TEST FOR DELAYING THE SENESCENCE OF TOMATO (Lycopersicum esculentum) USING DIFFERENT LEVEL OF POTASSIUM ALUMINUM SULFATE (ALUM)

CHAPTER 1

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.

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)-2benzimidazole 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 stolonifer and to evaluate the potential application of alum to control postharvest spoilage on tomatoes during storage (Turkhan, 2010)

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 of Rhizopus stolonifer

Alternative Hypothesis: Alum inhibit the growth of Rhizopus stolonifer

1.6 Significance of the Study

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

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

Inoculum

Chapter 2

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.

Alum sulfate

Rhizopus stolonifer

In Vivo

In Vitro a. Growth Inhibition of Rhizopus stolonifer on Commercial Tomato b. Color and c. Firmness

Anti-fungal Screening of Alum Sulfate

Recommending the Use of Potassium Alum Sulfate Against Rhizopus stolonifer Figure 1. Theoretical Framework
This portion showed the conceptual framework of the research, the major steps undertaken towards the desired result.

Related Literature

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.

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. stolonifer var. stolonifer produces straight, erect sporangiophores, whereas those of R. stolonifer var. lyococcos are curved. A closely related species, Rhizopus sexualis, differs primarily in being homothallic (self-compatible).

The Rhiopus stolonifer are 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).

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).

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 [T 1 as negative control, water; T 2 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.

Table 1.0 Experimental Design Treatments Water 1.0 mg./L Alum 2.0 mg./L Alum 3.0 mg./L Alum Baking Power 1 Replication 2 3 Total Mean

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

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)

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. 1.0mg/L, Each concentration of alum sulfate at

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 of R. stolonifer and 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

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

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.

Table ____. Relationship between Subjective Firmness Measurements for Whole Tomato 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 oneway 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

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 Treatment Water 0.5 mg/L 1.0mg/L 1.5mg/L Baking Powder Total 1 Replication 2 3 Total Mean

ANOVA

In Vivo Screening Result A. Mould Growth Inhibition of Rhizopus stolonifer Table ____. Radius of Rhizopus stolonifer Growth (mm) Replication Treatment Water 0.5 mg/L 1.0mg/L 1.5mg/L Baking Powder 1 2 3 Total Mean Index

B. Color Change Table _____ Tomato Fruit Color Index Day 1 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

Table _____ Tomato Fruit Color Index Day 3 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

Table _____ Tomato Fruit Color Index Day 5 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

Table _____ Tomato Fruit Color Index Day 7 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

Table _____ Tomato Fruit Color Index Day 9 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

Table _____ Tomato Fruit Color Index Day 11 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

C. Firmness Index Using Modified Penetrometer Deformation Apparatus Table _____ Tomato Fruit Color Index Day 1 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

Table _____ Tomato Fruit Color Index Day 11 Replication 2

Treatments Water

Total

Mean

1.0 mg/L

2.0 mg/L

3.0 mg/L

Baking Soda

Spearmans Rho

Fungal growth inhibition of citronella oil against Rhizopus stolonifer are shown in Table 1. The MIC of citronella oil against R. stolonifer on agar was 5L/mL. Fungal resistance of tomatoes dip-treated with various concentrations of citronella oil against R. stolonifer is 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. stolonifer on 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. stolonifer for 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

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:

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

Journals Bartz, J.A, Steven, R.A and Mahovic, M. Guide t0 Identifying and Controlling Postharvest Tomato Diseases in Florida, J, p.23 Barrett, D. M.et all. 1998.Textural Modification of Processing Tomatoes, 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 in Korea, Mycobiology 176-178. Matan, N, W., 2011. Postharvest Control of Rhizopus stolonifer on Tomato by Citronella Oil, 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 of filed Crops Vol. 15(1), p. 94 www.lagorio.com, California Tomato Commission 185