Seed health testing of selected forest tree seeds and their control using sodium hypochlorite and capsicum extract soap

Corpuz Student ID DTN1454290095 Thesis Title Seed Health Testing of Selected Forest Tree Seeds and Their Control Using Sodium Hypochlorite and Capsicum Extract Soap Supervisors Marceli

Study Mode: Full-time

Major: Environmental Science and

Management Faculty: Advanced Education Program

Batch: 2015 – 2018

Type: On- The Job Training (OJT)

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

JERZYNE ANNE ABRERA CORPUZ

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Environmental Science and Management

Student Name Jerzyne Anne A Corpuz

Student ID DTN1454290095

Thesis Title Seed Health Testing of Selected Forest Tree Seeds and Their Control Using Sodium Hypochlorite and Capsicum Extract

Soap Supervisor(s) Marcelina V Pacho (M.A.) &Ly Thi Duong (M.A.)

The result of this study showed that Sodium Hypochlorite (NaOCl) and Capsicum Extract Soap Solution (CESS) is effective in reducing the fungal infection on the forest tree seeds However in terms of the Molave seeds there is no significant effect

on reducing the fungal infection.There is a possibility that the level of concentration

of the two treatments need to be increased But still the seed borne pathogen of Molave seed was slightly reduce some fungi species based on the results

Keywords: Seed health testing, fungal infection, Sodium Hypochlorite,

Capsicum Extract soap solution, seed borne pathogens Number of Pages 86

Date of Submission September 15 , 2018

Hypochlorite and Capsicum Extract Soap Solution) of Ecosystem Research

Development Bureau (ERDB) as well as Mrs Ly Thi Duong which also help me

to improve my research topic and I came to know about so many new things I am really thankful to them

Secondly I would also like to thank my parents “Daddy” Virgilio and Ledivina and loving brothers “Kuyas” Jefferson and Jay Mark, to my Aunt Evangelina I wouldn’t be able to achieve this success without your financial support, love and guidance

To all friends, Especially Hannah Ayson, Nicole de Castro, Alexis Alcantara, Mark Glodo, Angelika Aragones, Ghia Sarmiento, Colleene Lanzador, Mishel Ranada, Kenneth Kua and ERDB family for the ideas, support and modivation

To the Advance Education Program Thai Nguyen University of Agriculture and Forestry for providing the opportunity of study abroad and grateful to the Ecosystem Research Development Bureau (ERDB) for accepting

me to conduct my thesis/ OJT and whose made all the laboratory experiment possible without further expenses.To batch, K46-A AEP, for sharing knowledge and crazy memories along the journey in Vietnam

Dear God from the bottom of my heart I want to thank you for being with

me all the way

Jerzyne Anne A Corpuz

TABLE OF CONTENTS

List of Figures 1

List of Tables 2

List of Abbreviations 3

CHAPTER I INTRODUCTION 5

1.1.Rationale 5

1.2.Objectives 6

1.2.1 General 6

1.2.2 Specific 6

1.3.Content of Work 7

1.4.Profile of the Cooperating Agency 8

1.4.1 Vision 8

1.4.2 Mission 8

1.4.3 ERDB Main Office Function 8

1.4.4 ERDB Research Centers Function 9

1.4.5 ERDB’S Research, Development and Extension Program 9

1.4.6 Special Projects 10

1.4.7 Adviser 11

1.5.Description of the Activity 11

1.6.Timeline 11

CHAPTER II: REVIEW OF RELATED LITERATURE 13

2.1 Seedborne Pathognes 13

2.2 Seed Treatments 14

2.3.Methodology 17

2.4.Seed Borne Fungi of Some Forest and Their Control 19

2.5.Impact of Seed Mycoflora and Forest Seed on See Quality anf Their

Management 20

CHAPTER III STATUS OF THE CONSIDERED ISSUE AT THE TRAINING AGENCY 21

3.1 Title 22

3.2 Time and Place 22

3.4 Statement of the Problem 22

3.5 Hypothesis 23

3.6 Scope and Limitation 23

3.7 Materials 23

3.7.1 Four selected forestry seeds species 24

3.7.2 Petri Plates 24

3.7.3 Autoclave 24

3.7.4 Compound Microscope 24

3.7.5 Hot air oven 24

3.7.6 Distilled water 24

3.7.7 Filter paper 24

3.7.8 Forceps 25

3.7.9 De- natured Alcohol and Alcohol lamp 25

3.7.10 Glass slides and Glass cover 25

3.8 Methods 25

3.9 Research Approach 26

3.10 Research Design 27

3.11 Data Gathering 27

3.12 Statistical Analysis 28

3.13.1 Seedborne Pathogens 28

3.13.2 Fungal Infection 29

3.13.3 Bacteria 31

3.13.4 Aspegillus flavus 33

3.13.5 Verticillium sp 35

3.13.6 Fusarium spp 37

3.13.7 Asperillus niger 39

3.13.8 Chaetonium sp 41

3.13.9 Germination Rate 43

3.14 Discussion 45

4.1 Discussion 49

4.2 Lesson Learned 51

CHAPTER V: CONCLUSION 53

References 54

APPENDICES 58

LIST OF FIGURES

Figure 1: Research Approach 26 Figure 2: Research Design 27

LIST OF TABLES

Table 1: Timeline of Working Days of On-The-Job Training 12 Table 2: List of Seedborne Pathogens/Contaminants 28 Table 3: Percentage of infection of Fungi on four slected forestry Seeds and Effect of Pre-treatment 29 Table 4: Percentage of Bacteria of Four Selected Forestry Seeds with Pre-treatment Using NaOCl and CESS 31 Table 5: Percentage of As flavus of four selected Forestry Seeds and effect of Pre-treatment Using NaOCl andd CESS 33 Table 6: Percentage of Verticilium sp on Four Seleceted Forestry Seeds and Effect of Pre-treatment Using NaOCl and CESS 35 Table 7: Percentage of Fusarium ssp on Four Selected Forestry Seeds andEffect

of Pre-treatment Using NaOCl and CESS 37 Table 8: Percentage of As niger on Four Selected Forestry Seeds and Effect of Pre-treatment Using NaOCl and CESS 39 Table 9: Percentage of Chaetonium sp on Four Selected Forestry Seeds and Effect of Pre-treatment Using NaOCl and CESS 41 Table 10: Percentage of Germiantion Rate of Four Selected Forestry Seeds as Affected by NaOCl and CESS 43

LIST OF ABBREVIATIONS

ATISC Alberta Tree Improvement and Seed Center

DENR Department of Environment and Natural Resources

ENR-RDE-PAPs Environment and Natural Resources-Research,

Development and Extension Program/ Activities/ Project

PQPM Production of Quality Planting Materials

ISTA International Seed Testing Association

UPLB-CFNR University of the Philippines at Los Banos, College of

Forestry and Natural Resources

SAS Statistical Analysis System

CHAPTER I INTRODUCTION 1.1 Rationale

Seed is the fertilized- ripened ovule of a flowering plants containing an embryo and capable normally of germination that produce new plant To produce

or reproduce new and healthy plants, seeds must be genetically and physically pure, with high vigor and germination potential right moisture content and free from pest and diseases Seed contamination and seed-borne pathogens can cause infection that leads to reduction of plant growth /height This means, loss in the productivity both in terms of quantity and quality of the plant

Seed health testing is a procedure used to determine whether the seed is healthy or diseased Moreover it is also procedure used to detect the presence of absence of seed borne pathogens in a seed lot Seed health test concerns the overall condition of the seed such as the germination capacity of the seeds, the diseases tolerance level or seed standard

Seed technology takes care of asserting the fitness of seed in respect of its genetic nutritive value, high yield of quality production and good health Seed health test (SHT) is a procedure to detect seed borne pathogens ,insect/pest and

or other contaminants that came from the mother tree during harvesting, drying and storage These pest (insect & microorganism) effect can be the reduction of plant germination, i.e poor seedling growth or stunting A number of methods was set by International Seed Testing Association (ISTA) and these are useful whether for both agricultural or forestry seed species found on tropical or temperate conditions

Therefore the objective of this study is to know the health status of the selected forest tree seed species and to disinfect the seed or eliminate

microorganisms on the seed with the use of Sodium hypochlorite and Capsicum

extract soap solution This can help to reduce the microorganisms infecting the seeds and increase the quantity and quality of the crop production and germination capacity of the seeds.instead of fungicides disinfectants were used Lesser chemical affect both user and environment was priorities (Sauer& Burroughs, 1986)

1.2 Objectives

1.2.1 General

To determine the different types of seed pathogen of four (4) selected forestry seed species and their control/ fungus contamination reduction using Sodium hypochlorite and Capsicum Extract Soap Solution

1.3 Content of Work

 Petri dish sterilization in hot air oven at 80 degree Celsius for 2 hours Distilled water sterilization and filter paper sterilization in automatic autoclave at

212 degree Celsius, 120 Pressure per square inch (psi) for one (1) hour

 Prepare Capsicum Extract Soap Solution using 25 pieces hot chili sieved

in gauze, mixed with on teas spoon liquid soap 1 L distilled water; and steam sterilized at 212 degree Celsius 120 psi for one hour

 Prepare four (4) kinds of forestry seeds that will be tested;

 Plant the seed onto Petri dish with three layers of filter paper that can hold 5ml distilled water

 Incubate the plated seeds under height (12 inches space between) day light tubes Petri dish for 7- 10 days under alternating light (12 hours) and 12 hours of darkness

 Examined the seed with the naked eyes 7 days after incubation, first under binocular microscope (10-4-x magnification) Those with microbial growth were further examined under the compound microscope 100 to 400 x magnifications with high magnification, some fungi producing spores were identified/ suspected

up to species level

 Other data collected were bacterial contamination and rate of seed germination

 Data were tabulated and statistically analyzed

 Report was then prepared

1.4 Profile of the Cooperating Agency

The Ecosystem Research and Development Bureau (ERDB) is the principle research arm of the Department of Environmental and Natural Resources (DENR) Its R&D and extension activities are focused on the five major ecosystem of the Philippines which include forest, upland farms, grassland and degraded area coastal zone and fresh water and urban area

The ERDB was created June 1987 under Executive Order (EO) 192 that merged the function of the former Forest Research Institute (FORI) and the National Mangrove Committee It’s headquarter is located at the university of the Philippines Los Banos, College of Forestry and Natural Resources (UPLB-CFNR) campus in the municipal of Los Banos, Province of Laguna, Philippines

about 65 kms south of Manila

1.4.3 ERDB Main Office Function

 Formulate, implements, coordinates and evaluated integrated research, development and extension agenda/ programs on Environment and Natural

Resources (ENR) and other related cross-cutting concern to address the need of the various sector in support of the thrusts and program of the Department

 Conduct basic research and develop standard, protocol and science- based technologies to address the need of the different DENR Bureaus, end-user and stakeholder

 Provide technical assistance by developing and maintaining network and linkages with DENR Bureaus and office, local, and international institution, other stakeholder and civil society; and

 Perform other functions that may be assigned by higher authorities

1.4.4 ERDB Research Centers Function

 Formulates, implement and monitor and evaluate applied and oriented Environment and Natural Resources-Research, Development and Extension Program/ Activities/ Project (ENR-RDE-PAPs) on the watershed, water resources, land, agroforestry and upland farming system, biodiversity, coastal areas, wetland and ecotourism, mining and degraded areas, forest and timber resources and toxic wastes and management of hazardous wastes

action- Pilot test and verify the standards and protocol developed by ERDB-MO;

 Provide the latter feedback towards their improvement;

 Conduct coordination and networking services with RDE stakeholder; and

 Provide technical assistance and extension services on ENR technologies

1.4.5 ERDB’S Research, Development and Extension Program

 Forest Ecosystem Resiliency and Sustainability

 Ecosystem dynamics and Sustainable of Urban Ecosystem

 Sustainability of Laboratory and Experimental/ Demonstration Service in Support to RDE Program, Activities and Projects of ERDB Main Office and Research Centers

 Client- Based Technology Transfer and Extension Service of ENR Management

 Addressing Hazard in the Watershed for Water Supply Sufficiency and Resources Availability

 Land Management and Sustainability and Agroforestry and Upland Farming System

 Enhancing Resilience of Wetlands and Coastal Areas to Support Biodiversity Conservation and Ecotourism Development

 Conservation and Sustainable Management of Natural and Plantation Forest

 Action Program to Stabilize Mined- Out and Degraded Areas through Rehabilitation Technologies

 Pollution Abatement though Integrated Management of Toxic Substances, Hazardous Wastes in Highly Urbanized Areas

 Vulnerability Assessment of Priority Watershed

 Integrated Pest Management

 An Integrated Science Based Approch Rehabilitation of /mined- out and Wasted Dump areas, in Bagacay, Hinabangan, Western, Samar

 Development of Micro- Propagation Protocol for Four Economically Important Bamboo Species in the Philippines

 Rubber, Coffee and Cacao: Building Site Matching Function for Improved Farm and Agroforestry Development

1.4.7 Adviser

Mrs Marcelina V Pacho studied at the University of the Philippines at Los Banos Laguna, Bachelor of Science in Agriculture Major in Microbiology; continued her study at the Graduate School UP at Los Banos Science in Forestry Major in Forest biological Science Minor in Plant Patholoogy; also trained in Institute of Seed Pathology in Hellerup, Denmark and Tree Surgery in College of Forestry and Natural Resources, UP at Los Banos

1.5 Description of the Activity

In this study the main task was given to test the health of the four forestry Lanete, Taluto, Ipil- ipil, and Molave seed species The main objective is to identify what types of pathogens/pest and other microorganisms associated with the seeds, and the level of effectiveness of the two (2) kinds of treatment was applied: Sodium hypochlorite and Capsicum Extract Soap Solution to disinfect the contamination of the seeds By the help of the instructor she trained of the student to familiarize with various organisms associated with the test seeds

1.6 Timeline

The internship training started on March 5 to July 4, 2018; committed to work 8 hours per day and 5 days per week at the ERDB Los Baňos Laguna

Philippines Log book was be used for recording of work time and working days/ hours were completed Field work activities were counted as a working day or

OB (Official Business) trip that is a part of the project or an order which allows

an employee to leave the office and continue to undertake errands outside of the office (See Table 1)

Table 1: Timeline of Working Days of On-The-Job Training

Weeks 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Laboratory

CHAPTER II: REVIEW OF RELATED LITERATURE 2.1 Seedborne Pathognes

Plants are commonly produced through seeds.The seeds of forest tree species like other seeds are susceptible to pathogens and thus affecting its viability The seeds are the most important input for crop production Quality in terms of uniformity on seed sizes, proper moisture content per species and healthy seeds are the crying need of the day (ISTA, 1985) Pathogen free seeds are considered as the vital factor for plant reproduction and good harvest The health of the seeds are affected by direct infection of pathogens or though contamination of storage fungi Infection of the seed by a pathogenic organism and the presence of pathogens in a seed lot may fail the germination (Fisher,

1941 & Epner, 1964) because of infection to seedlings and growing plants Healthy seeds are considered as an important factor for successful crop

production

So Seed health testing can help detect the seed pathogens and control them from infection /diseases The International Seed Health Testing Association (ISTA) showed interest since1924 in seed gene purity and germination aspect Seed testing are closely aligned with the International Rule for Seed Testing (ISTA, International Rules for Seed Testing 2015, 2015) and follow this procedure from sampling, moisture content determination, purity analysis and seed weight determination The germination testing standard depends on protocol developed at the Petawawa National Forestry Institute, the British Columbia Tree Seed Centre and the Alberta Tree Improvement and Seed Center (ATISC) and

has been tested for highest performance at ATISC Procedures and standards for the determination of seed equilibrium and relative humidity are supported by the Genebank Standards for Plant Genetic Resources and Food & Agriculture (FAO 2013) and research at the Royal Botanic Gardens, Kew (Smith et al., 2003, Gold

& Manger, 2008)

2.2 Seed Treatments

Seed treatments were currently used in United State (US) for the control

of seed borne pathogens which may include chemical, physical, and mechanical control We know that seed borne pathogens have the potential to damage seeds Therefore seed treatment has the ability to disinfect contamination of seeds through chemical, physical and mechanical control to increase the germination rate of seeds

Chemical seed treatment currently practiced in the United States to control seed borne pathogens using thiram and seed disinfectant Thiram is usually used

in nurseries as a bird and animal repellent, as well as fungicide This treatment

has been effective against F oxysporum on ponderosa pine and Douglas-fir seeds (Littke 1997), and against Fusarium spp on longleaf pine seeds (Barnnetth &

Valerla, 2003).However thiram has a toxic effect on seed of many conifers, germination under operational condition species especially in high dosage (Abbott, 1958) (Nolte & Barnett, 2000) Other fungicide not specifically labeled for use on tree seed have been tested with some positive result, but so far this treatment is not significantly better control of fungal infestation or germination or the disinfectant (Barnett & Mcgilvray, 2002; Barnett & Varela,

2003).Disinfectant such as sodium hypochlorite, hydrogen peroxide and hydrogen dioxide (Zero Tol®) can be used to reduce fungal infection and improve seed germination Hydrogen peroxide has the ability to eliminate seed borne mycoflora and to stimulate seed germination of conifer seeds with a 30- percent concentration (Trappe, 1961), and can virtually eliminate seed coat

contamination of long leaf pine seed by F circinantum and other Fusarium spp

(Fraedrich, 1997).Hydrogen peroxide have also been effective for reducing seed

borne contamination of Fusarium spp.with 3 percent of hydro peroxide are

effective for eliminating seed borne inoculum and maintain high germination (Dumroese et al, 1998) Sodium hypochlorite also can reduce fusarium contamination and can increase germination in several western conifers (Wenny

& Dumroese, 1987) Pretreatment of seeds with ethyl alcohol can increase the efficacy of sodium hypochlorite in agricultural crops (Sauer & Burroughs, 1986)

Physical Seed Treatment Water rinses can be used to reduce seed borne pathogens and improve germination (Riffle & Springfield, 1968) Stratification can increase the presence of fungi in some seed lots and running water can help

to decreased surface contamination (Alxelrood & et al, 1995).However, water rinses will not be able to eliminate contamination of the fungi from the seeds (Riffle & Springfield, 1968).Seed lots have been suspected of being contaminated with a seed seedborne pathogens should also be treated with a disinfectant or fungicide (Campbell & Landis, 1990)

Heat treatment is used to control certain seed borne pathogens without affecting the viability Heat treatments include hot water; aerated steam and

microwave radiation are usually used on seed of agricultural crops in numerous studies (Agarwal & Sinclair, 1997) By the use of the microwave hot water

treatment they’ve controlled Fusarium spp on DouglƯDas-fir seed without

significantly affecting the germination rate of the seeds (James, Gilligan, Dumroese, & Wenny, 1988), but will require further information or additional research

Mechanical Seed Treatment Mechanical methods have been used to

remove dead and fungus-damage seed from healthy, and in order to increase germination of seed lots and reduce inoculum of seed borne pathogens Gravity table can be used to separate fungus damage seed from seed lots (Karrfalt, 1983) this strategy has been used by several organizations with good results The Incubation, Drying, Separation (IDS) system is another procedure for separating viable from filled-dead seeds (Simak, 1984),this has been successfully conducted removed damaged seeds from seed lot (Donald, 1985, Downie & Wang, 1992)

on Plantanus x acerifolia (Ait.) Willd (Falleri & Pacella, 1997)

However through the effective and efficient control of seed borne fungi can be achieved by chemical fungicide, the same cannot be applied to grains for reasons of pesticide toxicity (Ferrer, 1991; Harris, 2001; Dukic, 2004) To store seed for safe human consumption there is still a need to search for alternative approach that are cheap and ecofriendly They used organic soap solution to reduce the seed contamination/ infection from seeds National Academy of Science (1986) report of pesticide residues on food indicate that fungicide pose more of a carcinogenic risk than insecticide and herbicide Synthetic fungicide is

detected in food chain, safer and alternative should be found immediately Over the years much effort has been devoted to to the search for new antifungal material from natural sources for food preservation The principal aim to the

present work is to test the antifungal activity of aqueous Capsicum ‘frutencens (Hot chilli pepper) extract against the important seed borne pathogens Capsicum frutescens has an positive effect to reduce A.flavus and A.niger at a

comparatively lower concentration However (MVP, 1985) stated that not chemical or organic herbal treatment are effective to reduce contamination infection that can kill the seeds and also the seeds has a possible the seed coat might too hard to break so the treatment will not able to reduce the infection or the increased the dosage of the pre-treatment or the storage period might be too long and so the radicle is already dead

2.3 Methodology

Standard blotter method by (International Rules fore Seed Testing Science and Technology, 1996) was conducted, seed were washed /shaken with 0.1% Sodium Hypochlorite for 10 minutes then placed on the three layers of moistened blotter were, 10 seeds per Petri dish (depends on seed size) the plate were incubated in B.O.D incubator at 25± 2˚C for 7-10 days under 12 hours of altering cycle of light and darkness On the 8th day seeds were examined under stereo

binocular microscope for presence of seed borne fungi There were identified eater using compound microcope pathogens found recorded The percentage seed infection and germination of the seeds were calculated Isolated fungi were identified with the help of the key monograph and literature provided by

(Fennell, 1965) (Booth, 1971) (Ellis, 1971) (Hunter, 1972)the untreated seeds were used as control All experiments were carried out in triplicate However

(1982) reported from the seeds of Shtsjorea robusta, 21 fungal species in blotter

method and only 5 fungal were recorded on PDA Later also Mittal (1983) recorded 26 fungal species in blotter method and only 10 species on seed of

Cedrus deodara in PDA method

The health of the seeds is of utmost importance as might be possibility of the survival and transmission of pathogens The microorganisms are present as pathogens/disease causing agent within or contaminant on the surface of the seeds and sometimes even in the seed lot, thereby introducing the diseases into previous clean areas (Henning, 2005) According to Souza et al (2007), fungi are the biggest group of pathogenic agents associated with seeds and grains They used sodium hypochlorite (2.0-2.5% active chlorine) for treatment

Sodium hypochlorite completely (5.0% NaOCl) inhibited the in vitro

mycelia growth of Cercospora longissima Similarly, other authors have also

evaluated allelopathic activity of plant extracts using lettuce (MAGIERO, 2009) Based on the result there is no mimoreduction on gemiantion time at any of the tested concentration

Fungi associated with seeds of Dalbergia sissoo, Azadirachta indica, Paulownia fortunei, Albizia procera, Strychnos nux-vomica and Plantago ovate

from India were numerous In all 22 were fungi belonging to 15 genera were recorded on the seeds studied; detail is tabulated of the fungal species found on seeds of each tree species Wide variability was observed in seed mycoflora and stored and freshly collected seeds Stored seeds in general had greater numberof

fungi in comparison with freshly collected seeds Seed treatment with fungicides (Thiram, Dithane M-45[mancozeb] Bavistin [carbendazim] and Emisan [2-methox yethylmercury chloride]) was found effective in controlling the seed mycoflora The fungicide differe in their order of efficiency depending on the comparison of the seed mycoflora

2.4.Seed Borne Fungi of Some Forest and Their Control

The study was undertaken of the mycoflora of seeds (Singh & Khann,

1999)of Prosopis chinensis [p chilensis], Albizia procera, Michelia champaca fortunea, produce from the forest Research institute at Dehra Dun, Uttar Pradesh

(India) In all 16 fungi belonging to 8 genera were recorded of which moulds

were predominant The maximum number of fungi (8) appeared on seeds of P fortunea and the minimum (4) was recorded on Prosopis chilensis Aspergillus spp were present on the seeds of all the tree species except for Albizia procera whereas Penicillium citrium was detected on M champaca, Albrizia procera, Paulownia fortune and B purperea and was absent on the other tree species Three species of Phoma were detected on P.fortunei only, while Trichohecium roseum was present on seeds of Albrizia procera only Fusarium avenaceum [Gibberella avenacea] was present on seeds of Albizia procera, Accia nilotica, Acia catechu and B purrurea whereas it was absent in M champaca and Paulownia firtunei Application of Emisan [2-methoxythylmercury chlorite],

Topsin [thiophanate-methyl] and Bavistin [carbendazim] at 2g/kg and Dithane M-45 [Mncozeb], copper ox chlorite and Thiram at 4g/kg of efficacy Emisan was

followed by Thiram in M champaca, Albizia procera and Acacia nilotica, B purpurea and Dithane M-45 in case of Acacia catechu Topsin and copper ox

2.5 Impact of Seed Mycoflora and Forest Seed on See Quality anf Their Management

Seed ( Mamatha et.al, 2000) of Dendrocalamus strictus Phyllanthus emblica Hardwickia binata and Dalbergia latifolia were screened for myccoflora

incidence and showed both field and storage fungi Dominant mycoflora were isolated and their effect was studied on quality aspect like germination and vigour Seeds inoculated with dominant fungi showed significant decrease in germination and seedling vigour Seed samples were subjected to four different

pre-treatments Treatments with Trichoderma spp was most bith in reducing the

incidence of mycoflora and enhancing the germination and vigour

The purpose of this study is to address the issue of the research priorities with the science of seed pathology Seeds are important to be tested because catastrophic losses may occur in the event of diseases outbreak which may prove

to be seed-borne So protection measurement on forestry seed species must be givens purify Since forestry studies of seed borne transmission may have long been neglected Therefore seed health testing can reveal seed borne pathogens thus needs seed treatment (Nene, 1979) such as Sodium hypochlorite and /or the

used of plant extract like Capsicum sp this pre- treatment can be an effective

treatment that can reduce seed contamination and increase the germination, The study reflects to determine the seed pathogens and also the level of effectiveness

of the treatment to reduce the infection on forestry species seed

CHAPTER III STATUS OF THE CONSIDERED ISSUE

AT THE TRAINING AGENCY

The Ecosystems Research Development Bureau in Los Banos Laguna, Philippines is mandated to protect and conserve the environment and its natural resources Researcher’s help the society on what will be the possible solution on how to protect the environment in hazardous, pollution and other things that can affect the country’s the natural resources One of the projects of the ERDB is the proper care of the seeds coming from seed production areas (SPAs) to ensure the good quality and maximum quantity of seed produced seeds Quality planting material should come from good mother tree, proper harvesting or seed collection free from impurities including microbial contamination and pathogens when sorted, seeds are checked of moisture content (MC) That is right quantify of MC require per species

The quantity of the seeds/seed lots (total seed collection per species per site) are checked both in terms of health though seed and health test inherit quality thought seed technology These include pest (insects & pathogens) inert matters MC, germination rate, size and weight

One of the management strategies prior for plantation establishment is the seed health test Doing such will ensure the reduction of its total elimination of seeds contaminants and pest/ pathogens or seed storage microorganism Seed pathology complementing seed technology will produce therefore lead to increased rate of germination, good seedling quality health matured tree

To learn seed health test, a simple experiment was conducted in the Forest

Pathology Laboratory (also in ERDB) Seed health testing is one of the solutions that can be used to test if the forestry seeds are positive or seed are contaminated Application of pre- treatments to disinfect the seed borne pathogens (Fraedrich

M C., n.d.)such as Sodium Hypochlorite (Clorox) and Capsicum Extract Soap Solution (Chili Soap) was conducted

3.1 Title

Seed Health Testing of Selected Forestry Seeds and Their Control Using Sodium Hypochlorite and Capsicum Extract Soap Solution

3.2 Time and Place

The actual experiment was conducted at the ERDB seed center and in the Seed Pathology Laboratory and Tree Seed Center of Environmental Research and Development Bureau (ERDB) 100 Makiling, Los Baños, Laguna, Philippines form April 2018 to June 2017 The experiment area has normal humidity After 7-10 days check the seeds in the incubator for the presence of microorganisms

3.3 Objectives of the Study

 To determine different kinds of seed pathogens/other microorganism

contaminating or infecting four (4) kinds of forestry seeds

 To determine the level/percent seed pest or microbial infection

 To determine the effectiveness of sodium hypochlorite and capsicum

extract -soap solution as seed disinfectant

3.4 Statement of the Problem

 What are the seed borne pathogens found in four selected forestry seed species?

 What are the level/percentage of seed pests or microbial infection?

 Which is more effective treatment, Sodium Hypochlorite or Capsicum Extract-Soap Solution?

3.5 Hypothesis

Null Hypothesis

 Percentage fungal seed infection/contamination is low

 The treatments are not effective as fungal control

Alternative Hypothesis

 Percentage fungal seed infection/ contamination is high

 The treatments are effective as fungal control

3.6 Scope and Limitation

There were several notable limitations in the research and time allotted on the study is not enough to evaluate the long-term growth of the seeds and its performance in the nursery The experiment was conducted in the laboratory only Not too many forestry species are available in ERDB seed center Some species have limited number of the desire experiment The study focuses to determine the fungal diversity/ contamination of the seeds and its control Bacterial infection was included but not characterized and identified due to absence of facilities including chemical reagent/culture media for physiological study Germination is the lone parameter considered as affected by microorganism infection Seed moisture, weight, size and color were not included

3.7 Materials

All the materials and equipment are provided by Ecosystem Research Development Bureau, University of the Philippines, Los Banos Laguna Philippnes

3.7.1 Four selected forestry seeds species

 Lanete seeds (100 seeds per treatment)

 Taluto seeds (100 seeds per treatment)

 Ipil-ipil seeds (100 seeds per treatment)

 Molave seeds (100 seeds per treatment)

3.7.2 Petri Plates

Shaped like cylinder they are usally made of glass or plastic That Scientist uses it to grow cell from fungus and diseases of the seeds

3.7.3 Autoclave

Heated container used for chemical reaction and other processes using

high pressure and temperature e.g., steam sterilization

3.7.4 Compound Microscope

Optical instrument for viewing sample at high magnification has three, four or five objective lenses that range in power from 4x too 100x

3.7.5 Hot air oven

Is an electrical device which use dry heat to sterilize Petri dish and othe equipment was use in the experiment

3.7.8 Forceps

Are a handheld, hinged instrument used for grasping and holding object This is used to make sure that seeds are protected for other microorganisms

3.7.9 De- natured Alcohol and Alcohol lamp

Is a piece of laboratory equipment used to produce an open flame This is used for sterilization of the disease

3.7.10 Glass slides and Glass cover

This is used to hold objects for examination under a microscope Typically the object is mounted on the slide, and then both are inserted together in the microscope for viewing

3.8 Methods

 Petri dish sterilization in hot air oven at 80 degree Celsius for 2 hours Distilled water sterilization and filter paper sterilization in automatic autoclave at

212 degree Celsius, 120 Pressure per square inch (psi) for one (1) hour

 Prepare Capsicum Extract Soap Solution using 25 pieces hot chilli sieved

in gauze, mixed with on teas spoon liquid soap 1 L distilled water; and steam sterilized at 212 degree Celsius 120 psi for one hour

 Prepare four (4) selected forestry seeds that will be tested;

 Plant the seed onto Petri dish with three layers of filter paper that can hold

5 mL distilled water

 Incubate the plated seeds under (12 inches space between) day light tubes for 7- 10 days under alternating light (12 hours) and 12 hours of darkness

 Examine the seed with the naked eyes 7 days after incubation, then under binocular microscope (4-10-x magnification) Those with microbial growth were further examined under the compound microscope 100 to 400x magnification with high magnification, some fungi producing spores were identified/ suspected

up to species level

 Other data collected were bacterial contamination and rate of seed germination

 Data were tabulated and statistically analyzed

 Report was then prepared

3.9 Research Approach

Figure 1: Research Approach

3.10 Research Design

The research design that was used is Complete Randomized Design (CRD) as layout in the table below

Treatment 1(T1) - Control (No Treatment)

Treatment 2(T2) - Sodium Hypochlorite (NaOCl)

Treatment 3 (T3) - Capsicum Extract Soap Solution (CESS)

Replication (R)

3.11 Data Gathering

The data gathering will be gathered after seven (7) days of incubation (baseline data) and seven days after the treatment were applied For this study, seed were assessed using the parameter such us number of infected seeds, germinated seeds, number of seeds with fungal growth and with bacterial contamination, The fungi were identified using various references while bacteria will be recorded as presence of bacterium only Through physiological test which cannot be done in the laboratory due to unavailability of reagents and other

Figure 2: Research Design

3.12 Statistical Analysis

The statistical analyses of the data were performed using the Multiple Comparison of Turkeys of Statistical Analysis System (SAS) was carried out to determine if there were a high percentage of infected seeds between the germination of the seeds of the four selected forestry seeds with in different treatment to disinfect the fungus The Multiple Comparison will be specifically used with the growth parameter of the fungal infection, bacteria, fungi spicbes and germination rate

3.13 Results

This chapter summarizes the results of data analysis Tables were used to present the data of infected seeds, germinated; bacteria and numbers of fungus were found.The study used the following labels to indicate different levels of treatemts T1- Control (No Treatment)

T2- Sodium Hypochlorite /NaOCl

T3- Capsicum Extract Soap Solution Soap/CESS

3.13.1 Seedborne Pathogens

Table 2: List of Seedborne Pathogens/Contaminants

Forest Tree Seed Species Type of Seed Borne Pathogens

sp.Verticillium sp

Aspergillus niger

This seed borne pathogens were identified based on the characteristic of the

pathogens.Using references: Monograph on Fusarium: Illustrated genera of fungi

imperfecti, Recent studies on Forestry species Seedbrone fungi

3.13.2 Fungal Infection

Table 3: Percentage of infection of Fungi on four slected forestry

Seeds and Effect of Pre-treatment

Fungal Infection Type of

Seeds Treatments Percentage Std Err

Trurkeys Group p-value

treatment to fungal infection of Apocynacea wrighita pubescens R.Br (Lanete)

from treatment 1 and treatment 2 while treatment 1 and treatment 2 has no significant difference P-value is <0.0001 which is less than the significance value of 0.05, which indicate that there is no significant evidence to reject the null hypothesis (Percentage fungal seed infection/contamination is low and the treatments are not effective as fungal control)

On Ipil ipil seeds as the results showed in the table Control Treatment has the highest average value with the mean of 77% Followed by Capsicum Extract Soap Solution (CESS) with average value mean of 29% and Sodium Hypochlorite (NaOCl) has lowest value with a 14% This means that treatment 2 has the most effective treatment to reduce the infection on the seeds Statistical analysis indicated that all Treatment has significantly difference from each other P-value is <0.0001 which is less than the significance value of 0.05, which indicate that there is no significant evidence to reject the null hypothesis

In terms of fungal infection of Taluto seeds as shown in the table Control Treatment have the highest seeds infection with the mean of 35% (at 100 seeds per treatment replicated 3 times) Followed by NaOCl with the mean of 1%and CESS has the lowest value with the mean of 6% This means that treatment 3 is the most effective treatment to reduce the level of infection on the seeds On the statistical analysis treatment 2 and treatment 3 has no significant difference However treatment 1 has significant difference from treatment 2 and treatment 3 The results therefore showed that fungal infection can be controlled by NaOCl and CESS P-value is 0.0000 which is less than the significance value of 0.05, which indicate that there is no significant evidence to reject the null hypothesis

Since there was no great variation in the rawdata with the fungal infection

on Molave seeds were also consider the internal infection can no longer be determined and the seeds were stored for a long time so the seeds are too

infected (By component plating that is separation of seed coat drom cotyledons and radile) due to time contraints further study can only be recommended

3.13.3 Bacteria

Table 4: Percentage of Bacteria of Four Selected Forestry Seeds with

Pre-treatment Using NaOCl and CESS

Bacteria Type of

Seeds Treatments Percentage Std Err

Trurkeys Group

value

Treatment 3 but Treatment 1 has significant difference form Treatment 3 This means that CESS is a better control measure against bacteria P-value is 0.0486 which is less than the significance value of 0.05, which indicate that there is no significant evidence to reject the null hypothesis

In terms of the presence of bacteria on the Taluto seeds the results showed that Control (No-Treatment) has the highest value of bacteria with the average of 33%, followed by NaOCL with the average mean of 13% and CESS has the lowest value of bacteria with the average of 6% This means that treatment 3 is more effective treatment to reduce the bacteria on the seeds Statistical analysis indicated that NaOCl and CESS has no significantly difference, however treatment 1 has significantly difference between treatment 2 and treatment 3 P-value is 0.0000 which is less than the significance value of 0.05, which indicate that there is no significant evidence to reject the null hypothesis

In terms of the presence of the bacteria as the results showed in the tbale Capsicum Extract Soap Solution (CESS) has the highest value with a mean of 29% followed by Control Treatment with an average value mean of 25% and Sodium Hypochlorite (NaOCl) has the lowest with a mean of 14% This means that in terms on reducing the bacteria based on the analysis that in Treatment 3 increased the value of bacteria Statistical analysis that treatment 3 has no significant difference with treatment 1 but has significant difference with treatment 2 in this case treatment P-value is 0.015 which is less than the significance value of 0.05, which indicate that there is no significant evidence to reject the null hypothesis

In terms of Molave seeds, since there was no great variation in the rawdata with bacteria as response variables Was also considering internal infection can

no longer be determined