Allelopathic Activity of the Primary Methanolic Extracts from Mangrove Species, Excoecaria agallocha L.

(Buta-buta) and Rhizophora apiculata Bl. (Bakawan-lalake) Against Eichhornia crassipes (Mart.) Solms and Triticum aestivum L. Em Thell.
E. Palma, A.J. Paulo and A.L. Reyes
College of Science Bulacan State University Malolos City, Bulacan
The study was performed to test the allelopathic activity of the primary methanolic extracts (PME) from the mangroves E. agallocha L. (buta-buta) and R. apiculata Bl. (bakawan-lalake) against the weeds model organisms, water hyacinth and wheat grass. The PMEs were analyzed and determined to contain allelochemicals, then applied on the model organism in varying dosages (2.5%, 5%, and 10%). Results showed that the allelopathic activity of the PMEs were dose dependent, allelopathy was greater as the dosage increases, yet even on the treatment having lower dosage, allelopathy was also observed. The greatest allelopathy in water hyacinth was recorded to be 27% reduction in fresh weight (PRFW) and 100% loss of healthy leaves (PLHL). In the wheat grass, 88.89% and 89.29% growth inhibition (PGI) in shoot and root system, respectively, was observed. Generally, results showed that the produced extracts were capable of controlling the aquatic and terrestrial weeds through allelopathy. Keywords: mangroves, Primary Methanolic Extracts (PME), allelopathy, weeds INTRODUCTION The allelopathic effect of some plants against weeds shows a good option for the biological control against these noxious plant species (Kathiresan, 2000). Weeds are generally known as group of plants that grows in unwanted places. Weeds may be unwanted for a number of reasons; (1) they easily spread out on a certain area, (2) they compete with the nutrients of important plants like crops, (3) they transmit and spread pathogens affecting other plants and even human beings (Tu et al., 2001). Water hyacinth was common to be an alien invasive species on several countries that affects several bodies of water and cause various problems like depletion of dissolve oxygen, blockage of waterways, etc. Controlling these weeds becomes a big challenge to everyone, not only to avoid the negative effects but also to protect the local species, which results to the practice of various methods. The most common of which was the mechanical method using mechanical tools and man-power, but proven to be time consuming, expensive, and the mechanical weeders were known to reduce yields on important crops (Bayot et al., 1994). Chemical control also become an option in some cases, yet only offers practical, cost-effective and selective method of managing certain weeds, but due to their chemical components, issues on their adverse effect on the environment, animals and humans were still in protest (Paganelli et al., 2010). On the other hand, biological control becomes more familiar and was viewed to be the best option for an environment-friendly approach. The use of arthropods, fungus species and other living organisms was practiced to control and eradicate invasive species (Ndimele et al., 2011; Muniappan et al., 2009). Allelopathy also becomes an option for biological control for weeds (Kathiresan, 2010; Arumugam, 2008; Fujii, et al., 2008). In the Philippines, the conventional way being practiced was through mechanical methods and handweeding which consume intense amount of time and money yet only solve the infestation shortly because the invasive weeds may grow again in the vicinity after a period of time. Allelopathy was not common in the country due to lack of research and studies. The study tested the allelopathic activity of the mangroves present in the country against aquatic and terrestrial weeds, which aim to open the possibility for biological control. MATERIALS AND METHODS Preparation of the Extracts Plant Collection: The plant materials of the mangroves Excoecaria agallocha L. and Rhizophora apiculata Bl. were collected at Brgy. Babatnin, Malolos City, Bulacan and identified by the Botany

Division of the National Museum of the Philippines. The plant samples were sun-dried for 25days and oven dried for 1hour at 40C to 45C. After drying, the plant parts were ground into fine powder using an osterizer. Extraction: The powdered plant materials were soaked in 3000ml of 90% methanol in a container for six (6) days, separately. The primary methanolic extracts (PME) was obtained and concentrated in room temperature until reduced. Three (3) extracts were produced; A for E. agallocha, B for R. apiculata, and C for both mangrove species. Phytochemical Screening Test for Carbohydrates: Benedicts test was performed to confirm the presence of reducing sugars. A positive result shows brick red precipitate (Pascual and Politano, 2008). Test for Alkaloids: Wagners reagent was used to test the presence of alkaloids. (Guevarra et al., 2005) Test for Glycosides: Aqueous sodium hydroxide solution was applied in 1ml of each extract (Guevarra et al., 2005). A yellowish color reaction will signify the presence of glycosides. Test for Tannins: Ferric chloride test was performed and a deep blue or black precipitate indicates the presence of tannins (Guevarra et al., 2005). Test for Flavonoids: H2SO4 test and NaOH test was performed and yellow to orange color formations will indicate positive results (Guevarra et al., 2005). Test for Saponins: Froth test was performed in each extract (Guevarra et al., 2005). Test for Terpenoids: Salkowskis test using chloroform and concentrated H2SO4 will form a reddish brown color at the interface for the presence of terpenoids (Pascual and Politano, 2008). Liebermann-Burchard Test was also performed to test the presence of tritrpenes until color reaction is observed (Pascual and Politano, 2008; Guevarra et al., 2005). Allelopathy Bioassay Aquaria Set-up: Twenty (20) water hyacinths (Eichhornia crassipes (Mart.) Solms) of the same age were collected from a pond in Brgy. Sampaloc, Apalit, Pampanga, and were labeled and grown on plastic pails. The initial fresh weights of each plant

were recorded before the treatment. Each group receives different dosage (2.5%, 5%, and 10%) and type (A, B, and C) of extracts. The plants were observed daily, and the number of healthy leaves fresh weights were recorded in four (4) days interval for twelve (12) days. The PRFW and PLHL were computed as a basis of allelopathy and analyzed using ANOVA and DMRT to know the variance of the effects in each treatment group. Petri-dish Set-up: Five wheat grass (Triticum aestivum em Thell.) seeds were selected at random and placed on filter paper in a Petri dish containing 5ml of the PMEs (A, B, and C) solutions of different concentrations (2.5%, 5%, and 10%). The Petri dishes were incubated for seven (7) days under normal condition in the laboratory. The shoot and root length of the seedlings after incubation were used in computing the PGI to test the allelopathic activity of each extracts in varying dosages. The data computed were then analyzed using ANOVA and DMRT calculations. RESULTS AND DISCUSSIONS Phytochemical Screening From the phytochemical screening, the researchers observed that the PMEs contain, at least, the three main groups of allelochemicals; phenols, alkaloids, and terpenoids. Extract A gave positive results with Wagners reagent, glycoside test, FeCl3 test, Froth test, NaOH test, and Salkowskis test and Liebermann-Burchard test. Extract B and C were determined to contain reducing sugars, alkaloids, glycosides, tannins, saponins, flavonoids, and terpenoids. These secondary metabolites do not have apparent function on growth of the plant, but mainly involved in many functions like allelopathy and other plant defense mechanism (Anonymous, 2011; Schultz, 2011) Table 1. Results of the Phytochemical Screening
PHYTOCHEMICALS POSITIVE RESULT

A B C

Carbohydrates Alkaloids Glycosides Tannins Saponins Flavonoids Terpenoids

brick red ppt. brownish color yellowish color deep blue or black Froth Formation orange color yellow-orange red-brown int. pink, red, or green

+ + + + + + +

+ + + + + + + +

+ + + + + + + + +

Allelopathic Activity on the E. crassipes Percent Reduction in Fresh Weight: The PMEs A, B, and C shows allelopathic activity on the water hyacinths. It reduces the fresh weight of the model organisms by a certain rate. The recorded PRFW on the treatment groups for water hyacinths were dose dependent and the greatest allelopathy of 27% and 26% were observed in groups A100 and B100, respectively, recorded in day12. Groups A50 and B50 also shows significant reduction in fresh weight of 23% and 24%, respectively. The water hyacinths from groups A25 and B25, despite of minimal dosage of extracts, shows 10% and 9% PRFW, respectively. The treatment groups with extract C also shows reduction in fresh weight on the water hyacinths, but in minimal rate compared to those with extract A and B. The recorded PRFW from groups C50 and C100 were 12% and 15%, whereas, group C25 shows no reduction in fresh weight rather an increase of 1% in its fresh weight after the twelfth day of the experiment, but still an inhibition on the growth was recorded. Table 2. Percent Reduction in Fresh Weight of water hyacinths from Day 4 Day12 GROUP A25 A50 A100 B25 B50 B100 C25 C50 C100 DAY4 12% 21% 17% 6% 10% 20% -11% 1% 6% DAY8 10% 21% 18% 8% 16% 24% -11% 5% 10% DAY12 10% 23% 27% 9% 24% 26% -1% 12% 15%

researchers recorded a significantly greater allelopathy on the groups with extracts B and C, killing the plant having 100% PLHL on the highest dosage in day4. Besides, 67% and 86% was observed from B25 and B50, and for the groups C25 and C50, the recorded PLHL were 59% and 83%, respectively. Table 3. Percent Loss of Healthy Leaves of water hyacinths from Day 4 Day12 GROUP A25 A50 A100 B25 B50 B100 C25 C50 C100 DAY4 29% 54% 83% 36% 45% 100% 42% 66% 100% DAY8 47% 62% 100% 54% 72% 100% 59% 83% 100% DAY12 42% 67% 100% 67% 86% 100% 59% 83% 100%

7% 28% 41% CONTROL The recorded 100% PLHL can be an indication that the plant cannot perform its full capacity for photosynthesis for it to survive, due to the great loss of chlorophyll in the leaves (Pandey, 1996), and is considered dead. Aside from the leaves, petiole and roots also starts to wilt and dries out during the experiment. Allelopathic Activity on the T. aestivum Percent Growth Inhibition: The PMEs have a potent growth inhibitory effect on both the wheat shoots and roots. The greatest inhibition recorded on the shoots was 88.82% both observed on the seedlings from A100 and C100. Besides, the 10% concentration of extract B shows great inhibition of 64.12%. Even at the lower dosage of 5%, there were observed inhibition on the shoot length of the model organisms of about 62.16%, 19.61%, and 75.29% from groups A50, B50, and C50, respectively. At the lowest dosage, Extract A, B, and C also inhibit the growth of the shoots at 37.06%, 6.86%, and 16.67%, respectively. The results are comparable to the growth inhibition bioassay by Shahid-ud Daula and Basher (2009) using the mangrove Xylocarpus granatum Koen., having 89.4%, 96.3%, 98.8% and 100% shoot inhibition in 250 g/ml, 500 g/ml, 1000 g/ml, and 1500 g/ml concentration, respectively.

Reduction in fresh weight in plants indicate allelopathic activity not only to the loss of water in plants but also other physiological effects (Kathiresan, 2000), where in the higher the PRFW, the greater the allelopathic exhibited by the extract. Percent Loss of Healthy Leaves: Another basis on the allelopathic activity by the PMEs was the loss of healthy leaves, wherein, the initial number of healthy leaves before the treatment was recorded and compared until the twelfth day. Extract A shows 100% PLHL in the highest dosage at day8 and kill the whole plant. On the other hand, 42% and 67% were recorded in A25 and A50, respectively. The

2.

Figure 1. Percent Growth Inhibition of the Shoot System of wheat grass seedlings after incubation The greatest inhibitions of the extracts on the rootlets were also recorded in groups A100 and C100 as 89.29% and 88.72%, respectively. The roots from B100 were inhibited by 59.27%, quite lower compare form the previous values. A25, B25 and C25 were observed to have 42.45%, 4.78%, and 1.91% inhibition on the root system, respectively. The 5% concentration of the extracts, A50, B50, and C50, inhibited the wheat seedlings roots by 47.8%, 16.25%, and 75.33%, respectively.

b. Extract A and B shows greater allelopathic activity to water hyacinths based on PRFW compared to extract c. High dosages of each extract shows great loss of healthy leaves compare to the lowest dosage. d. The treatments having equal dosages show no significant difference on the PLHL on the water hyacinths. The extracts also show allelopathic effect on the shoot and root system of the model organism, wheat grass. a. Extracts A and B shows greater allelopathic activity compared to extract C based on the growth inhibition of the shoot system in wheat grass. b. Extracts A and B shows greater allelopathic activity compared to extract C based on the growth inhibition of the shoot system in wheat grass.

ACKNOWLEDGEMENT The authors would like to thank the Almighty God who guides us in all the days of our life, especially during the hardest time in doing this research. We wish to thank all the persons behind the success of this study; our family, classmates, friends and our instructors and mentors. We would like also to express our gratitude to Mr. Richard F. Clemente, our Bioresearch instructor and to our adviser, Mr. Edwin C. Reyes, for their kindness assistance in finishing this paper.

REFERENCES Kathiresan, R.M., 2000. Allelopathic potential of native plants against water hyacinth. Journal of Crop Protection, Vol. 19, Issues 8-10: 705-708. Tu, M. et al., 2001. Glyphosate. UCD Weed Control Methods Handbook, The Nature Conservancy. Volume 7e:1-10. Bayot, R.G., Watson, A.K. and Moody, K., 1994. Control of Paddy Weeds by Plant Pathogens in the Philippines. National Crop Protection Center, UPLB, Los Baos, Laguna. pp. 139143. Paganelli, A. et al., May 2010. Glyphosate-Based Herbicides Produced Teratogenic Effects on Vertebrates by Imparing Retinoic Acid

Figure 2. Percent Growth Inhibition of the Root System of wheat grass seedlings after incubation

CONCLUSIONS 1. The study showed that the extracts show allelopathic activity on the water hyacinth based on percentage reduction in fresh weight (PRFW) and percentage loss of healthy leaves (PLHL). a. The allelopathic activities of each extracts on water hyacinth based on PRFW were dose dependent.

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