycota. (http://www.yourdictionary.com/teleomorph)
Virulence.It refers to the degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. (Medical Subject Headings, Karolinska Institute, 13 April 2010)
Zone of inhibition.In this study, it refers to an area around where the fungi have not grown enough to be visible (http://medical dictionary.thefreedictionary.com/zone+of+inhibition)
REVIEW OF RELATED LITERATURE AND STUDIES
Related Literature Stępień et al. (2011) found that Fusarium proliferatum (Matsushima) Nirenberg is a common pathogen occurring in various climatic zones infecting numerous crop plants. Fumonisin biosynthesis is determined by the presence and activity of the FUM cluster, several co-regulated genes with a common expression pattern. In the present work, they analyzed 38 F. proliferatum isolates from different host plant species, through phylogenic analysis based on FUM1 and tef-1a sequences. They also studied growth rates across different temperatures and sample origin and tried to establish the relationships between DNA sequence polymorphism and toxigenic potential. The results indicated the greatest variations of both toxigenic potential and growth patterns found across the wide selection of isolates derived from maize. A weak negative rank correlation between fumonisin content and isolate growth rates was observed. Interestingly, isolates with the fastest growth patterns were also grouped together and included both isolates originating from rice. The sequencing of elongation factor (Ef1-a) gene from two isolates was used to confirm the identity of species specifically correlated with the host plant.
Díaz Arias, et al. (2011) stated that Fusarium spp. are widespread soil borne pathogens that cause important soybean diseases and sudden death syndrome. In collaboration with 12 Iowa State University extension specialists, 12 species of F.proliferatum have been associated with soybean roots were arbitrarily sampled from three fields in each of 98 Iowa counties from 2007 to 2009. Symptomatic and asymptomatic root pieces were sterilized. The colonies were transferred to carnation leaf agar (CLA) and potato dextrose agar and later identified to species based on cultural and morphological characteristics. Of the 1,230 Fusarium isolates identified, 50 were recognized as F. proliferatum based on morphological characteristics. F. proliferatum isolates produced abundant, aerial, white mycelium and a violet-to-dark purple pigmentation. On CLA, microconidia were abundant, single celled, oval, and in chains on monophialides and polyphialides. Fusarium crown and root rot is a destructive disease of asparagus that are caused by the agents, F.proliferatum and F.oxysporum. To complete Koch's postulates, (4) four F. proliferatum isolates were tested for pathogenicity on soybean in a greenhouse. Infected plants showed dark brown lesions in the root system as well as decay of the entire taproot. F. proliferatum was reisolated from symptomatic root tissue of infected plants but not from similar tissues of control plants.
Related Studies
Mule et al. (2003) found that the Fusarium crown and root rot is a destructive disease of asparagus that are caused by the agents, F. proliferatum and F. oxysporum. Polymerase chain reaction (PCR) assays were developed for the rapid identification of pathogens.
Hawksworth et al. (1995) stated that conidia were produced a million times from F.proliferatum with conidiophores and microconidia that germinate in suitable substrates and is favorable in the temperature of 25-31̊C.
Nelson,et al. (1983) stated that both young and older shoots are prone to infection and raid disease development that may cause the leaf, sheath, shoot and flower rots. Wind, splashing water, insects, snails, and slugs are the main reasons of the movement of spores. Fusarium survives for months and will contaminate greenhouse walls, benches, and floors, as long as it is present.
Uchida, et al. (1966) found that watering in the early evening is not advisable as it caused a long period of wetness during the night. They suggested that plants be watered at day time so that the heat from the sun keeps the leaves dry. In effect, the growth of the fungi can be prevented.
Quazi et al. (2013), F. proliferatum is one of the pathogens responsible for bakanae disease of rice. It is a pathogen which caused in the decrease of plant height (%), decrease in main root length (%) and decrease in lateral roots number (%) of inoculated plants compared to control plants.
RESEARCH METHODOLOGY
This section includes the design and procedure of the study. Research Design
The Randomized Complete Block Design was utilized in this study, wherein the control setups included the Positive (Commercial Solution) and Negative (Distilled Water) while the experimental setups were those which were treated with different concentrations of wood vinegar extracts (10%, 50%, and 100%). The dependent variable of this study was the change in mm of the zone of inhibition while the independent variables were the types of treatments and the varying time of exposure of the pathogen to the treatments. Below are the controlled variables in each treatment.
Table 1.Controlled Variables in Each Treatment Treatments | T1= Treated with + Control (Commercial Fungicide) | T2= Treated with - Control (Distilled Water) | T3= Treated with 10% Wood Vinegar Extracts | T4= Treated with 50% Wood Vinegar Extracts | T5= Treated with 100% Wood Vinegar Extracts | Kind Of Agar Used | Potato Dextrose Agar | Potato Dextrose Agar | Potato Dextrose Agar | Potato Dextrose Agar | Potato Dextrose Agar | Type of Fungus Used | FusariumProliferatum | FusariumProliferatm | FusariumProliferatum | FusariumProliferatum | FusariumProliferatum | Incubator TemperaTure | 370C (98.60F) | 370C (98.60F) | 370C (98.60F) | 370C (98.60F) | 370C (98.60F) | Storage | Properly Sealed | Properly Sealed | Properly Sealed | Properly Sealed | Properly Sealed | Filter Paper Disk (Size) | Puncher Size | Puncher Size | Puncher Size | Puncher Size | Puncher Size | InoculaTion | 0.5McFarland Standard | 0.5McFarland Standard | 0.5McFarland Standard | 0.5McFarland Standard | 0.5McFarland Standard |
Research Procedure
A. Collection of Fusarium proliferatum Rice grains, leaves and leaf sheath were collected from the rice field in Barangay Pulangbato, Cebu City. They were placed together in a 10”x 8” resealable and air tight plastic to ensure that no other pathogen can contaminate. Two cotton balls which were moistened with distilled water were also placed inside the resealable and air tight plastic to keep the fungi alive.
B. Isolation of the Pathogen
The above mentioned parts of the rice were brought to the Department of Agriculture, Research Field Office 7 Laboratory, in Maguikay, Mandaue City
In the lab, they were sorted. The rice grains, leaves, and leaf sheath were placed separately. They were examined for signs of the disease. The collected diseased rice specimen was cut using a sterile scalpel at the advancing margin with a size of 1mm leaf tissue. The surface of the cut leaf tissues was sterilized with the prepared 1% Sodium hypochlorite for 1 minute and washed three times with distilled water. The sterilized surface leaf tissues were placed on top of the sterile tissue paper so that any amount of distilled water would be absorbed completely.
The specimens were placed in 3 Petri dishes with plated PDA for growth. The said Petri dishes were sealed with parafilm.
They were set aside to allow the growth of the fungi. The fungal growth was transferred to an agar slant for pure culture. Samples were taken from the agar slant and were dyed with methylene blue. Once dyed, each of them was mounted on a compound light microscope. Pure cultures which were positive of F.proliferatum were then placed into previously plated PDA and were allowed to grow for about two weeks. The said petri dishes were sealed again with parafilm to prevent contamination (Libato et. al, 2015).
C. Preparation of treatments The wood vinegar extracts were diluted into 10% and 50% concentrations with distilled water. For 10% concentration, 4.5ml of wood vinegar extract was added to 0.5ml distilled water to come up with 5mL solution. It was transferred in a sterilized 50 mL beaker and was labeled. For the 50% concentration, 2.5mL of wood vinegar extracts was measured in a graduated cylinder and was added to 2.5ml distilled water to come up with 5mL solution. It was then transferred to the second sterilized 50mL beaker and was labeled. The 100% concentration of wood vinegar was taken from the stock solution which was provided for by DA RFO 7. Five (5) mL of the wood vinegar extract was measured in a graduated cylinder. It was also transferred in the third 50mL beaker and was labeled. For the positive control preparation, 0.06 grams of commercial fungicide was measured using the digital kitchen scale and was added to 5mL of distilled water. It was transferred in a 50mL beaker and was also labeled. The negative control preparation was done by measuring 5ml. It was transferred into the 5th 50mL beaker and was also labeled. The opening of each beaker was covered with tin foil. The 5 treatments were stored until they were used.
D. Preparation of the Culture Media
The Potato Dextrose Agar (PDA) used as the culture medium for growth of the pathogen was also provided for by the Department of Agriculture Research Field Office 7. It was prepared by dissolving 39 g of PDA in 1 liter of hot distilled water. Fifteen (15) mL was poured in each of the 10 test tubes. Each of them was plugged with cotton and was sterilized for about 15pound per square inch (psi) for 15minutes. The sterilized PDA in tubes was set aside to solidify.
E. Preparation of Fungal Standard Solution Exactly 0.5 McFarland equivalent turbidity standards were prepared. Suspension of Fusarium proliferatum was prepared by aseptically inoculating the sterile saline solution. The inoculated tube was swirled and it was compared to 0.5 McFarland Standard tube against a black and white line. The sample of F.proliferatum was transferred to the test tube containing 10ml of distilled water. Then, it was transferred in a 30ml PDA in Erlenmeyer flask and it was incubated for 24 hours to produce the active strain.
F. Impregnation of Filter Paper Disk The filter paper discs were used in this study. The previously prepared three concentrations of wood vinegar extracts, the positive control (Commercial fungicide) and the negative control (distilled water) were taken for investigation. Then, 50 pieces of punched filter paper were produced. The ten (10) discs were soaked into each desired different treatments for 12 hours inside the chiller. After the 12th h , the soaked filter paper discs were removed from each treatment and placed into the designated area in the Petri dish.
G. Application of Discs on PDA Using a pen, the dishes were divided into 5 and labeled into I- (+ ctrl), II- (-ctrl), III- (10%), IV-(50%), V- (100%). The forceps were soaked first in 80% ethanol solutions and passed onto the flame to kill microorganisms. It was left to cool for 5 seconds. With the sterilized forceps, the discs were taken from each treatment and placed into its designated areas in the previously inoculated plates. Each of them was pressed gently until it was in full contact with the PDA surface. It was placed equidistant from each for at least 10mm apart and the opening of the plate should at 450 to avoid contamination.
H. Incubation of F. proliferatum with the treatments After the application of discs, the 9 plates were incubated 24 hours at 37.5oC (98.6OF) before observations and measurements were taken.
I. Data Gathering Technique The diameter of the zone of inhibition around each of the puncher size filter paper which was soaked for each treatment, 10% wood vinegar, 50% wood vinegar, 100% wood vinegar, commercial fungicide (+ control) and the distilled water (- control) during the time of exposure was measured with a ruler and was recorded separately after the 6th, 12th, 18th and 24th h. The zone of inhibition was observed to be colorless and clear not hazy. The method used to test the wood vinegar to F. proliferatum was the filter paper disc method.
J. Disposal of PDA of F.proliferatum After observation and measurements were taken, dishes were secured in a plastic autoclave bag and were sterilized for 15 minutes in the autoclave for 37o to kill the F. proliferatum to prevent any accidental contact with the host plant. After sterilizing, the PDA from each Petri plates was removed and was thrown in a laboratory wastebasket. Disinfectant liquid soap was used for the Petri plates. They were rinsed with water and were left to drip dry before wiping each of them with a clean laboratory cloth.
K. Statistical Treatment
The data for every treatment in each replicate of the filter paper disc method were recorded, statistically treated, analyzed and interpreted. The mean for each treatment and exposure were computed. The variance was determined through the use of two-way ANOVA with repeated measures since the inhibition of F. proliferatum was observed with five (5) treatments at different time and that RCBD was used as its research design.
RESULTS AND DISCUSSION
To achieve the objectives of this investigatory project, a laboratory experiment was conducted in the Department of Agriculture RFO 7 in Maguikay, Mandaue City and the University of San Carlos (USC)-Talamban Campus. The subsequent findings were presented under headings that coincide to the various points of the problem.
Table 2
Average zone of inhibition (mm) of Fusarium proliferatum in each treatment (Trials 1-3) in Different Time Time of ExposureTreatments | Zone of inhibition (mm) | | 6th Hour | 12th Hour | 18th Hour | 24th Hour | | T1 | T2 | T3 | T1 | T2 | T3 | T1 | T2 | T3 | T1 | T2 | T3 | Positive Control (Commercial Fungicide) | 9 | 8.17 | 8.33 | 15.17 | 15 | 12.83 | 15.17 | 20.17 | 13.17 | 15.5 | 23.33 | 14. | Negative Control (Distilled Water) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 10% Wood Vinegar Extract | 0 | 0 | 0 | 2.33 | 0 | 3.5 | 2.67 | 0 | 3.67 | 2.67 | 0 | 3.83 | 50% Wood Vinegar Extract | 0 | 0 | 0 | 2.33 | 0 | 0 | 2.33 | 0 | 2.33 | 2.83 | 0 | 2.33 | 100 % Wood Vinegar Extract | 0 | 0 | 0 | 0 | 2.33 | 7 | 0 | 2.5 | 7.33 | 0 | 2.67 | 7.67 |
Table 2 above shows that the highest average zone of inhibition was obtained by the positive control for it has been proven and tested to have an antifungal remedy while the negative control had no zone of inhibition for it was proven that water has no antifungal effect on Fusarium proliferatum.However, from the different concentrations of the experimental setups the highest average zone of inhibition was attained by the 100% concentration of wood vinegar while the least average zone of inhibition was obtained by the 50% concentration of Wood Vinegar. Studies had shown that the acid on wood vinegar have inhibitory effect on Fusarium proliferatum fungus. Furthermore, the data also showed that longer the fungi were exposed to the treatments, the bigger the zone of inhibition.
Table 3 Sources of Variation | Sum of Squares | Degree of Freedom | Mean Square | F-Value | | | | | Computed | Tabular | Interpretation | Treatments | 546.45 | 4 | 136.61 | 84.85 | 5.41 | Significant | Timezones | 151.77 | 3 | 50.59 | 18.33 | 5.95 | Significant | Errors | 19.28 | 12 | 1.61 | | | | Total 698.22 7 |
Significant Mean Squares of the Treatment Made for the Average Zone of Inhibition in
Fusarium proliferatum
Table 3 reveals the Significant Mean Squares of the Treatments made for the Average Zone of Inhibition in Fusarium proliferatum. Since the computed F-value of the treatments, rows and interaction were greater than the F-tabular value, the interpretation would be significant at 1 % level of confidence and the null hypotheses were rejected saying that that the computed F-value (treatment) of 84.85 computed to the F-tabular value of 5.41 with dF of 4 at 0.01 level of significance, the null hypotheses were rejected in favor of the research hypotheses which means that there was a significant difference between the different treatment, varying time zones and the interaction between the treatments and the time zones is present in inhibiting the growth of Fusarium proliferatum. It implied that the Positive Control (Commercial Fungicide) had greater antifungal power compared to the experimental treatments. It also implied that the time of exposure played a vital role in inhibiting the growth of F. proliferatum. The longer the time the fungi were exposed to the positive control and the experimental treatments, the greater was the zone of inhibition.
CONCLUSIONS and RECOMMENDATIONS
Conclusions
Based on the experimental and statistical results the researchers have concluded that:
1. The Commercial Fungicide (+ control) had the longest diameter in the zone of inhibition. However, among the 3 experimental set-ups, the 100% concentration of wood vinegar had the longest diameter in the zone of inhibition.
2. The treatments exhibited some inhibition in the growth of F. proliferatum except for the negative control. The highest average zone of inhibition was observed in the positive control for it is already proven and tested to have an antifungal remedy while the negative control had none for there was no treatment being used .From the different concentrations of the experimental setups , however, the highest average zone of inhibition was attained by the 100% concentration of wood vinegar while the least average zone of inhibition was exhibited by the 50% concentration of wood vinegar.
3. Among the 4 different time of exposure, the 24 hour of exposure of F.proliferatum to the treatments showed the biggest diameter in the zone of inhibition.
4. The 100 % concentration of wood vinegar was the best among the 3 concentrations in stopping the growing of F. proliferatum but it was not comparable to the commercial fungicide. Hence, none of the experimental setups was comparable to the commercial fungicide.
Recommendations Since the study is curative in nature because F. proliferatum was allowed to grow before the application of the filter paper disc method, it is recommended that preventive study should be done. It means that the application of the treatments should be made before the inoculation and incubation of the pathogen in PDA. This is important to determine if the presence of treatment prevents the growth of pathogen. As an application, the farmers can spray the rice before the F. proliferatum can do any damage to the grains. It is further recommended that there should be a field application after the lab tests to determine if the presence of environmental factors such as temperature, wind and rain can affect the ability of the 100% wood vinegar in inhibiting the growth of F.proliferatum. In so doing, the results of the study can be submitted to the Department of Agriculture so that small scale Filipino rice farmers can use the mentioned technology.
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APPENDICES
APPENDIX A1
Transmittal letter for the principal
Republic of the Philippines
Department of Education
TALAMBAN NATIONAL HIGH SCHOOL
Talamban, Cebu City
April 10,2015
DR. MARIA JESSICA N. ABAYON
Principal III
Talamban National High School
Talamban Cebu City
Dear Madame, Good Day! We, the students of Grade 8- Ceniza, are required to make an investigatory project as a requirement for our Research II. We have decided to conduct our study which is entitled,“EFFECTIVENESS OF WOOD VINEGAR (Pyroligneous acid)AGAINST Fusarium proliferatum.” In this connection, we would like to ask your permission to conduct our study in the University of San Carlos- Talamban Campus, Biology Laboratory this summer.
We are hoping for your approval regarding this matter. Thank you and God bless! Sincerely,
JHAEL HAZEL MARIE L. ABUCEJO KIM CARLO L. TUDTUD
Noted by: FLORENCE MARIE M. VILLAR CELIA C. GEPITULAN,M. Ed.
Research Teacher Approved by:
MA. JESSICA N. ABAYON Principal III.
APPENDIX A2
Transmittal Letter for the Biology Department
Talamban National High School
Talamban, Cebu City
April 15,2015
DR. JULIE B. OTADOY
Department Head
Biology Department
University of San Carlos-Talamban Campus
Nasipit, Talamban Cebu City
Dear Madam:
Good Day!
We, the Grade 8 researchers of Talamban National High School, would like to conduct our study entitled, “EFFECTIVENESS OF WOOD VINEGAR (Pyroligneous acid) AGAINST Fusarium proliferatum.”It is the purpose of this study to test the effectiveness of Wood Vinegar on the said fungi. Yet, our school does not have the necessary facilities and apparatuses needed to perform the experiment.
In this connection, we would like to ask your permission for the use of facilities of the Biology Laboratory for the experimentation for 1 month.
We are willing to pay for the services, lab equipments an chemicals which we will be using. Attached herewith is our research methodology for your perusal.
We are hoping for your approval regarding this matter. Thank you and God bless!
Noted: Respectfully yours, CELIA C. GEPITULAN, M.Ed. Research Teacher JHAEL HAZEL MARIE L. ABUCEJO
MA. JESSICA N. ABAYON, Ed. D. KIM CARLO L. TUDTUD
Principal III FLORENCE MARIE M. VILLAR
Approved:
JULIE B. OTADOY
Department head of Biology Department
APPENDIX A3
Transmittal letter for the Department of Agriculture, Regional Field Office 7
TALAMBAN NATIONAL HIGH SCHOOL
Talamban, Cebu City
April 10, 2015
MS. ANGEL C. ENRIQUEZ, CESO IV
OIC, Regional Executive Director
Department of Agriculture, Regional Field Office 7
Maguikay, Mandaue City Cebu
Attention: BIENVENIDO D. ACABAL JR. (Chief,RCPC)
Dear Miss Enriquez,
Good Day!
We, the undersigned Grade 8 researchers from Talamban National High School under the Special Science Curriculum, are required to make investigatory projects. Since our staple food is rice, we tried to determine the diseases which may lower its harvest. We found that Fusarium proliferatum can cause the rotting of rice grain. Thus, we decided to study it with the use of wood vinegar (Pyroligneos acid) as an experimental setup at 10%, 50% and 100%. We will also need PDA as growth medium of the mentioned pathogen.
Since your good office has an experiment station, we would like to ask for assistance on the isolation of the mentioned fungi and the availability of the wood vinegar. Moreover, we would like to also seek permission from your good office that we may be allowed to perform the necessary procedures guided by the lab technician.
We are willing to pay for the services and the materials which we will use.
Your approval on this matter can help a lot on our study and can hone our skills in research as beginners
Very respectfully yours, Approved:
JHAEL HAZEL MARIE L. ABUCEJO
KIM CARLO TUDTUD ANGEL C. ENRIQUEZ, CESO IV
FLORENCE MARIE M. VILLAR OIC-Regional Executive Director
Noted:
CELIA C. GEPITULAN, M. Ed.
Research Teacher
APPENDIX B
Data Results on the Zone of Inhibition of Fusarium proliferatum during 6th, 12th, 18th and 24thhour of Exposure to the Wood Vinegar
Table 5. Data Result on the Zone of Inhibition of Fusarium proliferatum during the 6th hour
APPENDIX C
Hypotheses Testing on the Different Treatments (Positive Control, Negative Control, 10%, 50% and 100% concentrations of Wood Vinegar) and Timezones
Solving by the Stepwise Method: I. Problem:
1. Which of the following treatments has the longest diameter in the zone of inhibition:
1.110% of wood vinegar only
1.250% of wood vinegar only
1.3100% of wood vinegar only
1.4 Commercial Fungicide (positive control)
1.5 Distilled Water (negative control)
2. What are the effects of the various treatments, (Commercial Fungicide, 10% of wood vinegar, 50% of wood vinegar, 100% of wood vinegar, and distilled water) in its:
2.1 Diameter of the zone of inhibition
3. How effective is the wood vinegar in inhibiting the Fusariumproliferatumin:
3.1) 6 hours
3.2) 12 hours
3.3) 18 hours
3.4) 24 hours
4. Which experimental treatment is comparable to the commercial fungicide in terms of the size of the diameter of the zone of inhibition? II. Hypotheses
Ho1: The different treatments have no significant difference in the diameter of the zone of inhibition of Fusarium proliferatum.
Ho2: The diverse treatments have no significant effect in the appearance of the zone of inhibition in days and the diameter of the zone of inhibition.
Ho3: The number of hours of exposure has no significant difference in inhibiting the growth of Fusarium proliferatum.
Ho4: The experimental treatments with the highest zone of inhibition have no significant difference to the commercial fungicide in the size of the diameter of the zone of inhibition. III. Level of Significance α= 0.01 dfTrt= N(row)-1 dfp= N(column)-1 dfT= N-1 dfE= dfT-(dfTrt+dfP)
A. Collection of the fungi B. Setting aside to allow the growth of the fungus
C. Transferring to an agar slant D. Dyeing with methylene blue for pure culture
E. F. proliferatum under the microscope F. Preparation of treatments
G. Preparation of the Culture Media H. Preparation of Fungal Standard Solution
I. Comparing the Fungal Standard J. Impregnation of Filter Paper Discs Solution to the McFarland Standard
K.. Labeling of petri plates L. Application of Disks on PDA
M. Disposal of PDA with F.proliferatum
Photo credits: Florence Marie M. Villar
APPENDIX E
EXPENSES
Laboratory Room Use P350
Autoclave P110
Clean Bench P300
Dry- Air Oven P350
Glasswares/ Materials P250
Laboratory Technician Honorarium P500
¼ kgs. Of Daconil P200
TOTAL: P2060.00
ACKNOWLEDGEMENT
The researchers would like to express their sincerest gratitude to all the people who made this research paper complete and possible: First and foremost, to the God Almighty, who in heaven never left the researchers in accomplishing things all throughout their study and giving the wisdom, strength and endurance to carry on. To Dr. Julie B. Otadoy, Department Head of Biology, University of San Carlos- Talamban Campus, for her valuable and considerate approval for the researchers to conduct their experiment in the Biology Department Laboratory; To Miss Angel C. Enriquez, OIC Regional Executive Director, DA RFO 7 Maguikay Mandaue City for the approval and trust given to the researchers despite of their age to work in the laboratory of the mentioned department during the isolation of the pathogen;
To Mr. Wilberto O. Castillo, Research Department Head DA RFO7, Maguikay Mandaue City for entrusting them to Mr. Bienvenido D. Acabal Jr. and Ms. Christine T. Advincula who supervised in the isolation of the fungi.
To Mr. Bienvenido D.Acabal Jr., Chief Regional Crop Center, for letting them do the isolation of the fungi in laboratory and for providing them the wood vinegar extracts and PDA (Potato Dextrose Agar) which were valuable in the bioassay;
To Ms. Christine T. Advincula, laboratory technician and plant pathologist of the Department of Agriculture - RFO 7 for patiently teaching them the processes in the isolation of fungi; To Mr. RollyViescel, Microbiologist, University of San Carlos- Talamban Campus, for being the lab facilitator, orienting the researchers on their work plan, and for providing all the necessary materials and apparatuses which were needed to complete their experimental work; To Ms. Celia C. Gepitulan, Research Adviser, for the advice and guidance in giving the proper direction for this study; for her patience in accommodating consultations to help make this research paper as accurate as possible. And to their families who had been giving them endless support both financially and morally; for their inspiration and endless dedication.
JHAEL HAZEL MARIE L. ABUCEJO KIM CARLO L. TUDTUD
FLORENCE MARIE M. VILLAR
Researchers
CURRICULUM VITAE
I. PERSONAL BACKGROUND
Name: Jhael Hazel Marie L. Abucejo
Age: 13 years old
Birth date: September 12, 2001
Address: VLT-F blk. 3 lot 6 Bacayan, Cebu City
Gender: Female
Religion: Roman Catholic
Name of Father: Jereme L. Abucejo
Name of Mother: Julifer L. Abucejo
II. EDUCATIONAL BACKGROUND
Level | School | Year | Kinder (I-II) | Maeryll Tutorial School | 2005-2007 | Elementary (I-VI) | Talamban Elementary School | 2007-2013 | High School (VII-IX) | Talamban National High School | 2013- present |
CURRICULUM VITAE
I. PERSONAL BACKGROUND
Name: Kim Carlo Tudtud
Age: 14 years old
Birth date: June 29, 2000
Address: Tintay, Talamban, Cebu city
Gender: Male
Religion: Roman Catholic
Name of Father: WelfredoTudtud
Name of Mother: Susana Tudtud
II. EDUCATIONAL BACKGROUND
Level | School | Year | Kinder ( I-II) | Talamban Day Care Center | 2005-2007 | Elementary (I-VI) | Talamban Elementary School | 2007-2013 | High School (VII-IX) | Talamban National High School | 2013- present |
CURRICULUM VITAE
I. PERSONAL BACKGROUND
Name: Florence Marie Villar
Age: 14 years old
Birth date: October 26, 2000
Address: Burgos, Pulangbato, Cebu City
Gender: Female
Religion: Roman Catholic
Name of Father: Erwin Villar
Name of Mother: ErlindaVillar
II. EDUCATIONAL BACKGROUND
Level | School | Year | Kinder (I-II) | Pulangbato Day Care Center | 2005-2007 | Elementary (I-VI) | Pulangbato Elementary School | 2007-2013 | High School (VII-IX) | Talamban National High School | 2013- present |
