WO2011099878A2 - Organic pesticide - Google Patents

Abstract

This invention pertains to a composition, which is a natural and organic pesticide, specifically with fungicidal and bactericidal action against pathogenic microorganisms in agricultural products such as rootcrops, fruits and vegetables. Examples of such disease-causing microorganisms are Mycosphaerella fijensis causing black Sigatoka disease in Cavendish (banana), Ralstonia solanacearum causing Moko disease in Cavendish (banana), Colletotrichum gloespoiroides and Botryodiplodia theobromae causing anthracnose in crops and fruits, tomato yellow leaf curl virus (TYLCV) in tomatoes, Lasiodiplodia theobromae causing soft rot or fruit rot in crops and fruits, Fusarium oxysporum causing Panama wilt in fruits and crops, and many others. The composition, which is an extract of a fermented concoction of tropical plants, carbon source, protein (nitrogen) source, and a carrier agent, is mainly composed of alpha-hydroxy acids (AHAs) as the active ingredients, with lactic acid as the most dominant AHA, and other natural nutrients, all of which may help strengthen the plant's immune system to fight pathogenic diseases. Since all the major constituents of the composition are generally regarded as safe, this natural pesticide is found to be non-toxic and safe to humans and animals, and environmentally benign.

Description

ORGANIC PESTICIDE

Description

Over the years, the demand for agricultural fruits, vegetables, and crops such as banana, mango, sweetpotato, cassava, and yam in the world market increased due to rising population and the influx of more novel applications in the food industry. Meanwhile, environment-friendly sustainable agricultural practices are getting more attractive to farmers since they have more benefits in doing such than the usual chemical control. The use of chemical agents for pests and diseases is disadvantageous because of high power consumption, soil and environment pollution, and the presence of pesticide residues, which are usually harsh chemicals, in fruits, vegetables, and crops harmful to humans and animals. The discovery of natural products that are beneficial to agriculture is highly important in addressing the concerns of farmers such as in increasing fruit, vegetable, and rootcrop yield and productivity.

This invention presents an alternative product to chemical pesticides, which is a natural organic composition derived from a fermented mixture of tropical plants, sugar, and milk. The antimicrobial activity of this composition is investigated against Mycosphaerella fijensis causing Sigatoka disease in Cavendish (banana), Ralstonia solanacearum causing Moko disease in Cavendish (banana), Colletotrichum gloespoiroides and Botryodiplodia theobromae causing anthracnose in crops and fruits, tomato yellow leaf curl virus (TYLCV) in tomatoes,

Lasiodiplodia theobromae causing soft rot o fruit rot in crops and fruits, Fusarium oxysporum causing Panama wilt in fruits and crops, and many others.

Lactic acid, as the major constituent in the composition of the present invention, is known to be Generally Recognized As Safe (GRAS) for use in food. No human health concerns and environmental concerns ever arise with the use of lactic acid in pesticide products. Lactic acid undergoes rapid aerobic and anaerobic biodegradation in both soil and water with an estimated half-life of 5 days or less. Mineralization and degradation to form C0₂, H₂0 and other minor constituents, usually occur in days to weeks; thus, it is classified as readily biodegrable.

From literature, a study by Trias et al. (Int Microbiol 2008; 11(4):231-236) evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. Furthermore, in this study, cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when some strains were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot.

Extracts from microbial fermentations are found to exhibit anti-fungal, anti-bacterial or insecticidal activity. A United States Patent 6,417,163 by Heins, et al. filed on July 9, 2002 showed an invention on the compositions and methods for controlling plant pests, which relates to a novel antibiotic-producing and metabolite-producing Bacillus subtilis strain that exhibits insecticidal, antifungal and antibacterial activity. The supernatant of this novel strain contains effective insecticidal, antifungal and antibacterial agents. Also included in the invention is a solvent extractable, small molecular weight (<10,000 daltons) corn rootworm-active metabolite produced in the supernatant. Also included in the invention are methods of protecting or treating plants from fungal and bacterial infections and corn rootworm infestations comprising the step of applying to the plant an effective amount of the antibiotic/metabolite-producing novel Bacillus subtilis strain, the antibiotic/metabolite produced by the novel Bacillus subtilis strain or a combination thereof, optionally further comprising another antibiotic-producing bacterial strain and/or a chemical pesticide. The invention also includes methods of preventing or treating fungal and bacterial infections using whole broth cultures or supernatants obtained from cultures of the novel Bacillus subtilis strain alone or in ctfmb¾ation with chemical pesticides and/or other biocontrol agents. The Invention

The invention is a composition, which is the broth extract of a fermented concoction of tropical plants, carbon source, protein (nitrogen) source, and a carrier agent. The broth extract is derived from the fermentation of one or any combination of plant extracts from any flowering plant of the Order Brassicales belonging to the eurosids II group of dicotyledons under APG II system with any or a combination of the following families: Family Brassicaceae (mustard and cabbage family which may include the Cleomaceae plant) and Family Caricaceae (papaya family). The extracts of any or combination of these plants can be derived from fruits, leaves, bark, roots, and seeds. In the preparation of these plant extracts, the weight ratio of plant from Family Caricaceae to plant from Family Brasicaceae is 1:1 to about 100:1, specifically at a ratio of 50:1 to about 5:1, and more specifically at a ratio of about 3:1. Alternatively, a 100% pure plant extract from Family Caricaceae or 50% thereof in combination with any other member of the family Brassicales may be used, specifically of the family Brasicaceae. In the same manner, a 100% pure plant extract from Family Caricaceae or 50% thereof in combination with any other member of the family Brassicales may be used, specifically of the family Brasicaceae and family Caricaceae.

The carbon source, which is mixed with the plant extracts, comprises simple sugars such as glucose, fructose, and dextrose or a combination thereof. Alternatively, complex carbon sources can also be used such as molasses, refined sugar, honey or a combination thereof. In this invention, glucose is employed as carbon source with water as carrier agent at a ratio of 1:1 to about 1:1000, preferably at a ratio of 1:3 to about 1:100, most preferably at 1 :5 to about 1:80, and best at 1 :50.

The protein (or nitrogen) source can be any or combination of processed milk, fresh milk, skimmed milk, UHT milk, powdered milk, yoghurt milk, and full cream milk at a ratio of 1 : 10 to 1:1000, preferably at a ratio of 1:100 to about 1:150, most preferably at 1:150, with water as carrier agent.

Water, with or without wheat or corn or rice flour to promote the powderized form of the composition, can be used as carrier agent in the process of fermentation, employing a commercially available lactic acid producing bacteria as the fermenting agent. In the preparation of the composition, first, the plant extracts are locally collected. These must be well-chosen such that these are firm and solid and have no unnecessary lacerations. These are further washed and sliced into smaller pieces or cubes. These plant extracts are cooked in boiling water for about one hour, after which it is cooled down and covered tightly with plastic wrap and set aside for seven (7) days to ferment at ambient temperature and pressure. In the meantime, a mixture of carbon source (e.g. glucose), protein source (e.g. skimmed milk) and water (as carrier agent) is prepared in known proportions described above. The mixture is placed in a glass vessel, added with the fermenting agent (Lactic acid producing bacteria) and the supernatant liquid from the fermented plant extract prepared as described above, covered tightly with plastic wrap, and set aside for another seven (7) days to ferment at ambient temperature and pressure.

After fermentation, the mixture is filtered and the supernatant liquid is collected as the product extract, which is referred to as the composition of the present invention. This is further formulated and packed in appropriate bottle containers ready for use.

All ingredients are locally supplied and readily used in the process, as these do not require further pre-treatment and quality control. However, the workplace must be clean and free from dusts, and all containers and vessels are thoroughly washed.

A natural fermentation process employing the commercially available lactic acid producing bacteria is adopted in the manufacturing procedures, and as such no other biochemical protocols are carried out. The production process does not involve any toxic or sentisizing substances.

From the analysis of samples, the product extract is mainly composed of alpha-hydroxy acids (AHAs) as the active ingredients, such as lactic acid, acetic acid, oxalic acid, and glycolic acid (see Table 1). These acids can actually be traced back to the plant extracts as its natural constituents. These acids are reported to be safe for humans and are easily assimilable in the environment.

Specifications

The composition of the present invention, which is the extract from the fermentation process described earlier is analysed for its composition using the High Performance Liquid Chromatography (HPLC-VP series, Shimadzu, Japan), equipped with a tJV-VIS detector (SPD- 10AV) 210 nm and an Inertsil ODS-3V column (5 μπι, 250 x 4.6 μίη I.D.). The oven temperature is set at 40°C. The eluent used was 0.1 M ammonium dihydrogen phoshate + phosphoric acid (pH - 2.58) with a flow rate of 1.0 ml/min. A sample volume of 20 μί, is injected into the column. Triplicate runs of samples were done and the results obtained are the average of these triplicates, with standard deviations of ± 0.005. Table 1 shows the results of the analysis. Results indicate that the extract is mainly composed of alpha-hydroxy acids (AHAs) as the active ingredients.

There are two active ingredients of the product extract, which are classified as the alpha-hydroxy acids (AHAs). These ingredients are all dissolved in water solution, as originally prepared during the fermentation process and are produced according to the composition shown in Table 1. Impurities of the composition are the residual sugar and the colloidal suspension in the extract after the fermentation process, which are totally organic in nature.

The active ingredients in the product mixture are determined to be in small amounts as shown in Table 1. Repeated batches of the product also have reproducible contents of the organic acid as shown in Table la. The total organic acid is within the range of 80-110 g/kg lactic acid, 4-8 g/kg acetic acid, and negligible amounts of oxalic acid and glycolic acid. Bicinchoninic protein assay of the product composition shows an average protein composition of 3-5 mg per mL. These levels in the product mixture are completely safe for humans, animals, and the environment.

The physical properties of the product are shown in Table 2, its nutritional supplement specifications in Table 3, and heavy metal composition in Table 4.

Table 1. Product Composition

Components Content in g/kg

Lactic acid 80-110

Acetic acid 4-10

Oxalic acid 0.01 - 1.0 (negligible)

Glycolic acid 0.02 - 0.08 (negligible)

Table la. Product Composition of some batch samples.

Batches Organic acid Composition, g/kg Remarks

Batch 001 Lactic acid 94.3 Within specifications

Acetic acid 9.5 Within specifications

Oxalic acid negligible Within specifications

Glycolic acid negligible Within specifications

Batch 005 Lactic acid 98.44 Within specifications

Acetic acid 6.12 Within specifications

Oxalic acid negligible Within specifications

Glycolic acid negligible Within specifications

Batch 006 Lactic acid 87.06 Within specifications

Acetic acid 7.54 Within specifications

Oxalic acid negligible Within specifications

Glycolic acid negligible Within specifications Table 2. Physical Properties of the Product

Physical property description

Color Yellowish solution with white colloidal suspension

Odor Sour odor

Flammability test Non-flammable

Solubility in water Suspended particles not soluble in water

Density at 25°C, g/ml 1.000 - 1.01

Viscosity at 25°C,cp 0.10 - 0.2

pH at 25°C 2.5 - 3.0

Table 3. Nutritional Supplement Specification of the Product

Parameter *Content range

Calcium (mg Ca/ L) 25- 30; 100-140

Iron (mg Fe/L) 0.04- 0.08

Sodium (mg Na/L) 10 - 15; 50-70

Potassium (mg K/L) 45 - 55; 60-85

Magnesium (mg Mg/L) 4 - 8; 25-30

Manganese (mg Mn/L) 0.05- 1.0

Zinc (mg Zn/L) 0.05 -1.0

*Second set of content range indicated are results of best preferences.

Table 4. Heavy Metals Specification of the Product

Heavy Metal Content

Cadmium (mg Cd/L) Less than 0.005

Chromium (mg Cr/L) Less than 0.03

Cobalt (mg Co L) Less than 0.05

Copper (mg Cu/L) Less than 0.006

Lead (mg Pb/L) Less than 0.10

Nickel (mgNi/L) Less than 0.01

Mercury ^g Hg/L) Less than 0.06

Toxicity

The composition of the present invention was tested for its toxicity in terms of lethal dose (LD₅o). The results indicated that the composition did not cause death in the test animal even up to a high dosage of 160 ml/kg of the composition. This is consistent with reports that acute oral and inhalation toxicity of lactic acid to rats and acute dermal toxicity of lactic acid to rabbits were already established to be very low. Repeated oral exposure of rats to lactic acid for 90 days produced no toxicity. A developmental study in mice showed no toxicity at the only dose tested. A 2-year bioassay study of calcium lactate in rats showed no evidence of carcinogenicity. Lactic acid did not induce gene mutations in bacteria or induced chromosomal aberrations in mammalian ceils in vitro. No data were provided for reproductive toxicity, but testing is not deemed necessary because the substance is a normal component of human intermediary metabolism. Based on safety data reports, the lactic acid-based products are completely safe for beneficial living organisms or non-target organisms including humans, animals, plants and other micro-organisms and the environment in general.

Technical effects

Based on laboratory assays and field tests, the composition of the present invention, which contains AHAs significantly, inhibited the growth of the fungal pathogen Mycosphaerella fijiensis causing black Sigatoka disease in banana {Musa cavendish). The efficacy is comparable to Mancozeb, which is the standard fungicide check after 48 and 72 hours of incubation. From 64 to 150 mL of the composition per 16 liters of water, the product showed significant efficacy against the fungal pathogen.

Similar results were observed during laboratory assays using the same composition of the present invention against Ralstonia solanacearum causing Moko disease in banana {Musa cavendish), in which no growth of the bacterium is observed after a week of incubation. This product containing lactic acid was effective against the pathogen beginning at 50 mL per 16 liters of water, and was significantly superior when compared to the bacterial antibiotic check streptomycin and also better than brine solution, which is commonly used by farmers to control the disease.

The composition of the present invention was also found to inhibit the growth of Colletotrichum gloespoiriodes and Botryodiplodia theobromae causing anthracnose in crops and fruits, tomato yellow leaf curl virus (TYLCV) in tomatoes, Lasiodiplodia theobromae causing soft rot or fruit rot in crops and fruits, Fusarium oxysporum causing Panama wilt in fruits and crops, and many others.

The bio-efficacy of the composition of the present invention was evaluated in a 26-hectarp banana plantation for 8 straight cycles at 6 days interval against black leaf streak or black Sigatoka disease of Cavendish banana in comparison with plantation Standard spraying scheme of systemic and contact fungicides and their combinations, which showed unique results. The treatment using the composition showed better performance over the standard as it showed higher number of functional leaves at harvest, which is considered as the most important parameter in efficacy evaluation of the product against Sigatoka disease in banana. Although it showed higher earlier infections, these did not translated into corresponding losses of leaves but retained these up to harvest. The results confirm the booster effect of the composition on the leaves aside from its fungicidal activity of containing the disease infection, it has tolerated the plants the impact of infection. The build-up of bioactive substances supplied by the macro and micronutrient components of the composition directly feed to the leaves might have prevented the loss of leaves. Therefore, the composition of the present invention at a rate of 0.75 to 1.0 L/ha can be used as an alternate pesticide in the black Sigatoka control program in Cavendish banana plantations.

Application method

Recommended spray dilution rate is from 64 to 150 mL of the product composition per 16 liters of water for the control of the fungal pathogen Mycosphaerella fijiensis causing black Sigatoka disease. For Ralstonia solanacearum causing Moko disease, formulation can start at 50 mL of product composition per 16 liters of water. To translate this into farmer's practice, a spraying program at a rate of 0.75 to 1 L per hectare of plantation can be employed using the composition of the present invention to control the black Sigatoka disease in banana.

A regular spray interval of 6-7 day-cycle is recommended starting with one-month old banana seedlings for treatment with banana plants. A dilution rate starting at 50 mL of the product composition per 16 L of water is best employed against the Moko disease. With this rate, the bacterium Ralstonia solanacearum causing Moko disease, can be controlled. Increasing the formulation to at least 64 mL per 16 liters of water can also significantly control the other fungal pathogen Mycosphaerella fijiensis causing black Sigatoka disease in banana.

Foliar spraying is the best and preferred method of application to the various parts of the banana plant. The same is employed to other fruits, crops, and vegetables.

EXAMPLES

EXAMPLE 1: Efficacy against Mycosphaerella fijiensis causing Black Sigatoka disease

A study was conducted to determine the efficacy of the composition of the present invention against the fungal pathogen Mycosphaerella fijiensis causing black Sigatoka disease in Cavendish banana. Laboratory bioassays and screenhouse tests were done.

Black sigatoka caused by Mycosphaerella fijiensis is widespread in banana growing regions. Cavendish banana is one of the highly susceptible cultivars to this disease. It causes considerable economic loss because the fruits of affected plants ripen early, are smaller and lighter, resulting to low production yields. Although it is eaten only as a dessert or an addition to breakfast cereal in most developed countries, it is actually a very important agricultural product. After rice, wheat, and milk, it is the fourth most valuable food. In the export market, it ranks fourth among all agricultural commodities and is the most significant of all fruits. Thus, the control of the disease-causing fungus Mycosphaerella fijiensis causing Black sigatoka on Cavendish banana is an important issue to consider.

1A. Bioassay test

The bioassay test was done using agar disc method. Petri plates were poured with 10 mL of melted potato sucrose agar (PSA) and 10 mL of different dosages of the composition samples and rotated to allow thorough mixing before congealing. Agar planting was done using cork borer to cut agar with fungal growth from the two-week-old pure culture of M. fijiensis. Plates were labeled properly and were incubated in inverted position at room temperature. Observations were done after 48-72 hours.

Another bioassay test was done using the paper disc method. One mL of the fungal suspension was poured into each of the sterile Petri plates after which 10 mL of culture media was added. The mixture was rotated to ensure thorough mixing of the culture medium and the test pathogen and allowed to congeal. With the sterile pair of forceps, sterilized paper discs were dipped into different levels of the composition of the present invention and were planted at the center of each of the Petri plates, labeled, and incubated in upside down position at room temperature.

Zones of growth of M. fijiensis causing Sigatoka of Cavendish banana after 48 hours of incubation (Table 5) were significantly different from each other. Statistical analysis revealed that treatment with a ratio of 1 :1 gave the lowest mean of zone of growth of the pathogen which was 6.63 mm, followed by the chemical check (Mancozeb) with recommended dosage which had a mean of 10.75 mm and were comparable to each other. Other treatments have means of zones of growth that ranged from 22.88-24.88 and the ratio of 0.25:40, which were comparable to the untreated control with respective means of 28.00 and 32.50. Furthermore, after 72 hours of incubation, the results showed the same trend as that at 48 hours of incubation (Table 6).

Table 5. Zones of growth (mm) of M. fijiensis causing Sigatoka of Cavendish banana at different dosages of the composition of the present invention, after 48 hours of incubation.

Treatments / II III IV Mean

1:1 6.0 6.0 8.50 6.0 6.63^a

1:40 (400 mL/16 L) 28.5 25.5 23.5 22.0 24.88"

0.50:40 (200 mL/16 L) 23.0 23.5 25.5 27.5 24.88^b

0.25:40 (100 mL/16 L) 30.0 25.0 29.5 27.5 28.00"°

0.125:40 (50 mL/16 L) 23.0 27.5 23.0 25.0 24.63^b

0.063:40 (25 mL/16 L) 23.0 20.5 21.0 27.0 22.88^b

0.031:40 (12.5 mL/16 L) 20.0 23.0 22.5 28.0 23.38^b

Mancozeb 8.0 10.0 14.0 11.0 10.75^a

Untreated control 30.0 33.0 32.0 35.0 32.50^c

* Means with common letter do not differ significantly at 1% level (DMRT). Table 6. Zones of growth (mm) of M. fijiensis causing Sigatoka of Cavendish banana at different dosages of the composition of the present invention, after 72 hours of incubation.

Treatments J II III IV Mean

1 :1 15.0 18.5 23.0 14.5 17.75"

1 :40 (400 mL/16 L) 44.0 37.5 37.5 33.5 38.13^b

0.50:40 (200 mL/16 L) 39.5 34.0 43.0 38.0 38.63^b

0.25:40 (100 mL/16 L) 41.5 38.5 36.0 41.0 39.25^b

0.125:40 (50 mL/16 L) 33.5 40.0 33.0 38.5 36.25^b

0.063:40 (25 mL/16 L) 28.5 27.5 32.0 33.5 30.38^b

0.031 :40 (12.5 mL/16 L) 31.5 30.0 34.0 47.0 35.63^b

Mancozeb 13.0 17.5 24.5 13.5 17.13"

Untreated control 51.0 48.0 50.0 49.0 49.50°

*Means with common letter do not differ significantly at ]% level (DMRT).

The zones of inhibition of M. fijiensis, which were affected by the different dosages of the composition of the present invention, were significantly different from each other (Table 7). The zones of inhibition with the composition at the dosage of 1:1 was comparable to that of the chemical check (Mancozeb) with a mean zone of inhibition of 21.25 mm and 23.25 mm, respectively, followed by the treatment at the ratio of 1 :2 with a mean zone of inhibition of 19.50 mm, then by the treatment at the ratio of 1 :40 at 9.75 mm.

The lengths of germ tubes of M. fijiensis causing Black sigatoka disease in banana were also measured using a calibrated microscope and the results in Table 8 showed that the different treatments were significantly different from each other. The chemical check (Mancozeb) suppressed germ tube germination after 72 hours of incubation while the shortest germ tube was noted with the treatment using the composition of this invention at a ratio of 1 : 1 with water. The solution with less dosage of the composition (12.5 - 400 mL in 16 L water) had high germ tube lengths, however, the untreated control had the highest at 51.53 mm. On the other hand, statistical analysis revealed that the weights of ascospores (g) of M. fijiensis as influenced by the different treatment dosages of the composition of this invention shown in Table 9 were not significantly different from each other.

Table 7. Zones of inhibition (mm) of M. fijiensis causing Sigatoka of Cavendish banana at different dosages of the composition of the present invention, after 72 hours of incubation.

Treatments J II III Mean

1 : 1 21.5 19.0 23.25 21.25"^b

1 :2 15.5 24.0 19.0 19.50

1 :40 (400 mL/16 L) 10.25 9.0 10.0 9.75°

0.50:40 (200 mL/16 L) 0 0 0 0^d

0.25:40 (100 mL/16 L) 0 0 0 0^d

0.125:40 (50 mL/16 L) 0 0 0 0^d

0.063:40 (25 mL/16 L) 0 0 0 0^d

0.031:40 (12.5 mL/16 L) 0 0 0 0^d

Mancozeb 22.5 24.0 23.25 23.25"

Untreated control 0 0 0 0^d

*Means with common letter do not differ significantly at 1% level (DMRT). Table 8. Lengths (micron) of germ tubes of M. fijiensis causing Sigatoka of Cavendish banana at different dosages of the composition of the present invention, after 72 hours of incubation.

Treatments / II III Mean

1 :1 9.8 9.6 9.7 9.70^b

1 :40 (400 mL/16 L) 21.0 22.0 20.7 21.23^c

0.50:40 (200 mL/16 L) 33.0 34.6 30.9 32.83^d

0.25:40 (I00 mL/I6 L) 34.0 33.6 30.5 32.70^d

0.125:40 (50 mL/16 L) 38.9 40.0 40.0 39.63^e

0.063:40 (25 mL/16 L) 22.7 24.0 25.0 23.90°

0.031:40 (12.5 mL/16 L) 35.0 34.0 30.0 33.00^d

Mancozeb 0 0 0 0^a

Untreated control 52.0 49.9 52.7 51.53

*Me ns with common letter do not differ significantly at 1% level (DMRT).

Table 9. Weights (mg) of ascospores of M. fijiensis causing Sigatoka of Cavendish banana at different dosages the composition of the present invention, after 72 hours of incubation

Treatments / II III Mean

1 :1 1 1.4 14.3 7.8 1 1.17

1:2 8.9 8.4 13.9 10.40

1:40 (400 mL/16 L) 6.4 12.9 9.9 9.73

0.50:40 (200 mL/16 L) 5.5 12.9 0.9 6.43

0.25:40 (100 mL/16 L) 4.8 5.7 9.2 6.57

0.125:40 (50 mL/16 L) 7.5 6.7 5.7 6.63

0.063:40 (25 mL/16 L) 10.2 9.5 6.1 8.60

0.031 :40 (12.5 mL/16 L) 8.2 10.6 8.7 9.17

Mancozeb 6.4 3.2 6.6 5.40

Untreated control 9.8 8.3 4.2 7.43

Screenhouse test results

Statistical analysis revealed no significant differences were observed among treatments after the first, second, and third spray of applications at one-week intervals, as shown in the succeeding tables 10-12. However, it is also noted that after the third week of application, the trend of disease infection was lowest than in the first and second week of applications.

Table 10. Disease severity of in vivo (screenhouse) test of Cavendish banana seedlings at different dosages of the composition of the present invention, after first week of application.

Treatments / II III Mean

1: 1 25.93 22.22 22.22 23.46

1 :2 20.37 25.93 22.22 22.84

1 :40 (400 mL/16 L) 16.67 14.81 18.52 16.67

0.50:40 (200 mL/16 L) 18.52 18.52 18.52 18.52

0.25:40 (100 mL/16 L) 20.37 16.67 14.81 17.28

0.125:40 (50 mL/16 L) 20.37 22.22 16.67 19.75

0.063:40 (25 mL/16 L) 16.67 24.07 16.67 19.14

0.031 :40 (12.5 mL/16 L) 14.81 14.81 16.67 15.43

Mancozeb 27.78 16.67 18.52 20.99

Untreated control 18.52 16.67 16.67 17.28 Table 11. Disease severity of in vivo (screenhouse) test of Cavendish banana seedlings at different dosages of the composition of the present invention, after second week of application.

Treatments / II III Mean

1 :1 27.78 24.07 24.07 25.31

1:2 20.37 20.37 42.59 27.78

1 :40 (400 mL/16 L) 24.07 14.81 24.07 20.99

0.50:40 (200 mL/16 L) 25.93 24.07 18.52 22.84

0.25:40 (100 mL/16 L) 29.63 22.22 20.37 24.07

0.125:40 (50 mL/16 L) 18.52 18.52 22.22 19.75

0.063:40 (25 mL/16 L) 18.52 18.52 22.22 19.75

0.031:40 (12.5 mL/16 L) 18.52 20.37 25.93 21.60

Mancozeb 22.22 22.22 25.93 23.46

Untreated control 25.93 29.63 18.52 24.69

Table 12. Disease severity of in vivo (screenhouse) test of Cavendish banana seedlings at different dosages of tl composition of the present invention, after third week of application.

Treatments / // III Mean

1 : 1 12.96 14.81 14.81 14.20

1 :2 11.11 12.96 16.67 13.58

1 :40 (400 mL/16 L) 7.41 9.26 7.41 8.02

0.50:40 (200 mL/16 L) 5.56 5.56 7.41 6.17

0.25:40 (100 mL/16 L) 7.41 12.96 14.81 11.73

0.125:40 (50 mL/16 L) 14.81 11.1 1 12.96 12.96

0.063:40 (25 mL/16 L) 9.26 14.81 11.11 11.73

0.031 :40 (12.5 mL/16 L) 14.81 9.26 7.41 10.49

Mancozeb 9.26 7.41 9.26 8.64

Untreated control 12.96 20.37 7.41 13.58

IB. Single Leaf Test

Another study was conducted to evaluate the efficacy of the composition of the present invention against black leaf streak or Sigatoka disease of Cavendish banana by Single Leaf Test.

Results of the single-leaf test of the composition against BLS (Black Leaf Streak) or Sigatoka disease of Cavendish banana showed the lowest rate @ 0.187 li/ha as the most effective among the rates evaluated comparable with Standard, Dithane M-45 @ 2.0 kgs/ha and much better than the Untreated Control.

There was no phytotoxicity observed on young and old leaves of banana nor on young baggable fruits. Stability of spray solution mixtures were comparable with the Standard, Dithane M-45 or numerically even better. EXAMPLE 2: Efficacy against Ralstonia solanacearum causing Moko disease

Moko wilt disease caused by Ralstonia solanacearum is one of the most serious diseases of bananas and plantains. It has been recorded as a continuing problem worldwide. Reduction in banana yield of up to 70% due to Moko disease had been reported in Guyana. A single localized root invasion by Moko can soon become an epidemic if flowers are being produced at the time and if the bacterial pathogen begins to ooze from the male buds. R. solanacearum, the culprit of bacterial infection on bananas is a soil-borne pathogen that is a major limiting factor in the production of many crop plants around the world. However, good management practices can help to reduce the risk of spreading this disease. The anti-fungal activity of the present invention against Ralstonia solanacearum causing Moko disease in banana was studied and results are presented in this example. Studies comparing anti-fungal activity of the present composition to activity of known fungal pesticides can further prove the efficacy of the composition against Moko disease.

Bioassay tests

Bioassay tests of different concentrations of the composition of the present invention were done against Ralstonia solanacearum, employing Kocide (RR), alum, streptomycin, brine solution, three levels of AZ41 (an organic foliar fertilizer with pesticidal properties) at 45, 60, 75 mL per 16 liters of water, and the untreated control. The experiment was repeated three times in completely randomized design.

The bacterium Ralstonia solanacearum was sourced out from a moko-infected banana from an established banana plantation. The diseased specimens were brought to the laboratory. Using a portion of the infected tissue, the same was allowed to ooze out in clean distilled water inside a test tube. From this suspension, the pathogen was isolated by streaking onto freshly plated potato dextrose peptone agar (potato 200 g, dextrose 10 g, peptone 8 g, and agar 20 g). The pure culture was maintained until needed. R. solanacearum is a fast growing bacterium with whitish to slimy yellowish colonies in culture medium.

Using strict aseptic means, one mL of each formulated treatments was poured into each of the sterile Petri plates, and after which ten mL of culture medium was added. The plates were then rotated to ensure even distribution of the mixture and were allowed to congeal. With a pair of sterile forceps, sterilized paper discs were dipped into the bacterial suspension taken from a pure

— R

culture (calibrated at about 10 cells per mL of H₂0) and were planted at the center of the Petri plates. All plates were labeled and incubated in upside down position. Clear zones of inhibition (mm) were measured after three days of growth and at one week (7 days) of growth of the pathogen. This served as basis to measure the efficacy of the various treatments at difference concentrations of the composition of the present invention including the bactericidal check - Kocide (RR), streptomycin, brine solution (which is a common farmer's practice), and the untreated control.

Bioassay Test Results

Table 13 shows the various zones of growth of the pathogen Ralstonia solanacearum as affected by the different treatments after three days of incubation. Results showed highly significant differences among the various treatments used. The composition of the present invention at a concentration range of 45-60 mL per 16 liter of water showed very minimal zones of growth at 1.3-1.8 mm, which is not significantly different from each other. However, increasing the composition concentration to a range from 70-150 mL per 16 liter of water led to clearly minimum zones of growth of the pathogen at 0.7-0.9 mm. These values are not significantly different from each other, which suggests that increasing the composition concentration beyond 70 mL per 16 liter of water would result to the same efficacy.

Table 13. Bioassay results of the composition of the present invention against Ralstonia solanacearum causing moko disease in banana showing zones of growth at three days of incubation.

Treatment / II III *Mean

Composition concentration (this invention)

10 mL per 16 L water 3.2 3.9 2.1 2.6^C

15 mL per 16 L water 2.9 3.4 2.9 2.7^f

20 mL per 16 L water 2.6 2.6 2.7 2.5^de

30 mL per 16 L water 2.1 2.4 2.1 2.3^d

40 mL per 16 L water 2.4 2.1 2.1 2.0°

45 mL per 16 L water 2.4 1 .7 1.7 J gbcd

50 mL per 16 L water 1 .6 2.1 1.6 1 .6^bc

60 mL per 16 L water 1 .4 1 .2 1.4 1.3^b

70 mL per 16 L water 1 .2 0.7 1.2 0.9^a

75 mL per 16 L water 1 .2 1.2 0.7 0.9^a

80 mL per 16 L water 0.7 1.2 0.7 0.7^a

90 mL per 16 L water 1.2 0.7 0.7 0.7^a

100 mL per 16 L water 0.7 1 .2 0.7 0.7^a

105 mL per 16 L water 0.7 0.7 0.7 0.7^a

1 10 mL per 16 L water 0.7 0.7 0.7 0.7^a

150 mL per 16 L water 0.7 0.7 0.7 0.7^a

Kocide (RR) 0.7 0.7 0.7 0.7^a

Alum 0.7 0.7 0.7 0.7^a

Streptomycin 2.3 2.3 2.3 2.3^a

Brine solution 2.4 2.2 2.1 _j gbcd

AZ 41 concentration

45 mL per 16 L water 2.1 2.7 1.7 2.0^C

60 mL per 16 L water 1.2 1.7 1.7 1 .7^ed

75 mL per 16 L water 1 .2 0.7 0.7 0.7^a

Untreated Control 8.9 8:9 8.2 7.8^g

*Means with common letter do not differ significantly at 1% level (DMRT). Furthermore, it must be noted that at the composition concentration range of 70-150 mL per 16 liter of water, comparable results were observed using Kocide (RR), Alum, and AZ 41 at 75 mL per 16 liter of water (the highest concentration tested in this study). Streptomycin and the brine solution have comparable results of zones of growth with the composition concentration range of 10-40 and 45-60 mL per 16 liter of water, respectively.

Performing the bioassay tests at seven days of incubation of the pathogen Ralstonia solanacearum showed similar results as shown in Table 14. These results strongly indicate that the composition of the present invention at a concentration of at least 70 mL per 16 liter of water is effective in inhibiting the growth of Ralstonia solanacearum causing moko disease in Cavendish banana. Its efficacy is comparable to that of the chemical pesticides currently used by the banana farmers.

Table 14. Bioassay results of the composition of the present invention against Ralstonia solanacearum causing moko disease in banana showing zones of growth at seven days of incubation.

Treatment / II III *Mean

Composition concentration (this invention)

10 mL per 16 L water 2.7 2.3 3.5 3.6^efg

15 mL per 16 L water 2.6 2.7 3.1 3.1^cf

20 mL per 16 L water 2.6 2.4 3.1 2.7*=

30 mL per 16 L water 2.1 2.6 2.6 2.3^C

40 mL per 16 L water 2.1 1.7 2.6 2.3^C

45 mL per 16 L water 2.1 1.4 2.1 2.1^b

50 mL per 16 L water 1.4 1.9 1.7 1.8"

60 mL per 16 L water 1.0 1.4 1.4 1.3'

70 mL per 16 L water 0.7 0.7 1.2 1.1"

75 mL per 16 L water 1.2 0.7 0.7 1.0^a

80 mL per 16 L water 0.7 0.7 1.2 1.0^a

90 mL per 16 L water 0.7 0.7 0.7 0.9^a

100 mL per 16 L water 0.7 0.7 0.7 0.9^a

105 mL per 16 L water 0.7 0.7 0.7 0.7^a

110 mL per 16 L water 0.7 0.7 0.7 0.7^a

150 mL per 16 L water 0.7 0.7 0.7 0.7^a

Kocide (RR) 0.7 0.7 0.7 0.7^a

Alum 0.7 0.7 0.7 0.7^a

Streptomycin 4.4 4.5 4.3 4.4^fg

Brine solution 1.8 1.7 3.7 2.4^d

AZ 41 concentration

45 mL per 16 L water 2.1 2.1 2.7 2.7^f

60 mL per 16 L water 1.6 1.7 2.4 2.1^b

75 mL per 16 L water 0.7 0.7 1.2 1. Γ

Untreated Control 7.5 7.8 8.9 8.9⁸

*Means with common letter do not differ significantly at 1% level (DMRT). EXAMPLE 3: Efficacy against CoUetotrichum gloesporioides causing anthracnose

A study was conducted on the inhibitory effect of the composition of the present invention against CoUetotrichum gloespoiroides causing anthracnose in many fruits, vegetables, and crops. These include mango, banana, tomato, yam, potato, cassava and other tropical fruits and vegetables with high-value in the market.

For instance, mango production is confronted with the occurrence of pests and diseases. Infection with a disease like "anthracnose" caused by the fungus Collectotrichum gloesporioides is common during the flowering stage until post-harvest period; although, anthracnose together with stem-end rot are the two common diseases at post-harvest stage. Regarded as a serious problem for farmers and fruit exporters, chemical checks which are highly toxic in nature are currently used for the control of anthracnose.

Bioassay tests

One and a half milliliters of samples of the composition were centrifuged twice (Desk-top High Speed Centrifuge Model TGL-14Gt, China) at 13,500 rpm for 15 minutes. The resulting supernatant was taken and filtered using an Acrodisc^® microfilter (CR 13 mm syringe filter, 0.2 μπι PTFE membrane, PALL-Gelman Laboratory, USA). Another set of samples of the composition were used directly for the bioassays. Anti-fungal activity was determined using the Nathan's agar well method and alternatively Kirby-Bauer method (paper disc method). The agar plates were inoculated with pure culture of CoUetotrichum gloespoiroides, which was aseptically suspended in phosphate buffer and inoculated in potato dextrose agar (0.67 % v/v). Eighty microliters of the final supernatant was loaded onto the sterile 6.7-mm agar wells (or paper discs with 6-mm dia) mounted on agar plates and dried for about 10 minutes. The agar plates were sealed, inverted, and incubated at 30°C for 5 days or longer. For each test, the zone (or diameter) of inhibition (mm) was measured. Streptomycin, a known classical antibiotic and pesticide against fungi and bacteria and Antracol^® (Bayer, Germany), a known chemical pesticide against anthracnose, were used as controls at the recommended dosages. Four replicate experiments, with two agar wells each, were done for each test. Results were presented as average of all tests,, if results have differences of ±10%. An aseptic condition is always ensured during the experiments.

Bioassay Test Results

Results from bioassay tests show that the unfiltered composition of this present invention gave a consistently strong inhibition against CoUetotrichum gloeosporioides, which causes anthracnose. Its zone of inhibition reached an average of 24.19 mm during the five-day incubation period, and was well-sustained during an extended period of ten incubation days (see Figures 1A and IB). This indicates that the unfiltered and undiluted composition of the present invention can inhibit the growth of CoUetotrichum gloeosporioides, which causes anthracnose in agricultural fruits, vegetables, and crops. A concentrated sample of the composition (coded WT002) shows total inhibition against Colletotrichum gloeosporioides as no growth of the mold was observed during the extended 10-day incubation period (Figure IB).

Results using streptomycin at 125 mg per liter (125 ppm) shows that it has a comparatively lower inhibitory activity against C. gloesporioides than the unfiltered composition of the present invention (Figure 2). The latter is far better than the known antibiotic and pesticide (Streptomycin) in inhibiting the growth of C. gloesporioides. However, when the composition is diluted at a volume ratio of 1:1 and 1:3 with water, the zones of inhibition were comparable with Streptomycin at 125 ppm (Figure 2), but the inhibitory activity of the latter did not persist as it started to drop to almost zero activity on the 4^th day of incubation. This suggests that the pathogen (C. gloesporioides) can counteract the inhibitory activity of streptomycin by continuing to grow further, thus reducing the zones of inhibition by this antibiotic. In the end, Streptomycin showed negative inhibitory activity against C. gloeosporioides, which suggests that it is inferior to the composition of the present invention in inhibiting the growth of such pathogen.

□ Unfiltered Composition Q Unfiltered Composition

Day 5 Incubation Day 10 Incubation

□ Concentrated Composition (WTO02) o Concentrated Composition (WT002) o WTD02 sample (1 :1) (extended period) Q WTO02 sample (1 :1)

Samples B Samples

Figure 1. Zones of Inhibition of Unfiltered Composition as compared with its concentrated version coded as WT002 sample against C. gloeosporioides during the 5^th (A) and 10 day (B) of incubation. Zones of inhibition of

diluted WT002 sample at a ratio of 1 : 1 with water (by volume) is also shown.

Furthermore, the inhibitory activity of the composition of the present invention against C. gloesporioides was compared with lactic acid at 10 ppm (which is the average concentration in composition), 5 ppm, and 2.5 ppm. Results as seen in Figure 2 show that the undiluted composition performed better than lactic acid in inhibiting the growth of C. gloesporioides. However, comparable inhibitory activities were observed with lactic acid when the composition is diluted with water at a ratio of 1 : 1 and 1 :3; but its activity did not persist as it dropped to zero on the fourth day. The same observations were noted for both lactic acid and streptomycin.

It must be noted further that the composition of the present invention is an extract from a fermented concoction of papaya and sugar, composed of alpha-hydroxy acids of which lactic acid is among them. With the poor inhibitory performance of pure lactic acid during the bioassays against C. gloesporioides, it means that the inhibitory activity of the composition cannot be attributed solely to the presence of lactic acid, but most likely to the whole cluster of alpha-hydroxy acids present in the composition which acted synergistically to inhibit the growth of such disease-causing microorganisms in fruits, vegetable, and crops. a Undiluted Composition D Composition (1 :1) □ Composition (1 :3)

D Lactic Acid (10 ppm) □ Lactic Acid (5 ppm) B Lactic Acid (2.5 ppm)

D Streptomycin (125 ppm)

Days of Incubation

Figure 2. Zones of Inhibition of the Composition of the present invention as compared with Lactic acid and Streptmycin against C. gloeospohoides during the 5-day incubation period.

In addition, Antracol , which is a known chemical fungicide for anthracnose, is used as positive control in this work, and its inhibitory activity against C. gloesporioides is compared with the composition of the present invention. Results show that the zones of inhibition of the composition is only half (average = 21.02 mm) that of the zone of inhibition of Antracol^®, which is on average 42.13 mm, at the highest formulation of Antracol^® at 65 g per 16 liter of water (see Figure 3). Diluting Antracol^® to 50g per 16 liter water and 58g per 16 liter water would still give very high zones of inhibition against C. gloesporioides, which is on average 38.55 mm and 38.77 mm, respectively. During the onset of the incubation, it is clear that Antracol^® is the better fungicide against C. gloesporioides as it has a very clear halo of inhibition against the pathogenic mold as compared with the composition of the present invention. However, it was observed that everyday thereafter during the incubation period, the zones of inhibition of Antracol decreased as the pathogen persisted to grow further leading to the decrease in its zones of inhibition. On the other hand, in the bioassays with the composition of the present invention, the zones of inhibition were constant, which suggests that it could have kept its efficacy during the entire five-day incubation period. This results in comparatively better zones of inhibition for the composition than Antracol^® at the end of the five-day incubation period. These observations suggest that Antracol^® may be an effective pesticide against C. gloesporioides on the onset, but it does not keep its efficacy for a longer period of time, which gives the pathogen the chance to persist itself and grow rapidly by then.

This observation resulted in a comparable inhibitory activity of Antracol^® with the composition of the present invention during the five-day incubation period. Extending the incubation period to 10 days led to the decrease in the zones of inhibition with Antracol^®, while that of the composition of the present invention, the zones remain constant (Figure 3). This indicates that the composition can inhibit the growth of C. gloesporioides better than Antracol^® during long incubation periods as it can keep its efficacy constant in time.

Figure 3. Zones of Inhibition of Antracol as compared with the Composition of the present invention and its concentrated verion (WT002 samples) against C. gloeosporioides

during the extended ten-day incubation period. Furthermore, Figure 3 shows the comparison between the zones of inhibition of Antracol , unfiltered composition, and the concentrated samples of the composition coded WT002 sample and diluted WT002 sample (1:1). From this figure, it is clear that the unfiltered composition of the present invention has comparable performance with Antracol^® in inhibiting the growth of the pathogenic mold during long incubation periods. In addition, the composition's concentrated samples coded as WT002 samples and its diluted form WT002 (1:1) samples in these tests are best in inhibiting the growth of the pathogen.

EXAMPLE 4: Efficacy for the control of post harvest diseases in mangoes

A study was conducted to evaluate the efficacy of the composition of the present invention in controlling the post-harvest diseases in mangoes especially anthracnose, caused by the mold Colletotrichum gloesporioides. Newly harvested mangoes were collected and simply dipped for 10 minutes in prepared treatment solutions (referred to in here as the composition of the present invention) in order to decrease if not eliminate the occurrence of anthracnose in mangoes at different ripening stages. A set of untreated mangoes was also included in the study as control. The first treatment solution was a suspension of 500 mL of the composition of the present invention mixed in 16 liters of water. The second treatment solution was a common chemical control (fungicide) at the recommended dosage. The third treatment solution was simply water heated up to about 40-50°C. During the test, a known number of newly harvested mangoes were dipped in each treatment solution and mangoes infected with anthracnose during the early ripening and late ripening stages were counted. Table 15 shows the results during this simple dipping test. The percentage of mangoes infected with anthracnose after dipping in a suspension of 500 mL product composition in 16 liters of water is comparable with that percentage dipped in a chemical control fungicide. This suggests that the composition of the present invention can be a good alternative to this chemical control as it is organic, natural and non-toxic.

Table 15. Disease infection of mangoes after the dipping test with different treatment solutions.

Treatment solution % Infection during % Infection during

Early Ripening Late Ripening

Composition of this invention (500 mL/16 liter water) 22.50 51.25

Chemical control fungicide 18.75 50.00

Hot water treatment 33.75 60.00

Untreated control 36.25 72.50

EXAMPLE S: Efficacy for the control of post harvest diseases in bananas

A study was conducted to evaluate the efficacy of the composition of the present invention in controlling the post-harvest diseases in banana^ (Musa cav f(dish) especially crown rot or anthracnose, caused by the molds Colletotrichum gloesporioides and Botryodiplodia theobromae and soft rot or fruit rot caused by Lasiodiplodia theobromae. Newly harvested Cavendish bananas were collected and simply dipped with the prepared treatment solutions (referred to in here as the composition of the present invention) in order to control the post-harvest disease in bananas at different ripening stages. A set of untreated bananas was also included in the study as control. The first treatment solution was a set of suspensions of 10 mL, 20 mL, 30 mL and 40 mL, respectively, of the composition of the present invention mixed in 1 liter of water. The second treatment solution was a common chemical control at the recommended dosage of 1 g alum per liter of water.

The first part of the results shows the percentage infection and severity of anthracnose disease caused by Colletotrichum gloesporioides during post-harvest of Cavendish bananas treated at different levels of the extract (composition of the present invention) at 7, 14, and 21 days after treatment referred to in Tables 16-18. The percentage infection and severity of disease usually decreased with the increase of the composition in the treatment solution applied. The percentage of bananas infected with anthracnose after dipping in a suspension of 40 mL composition in 1 liter of water, is most often comparable with the samples dipped in a chemical control fungicide after 7 to 21 days of treatment. Based on the results (see tables 16-18 below), the untreated control has always the highest percentage of infection and severity of disease, which indicates that post-harvest treatment is necessary to prolong the quality of the fruit.

Table 16. Percent Infection and Percent severity of anthracnose caused by Colletotrichum gloesporioides

Cavendish bananas 7 days after treatment (post-harvest dip).

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 33.33 1 1.11 Slight

20 ml composition /li H₂0 0.00 0.00 No Disease

30 ml composition /li H₂0 0.00 0.00 No Disease

40 ml composition /li H₂0 0.00 0.00 No Disease

1 g Alum/1 li H₂0 0.00 0.00 No Disease

Untreated Control 66.67 22.22 Slight

Table 17. Percent Infection and Percent severity of anthracnose caused by Colletotrichum gloesporioides

Cavendish banands 14 days after treatment (post-harvest dip).

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 100 33.33 Moderate

20 ml composition /li H₂0 66.67 22.22 Slight

30 ml composition /li H₂0 66.67 22.22 Slight

40 ml composition /li H₂0 33.33 11.1 1 Slight

l Alum/1 li H₂0 0.00 0.00 No Disease

Untreated Control 100 66.67 Severe Table 18. Percent Infection and Percent severity of anthracnose caused by Colletotrichum gloesporioides in Cavendish bananas 21 days after treatment (post-harvest dip).

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 100 77.78 Severe

20 ml composition /li H₂0 100 66.67 Severe

30 ml composition /li H₂0 100 55.56 Severe

40 ml composition /li H₂0 100 33.33 Moderate

1 g Alum/1 li H₂0 100 33.33 Moderate

Untreated Control 100 100 Severe

The second part of the results shows the percentage infection and severity of anthracnose disease caused by Botryodiplodia theobromae during post-harvest of Cavendish bananas treated at different levels of the extract (composition of the present invention) at 7, 14, and 21 days after treatment referred to in Tables 19-21.

Table 19. % Infection and % Severity of anthracnose by Botryodiplodia theobromae in Cavendish banana 7 days after treatment.

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 33.33 11.11 Slight

20 ml composition /li H₂0 0 0 No Disease

30 ml composition /li H₂0 0 0 No Disease

40 ml composition /li H₂0 0 0 No Disease

1 g Alum/1 li H₂0 0 0 No Disease

Untreated Control 100 33.33 Moderate

Table 20. % Infection and % Severity of anthracnose disease caused by Botryodiplodia theobromae of Cavendish Banana Fourteen (14) days after treatment.

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 100 44.44 Moderate

20 ml composition /li H₂0 100 33.33 Moderate

30 ml composition /li H₂0 100 33.33 Moderate

40 ml composition /li H₂0 33.33 1 1.11 Slight

1 g Alum/1 li H20 0 0 No Disease

Untreated Control 100 66.67 Severe

Table 21. % Infection and % Severity of anthracnose caused by Botryodiplodia theobromae in Cavendish banana twenty-one (21) days after treatment.

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 100 88.89 Severe

20 ml composition /li H₂0 100 66.67 Severe

30 ml composition /li H₂0 100 66.67 Severe

40 ml composition /li H₂0 100 55.56 Severe

1 g Alum/1 li H20 100 44.44 Modrate

Untreated Control 100 100 Severe The third part of the results shows the percentage infection and severity of soft rot or fruit rot postharvest disease caused by Lasiodiplodia theobromae on Cavendish banana at different levels of the composition of the present invention at 14 and 21 days after treatment referred to in Tables 22-23.

Table 22. % Infection and % Severity of soft rot caused by Lasiodiplodia theobromae in Cavendish banana

Fourteen (14) days after treatment with the composition of the present invention.

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 100 33.33 Moderate

20 ml composition /li H₂0 100 33.33 Moderate

30 ml composition /li H₂0 33.33 11.1 1 Slight

40 ml composition /li f¾0 0 0 No Disease

l g Alum 1 li H20 0 0 No Disease

Untreated Control 100 55.56 Severe

Table 23. % Infection and % Severity of soft rot caused by Lasiodiplodia theobromae in Cavendish banana Twen one (21 ) days after treatment.

Treatment % infection % severity Description of samples

10 ml composition /li H₂0 100 66.67 Severe

20 ml composition /li H₂0 100 66.67 Severe

30 ml composition /li H₂0 100 55.56 Severe

40 ml composition /li H₂0 100 44.44 Moderate

1 g Alum/1 li H20 100 33.33 Moderate

Untreated Control 100 100 Severe

Table 24. Visual quality rating (VQR) of Cavendish banana fruits treated at different levels of the composition of the present invention collected at 7, 14, and 21 days after treatment.

Treatment 7 days after treatment 14 days after treatment 21 days after treatment

10 ml composition /li H₂0 8.33 6.33^bc 3.67^c

20 ml composition /li H₂0 9.00 7.00^b 5.00^b

30 ml composition /li ¾0 9.00 7.67^ab 5.67^ab

40 ml composition /li ¾0 9.00 7.67^ab 5.67^ab

1 g Alum/1 li H₂0 9.00 9.00^a 6.33°

Untreated Control 7^67 4 3^ l.OO"

**Means with common letter do not differ significantly at 5% level (DMRT); ns - not significant

Table 25. Firmness of Cavendish banana fruits treated at different levels of the composition of the present invention at 7, 14, and 21 days after treatment.

Treatment 7 days after treatment ^m 14 days after treatment 21 days after treatment

10 ml composition /li H₂0 L3 2.67^ab 3.67^{ab ~}

20 ml composition /li H₂0 1.3 2.33^b 3.67^ab

30 ml composition /li H₂0 1.0 1.67^bc 3.33^abc

40 ml composition /li H₂0 1.0 1.00^c 2.67^b

1 g Alum/1 li H₂0 1.0 1.00^c 2.33^bc

Untreated Control 1.7 3.33^a 4.00^a

**Means with common letter do not differ significantly at 5% level (DMRT); ns - not significant Table 26. Storage life as affected by different levels of treatment with the composition of the present invention.

Treatment Storage life (days)

10 ml composition /li H₂0 14.7^de

20 ml composition /li H₂0 15.0^d

30 ml composition /li H₂0 19.3^abc

40 ml composition /li H₂0 20.7^ab

1 g Alum/1 li H₂0 22.0^a

Untreated Control 12.7^def

**Means with common letter do not differ significantly at 5% level (DMRT).

The same observations and results were seen in treatments with infection caused by Botryodiplodia theobromae and Lasiodiplodia theobromae. These suggest that the composition of the present invention can be a good alternative to the chemical control currently used as pesticide as it is organic, natural and non-toxic.

EXAMPLE 6: Efficacy against Tomato Yellow Leaf Curl Virus

A preliminary study on the efficacy of the composition of the present invention as viral control against Tomato Yellow Leaf Curl Virus (TYLCV) in tomato was done. The high risk of pest infestation in vegetables, specifically tomato is a never-ending problem of farmers nowadays. Thus, it is interesting to test quickly if the composition can be effective against such virus in tomato.

The study aimed to compare the degree of TYLCV pest infestation on tomato plants and growth performance of tomato in chosen areas treated and not treated with the composition (in this invention). An organic garden uniformly grown with tomato (in pots) was chosen for this test. The first set of 30 pots with One tomato plant per pot was treated with the composition (this invention) at a concentration of 5 mL per liter of water at weekly intervals and another set of 30 pots was not treated with the composition nor with any other pesticide.

Results

The tomato plants treated with the composition (of this invention) were observed to be healthy and robust as compared to the plants with no treatment. There was no sign of pest infestations in their leaves during the vegetative stage whereas untreated tomato plants slowly begin to show positive infestation with TYLCV.

During the reproductive stage, the treated tomato plants encountered a slight infestation as 20% of the plants were infected with TYLCV. However, the untreated tomato plants were severely infected as 90% of these plants were positively infected with TYLCV. As a result, more tomatoes were harvested from the treated plants than from the untreated plants. Although, further study needs to be done in order to compare efficacy of the composition of this invention with that of the commonly used pesticides for controlling TYLCV, this preliminary study strongly indicated that the composition of the present invention can already significantly reduce the infestation of TYLCV in tomato plants.

EXAMPLE 7: Efficacy against Fusarium oxysporum causing Panama wilt in Cavendish banana

A study was conducted to determine the efficacy of the composition of the present invention against the fungal pathogen Fusarium oxysporum causing Panama wilt in Cavendish banana. Laboratory bioassays were done in which zones of growth of the pathogen were measured. Six treatments were employed in the assays. The first (Tl) was the untreated control, the second (T2) treatment used a standard check (Antracol 50WP), the third (T3) used AZ41 (an organic foliar fertilizer with pesticidal properties) and the last three treatments (T4, T5, and T6) used the composition of the present invention at ratios of 1 :2 (1 mL composition in 2 mL water), 1 :5 (1 mL composition in 5 mL water) and 1:10 (1 mL composition in 10 mL water), respectively.

(A) First set of experiments:

For the first set of experiments, Table 27 presents the zones of growth of Fusarium oxysporum causing panama wilt of banana, after 4 days of incubation. The results showed that there were both significant and not significant differences between treatments. Among treatments with the composition of the present invention, the treatment at the ratio of 1 :5 (1 mL composition in 5 mL water) gave the lowest average mean of the zones of growth at 15.67 and it was found to be comparable to the standard check (Anthracol 50 WP) with an average of 12.17. Plates treated with the composition at the ratios of 1:2 (1 mL composition in 2 mL water) and 1:10 (1 mL composition in 10 mL water) however had higher zones of growth with an average mean of 21.50 and 23.83, respectively, which were comparable with the zones of growth using the treatment with AZ41 and the untreated control, having an average mean of 24.67 and 25.17, respectively.

However, after 10 days of incubation (Table 28), results revealed that the growth of the fungus was inhibited as shown in treatment T4 using the composition at a ratio of 1:2 (1 ml of composition in 2 ml water), in which the average mean of the zones of growth approached zero. But in other composition treated plates (T5 and T6), there was so much growth observed but both of them are comparable with the zones of growth of the standard check (T2 using Antracol 50WP). Treatment with AZ 41 gave higher zones of growth while the untreated control gave the highest zones of growth at 53.17 mm. Table 27. Zones of growth (mm) of Fusarium oxysporum causing Panama wilt in Cavendish banana after 4 days of incubation.

Treatment I // III Total Mean (mm)^*

Tl- Control 24.00 25.00 26.50 75.50 25.17b

T2- Stn ck 14.00 10.50 12.00 36.50 12.17a

T3- AZ41 22.50 25.00 26.50 74.00 24.67b

T4- 1 :2 18.00 13.50 33.00 64.50 21.50b

T5- 1 :5 19.50 16.50 11.00 47.00 15.67a

T6- 1 : 10 20.50 27.50 23.50 71.50 23.83b

*Means with common letter do not differ significantly at 5% level (DMRT).

Table 28. Zones of growth ( mm) of Fusarium oxysporum causing Panama wilt in Cavendish banana after 10 da; of incubation.

Treatment // III Total Mean (mm/

Tl- Control 50.00 55.00 54.50 159.50 53.17^d

T2- Stn ck 22.00 18.50 27.50 68.00 22.67^b

T3- AZ41 23.00 32.00 34.50 89.50 29.83°

T4- 1:2 0.00 0.00 0.00 0.00 0.00^a

T5- 1 :5 20.50 19.00 13.00 52.50 17.50^b

T6- 1: 10 20.00 30.00 21.50 71.50 23.83^b

*Means with common letter do not differ significantly at 5% level (DMRT).

(B) Second set of experiments

For the second set of experiments as shown in Tables 29 and 30, the zones of growth (mm) of Fusarium oxysporum causing panama wilt of Cavendish banana after 72 hours of incubation were presented. Statistical analysis revealed that that there was a significant effect between treatments after 72 hours (3 days) of incubation. The treatments (T4 and T6) using the composition at ratios of 1:2 (1 mL composition in 2 niL water) and 1 :10 (1 mL composition in 10 mL water), showed no growth of F. oxysporum, which were the same in the treatment with the chemical check (T2). The treatment (T5) at a ratio of 1:5 (1 mL composition in 5 mL water) gave an average mean zone of growth of 1.83 mm, followed by the treatment (T3) using AZ41 which gave an average mean zone of growth of 3.33 mm.

These treatments were all comparable to each other but numerically better than AZ41 (as the standard organic check). After one- week of incubation (see Table 30), Treatment 4 had no more growth of the fungus. However, treatment T5 exhibited a little zone of growth, followed by the treatment using a standard chemical check (T2), and treatment (T3) using AZ41 with the zones of growth ranging from 2.50- 10.00 which were nearly comparable to each other. Furthermore, after 15 days of incubation (Table 31), treatment T4 using the composition at a ratio of 1 :2 had no growth of the mycelium; while treatment T5 using the composition at a ratio of 1 :5 had an average mean zone of growth of 3.50 mm, which was nearly comparable to the result with treatment T2 using the standard chemical check which had an average mean zone of growth of 8.33.

The rest of the treatments gave much higher zones of growth after 15 days of incubation, with the untreated control registering the highest average mean zone of growth at 53.33 mm. The above results imply that the use of the composition of the present invention at ratios of 1:2 (1 mL composition in 2 mL water) and 1 :5 (1 mL composition in 5 mL water) were more effective in inhibiting the growth of Fusarium oxysporum causing Panama wilt in bananas than the use of standard chemical check and organic check.

Table 29. Zones of growth (mm) of Fusarium oxysporium causing panama wilt in Cavendish banana at different treatments after 72 hours (3 days) of incubation.

Treatment I // Ill Total Mean (mm)^*

Tl - Control 12 13.5 12 37.5 12.50^b

T2 - Stn ck 0 0 0 0 0^a

T3 - AZ4J 10 0 0 0 3.33^a

T4 - 1 :2 0 0 0 0 0^a

T5 - 1 :5 5.5 0 0 5.5 1.83^a

T6 - 1:10 0 0 0 0 0^a

*Means with common letter do not differ significantly at 5% level (DMRT).

Table 30. Zones of growth of Fusarium oxysporium causing panama wilt of Cavendish banana as affected by different treatments after 7 days (one week) of incubation.

Treatment / II III Total Mean (mm)^*

Tl - Control 30.00 22.00 19.00 71.00 23.67°

T2 - Stn ck 0.00 0.00 19.00 19.00 6.33^b

T3 - AZ41 30.00 0.00 0.00 30.00 10.00^b

T4 - 1 :2 0 0 0 0 0.00^a

T5 - 1 :5 7.5 0 0 7.5 2.50^a

T6 - 1:10 15.00 14.50 26.50 > 56.00 18.67^c

*Means with common letter do not differ significantly at 5% level (DMRT).

Table 31. Zones of growth of Fusarium oxysporium causing Panama wilt in Cavendish banana as affected by different treatments after 15 days (two weeks +) of incubation.

Treatment / // III Total Mean (mm)^*

Tl - Control 60.00 45.00 55.00 160.00 53.33^d

T2 - Stn ck 0.00 0.00 25.00 25.00 8.33^b

T3 - AZ41 35.00 12.00 14.00 61.00 20.33°

T4 - 1:2 0.00 0.00 0.00 0.00 0.00^a

T5 - 1 :5 10.50 0.00 0.00 10.50 3.50^Λ

T6 - 1 :10 23.00 17.50 35.00 75.50 25.17°

*Means with common letter do not differ significantly at 5% level (DMRT). EXAMPLE 8: Efficacy against Pseudoperotitispora cubensis causing downy mildew in fruits and vegetables

A short study was done voluntarily by some farmers in the countryside to check the efficacy of the composition of the present invention against Pseudoperonospora cubensis causing downy mildew, which is a very common disease in various fruits and vegetables of Cucurbits family such as honeydew, melon, and ampalaya. Farm plots planted with specific fruit or vegetable were prepared. At certain stages, according to normal farming practice, the plots were treated by spraying with the standard chemical pesticide to control the incidence of downy mildew disease. In this study, some plots were treated by spraying with the composition of the present invention and incidence of infection was compared with the farm plots sprayed with chemical pesticide as the normal farming practice. Determination of the incidence of infection and severity of disease was done by ocular inspection by the farmers themselves.

In plants, the infection of Pseudoperonospora cubensis causing downy mildew is characterized by the change of the pale green areas of the leaf to yellow angular spots. The spot is brighter on the upper leaf surface than on the lower leaf surface. The spots may turn brown or may remain yellow depending on the severity of the disease. During moist or rainy weather, the undersides of the spots are usually covered with the layers of the fungus and with such condition the entire leaf dies quickly. This leads to poor production or yield of the fruits or vegetables.

Table 32 below shows the percentage of crops infected by downy mildew before treatment. Results of the study show that at most 65% of the plants were infected by downy mildew prior to treatment with the appropriate pesticide. Table 33 showing the results in three days after the first spraying with the composition of the present invention indicates a slight decrease in the percentage of infection of the disease in various crops. Furthermore, as shown in Tables 34 and 35, the disease was dramatically controlled and after a week from the third spraying, the incidence of infection was reduced to practically zero (see Table 35). This indicates that the growth and proliferation of the causative agent of the downy mildew disease was inhibited by the treatment of plants with the composition of the present invention at the dosage indicated therein.

Furthermore, while there was infection of the disease in plants prior to treatment, this disease was controlled and the plants eventually regained its healthy characteristics upon treatment with the composition. This suggests that the composition, which also contains some nutrients as shown in Tables 1 and 3, may have some strengthening effect on the plants. These nutrients in the composition may have contributed to the repair and regeneration of new plant cells and tissues after the treatment period, thereby improving its immune system in fighting pathogens and in inhibiting its growth upon infection. Table 32. Percentage Infection of plants by downy mildew before treatment.

Crops % Infection Description of infection

Honeydew 35% moderate

Melon 40% moderate

Ampalaya 65% Severe

Table 33. Percentage Infection of plants with Downy mildew 3 days after 1st spraying with the composition of the present invention.

Crops Treatment % Infection Description of infection

Honeydew 15 mL composition in 16 L water 31% moderate

Melon 15 mL composition in 16 L water 35% moderate

Ampalaya 15 mL composition in 16 L water 55% Severe

Table 34. Percentage Infection of plants with Downy mildew 7 days after 2nd spraying with the composition of the present invention.

Crops Treatment* % Infection Description of infection

Honeydew 15 mL composition in 16 L water 10% Slight

Melon 15 mL composition in 16 L water 13% Slight

Ampalaya 15 mL composition in 16 L water 23% Slight

*Treatment solution added with 10 mL All-Purpose Surface Adjuvant (APSA 80) as sticker.

Table 35. Percentage Infection of plants with Downy mildew 7 days after 3rd spraying with the composition of the present invention.

Crops Treatment* % Infection Description of infection

Honeydew 15 mL composition in 16 L water 0% no more fungus

Melon 15 mL composition in 16 L water 0% no more fungus

Ampalaya 15 mL composition in 16 L water 5% slight

"treatment solution added with 10 mL All-Purpose Surface Adjuvant (APSA 80) as sticker.

EXAMPLE 9: Efficacy against bacterial leaf blight in rice

Rice (Oryza sativa L.) is one of the most important crops of the world as it is the staple food for millions of people. Many farmers depend on rice for trading and source of livelihood. Rice is grown in practically any place with sufficient supply of water for irrigation. Although its species are native to south Asia and certain parts of Africa, centuries of trade and exportation have made it common place in many cultures.

Bacterial leaf blight (BLB) is a monsoon disease in rice and its incidence and severity is very much influenced by rainfall, number of rainy days, and susceptibility of the cultivar and nitrogen fertilizer application. Severe epidemic of BLB in rice can lead to drastic reduction in grain yields and productivity. Bacterial leaf blight appears on leaves of young plants after planting as pale-green to grey-green water-soaked streaks near the leaf tip and margins. These lesions coalesce and become yellowish- white with wavy edges. Eventually, the whole leaf may be affected, becomes whitish or grayish and then dies. Leaf sheaths and culms of the more susceptible cultivars may be attacked. Systemic infection results in desiccation of leaves and death, particularly of young transplanted plants. In older plants, the leaves become yellow and then die. In later stages, the disease may be difficult to distinguish from bacterial leaf streak.

This study was conducted to evaluate the efficacy of the composition of the present invention to inhibit the growth of Xanthomonas oryzae pv. oryzae (Xoo) causing BLB of hybrid rice (PSB Rc72H) in vitro and in vivo test, and to determine the most effective rate of the composition of the present invention in the management of BLB.

The bacterium Xanthomonas oryzae pv. oryzae causing bacterial leaf blight of rice are rods, 1.2 x 0.3-0.5 μιη. They are single, occasionally in pairs but not in chains. They are gram negative, non-spore-forming, and devoid of capsules. Their colonies on nutrient agar are pale yellow, circular, and smooth with an entire margin. They are convex and viscid. The bacterium or pathogen enters the leaf tissues through natural openings such as water pores on hydathodes or stomata on the leaf blade, growth cracks caused by the emergence of new roots at the base of the leaf sheath, and on leaf or root wounds. Once the bacterium enters the water pore or any openings, it multiplies in the epitheme, into which the vessel opens. When there is sufficient bacterial multiplication, some bacteria invade the vascular system and some ooze out from water pore.

The use of resistant varieties is the most effective and the most common management practices adopted by farmers in most growing countries in Asia. When different strains of bacteria are present, it is recommended to grow resistant varieties possessing field resistant genes. Fallow field which have provisions to allow to dry thoroughly is recommended. Chemicals such as the combination of zinc sulfate and copper sulphate are also suggested for use. Zinc sulfate also known as white vitriol is especially applied to crops as fertilizer. It is highly applied to crops as fertilizer. It is highly soluble which makes it favorable for delivering zinc values to crops. Seed treatment with 2% of this chemical is proven effective against BLB of rice. On the other hand, copper sulfate is a wide-spectrum fungicide and bactericide. This can denature cellular proteins and deactivate enzyme system, and also kills slugs and snails in irrigation and municipal water treatment systems.

Preliminary trials were conducted on ten antibacterial compounds for their efficacy against panicle blight. This includes antibiotics, copper and copper containing compounds, other organic compounds and fungicides used on rice. The pesticides that appeared to give measurable control either as seed treatments or foliar sprays were Axolinic acid, Top cop, Kocide 2000 and Tomaxil. Today, organic- based farming is gaining popularity. An example of this is the X-tekh which has the potential as a bactericide and fungicide. X-tekh contains many kinds of beneficial microorganisms. When the liquid fertilizer is applied in the soil, the microorganisms cluster around the root systems and symbiotically helps the introduce the macronutrients (nitrogen, phosphorus and potassium) as well as the micronutrients through nitrogen fixation, phosphate solubilization, and some other processes. At the same time, there are other important microorganisms that protect plants from parasitic fungi, bacteria, nematodes and other soilborne diseases.

It is further explained that X-tekh contains phosphate solubilizing bacteria which can solubilize thi insoluble phosphates, making them available for plant use. These microorganisms ( Pseudomonas, Bacillus, Aspergillus, and green Pennicillium) produce organic acids, i.e., lactic, gluconic, fumeric, succinic, and acetic acids which solubilize the insoluble phosphates.

Experimental Design: In vitro Test

The experiment was arranged in Completely Randomized Design (CRD) with 11 treatments replicated three times. Different rates of the composition was used both in vitro and in vivo test against Xoo causing BLB of rice. Treatment rates of application (mL composition per L of water) were Tl = 10, T2 = 20, T3 = 30, T4 = 40, T5 = 50, T6 = 60, T7 =^■ 70. Treatment T8 = 2 mL Cupric Hydroxide (as an inorganic standard check) per L water, T9 = 3 mL of X-tekh (as an organic standard check), and T10 as the untreated control using only sterile distilled water (SDW).

Collection of Specimens

Bacterial Leaf Blight (BLB) of rice was collected in the field affected with the disease and was brought to the Plant Pathology Laboratory for diagnosis of the causal pathogen. The specimens were examined under the microscope.

Preparation of Culture Media

Potato Dextrose Peptone Agar (PDPA) was prepared following the standard procedure using the following: 200 g potato, 10 g dextrose, 20 g agar, 10 g peptone and 1 L of distilled water. The flat bottles were filled with 20 mL of PDPA, plugged and autoclaved at 121°C and 15 psi for 20- 30 min, and then cooled to 40°C. The PDPA was allowed to congeal and stored at room temperature. Nutrient Agar (NA) was used for the mass production of the pathogen. This was prepared by using 1L Sterile Distilled Water (SDW), 17 g agar, 3 g beef extract and 10 g peptone. Isolation of the Pathogen

Isolation of pathogen was done by washing the infected leaves and allowed to ooze in SDW place in test tube for 3-5 min. The cut portion was observed against the light microscope to see the bacterial ooze streaming out from the cut ends into water. After slightly turbid, bacterial suspension was obtained. A loopful was streaked into plated PDPA medium in replicated plates. Fluidal single yellowish colonies was picked up after 3-5 days incubation and transferred to NA for mass production.

Preparation of Materials for Bioassay Test

Petri plates, wire loop, paper disk, beaker, pipettes and syringe was sterilized at 15 psi or 1200 °C for 20-30 min including the PDPA and NA before conducting bioassay test. A two day old culture of Xoo was used to prepare 106 cells/ml of suspension of the pathogen.

Bioassay of the Pathogen

Paper disc method was used. This was done by dispensing the NA in the plates in uniform thickness. One ml suspension of the pathogen was poured in the plate containing 20 ml agar and then partially rotated so that the medium and the pathogen was evenly distributed. Then, the paper disk was dipped into the treatments at different rates of concentration and then placed at the center of plates. Control was included for comparison. The plates was inverted and the incubated for 24-72 hours.

Experimental Design: In Vivo Test

The experiment was laid out in a Randomized Complete Block Design (RCBD). All treatments tested in vitro were further tested in vivo against Xoo causing BLB of rice. The ten treatments was replicated three times.

Land Preparation

The experimental area was ploughed two times and it was harrowed once to ensure good growth of rice and to control weeds. After which, granular fertilizer was applied at the rate of 500 kg per ha. A 50 cm alley was provided between plots for maintenance and ease in data collection.

Seed Preparation

The test seeds of hybrid rice (PSB Rc72 H) were soaked for 12 hours. Seeds were incubated for 24 hours before sowing in a 150 sq m elevated seedbed.

Pulling-out and Transplanting of S edlings

Seedlings were pulled out gently from the seedbed at 22 days after sowing and were transplanted at 20 cm x 20 cm. Crop Protection

Molluscicide was applied immediately after transplanting at the rate of 50 mL ICC perl 6 L water followed by pre-emergence herbicide (ROGUE) at the rate of 1.0 L per hectare at 3 DAT. Application of urea was done at 14 and 45 DAT and at 5% flowering. Before booting stage Mipcin and Furadan 3G at the rate of 20 kg per hectare was applied, at flowering stage spraying of Padan systemic insecticide was done. At milking stage, spraying of Magnum insecticide was done early in the morning or late afternoon. Lannate insecticide was applied. Spot weeding was done.

Application of Treatments

Eradicative method of application of treatments was done. First application was done at 14-21 DAT and every 5 days interval thereafter until the crop will reached its re-bloom and bloom stage. There were two applications of treatments when the crop is on ripening stage until harvesting.

Experimental Data Gathering

Diameter Zone of Inhibition. The measurement of diameter (mm) zone of inhibition (DZI) was taken after 24 hours of incubation. Efficacy of different levels of treatments was evaluated using the following arbitrary scale: 0-10 Not Effective (NE), 11-20 Moderately Effective (ME), 21-30 Effective (E), 31-above Very Effective (VE).

Number of days to symptom appearance. The natural occurrence of symptoms of BLB after transplanting of test plants were observed and recorded.

Severity infection. Severity infection of BLB was taken using the scale as Percent Infection (%): 0 no incidence, 1 less than 1%, 3 from 1 - 5%, 5 from 6 - 25%, 7 from 26 - 50%, 9 from 51 - 100%.

Number of productive and unproductive tillers/20 hills. Twenty hills per treatment were assessed at random in three replication. The number of productive and unproductive tillers per hill were recorded.

Number of filled and unfilled grain. Filled and unfilled grains was also assessed in twenty random hills of per treatment and in all three replications.

Grain Yield. The yield of rice was taken after harvesting. The weight of the seeds was taken according to treatments and converted to tons per hectare using the following formula: Yield (t/ha) = (kg of harvested grain per sq m area) x (1000 sq m/ha Area/1000 kg/t) In Vitro Test: Bioassay of Xoo

Table 36 presents the mean DZI of different rates of the composition and its degree of efficacy after 24 hr of incubation in vitro. Results revealed highly significant differences among treatment means. Plants applied with 10 ml of the composition per L water exhibited the highest DZI mean of 68.75 which was rated very effective (VE) and was comparable to plants apply with 40, 50, 60,70 mL composition per L and to Kocide (chemical check) with means ranging to 35.25 mm to 52.67 mm. Plants applied with 20, 30 mL of composition per L of water was rated Effective and comparable to results with X-tekh (an organic standard check) and to Kocide (a chemical standard check). The samples with untreated control failed to inhibit the growth of Xoo.

Results revealed efficacy of the six treatment rates of the composition as potential bactericide which are as effective as the treatment with organic and inorganic checks (X-tekh and Kocide) against Xoo causing BLB of rice. This result further confirms earlier studies (as shown in previous examples) that the composition of the present invention was effective against bacterial pathogens. This study also supports earlier in-house reports that lactic acid showed efficacy in inhibiting the growth of B. glumae causing panicle blight of rice and maybe utilized as substitutes to antibiotics and synthetic chemicals which are health hazardous and expensive.

Table 36. Mean DZI (mm) of Xoo causing BLB of rice at different treatments.

Treatment Diameter Zone of Inhibition (mm) * Degree of Efficacy*

10 mL composition per L water 68.75^a VE

20 mL composition per L water 27.08^bc E

30 mL composition per L water 23.91»^» E

40 mL composition per L water 44.17^ab VE

50 mL composition per L water 53.00^ab VE

60 mL composition per L water 35.25^ab VE

70 mL composition per L water 32.67^ab VE

Kocide (chemical check) 52.67^ab VE

X-Tekh (organic check) 29.67^bc E

Untreated control 0 -

^♦Means followed by a common letter superscript are not significantly different at 1% level of DMRT.

**Efficacy Rating: NE = Not Effective, ME = Moderately Effective, E = Effective, VE=Very Effective

DS A of BLB

The number of days to symptom aprearance (DSA) of bacterial leaf blight (BLB) on the hybrid rice (PSB Rc72H) as influenced by five round of eradicative application against Xoo is shown in Table 37. No significant differences among the treatments were observed. Test plant from all treatments exhibited similar number of DSA of the target diseased. This implies that a susceptible variety was subjected the natural BLB infections resulted to the even distribution of initial infection which warranted eradicative application of treatments. Symptoms appear on susceptible test variety (PSBRc72H) at the 8-DAT. % DI and % DC

Table 37 shows highly significant results with treatment at 40 mL of composition per L of water as it indicated lower percentage of disease incidence (% of DI), which are comparable to results of treatment with 10, 30, 50, 60, 70 mL of the composition per L water and to the treatments with X-tekh (as organic check) and Kocide (as chemical check) after five rounds of eradicative application. All treatments exhibited lower mean % DI of BLB with mean ranging from 52.96- 61.85% as compared to untreated control plants which had the highest mean of DI of 78.15 %. The above results support the outcomes of previous in-house studies in which rice treated with lactic acid manifested lesser severity of infection of panicle blight and comparable to treatments using a chemical standard check. The percentage degree of control (% DC) of BLB on PSBRc72H as influenced by eradicative application of the composition against Xoo is shown in Table 37, which indicated highly significant differences among treatments. Treatment with the composition at 40 mL per L of water showed highest % DC with a mean of 34.25%, followed by treatments with the composition at 10, 30, 50, 60, 70 mL per L water, with respective means of 30.08%, 30.09%, 31.02%, 29.18%, and 29.59%, and were all comparable to the plants treated with X-tekh and Kocide (as standard checks) with the means of 29.53% and 30.35, respectively. Treatment with the composition at 20 ml per L of water has lower degree of control with the mean 20.37%. The untreated plants have always the lowest degree of control of the disease. This also lend support to the findings that X-tekh stimulates natural biological activity in the soil introducing population of healthy microorganism breakdown and conversion of organic matter as source of nutrients for plants from disease caused by harmful conditions in the soil.

Table 37. Number of days to symptom appearance (DSA), initial and final % severity Infection of PSB Rc72H and percentage degree of control (DC) as influenced by five eradicative application of the composition of the present invention against Xoo causing BLB of rice.

Treatment DSA™ %D (initial)* % D (final)* %DC*

10 mL composition per L water 9.33 20.74^bc 54.07° 30.08³

20 mL composition per L water 10.67 20.74"° 61.85^b ' 20.37^b

30 mL composition per L water 10.67 22.96^b 55.93° 30.09³

40 mL composition per L water 10.33 52.96^c 52.96° 34.25^a

50 mL composition per L water 12.00 23.70^b 55.56° 31.02^a

60 mL composition per L water 11.67 21.11^bc 54.80° 29.18^a

70 mL composition per L water 11.33 24.07^b 54.44° 29.59^a

Cupric Hydroxide (chemical check) 10.67 17.41° 53.70° 30.55^a

X-Tekh (organic check) 11.00 \9.6?P* 55.56° 28.23^ab

Untreated control 8.67 32.59^a 78.25^a —

^♦Means followed by a common letter superscript are not significantly different at 1% level of DMRT; ns = not significant

Productive Tillers and Non Productive Tillers

Significant differences among the treatments were observed after five rounds of eradicative spraying for productive and non-productive tillers as shown in Table 38. All treatments with the composition have higher productive tillers which are comparable to both the treatments with organic and inorganic checks. Consequently, they give lower non-productive tillers as compared to the untreated control.

Filled and Unfilled grains

Table 38 showed the percentage of filled and unfilled rice grains (PSBRc72H) as influenced by the different treatments with the composition. The highest % filled grains was found on plants applied with Cupric hydroxide (an chemical check) and comparable to plants applied with 10 and 40 ml compositions per L of water. These were followed by plants applied with 60 ml composition per L of water which were comparable to plants applied with 30 mL and 50 mL of composition per L of water and to treatment with X-tekh (an organic check). The percentage filled grains have means ranging from 87.23-88.10%. Lowest filled grain was obtained from untreated plants.

Dry Weight (1000 grains) And Yield (t/ha)

Table 38 also reveals the mean dry weight and yield (t/ha) of rice grains. There were no significant differences among test treatments on dry weight (1000) of grains. Results imply that all treatments with the composition had not much influence on this parameter. The same results were observed on the grain yield in tons per hectare. All plants treated with the composition have the same high mean of grain yields and were comparable to plants treated with the standard checks with means ranging 4.45-6.10. Lowest yield was obtained from the untreated plants with a mean of only 1.73 t ha.

The results further imply a significant influence of the five eradicative applications of the seven treatment rates of the composition on yield performance of the treated plants as compared to the standard checks. Increasing yield was observed also in treated plants compared to standard checks and untreated control.

Occurrence of Other Pests and Diseases

Occurrence of other pests and diseases were observed during the whole duration of the study. Occurrences of rice black bug, rice stink bug, dead hearts and presence of leaf spot of rice were observed.

Cost - Benefit Analysis

Table 39 shows an attempt to conduct a cost-benefit analysis per hectare of rice production using the composition as treatment and corripared with the treatments with standard ciiecks as the normal farming practice. With all significant parameters considered, results *eveatecl that highest profit may be -realized from the treatment with the composition at 40 ml per L "water. The rest of the treatments showed lower net returns but are still significantly higher than with the untreated control. Results further imply that treatment with the composition of tne present invention, particularly at a treatment rate of 40 mL per L of water, was not only effective in reducing severity infection of BLB on PSB Rc72H but also significantly increase grain yield of rice.

Table 38. Mean number of productive tillers and unproductive tillers, percentage filled and unfilled grains, dry weight of 1 ,000 grains (g) and grain yield after five rounds of eradicative applications of different rates of the composition as compared to standard checks.

Treatment Productive Unproductive Filled Unfilled DW Grain tillers (%) tillers (%) Grains/1000 Grains/1000 seeds Yield seeds (%) seeds (%) ω (t/ha)

10 mL comp L H₂0 18.83^ab 1.93" 90.63"" 9.43"¹ 14.10³ 5.90³

20 mL comp/L H₂0 18.73^ab 1.92^b 85.90^d 14.10^b 16.00^a 4.70³

30 mL comp/L H₂0 18.98" 1.97^b 86.43"¹ _{12 J7}bc 12.77^a 5. IT

40 mL comp L ¾0 18.75^ab 2.00" 91.90^ab 8.70^de 15.37^a 6.10³

50 mL comp L H₂0 18.68^ab 1.95 88.07^te 11.60*^* 16.33^a 5.31"

60 mL comp/L H₂0 18.98^a 1.83^b 88.10^ _{13 j 7}bc 13.57³ 4.45³

70 mL comp/L H₂0 18.23^b 2.12^b 86.83^d 13.17 16.50³ 5.31³

Cupric Hydroxide 18.97³ 1.93^b 91.97^a 8.03^e 10.03³ 6.09³

(chemical check)

X-Tekh (organic 18.70^ab 1.97^b 88.03^bc 11.97^b 15.30³ 5.47^a check)

Untreated control 17.15^c 2.90^a 77.37^e 22.63^a 20.97 1.73^b

♦Means followed by a common letter superscript are not significantly different at 1% level of DMRT.

Table 39. Cost-benefit analysis for a hectare of production of rice (PSB Rc 72H) using treatments with the composition as compared to standard checks.

Particulars Treatments

1 2 3 4 5 6 7 8 9 10

Yield (t ha) 5.90 4.70 6.10 5.77 5.31 4.45 5.31 6.09 5.42 1.73

Gross Income 59.0 47.0 61.0 57.7 53.1 44.5 53.1 60.9 54.2 17.3

(Thousand Pesos)

MOOE (Thousand Pesos)

Land Preparation 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00

Pulling & 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00

Transplanting

Spraying 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00

Maintenance 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50

Fertilization 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20

Pesticides 3.68 3.68 3.68 3.68 3.68 3.68 3.68 3.68 3.68 3.68

Fertilizers 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0

Treatments 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6

Total Costs 25.98 25.98 25.98 25.98 25.98 25.98 25.98 25.98 25.98 25.98

Net Income 33.02 18.26 41.22 30.52 25.52 16.52 24.72 39.08 26.80 7.08

Assumption: Rice grain per Kilo = P 10.00; Treatments: Composition = ΡΙ,ΟΟΟ/L; X-Tekh = PI, 300/L; Cupric Hydroxide = P760/kilo; Fertilizer (Urea) = P 250/kg; Ammosol = P 40/kg; Manosul = P45/kg; Pesticide (Lannate, Furadan, Padan, & Magnum) = P 460/kg

Legend: Tl - 10 ml/L water; T2 - 20 ml/L water ; T3 - 30 ml/L water; T4 - 40 m L water; T5 - 50 ml/L water; T6 - 60 rnl/L water; T7 - 70 ml/L water; T8 - 2ml/L water (Cupric Hydroxide); T9 -X-tekh 3.00ml/L water; 10 - Untreated control Summary and Conclusions

The results revealed are summarized as follows:

1. In vitro test using the treatment with the composition at different rates against Xoo revealed comparable effects with the standard organic and inorganic check. All test treatments were found effective to very effective. These treatments exhibited significantly higher diameter zones of inhibition (DZI) means compared to the untreated control after 24 hr of incubation.

2. In Vivo test revealed the following:

a) No significant differences on the number of days to symptom appearance (DSA) on the test plants treated with different test treatments. Symptoms appeared simultaneously from 8-14 days after transplanting (DAT).

b) The percentage of disease incidence (% DI) of BLB on Rice (PSB Rc72H) showed highly significant differences in all treated plants with different rates of the composition. Highest % DI was expressed by the untreated control. Significantly lower % DI was observed on treatments using the composition at different rates which were comparable with the standard checks, except plants treated with the composition at 20 mL per L of water but lower than untreated control. Lowest % DI was observed with plants treated with the composition at 40 mL/L water.

c) Highly signinficant differences were observed on the percentage degree of control (% DC) of test treatments. Highest % DC was demonstrated by the treatments with the composition at 40 mL L water. Except with the treatment using the composition at 20 mL L water, all remaining treatments were comparable to standard checks.

d) Highest number of productive tillers was produce by plants treated with the composition at 60 mL/L water and the lowest tillers were produced by untreated plants. Highest mean number of unproductive tillers was produce by untreated control plants and conversely, plants treated with the composition at 60 mL/L water produced the lowest mean of unproductive tillers.

e) Highest % of filled grains was found on plants treated with cupric hydroxide and were comparable to plants treated with the composition at 10 and 40 mL per L of water.

f) Highest % mean of grain yield was obtained by the treatment with the composition at 40 mL/L water. All test treatments with the composition were comparable with the organic and inorganic checks. Lowest % mean of grain yield was obtained by untreated plants. No significant differences on dry weight of grains were observed among treated plants. Based on the results above, it could be concluded that BLB of PSB Rc72H caused by Xoo could be managed in both in vitro and in vivo by five eradicative applications of the composition at 5-7 days interval at the optimum rate of 40 mL L water; hence, would not only be a good substitute to synthetic chemicals that are hazardous to health but also considered as ecologically safe and nontoxic to human and animals. EXAMPLE 10: Field trials in 26-hectare plantation Against Black leaf streak or Black sigatoka disease in Cavendish banana

Sigatoka disease control in Cavendish banana production remains one of the major concerns in banana export industry. Thus, the search for cheaper and safer fungicides is a continuing effort of all plantations and growers. Environmental concern due to hazards of this pesticide is locally a growing controversy. ^"While globally, the demand for pesticide-residue free and organic agricultural food products is getting popular and priced higher. Hence evaluation of new, organic fungicide is recently becoming a must and a trend in the industry.

The composition of the present invention which is a fermentation extract from tropical fruits, previously found with fungicidal actions against Sigatoka pathogen, Mycosphaerella fijiensis under laboratory and field conditions is further evaluated under wider field conditions. It is noted that the composition is also known to contain some macro and micronutrients (see Table 3) like potassium, magnesium, calcium, and sodium, which are necessary nutrients for banana plantations. The composition is directly applied on the plant leaves, in which the components are directly absorb into these leaves, thus providing the necessary constituents in repairing and regenerating plant cells, thereby, boosting the immune system of the plants to tolerate the damage of disease infection. Thus this wider field trials in which the composition of the present invention is applied is seen to verify its efficacy against Sigatoka disease of Cavendish banana in a wider scale of field trials.

Materials and methods

The efficacy evaluation of the composition was done employing the rate of 0.75 L per hectare to control the black leaf streak (BLS) or Sigatoka disease of Cavendish banana. This was conducted in comparison with the standard aerial spraying scheme in the plantation whereby chemical pesticides are used.

The composition was applied employing 23 liters spray solution per hectare mixed with Banole at 7 L/ha and 1% Lutensol for eight (8) straight cycles at six (6) days interval. The standard spraying scheme used different fungicides and fungicide combinations with systemic and contact actions in the whole 8-cycle period, with the following sequence: Opal/Mancoseb combination, Antracol, Tridemorph/Mancoseb, Mancoseb, Antracol, Baycor Mancoseb, Mancoseb and Antracol. The interval of application was also usually 6 days except for 2 cycles with 8 and 10 days interval. Mixing of spray solutions and spray applications usually took place early in the mornings.

The area treated with the composition was isolated by two (2) big canals at both sides measuring 100 meters long with about 5-8 meters wide, while one end along banana area is bounded by an area about 340 meters long and 10 meters wide manually sprayed for Sigatoka disease control to minimize spray drift.

Pre-treatment data in Table 40 were taken on YLVS (youngest leaf with visible streaks), YLS (youngest leaf-spotted), transition period (EVS-YLS), number of functional leaves at shooting and at harvest, % infected leaf/plant and % rate of leaf loss, which are the parameters used in this wider field trial efficacy evaluation. These data were taken one (1) week after first application and weekly thereafter up to 2 and 3 weeks after last application. Sources of data were taken from fifteen (15) sample plants per station at 5 plants per row from a total of 3 sampling stations throughout the trial area. Data were subjected to statistical T-test analysis.

Results and discussions

From the collected results, the wider field trial efficacy evaluation of the composition showed significant outcomes in comparison with the standard spraying scheme of alternate use of different fungicides and their combinations to control black leaf streak (BLS) or Sigatoka disease of Cavendish banana.

Table 41 showed the averages of different Sigatoka parameters used in this evaluation between the treatments with the composition versus the Standard checks with statistical differences except for one (1) parameter. The data showed the Standard with significantly higher leaf numbers with early visible streak (EVS) and with early spots (YLS) of 3.97 and 12.38 respectively against 3.88 and 11.34 of the treatment with the composition. The higher leaf numbers in these parameters indicate better control of the disease. Furthermore, the transition period of the disease development from early visible streak to early spots symptoms took 59.34 days in the Standard which is significantly longer than 52 days in the treatment with the composition. Again, the longer the transition period of the disease from early visible streak to early spots, the more efficient is the control. The average percentage of infected leaf per plant is the only parameter with no significant difference from each other. Although the treatment with the composition has numerically higher infection of 14.20%, this is statistically comparable with 12.90% of the treatment with the standard, implying the efficacy of the composition in controlling the Sigatoka disease in the plantation.

The average functional leaves at harvest surprisingly gave 6.70 for the plants treated with the composition, which is significantly higher than 6.17 of the treatment with the standard. The data suggest better performance of the composition in controlling the Sigatoka disease, having tolerated the impact of the disease itself as manifested on younger leaves with visible streaks and early spots, shorter transition period of EVS - YLS, and lower functional leaves at shooting. Being considered the most important and ultimate parameter to determine the efficacy of the product, the treatment with the composition shows some unique action of tolerance on the plants against the damaging impact of the disease on the leaves. This is supported by statistically lower rate of leaf loss of 4.23% in the plants treated with the composition due to the disease against 5% of that in the plants treated with the standard. Figure 4 below strongly shows the increasing and higher number of functional leaves at harvest in the plants treated with the composition over that treated with the standard, even after the 8 cycles of its application. It is obvious that the residual activity of the composition on the leaves continuously influenced the increasing and higher number of functional leaves over that of the standard, even weeks after its last application. It is more obvious within 5 weeks after last application of the composition and beyond which remained higher over that treatment with the Standard spraying scheme. Figure 4a further confirms that the number of functional leaves at harvest of the treatment with the composition abruptly soared to 9.02 in Week 23 from only 7.78 in Week 22 after the treatment with the composition was re-applied in Week 20. It is obvious that re-application of the composition strongly affect the sharp increase in the number of functional leaves at harvest. It is noted in Figures 4 and 4a that increase in the functional leaves started 4 weeks after application of the composition, with the effect of the treatment with the composition manifested at least 4 weeks after application. The constituents found in the composition were deduced to have influenced the increase in the number of functional leaves at harvest as compared to the usual treatment with the Standard chemicals. The mineral nutrients in the composition such as potassium, manganese, calcium and magnesium could have boosted the repair and regeneration of plant cells which cause the leaves to tolerate the damaging impact of the disease. It might have strongly boost the immune system of the plants, hence retaining their leaves up to harvest which are crucially needed for its full fruit development into the desired maturity and quality acceptable in the market.

The effects of rainfall and number of rainy days on the trends of the different parameters are presented in the figures as well. With relatively lower rainfall and number of rainy days the effect are not strong and distinct. However, there are indications that at higher and increasing rainfall, the treatment with the composition seemed to have a weaker efficacy as indicated by increased infection and shorter transition period over the treatment with the standard chemicals as shown in the following figures, particularly in Weeks 9, 10 and 11.

Summary and Conclusions

The wider field trial efficacy evaluation of the treatment with the composition at 0.75 L per hectare was conducted in comparison with the treatment with the Standard chemical spraying scheme of the plantation consisting of different fungicides and their combinations against Black sigatoka disease of Cavendish banana by aerial application in 26 hectares of plantation.

Based from these field trials, the composition showed some unique performance against Sigatoka disease different from the conventional systemic and protectant fungicides. It has tolerated the impact of infection and retained more functional leaves at harvest which is the ultimate parameter for an effective Sigatoka control. Being an organic fungicide, it may not possess quick and direct action against the fungal pathogen but by direct application to the leaves it builds-up activity and boosts the immune system of the plants to tolerate the damage of infection. Hence plants retained the functional leaves at harvest which is the most important end result in controlling Sigatoka disease in Cavendish banana, for the full development of fruits into its desired quality acceptable in the market. The macro and micronutrient contents of the composition such as potassium, manganese, calcium and sodium maybe the factors which helped boost the immune system of the plants. The composition therefore in this wider field trial performed effectively against Sigatoka disease in Cavendish banana in containing the disease and maintaining the desired number of functional leaves at harvest.

Table 40. Pre-treatment data during the Comparison of Average Sigatoka disease control parameters of the treatment with the composition versus treatment with Standard chemical pesticides (as a normal famer's practice) in a 26-ha banana plantation.

Parameters T reatment with the Treatment with the

Composition Standard chemicals

1. Early Visible Streak (EVS) (Leaf Number) 3.80 3.97

2. Youngest Leaf Spotted (YLS) (Leaf Number) 11.00 11.19

3. % Infected Leaf/Plant (%) 13.00 16.00

4. Transition Period (EVS- YLS) (No. of Days) 47.37 48.97

5. No. of Functional Leaves at Shooting 12.80 13.41

6. No. of Functional Leaves at Harvest 6.60 6.03

7. Rate of Leaf Loss (%) 1.16 5.49

Table 41. Comparison of Average Sigatoka disease control parameters of the treatment with the composition versus treatment with Standard chemical pesticides (as a normal famer's practice) in a 26-ha banana plantation.

Parameters Treatment with the Treatment with the

Composition Standard chemicals

EARLY VISIBLE STREAK (EVS)

(Leaf Number) 3.88 3.97*

TRANSITION PERIOD (EVS-YLS)

(No. of Days) 52 59.34*

YOUNGEST LEAF-SPOTTED (YLS)

(Leaf Number) 1 1.34 12.38*

NO. OF FUNCTIONAL LEAVES

(At Shooting) 12.62 13.50*

INFECTED LEAF PLANT

(Percent) 14.20^ns 12.9

NUMBER OF FUNCTIONAL LEAVES

(At Harvest) 6.70* 6.17

RATE OF LEAF LOSS

(Percent) 4.23 5.00*

* Statistically significant; ns - not significant. Recommendations

From the study, it was recommended that the composition be used as an alternate product for use in the spraying program for Sigatoka control in Cavendish banana employing a rate of 0.75 L per hectare during low disease pressure and 1.0 L per hectare at high disease pressure. It is also recommended to verify further the booster effect of the composition in containing the disease and retention of the number of functional leaves at harvest in another site.

Claims

Claims

1. A composition, which is an extract derived from the fermentation of the following mixture comprising of (a) one or any combination of plant extracts from a flowering plant Order Brassicales belonging to the eurosids Π group of dicotyledons under the APG Π system, (b) a carbon source, (c) a protein (or nitrogen) source, (d) a carrier agent, and (e) a fermenting agent.

2. A composition, comprising predominantly of alpha-hydroxy acids with the following formulation: 80-110 g kg lactic acid, 4-10 g/kg acetic acid, and negligible amounts of oxalic acid and glycolic acid.

3. A composition, according to Claims 1 and 2, with natural supplementary nutrients such as calcium, iron, sodium, potassium, magnesium, manganese, and zinc.

4. A composition, according to Claims 1 and 2, wherein the plant extracts from the order of Brassicales are under any or a combination of the family Brassicaceae, and family Caricaceae.

5. A composition, according to Claims 1 to 3, wherein the ratio of the weight of plant extracts from family Caricaceae to Family Brassicaceae 1 :1 to about 100:1, preferably 50:1 to 5:1, and most preferably at 3:1.

6. A composition, according to Claim 4, wherein the ratio of the weight of plant extract from family Caricaceae to Family Brassicaceae is preferably 5:1 to about 50:1, and more specifically at 10:1.

7. A composition, according to Claims 1 to 3, wherein the plant extract is 100% pure Family Caricaceae or 50% in combination with any other member family of order Brassicacea.

8. A composition, according to Claims 1 to 3, wherein the plant extract is 100% pure or 50% in combination with any other member family of order Brassicaceae.

9. A composition, according to Claims 1 to 7, wherein the plant extracts are derived from fruits, leaves, bark, roots, and seeds of the plant.

10. A composition, according to Claims 1 and 2, wherein the carbon source comprises simple sugars such as glucose, fructose, and dextrose or a combination thereof.

11. A composition, according to Claims 1, 2 and 9, wherein the carbon source is glucose.

12. A composition, according to Claims 1 and 2, where in the carbon source comprises complex sugars such as molasses, refined table sugar, honey or a combination thereof.

13. A composition, according to Claims 1, 2, 9, 10 and 11, wherein the carbon source is at a ratio of 1:1 to 1:1000, preferably 1 :3 to 1:100, more preferably 1:5 to 1:80, and most preferably 1 :50, to water as carrier agent.

14. A composition, according to Claims 1 and 2, wherein the protein source is processed milk, fresh milk, skimmed milk, UHT milk, powdered milk, yoghurt milk, full cream milk or any combination thereof.

15. A composition, according to Claims 1, 2 and 12, wherein the protein source is skimmed milk.

16. A composition, according to Claims 1, 2, 13 and 14, wherein the protein source is at a ratio of 1:10 to about 1:1000, more preferably 1:100 to 1:150, and most preferably 1 : 150, to water as carrier agent.

17. A composition, according to Claims 1 and 2, wherein the carrier agent is water or water in combination with wheat or corn or rice flour.

18. A composition, according to any claims above, as an aqueous solution.

19. A composition, according to any claims above, in powdered form.

20. The use of the composition comprising predominantly of alpha-hydroxy acids with the following formulation (based on gram/kilogram): 10 - 150 gram/ kg lactic acid, 1-10 gram/kilogram acetic acid, 0.02 - 0.08 gram / kilogram glycolic acid, and 0.01- l.Ogram /kilogram oxalic acid.

21. The use of the composition which is an extract derived from the fermentation of the following mixture comprising (a) one or any combination of plant extracts from a flowering plant Order Brassicales belonging to the eurosids II group of dicotyledons under the APG II system, (b) a carbon source, (c) a protein (or nitrogen) source, (d) a carrier agent, and (e) a fermenting agent.

22. The use of the composition, according to Claims 19 and 20, as pesticide, and more specifically, as fungicide and bactericide, or a combination thereof.

23. The use of the composition, according to Claims 19 to 21, as pesticide, more specifically as fungicide and bactericide, or a combination thereof, for agricultural applications.

24. The use of the composition, according to Claims 19 and 20, as plant strengthened for agricultural applications.

25. The use of the composition, according to Claims 19 to 22, as fungicide against Mycosphaerellafijensis causing black Sigatoka disease in Cavendish banana.

26. The use of the composition, according to Claims 19 to 22, as fungicide against Ralstonia solanacearum causing Moko disease in Cavendish banana.

27. The use of the composition, according to Claims 19 to 22, as fungicide against Fusarium oxspoum causing Panama wilt in Cavendish banana and in other agricultural products .

28. The use of the composition, according to Claims 19 to 22, as fungicide against Colletotrichum gloespoiroides causing anthracnose in agricultural products.

29. The use of the composition, according to Claims 19 to 22, as fungicide against Botryodiplodia theobromae causing crown rot or anthracnose in agricultural products.

30. The use of the composition, according to Claims 19 to 22, as fungicide against Lasiodiplodia theobromae causing soft rot or fruit rot in agricultural products.

31. The use of the composition, according to Claims 19 to 22, as pesticide against downy mildew in agricultural products.

32. The use of the composition, according to Claims 19 to 22, as bactericide against bacterial leaf blight and bacterial leaf streak in rice and other agricultural products.

33. The use of the composition, according to Claims 19 to 22, as pesticide against tomato yellow leaf curl virus (TYLCV) in tomatoes and other agricultural products.

34. The use of the composition, according to Claims 19 to 20, as control agent against post-harvest diseases of agricultural products.

35. The method of application of the composition described in Claims 1 and 2, comprising

(a) dilution at a rate starting from 30 mL of the composition in 16 liters of water and

(b) direct spraying to the target plant or agricultural product, and any parts thereof.

36. The method of application of the composition described in Claims 1 and 2 comprising

(a) dilution at a rate starting from 100 mL of the composition in 16 liters of water and

(b) direct dipping of the agricultural products into it.