The allelopathic effects of tropical pioneer tree species on seed germination in chiang mai, thailand

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NEIL JOSHUA RENZO RIVERA VERIDIANO THE ALLELOPATHIC EFFECTS OF TROPICAL PIONEER TREE SPECIES ON SEED GERMINATION IN CHIANG MAI, THAILAND BACHELOR THESIS Study Mode: Major: Faculty: Batch: Full-time Environmental Science and Management Advanced Education Programs Office K46B – AEP (2014 – 2018) Thai Nguyen, 2018 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student name Neil Joshua Renzo Rivera Veridiano Student I.D DTN1454290093 Thesis Title “The Allelopathic Effects of Tropical Pioneer Tree Species on Seed Germination In Chiang Mai, Thailand” Supervisor Prof Dr Sutthathorn Chairuangsri and Dr Do Xuan Luan Supervisor’s signature Abstract: In the tropics, pioneer tree species successfully establish on open, weedy sites possibly by means of allelopathy - chemical inhibition of weedy herbs Therefore, pioneer tree species are a good place to search for natural herbicides, particularly those that might be used to facilitate forest restoration on such sites Therefore, the inhibitory effects of aqueous leaf extracts (ALE’s) of four pioneer tree species: Macaranga denticulata, Prunus cerasoides Gmelina arborea and Melisa toosendan on seed germination were tested, use lettuce (Lactuca sativa) as a model in vitro (on filter paper in petri dishes) Extracts from all three four species significantly inhibited germination compared to the control (water only with no extracts) Prunus cerasoides extract had the most potent effect Further tests on seeds germinating on natural substartes and on seeds of a wide range of weed species that typically hinder forest restoration on deforested sites are now needed, to develop effective and practical natural herbicides from these pioneer tree species Keywords Number of pages Date of submission allelochemical, allelopathy, weed management 45 ii ACKNOWLEDGEMENT I owe my deepest gratitude to my ever supportive parents, Aida R Veridiano and Noel T Veridiano Without your love, trust and support I wouldn’t be here on where I am standing right now To my brother and sister who have helped me grow, who has taught me to strive for more, to my best in everything that I do, thank you My research will not be successful without the help of the whole FORRU staff in Thailand especially Dr Steven Elliot who have helped me on every little thing that I needed throughout the months I did my research in Thailand To my adviser, Dr Sutthathorn Chairuangsri who inspired me to more and always believe in myself Her words of encouragement lead me to where I am now, she has been guiding me since day and I cannot thank her enough for everything that she has done for me I would also like to extend my sincere appreciation to Chiang Mai University, Biology Department for providing me the materials and laboratory equipment that I needed for my research Aom, pai, pi, som, aom and nok, this guys are the one that help me on my research, from collecting the leaves to doing the experiment on the laboratory I am so grateful to work with them in a short period of time .A special thanks to my partner, Alexis Alcantara who helped me to be a better person and for always believing in me.Thank you for always saying “kaya mo yan” which means “you can it” everytime I doubt myself Thank you for helping me whenever I ask questions about literally everything You never get tired of me, and I cannot thank you enough for that iii To Advance Education of Thai Nguyen University of Agriculture and Forestry and Chiang Mai University, thank you for giving me this opportunity to develop myself and increase my knowledge and skills And of course K46B, it has been a really great experience studying with you guys The friendships that we made, the memories that we share in long years I will forever cherish that and I will never forget you each and every one of you especially our monitor Above all, I want to thank God for all these people that I have met along the way throughout my journey in life I dedicate all my success to you Thank you for the unconditional love and blessings The researcher, Neil Joshua Renzo R Veridiano iv TABLE OF CONTENTS DOCUMENTATION PAGE WITH ABSTRACT ii Acknowledgement iii List of Tables vii List of Figures viii PART I Introduction 1.1 Research rationale 1.2 Research objectives .4 1.3 Research questions 1.4 Hypotheses 1.5 Limitations 1.6 Significance of the study .5 Part II Literature review Part III Methodology 10 3.1 Time and place of the study 10 3.2 Phase .11 3.2.1 Materials 11 3.2.2 Leaf collection of pioneer tree species 11 3.2.3 Preparation of leaf aqueous extract .12 3.2.4 Preparation of test specimen 13 v 3.2.5 Test procedure .14 3.3 Phase .17 3.3.1 Materials 18 3.3.2 Experimental layout 18 3.3.3 Test procedure 19 Part IV Results and Discussion 22 4.1 Phase 1: Germination rate 22 4.2 Phase 2: Germination rate 24 PART V Conclusion and Recomendations 29 Bibliography .31 Appendices 33 Appendix I – Leaf reference 33 Appendix II 35 vi LIST OF TABLES Table List of materials used for phase 11 Table Materials used for phase 18 Table Experimental layout for phase .19 Table Transformed binomial data 25 Table ANOVA – Two factor without replication: Results 26 Table t-Test: Paired two samples of means 27 Table T-Test: Paired two samples for means 27 vii LIST OF FIGURES Figure Control: germination percentage phase 23 Figure 2.Macaranga denticula: germination rates for phase 23 Figure Gmelina arborea: germination rates for phase 23 Figure Germination after 10 days: phase 24 viii PART I INTRODUCTION 1.1 Research rationale The Forest Restoration and Research Unit (FORRU) is situated in Chiang Mai, Northern Thailand within the Department of Biology of Chiang Mai University FORRU’s vision and mission is to reinstate the tropical forests of Northern Thailand in order to conserve and restore biodiversity as well as enhance environmental protection This is due to the fact that since 1960’s, Thailand nearly lost 65% of its forest coverage Deforestation was primarily driven by logging in the nineteenth century when Europeans arrived to obtain teak tree, a valuable resource (Eppard, et al 2005) In 1960, the Thai government granted logging under the condition of re-planting which was not properly managed After a terrible flooding disaster in 1988, caused primarily by deforestation, a total ban on all logging passed in 1989; however land clearing still occurs at a rapid rate today for agricultural expansion by converting forest to cropland for palm oil and rubber plantations (Yadav, et al 2016) This nonetheless helped the economic growth of Thailand extensively through export but impaired the natural ecosystems dramatically FORRU’s affirmative and progressive attitude is confident that the tropical forests can be restored Nevertheless, this assurance is based on the development of an established plant database, scientific report publications and the publication of six different books on restoring forests Therefore, FORRU aims to actively collaborate with locals in order to integrate conservation measures into the needs of communities and continue research for advancing forward with the replenishment of the forests of Thailand Herbicides produce a wide range of toxic side‐effects that pose a potential hazard to the environment The development of natural allelochemicals is one method of addressing these issues In the tropics, pioneer tree species successfully establish on open, weedy sites possibly by means of allelopathy-chemical inhibition of weedy herbs Therefore, pioneer tree species are a good place to search for natural herbicides, particularly those that might be used to facilitate forest restoration on such sites Therefore, the inhibitory effects of aqueous leaf extracts (ALE’s) of four pioneer tree species: Macaranga denticulata, Prunus cerasoides, Gmelina arborea Melisa toosendan on seed germination were tested, using lettuce (Lactuca sativa) as a model in vitro (on filter paper in petri dishes) Extracts from all tree species significantly inhibited germination compared to the control (water only with no extracts) Further tests on seeds germinating on natural substances and on seeds of a wide range of weed species that typically hinder forest restoration on deforested sites are now needed, to develop effective and practical natural herbicides from these pioneer tree species The study of allelopathy increased in the 1970s and has undergone rapid development since the mid-1990s, becoming a popular topic in botany, ecology, agronomy, soil science, horticulture, and other areas of inquiry in recent years The allelopathic interaction can be one of the significant factors contributing to species distribution and abundance within plant communities and can be important in the success of invasive plants Allelochemicals can stimulate or inhibit plant germination and growth, and permit the development of crops with low phytotoxic residue amounts in water and soil, thus facilitating wastewater treatment and recycling They are a suitable substitute for synthetic herbicides because allelochemicals not have residual or toxic effects, Control Germination % 80 60 40 67 70 After days After 10 days 45 20 After days Control Figure Control: germination percentage phase Gmelina arborea Germination % 25 20 22 20 15 10 After days 13 After days 2 1 2 After 10 days Concentration Figure Gmelina arborea: germination rates for phase Germination % Macaranga denticulata 40 35 30 25 20 15 10 34 27 After days 18 18 15 12 After days After 10 days Concentration Figure 2.Macaranga denticula: germination rates for phase 23 4.2 Phase 2: Germination rate All treatments substantially and significantly reduced germination percentage compared with the controls, even though concentrations of extracts used in phase II were 2x times lower than those used in phase I In comparison to phase I results, Macaranga and Gmelina again portrayed the highest germination with a decreasing germination percentage with increasing treatment concentrations The optimal concentration for Macaranga denticulata to inhibit seed germination completely would be 9.1% LAE concentrate For Gmelina arborea, none of the treatment concentrations completely prevented seed germination The Melia toosendan treatments showed the most consistent results with significantly lower germination percentages, which demonstrate constant effects of allelopathy Prunus cerasoides portrayed steady seed inhibition patterns except for an anomaly presented in concentration (3.61%) Overall, using Prunus cerasoides concentration treatment (1.23%) as a bio-herbicide would be most applicable since it inhibits seed germination completely using the least amount of resources to accomplish Phase II Germination after 10 days 47% Germination 50% 40% 30% 19% 20% 10% 11% 2%1%2% Conc.1 18% 0% 9% Conc.2 6%8% Conc.3 0%0% 0% Melia Macaranga Prunus toosendan denticulata cerasoides Gmelina arborea Control Water Specie Figure Germination after 10 days: phase 24 The data was prepared for binomial data for analysis of variance: for germinated and not germinated First arcsine transform the data, before carrying out the analysis for variance The top most rows in table shows original data of seeds germinated, the second and third table displays the transformed data The last set of rows in table was used for the ANOVA analysis Table Transformed binomial data Block Block Block Block Block Block Block Block Block Control Me.1 Me.2 Me.3 Pru.1 Pru.2 Pru.3 Ma.1 Ma.2 Ma.3 Gm.1 Gm.2 Gm.3 54 17 12 12 43 2 0 14 16 13 15 44 1 0 13 23 26 6 Control Me.1 Me.2 Me.3 Pru.1 Pru.2 Pru.3 Ma.1 Ma.2 Ma.3 Gm.1 Gm.2 Gm.3 54 17 12 12 43 2 0 14 16 13 15 44 1 0 13 23 26 6 Control Me.1 Me.2 Me.3 Pru.1 Pru.2 Pru.3 Ma.1 Ma.2 Ma.3 Gm.1 Gm.2 Gm.3 47.29 5.74 0.00 8.13 0.00 5.74 0.00 24.35 20.27 0.00 20.27 17.46 16.43 40.98 8.13 8.13 8.13 0.00 0.00 21.97 23.58 21.13 0.00 22.79 9.97 17.46 41.55 9.97 5.74 5.74 0.00 0.00 21.13 28.66 16.43 5.74 30.66 14.18 14.18 All ecological experiments generate highly variable results; therefore, the petri dishes were replicated three times The results are presented as mean and a measure of variation among replicates An experimental design was set up called the ‘randomized complete block design’ (RCBD), with the results analyzed by a two-way analysis of variance (ANOVA) followed by pair wise comparisons This experiment generated binomial data, which describes variables that only have two states, germinated and not germinated First, the data had to be arcsine transformed, before doing the analysis of variance ANOVA tests the null hypothesis that there are no real differences among 25 the control and the treatments tested and any variation between the mean values is just due to chance The important values to look at are the P-values, which quantify the probability that the null hypothesis is valid (Elliott, Restoring Tropical Forests, 2013) Tests were made to see if LAE treatments between species winthin concentrations are significant and to show the probability that differences between treatments not exist The calculations and tests can be found on the table below Table ANOVA – Two factor without replication: Results ANOVA Source of Variation Rows Columns Error SS 16.73362 364.3093 15.50159 Total 396.5446 df MS F P-value F crit 8.366811 3.238433 0.111208 5.143253 121.4364 47.00283 0.000146 4.757063 2.583599 11 The result for the treatments between species had a P-value of 0.000146% chance that differences between treatments exist and a 99.9998854% probability that they don’t However, within treatments the p-value was 0.111208 meaning it has no significant difference If the value is 0.05 or less, the difference is significant Now that the significant differences are confirmed by ANOVA, pair wise comparisons are required to determine which differences are significant The t-Test was used to determine this For these differences to be significant, the value of ‘t-Stat’ must be greater than the numbers of degrees of freedom All of the treatments satisfy this requirement Before carrying out the t-Test, the question that was addressed was, “do treatments significantly increase seed inhibition compared with the control?” (Elliott, Restoring Tropical Forests, 2013 26 Table t-Test: Paired two samples of means Control Mean Variance Observations Pearson Correlation Hypothesized Mean Difference df t Stat P(T