POTENTIAL

APPLICATION OF KAVA

(Piper methysticum F.)

IN NEMATODE CONTROL

A thesis submitted in partial fulfillment of the

requirements for the degree of

Master of Science

By

Ranjeeta D. Singh (BSc, PGDip)

School of Biological, Chemical and Environmental Sciences Faculty of Science and Technology

The University of the South Pacific

November 2006 Potential Application of Kava (Piper methysticum F.) in Nematode Control

DECLARATION

I declare that this submission is a result of my own investigation and has not been submitted in any form for another degree or diploma at any university. To the best of my knowledge, it contains no material written by another author or previously reported, except where due acknowledgement is credited in the text.

Candidate: RANJEETA D. SINGH

The current research was conducted under our supervision and we are certain that this is the sole work of Ms Ranjeeta D. Singh.

Supervisors:

Dr Mani Naiker Dr Uma Khurma

Chemistry Supervisor Biology Supervisor

School of Chemical Sciences School of Biological Sciences

Faculty of Science and Technology Faculty of Science and Technology

University of the South Pacific University of the South Pacific

ii Potential Application of Kava (Piper methysticum F.) in Nematode Control

ABSTRACT

Kava (Piper methysticum) has long history of its use in social and ceremonial gatherings

and for medicinal purposes by the Pacific islanders. Research has identified the active

ingredients as the kava lactones, which have been widely tested for various bioactivity

properties. The water soluble compounds on the other hand have not been scientifically

explored much. Work presented in this thesis reports for the first time the nematicidal

property of polar extracts of dried kava roots.

The water soluble compounds of dried kava root material referred to as sample X and the

highly polar extract, sample Y (sample X less the ethanol solubles) has been tested for juvenile mortality and suppression of egg hatching. Samples Y1, Y2, Y3 and Y4 obtained

by semi preparative HPLC separation of sample Y, were tested for juvenile mortality

only. The results of these experiments were compared with the activity of gallic acid as

the standard. Efficacy of kava powder was tested in pot experiments as soil amendment.

Samples X, Y, Y1, Y2, Y3, and Y4 showed significant activity towards juvenile mortality

and suppression of egg hatching. Samples X of FJ and VA kava showed some differences

in activity towards juvenile mortality but similar activity was noted for egg hatching

experiments for both FJ and VA kava samples. H-NMR analyses of the four fractions

isolated have shown the possibility of the presence of glucoside like compounds in the

polar extract of kava. Kava powder when mixed with soil significantly decreased the root

iii Potential Application of Kava (Piper methysticum F.) in Nematode Control galls on tomato plants. Allowing kava to degrade in the soil before planting seedlings minimized the phytotoxic effect while being effective in controlling nematodes.

These results have indicated that kava does have the nematicidal polar compounds.

Further purification of the extracts obtained will lead to the isolation of individual compounds. GC-MS and NMR analysis of individual compounds will further provide information on the structure of these compounds.

iv Potential Application of Kava (Piper methysticum F.) in Nematode Control

ACKNOWLEDGEMENTS

I gratefully take this opportunity to thank my supervisors Dr Mani Naiker and Dr Uma

Khurma (School of Biological, Chemical and Environmental Sciences) for their support,

guidance and criticism during my research.

Special thanks to Dr David Tucker of University of New England, Armidale, NSW,

Australia for his valuable time in performing NMR analysis.

The University Research Committee and the School of Pure and Applied Science

Research Committee are acknowledged for providing the necessary funds for the

operating costs of this research. The Scholarship Committee is duly thanked for awarding

me the Graduate Assistantship.

I am highly indebted to Dr Mathias Schmidt (HERB Research, Germany), Professor

Yadhu Singh (South Dakota State University, USA) and Dr Linton Winder, (School of

Biological, Chemical and Environmental Sciences, USP) for helping with obtaining journal papers. The assistance by Dr Linton Winder and Dr Badru Doza of the University

of the South Pacific in the statistical analysis of the results is also highly appreciated.

The technicians of School of Biological, Chemical and Environmental Sciences

especially Mr Vas Deo, Steve, Ragni and Dinesh are acknowledged for their help in

locating the necessities for this project. Praneel, Roneel and Abhinesh are acknowledged Potential Application of Kava (Piper methysticum F.) in Nematode Control for their support in carrying the experiments. Sincere thanks to Sachin and Kirti for their advice and guidance for the required instrumentation of this project.

I offer my heartfelt thanks to my parents (Mr & Mrs Vijay K. Singh), my brothers (Jagen and Rakesh) and my sister (Geeta) for their patience, encouragement and contribution to my education.

Finally my special thanks to my friends Kirti, Sachin, Modi, Ranjani, Vikashni,

Riteshma, Sunny, Babeeta, Pedro and Lawrence for helping with the writeup, giving enthusiasm and keeping me company when I needed them most.

vi Potential Application of Kava (Piper methysticum F.) in Nematode Control

PUBLICATIONS

Part of the work described in this thesis has been presented as:

1. Singh, R., Naiker, M. and Khurma, U. R. 2004. Evaluation of Nematode Suppressive

Potential of Kava. International Kava Conference, Suva, Fiji.

2. Khurma, U.R. and Singh, R. 2005. Effect of Kava as Soil additive on Root-knot

Nematode Population. ONTA 2005, Organization of Nematologists of Tropical

America at Viña del Mar, CHILE.

vi Potential Application of Kava (Piper methysticum F.) in Nematode Control

LIST OF ABBREVIATIONS

BOD biological oxygen demand

CCC countercurrent chromatography cm centimeters cv. cultivar

DCCC droplet countercurrent chromatography

DCM dichloromethane

FJ Fiji ft feet g grams g/mL grams per millilitre

GC-MS Gas chromatography coupled with mass spectroscopy

H-NMR proton nuclear magnetic resonance spectroscopy

HPLC High Performance Liquid Chromatography hrs hours in. inches

J1 first stage juveniles

J2 second stage juveniles

L litres

LC-MS liquid chromatography coupled with mass spectroscopy m metres min minutes

vi Potential Application of Kava (Piper methysticum F.) in Nematode Control mL milliliters mL/min milliliters per minute mL/kg milliliters per kilogram mm millimeters nm nanometers

NMR nuclear magnetic resonance spectroscopy ppm part per million

PPN plant parasitic nematodes

RLCCC rotational locular countercurrent chromatography

RKN root knot nematodes semi-prep semi preparative sp. Species

TLC Thin layer chromatography

UV-Vis Ultra-violet visual

VA Vanuatu w/v weight by volume w/w weight by weight

X-VA Freeze dried aqueous residue of Vanuatu Kava

X-FJ (stored) Freeze dried aqueous residue of Fiji Kava Stored at 4 °C for six

months

X-FJ (fresh) Freeze dried aqueous residue of Fiji Kava, freshly prepared

Y-FJ Freeze dried aqueous residue of Fiji Kava minus ethanol

extracts

ix Potential Application of Kava (Piper methysticum F.) in Nematode Control

Y-VA Freeze dried aqueous residue of Fiji Kava minus ethanol

extracts

YI-FJ First eluted fraction collected from Y-FJ in semi-preparative

HPLC

Y1-VA First eluted fraction collected from Y-VA in semi-preparative

HPLC

Y2-FJ Second eluted fraction collected from Y-FJ in semi-preparative

HPLC

Y2-VA Second eluted fraction collected from Y-VA in semi-preparative

HPLC

Y3-FJ Third eluted fraction collected from Y-FJ in semi-preparative

HPLC

Y3-VA Third eluted fraction collected from Y-VA in semi-preparative

HPLC

Y4-FJ Fourth eluted fraction collected from Y-FJ in semi-preparative

HPLC

Y4-VA Fourth eluted fraction collected from Y-VA in semi-preparative

HPLC uL microlitres um micrometres Potential Application of Kava (Piper methysticum F.) in Nematode Control

LIST OF FIGURES

Figure 1.1 Structures of six major kava lactones 4

Figure 1.2 Structure of Pipermethystine 5

Figure 1.3 Structures of pigment molecules 6

Figure 1.4 Structures of cinamic acid, pinostrobin and 5,7-dimethoxyflavone 7

Figure 1.5 Structures of phenolic compounds identified in kava…………………...... 8

Figure 1.6 Structure of 2-furfuraldehyde 23

Figure 1.7 Structure of 20-hydroxyecdysone 24

Figure 1.8 Structures of benzaldehyde, citral, furfural, menthol and

a-terpineol 24

Figure 2.1 HPLC chromatogram showing peaks of the ethanol solubles…………….34

Figure 2.2 HPLC chromatogram of the final ethanol extract from soxhlet…………..34

Figure 2.3 HPLC chromatogram for sample Y showing the groups of

peaks which were isolated 35

Figure 3.1 Tomato roots infected by RKN as viewed with naked eyes………………40

Figure 3.2 Live juveniles in motion in water as viewed under 40x magnification…...45

Figure 3.3 Dead juveniles viewed under 40x magnification (note the slightly

curved to almost straight posture) 46

Figure 3.4 Kava powder mixed in soil and left for degrading in the plot land……....50

Figure 3.5 The author planting the tomato plants in the pots after inoculating

with juvenile nematodes 50

xi Potential Application of Kava (Piper methysticum F.) in Nematode Control

Figure 3.6 Juvenile Mortality for FJ kava fractions 63

Figure 3.7 Juvenile Mortality for VA kava fraction 63

Figure 3.8 Treatment of egg masses with sample X-FJ (fresh)………………………67

Figure 3.9 Treatment of egg masses with sample X-VA…………………………...... 67

Figure 3.10 Treatment of egg masses with sample Y-FJ 69

Figure 3.11 Treatment of egg masses with sample Y-VA 69

Figure 3.12 Comparison of activity of samples X of FJ and VA kava

with Gallic Acid for egg hatching 73

Figure 3.13 Comparison of activity of samples Y of FJ and VA kava

with Gallic Acid for egg hatching 73

Figure 3.14 Zero weeks degradation FJ kava 75

Figure 3.15 Zero weeks degradation VA kava 75

Figure 3.16 Two weeks degradation FJ Kava 78

Figure 3.17 Two weeks degradation VA kava 78

Figure 3.18 Four weeks degradation FJ kava 79

Figure 3.19 Four weeks degradation VA kava 79

Figure 3.20 Six weeks degradation FJ kava 80

Figure 3.21 Six weeks degradation VA Kava 80

Figure 3.22 Root gall numbers against kava concentration for FJ kava………………83

Figure 3.23 Root gall numbers against kava concentration for VA kava…………….83

xi Potential Application of Kava (Piper methysticum F.) in Nematode Control

LIST OF TABLES

Table 3.1 FJ kava extracts tested for juvenile mortality at room

temperature 54

Table 3.2 Analysis of Variance Results for FJ Kava Samples

tested at room temperature 54

Table 3.3 FJ kava extracts tested for juvenile mortality at 28°C…………………...... 57

Table 3.4 Analysis of Variance Results for FJ Kava Samples tested at 28 °C…….....57

Table 3.5 VA kava extracts tested for juvenile mortality at 28°C……………………59

Table 3.6 Analysis of Variance Results for VA Kava Samples tested at 28 °C……...59

Table 3.7 Comparison of samples X of FJ and VA kava with gallic acid……………60

Table 3.8 Comparison of samples Y of FJ and VA kava with gallic acid……………61

Table 3.9 Analysis of Variance Results for FJ and VA Kava fractions tested

at 28°C 62

Table 3.10 Results for Sample X-FJ (fresh) 66

Table 3.11 Results for Sample X-VA 66

Table 3.12 Results for Sample Y-FJ 68

Table 3.13 Results for Sample Y-VA 68

Table 3.14 Observed plant heights for the various treatment experiments

of EJ and VA kava 81

Table 3.15 Observed root lengths for the various treatment experiments

ofFJ andVAkava 81

xi Potential Application of Kava (Piper methysticum F.) in Nematode Control

TABLE OF CONTENTS

Declaration ii

Abstract iii

Acknowledgements v Publications vii List of Abbreviations viii List of Figures xi List of Tables xiii 1.0 Introduction 1 1.1 Kava 1 1.2 Chemistry of Kava 3 1.3 Biological Activity of Kava 9 1.4 Toxic effects of other Piper spp 13 1.5 Nematodes 15 1.6 Effect of Meloidogyne spp on Plants 16 1.7 Nematode Management 18 1.7.1 Nematicides for Nematode Control 18 1.7.2 Natural products for Nematode Control 19 1.7.3 Compounds Isolated from Plants with Nematicidal Activity 22 1.8 Aims and Objectives of Present Study 25 2.0 Kava Extractions and Separation 26 2.1 Introduction 26 2.2 Methodology 27 2.2.1 General Methodology 27 2.2.2 Aqueous Extraction 28 2.2.3 Removal of Ethanol Solubles 28 2.2.3.1 HPLC analysis of Sample Y to verify the removal of Ethanol Solubles 29 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2.2.4 Separation of Sample Y 29 2.2.4.1 Identification of x max 30 2.2.4.2 Mobile Phase Composition Determination for separation of Sample Y using Thin Layer Chromatography 30 2.2.4.3 Gradient HPLC Analysis of Sample Y……………... 31 2.2.4.4 HPLC Separation of Potential Nematicidal Compounds from Sample Y 32 2.2.5 1H-NMR Analysis of Fractions of Sample Y…………………..33 2.3 Results and Discussion 34 2.3.1 HPLC analysis of Sample Y to verify the removal of Ethanol Solubles 34 2.3.2 1H-NMR Analysis of Fractions of Samples Y1,Y2, Y3and Y4 35 2.4 Conclusion 37 3.0 Bioassays 38 3.1 Introduction 38 3.2 Methodology 40 3.2.1 General Methodology 40 3.2.2 Nematode Culturing and Species Identification……………... 41 3.2.3 Sample Preparation 42 3.2.4 Laboratory Assays of Kava Extracts 43 3.2.4.1 Juvenile Mortality Tests 44 3.2.4.2 Egg Hatching Experiments 46 3.2.4.3 Comparison with standard (Gallic Acid)……………. 47 3.2.5 Pot Experiments (Soil Amendment Experiments)……………..48 3.2.5.1 Preparation of Pots and Soil for Experiments………...48 3.2.5.2 Experimental Setup 48 3.2.5.3 Planting of Seedlings and Inoculating………………..49 3.2.5.4 Plant Observations 51 3.3 Results and Discussion 52 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.3.1 Laboratory Assays of Kava Extracts 52 3.3.1.1 Juvenile Mortality Experiments 52 a) FJ Kava Extracts Tested at Room Temperature 52 b) FJ Kava Extracts Tested at 28 °C 55 c) VA Kava Extracts Tested at 28 °C………………...57 d) Comparison of sample X of FJ and VA Kava with the standard 59 e) Comparison of sample Y of FJ and VA Kava with the Standard 60 3.3.1.2 Juvenile Mortality Experiments of the Fractions 61 3.3.1.3 Egg Hatch Experiments 64 a) Activity of samples X and Y of FJ and VA kava 64 b) Comparison of Activity of Samples X and Y of FJ and VA Kava with Standard……….....70 3.3.2 Pot Experiments (Soil Amendment Experiments) for FJ and VA Kava 74 3.3.2.1 No Degradation Allowed (0 weeks)……………….....74 3.3.2.2 Two Weeks of Degradation 76 3.3.2.3 Four Weeks of Degradation 76 3.3.2.4 Six Weeks of Degradation 77 3.3.2.5 Comparison of Observed Results at Different Concentrations over various Degradation Periods 82 3.3.2.6 General Discussion 84 3.4 Conclusion 87 4.0 General Conclusions 89 5.0 Reference 91 Appendices 105 Potential Application of Kava (Piper methysticum F.) in Nematode Control

CHAPTER 1

1.0 INTRODUCTION

1.1 Kava

Piper methysticum Forst is a tropical shrub that grows throughout the Pacific Islands. It belongs to the pepper family (Piperaceae) and is also known as kava, asava pepper, or intoxicating pepper. It grows to an average height of 6 ft (1.83 m) and has large heart- shaped leaves that can grow to 10 in. (25.4 cm) wide. This plant is cultivated for commercial and social purposes and is harvested after 3 to 5 years from cultivation

(Davis and Brown 1999; Walji 1998). The roots and lower parts of the plant are dried, pounded and mixed with water to make an intoxicating drink that serves as a traditional beverage in Fiji and the South Pacific countries.

Various cultivars of kava are found in the Pacific. Fiji has eleven distinct cultivars while

Vanuatu, believed to be the origin of kava, has eighty-two different cultivars (Lebot and

Le'vesque 1989). Farmers distinguish them on the basis of morphological and ancillary characteristics, physiological effects on drinkers, and the region where the cultivar was thought to have originated (Walji 1998).

Kava drink is believed to help break social barriers, settle interpersonal conflicts and enhance social ties. The drink is prepared by mixing ground or masticated roots and stem Potential Application of Kava (Piper methysticum F.) in Nematode Control bases with water. Usually it is served in a half coconut shell, and the total contents are always drained in one draught (Davis and Brown 1999). The Pacific Islanders also offer it to gods, spirits and ancestors as a sign of respect, to obtain favor and to appease their resentment and anger if due respect has not been shown to them and to communicate with the supernatural world.

Various authors have described the effect of the drink differently. Some stated the thirst relieving potential of kava due to its pleasant, cooling, aromatic and numbing effect on the mucous membrane of the tongue (Singh 1986; Walji 1998). Te Rangi Hiroa a

Polynesian from New Zealand, who often drank kava found it cooling, refreshing, and stimulating without being intoxicating. Other reports describe it with bitterness and burning taste in the mouth but the first effect is a numbing and astringent effect on the tongue and the inner lining of the mouth (Singh 1986; Walji 1998).

Apart from being used as a beverage, kava is also used for medicinal reasons by the

Islanders. Gout, rheumatism, diarrhea, asthma, venereal diseases and convulsive disorders were some of the conditions, which were treated with kava (Duve 1976; Lebot et al., 1992, 1997; Singh and Blumenthal 1992). A tea made from the roots of kava is used as a diuretic for kidney and bladder ailments in Fiji. Coughs, colds and sore throats are treated with kava as well. A decoction of the root given to mothers who had given births is known to prevent them from getting pregnant again while leaves are chewed as a contraceptive measure. Wounds are known to be treated with juice extracted from fresh leaves (Forster 2000; Lebot et al, 1992, 1997). Potential Application of Kava (Piper methysticum F.) in Nematode Control

1.2 Chemistry of Kava

The medicinal uses of kava had initiated research into the chemistry and biological activity of kava. Many active compounds have been isolated from kava and the majority of the biological activity has been accredited to the kava lactones. Kava lactones are documented to have pharmacological properties such as anticonvulsive, antiepileptic, antifungal and local anesthetic effects (Cambie and Ash 1994; Cordell 1998).

Chemical analysis of the kava root stock has shown that fresh roots contain on average 80

% water, but when dried, consists of approximately 43 % starch, 20 % fibers, 12 % water,

3.2 % sugars, 3.6 % proteins, 3.2 % minerals, and 20 % kava lactones (can vary between

3 % to 20 % depending on the age of plant and the cultivar) (Lebot et al, 1992, 1997;

Singh 1986; Singh and Blumenthal 1992).

The kava lactones are a group of α - pyrones with a methoxy group at carbon 4 and an aromatic styryl moiety at carbon 6. Eighteen different lactones have been identified in kava of which the six major ones include kavain (1), methysticin (2), yangonin (3), 7,8- dihydrokavain (4), 7,8-dihydromethysticin (5) and desmethoxyyangonin (6). The lactones are lipid like compounds contained within oil cells and thus would be deemed to be generally insoluble in water, therefore the traditional preparation of kava drink produces an emulsion of the lactones (Basko 2002; Lebot et al., 1992, 1997; Walji 1998). Potential Application of Kava (Piper methysticum F.) in Nematode Control

CH3

,ChU

Figure 1.1: Structures of six major kava lactones

A novel pyridone alkaloid, pipermethystine (7) has also been identified in P. methysticum. This alkaloid was not present in the roots but formed a minor component of the stem and leaves (Cambie et al., 1997; Smith 1979, 1983; Dragull et al., 2003).

Pipermethysticine decomposes on standing at room temperature due to hydrolysis of the amide, to give 3-phenylpropionic acid and the dihydropyridone (Smith 1979). Other piperidine alkaloids found in leaves and stems of Piper methysticum include awaine and

3a, 4a-epoxy-5P-pipermethysticine (Dragull et al., 2003). N-cinnamoylpyrrolidine and N- Potential Application of Kava (Piper methysticum F.) in Nematode Control

[m-methoxycinnamoyl]-pyrrolidine were three other alkaloids isolated from the roots

(Sotheeswaran 1987).

Alkaloids are compounds of plant origin with complex structures having a nitrogen atom in the heterocyclic ring. So far over thousand alkaloids are known, and it is estimated that they are present in only 10-15 % of all vascular plants. They are found in cryptogamia, gymnosperms, or monocotyledons. They occur abundantly in certain dicotyledons. Well- characterized alkaloids have been isolated from roots, seeds, leaves or bark of plants

(Pelletier 1970).

CH

Figure 1.2: Structure of Pipermethystine

Glutathione is another compound of interest found in aqueous and 25 % ethanol extract of kava. Glutathione is soluble in high polarity solvents and the concentration of it increases with increasing polarity of the solvents. This compound has been found to be reacting with kava lactones. The decolourisation of total kava lactone solution in presence of glutathione supports this interaction. The loss of colour is due to the opening of the lactone ring (Whitton et al., 2003). The interaction is similar to those reported for Potential Application of Kava (Piper methysticum F.) in Nematode Control sesquiterpene lactones with glutathione. Such reaction of glutathione is important in converting the kava lactones into excretable waste products (Schmidt et al., 1999).

Other compounds isolated from kava are flavokawins, an alcohol, a phytosterol, ketones and organic acids (Lebot and Levesque 1989). Pigment materials, flavokawin A (8) and

B (9) were isolated and reported in 1963 and have been associated with the skin discoloration in chronic kava drinkers (Singh 1986).

H3C

Figure 1.3: Structures of pigment molecules

Analysis of water-soluble components of kava indicated the presence of glucose as the carbohydrate in addition to the pharmacologically active lactones. This was determined through thin-layer and paper chromatographic techniques (Sotheeswaran et al., 1998).

Treatment of the delactoned residue of kava (after the lactones and other ethanol solubles were removed) with ß-D-glucosidase showed regeneration of some of the lactones

(Naiker et al., 2006). Thus indicating the presence of glucosidic compounds together with carbohydrates in the highly polar water extract. Potential Application of Kava (Piper methysticum F.) in Nematode Control

Extraction of powdered kava root by steam followed by lyophilization produced the most pharmacologically active water-soluble component. Analysis of this fraction revealed presence of aldehydes and ketones but no nitrogen was detected (O'hara et al., 1965).

Hot water and methanol extraction of kava lactones revealed the presence of some other compounds of interest. The methanol extract yielded bornyl esters of 3,4-methylenedioxy cinnamic acid, cinnamic acid (10), pinostrobin (11), flavokawain B(9) and 5,7- dimethoxyflavonone (12). The aqueous extract contained the previously reported kava lactones upon TLC analysis (Wu et al, 2002).

OH

OH O CH

Figure 1.4: Structures of cinnamic acid, pinostrobin and 5,7-dimethoxyflavonone Potential Application of Kava (Piper methysticum F.) in Nematode Control

O O HO OH OH

HO "y" HO OH 13 OH 14

O

OH

HO 15 HO

O

OH

OH 18

19

Figure 1.5: Structures of phenolic compounds identified in kava Potential Application of Kava (Piper methysticum F.) in Nematode Control

High Performance Liquid Chromatography (HPLC) analysis of 80 % methanolic extract of kava revealed the presence of phenolic compounds. These were identified to be gallic acid (13), protocatechuic acid (14), p-hydrobenzoic acid (15), p-coumaric acid (16), ferulic acid (17), salicylic acid (18) and trans-o-coumaric (19). Trans-cinamic acid (10) was also reported together with these phenolic compounds (Xuan et al, 2003 ).

Elemental composition of the dried kava powder includes N: 0.37, P: 0.27, K: 0.63, Mg:

0.07 and Ca: 0.46 % (Xuan et al. 20031).

1.3 Biological Activity of Kava

The different lactones present in kava are said to be the psychoactive ingredients, which act as sedatives, soporifics, analgesics, anticonvulsives, local anesthetics, muscle relaxant and diuretics (Davis and Brown 1999; Walji 1998). Strong activity against fungi is also reported for these kava lactones (Duve 1976; Lebot et al., 1992; Singh and Blumenthal

1992). Minor skin infections are usually treated with kava in highlands of Indonesia and in Papua and New Guinea (Davis and Brown 1999).

Kava resin which contains the lactones is reported to have a weak sleep inducing action, paralyzing effect on sensory nerves and stimulating result on the smooth muscles before paralyzing them (Walji 1998). It was also noted that there was an increase in the activity of kava when mixed with saliva. Saliva reduces the starch component thereby increasing the concentration of the kava lactones (Walji 1998). The action of saliva can be compared with that of acid hydrolysis whereby kava lactones are regenerated from a totally devoid Potential Application of Kava (Piper methysticum F.) in Nematode Control sample (Naiker and Prasad 2005). The regeneration was explained as their release from precursors, which were postulated to be glucosides. The amylase enzyme in saliva could be behaving in a similar way thus increasing the concentration of kava lactones.

The water-soluble fractions of the steam distillate of the kava showed a depression of the spontaneous motor activity in albino mice without any effect on forced motor activity (O' hara et al., 1965). Kava resin as well as the water soluble, delactoned extracts of kava reduced amphetamine-induced hyper motility in mice (Duffield et al., 1989). The aqueous extract of kava, containing methysticin and dihydromethysticin showed enhanced spasmolytic activity (Sotheeswaran 1987). Tumor induced by okadaic acid on mouse was inhibited by methanol extract of kava as well as that of green tea

(Sotheeswaran 2002). This indicated that kava had the potential to prevent humans from tumors as effectively as green tea, which has well-known anticancer activity.

The ethanolic extract of kava produced dose-dependent anxiolytic-like behavioral changes and reduction in locomotor activity in mice. Flumazenil, a competitive benzodiazepine receptor antagonist was found to block the anxiolytic and sedative effects of diazepam but it did not have any effect on the behavioral actions of kava (Garrett et al., 2003). This indicates that the anxiolytic-like behavioral changes and sedation by kava is not mediated through the benzodiazepine-binding site on the GABA-A receptor complex.

10 Potential Application of Kava (Piper methysticum F.) in Nematode Control

There have not been any reports of any kind of toxic effects of kava among the Pacific

Islanders. The only detrimental effect noticed is the 'kava dermopathy' which is commonly known as 'kani' and is visible in heavy kava drinkers. In this condition the skin gets dry and is covered with scales. It is inferred that flavokawain is responsible for this condition. Flavokawain interferes with the normal uptake and metabolism of some of the B-group vitamins (Sotheeswaran 1987). Ruze (1990) carried out an examination on over 200 Tongans and came to the conclusion that the dermopathy was not due to the deficiency of niacin, as was the case for acquired ichthyosis. Postulations on interference of compounds present in kava with cholesterol metabolism have been made for this skin condition as well (Norton and Ruze 1994).

Kava has been linked to gastrointestinal complications in heavy consumers but there is limited scientific evidence for this (Malani 2002). Poor health has been associated with heavy kava drinkers (Mathews et al., 1988). This is mainly due to poor eating habits of these individuals. Other conditions associated with kava drinking are impaired vision, enlargement of pupils, disturbances in occulomotor equilibrium, worsening of

Parkinson's syndrome and interactions with central nervous system depressants especially benzodiazepines (Singh 2005).

Several pyridone alkaloids with structures similar to pipermethystine have been shown to be cytotoxic (Duh et al., 1990). The compounds 3-phenylpropionic acid and dihydropyridone exhibit structural features of 2,5- dihydroxypyridine, which has been shown to affect DNA integrity (Kim and Novak, 1990, 1991).

11 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Kava root extracts in 70 % methanol has been found to inhibit the growth of following

five fungi; Fusarium solani, Pyricularia grisea, Rhizopus stolonifer, Taphrina deformans

and Thanatephorus cucumeris. The highest effect was seen on R. stolonifer, where 100 %

inhibition was observed (Xuan et al., 20031). It inhibited mycoses (fungal infection) of

the skin, which is generally very resistant to treatment (Hansel and Klaproth 1966). The

kava pyrones have fungistatic properties against a wide range of fungi including many

which are pathogenic to humans (Singh 1986; Sotheeswaran 1987). Dihydrokavain (kava

lactone) was effective against Aspergillus niger (Hansel and Klaproth 1966).

Antibacterial properties of P. methysticum against Gonococcus sp. (the cause for

gonorrhea), Colon bacillus and Salmonella typhi have been reported as well (Walji

1998). The crude lactones showed antimicrobial properties against a number of bacteria

including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus,

Acinetobacter baumii, Salmonella typhi and Candida albicans (Lal 2003).

The kava lactone yangonin, is reported to have amoebicidal properties. In vitro tests

against Entamoeba histolytica resulted in 100 % elimination after 48 hours of incubation.

This activity was comparable to that of many commercial drugs like enteroviaform,

enteroquinol, clefamide and furamidazole (Sonfi et al., 1983). Another amoeba against

which 80 % ethanol extract of kava had detrimental effect was Acanthamoeba castellani

(Whitton et al., 2003). It was noticed that this extract had 100 % inhibition of amoeba but

the inhibition was reduced to 40 % when the ethanol extract was mixed with glutathione.

12 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Some herbicidal properties of kava have also been reported (Xuan et al., 20031). A strong inhibition of barnyard grass, monochoria, and knot grass was noted when kava was applied to soil. Eight phenolic compounds have been identified in kava which have also been isolated from many allelopathic plants (Xuan et al., 20032).

1.4 Toxic effects of other Piper spp.

Insect defense properties are common in the plants of the family Piperaceae, including the common spice black pepper, Piper nig rum L. and around 1000 other tropical species.

Research has shown that the plants of the Piper genus contain over 200 secondary compounds. The amides present are responsible for providing the 'hot pungent' taste and the biological activity to this genus. The most active piperamide discovered is pipericide

(Dyer et al, 2004).

The piperamides belong to the unsaturated isobutylamides which is a well known group of insecticidal compounds found in various Piper species. Various amides including pipericide have been isolated from the fruits, stem and leaves of P. nig rum, P. acutisleginum, P. khasiana, P. longum, P. pedicellosum and P. thomsoni. Having neurotoxic effect, this group of compounds has a knockdown as well as lethal action against insects which are resistant to pyrethroid insecticides (Parmar et al,. 1997). Piper extracts have the ability to anaesthetize adult and larvae of potato beetle. This activity on the beetle population was due to piperamides (Scott et al., 2003). Aqueous extract of

Piper colubrinum had nematicidal effect on root knot nematodes. Two crystalline

13 Potential Application of Kava (Piper methysticum F.) in Nematode Control

compounds identified in this plant with nematicidal properties were 5,3'-dihydroxy-7-

methoxy flavone and 5,3',4'-trihydroxy-7-methoxy flavone (Eapen and Kumar 2005).

In vitro testing of whole plant of Piper longum L. showed fungicidal activity against

phytopathogenic fungi namely Pyricularia grisea, Rhizoctonia solani, Botrytis cineria,

Phytophthora infestans, Puccina recondite and Erysiphe graminis. Hexane extract

contained piperoctadecalidine, a piperidine alkaloid which had a powerful antifungal

activity against Puccina recondite (Park et al., 2003).

Crude volatile oil and its petroleum ether and dichloromethane extracts of Piper betle L.

showed insecticidal and fungicidal effects on some pests of cotton (Solsoloy et al., 2001).

The crude oil controlled sucking insects such as Aphis gossypii and Amrasca bigutulla

and acted as ovicide against Helicoverpa armigera and Pectinophora gossypiella. The

two solvent fractions repressed growth of certain fungi (Sclerotium rolfsil, Fusarium

oxysporum and Rhizoctonia solani) in laboratory experiments. The fractions mainly

contained monoterpenes and sesquiterpenes. The crude oil was generally more effective

than the two fractions.

Piper sudangrass (Lb) is a summer crop and grows on hot, fertile and irrigated soil. It is

harvested for hay for horses and cattle. It is known to reduce populations of many species

of nematodes and symphylans when grown as a rotation crop. As it decomposes, it

releases cyanide from its roots, which has nematicidal properties (Chitwood 2002).

14 Potential Application of Kava (Piper methysticum F.) in Nematode Control

1.5 Nematodes

Nematodes are tiny worm like, unsegmented organisms. They play an important but unrecognized role in soil fertility and agricultural productivity. Many of the nematodes are free living found in the oceans, freshwater habitats and in soils. A small group of these are parasitic. Plant parasitic nematodes (PPN) make about 20 % of the species within the phylum Nematoda (Ferraz and Brown 2002). They live in soil where they infect plant roots and underground tissues. These nematodes are estimated to cause an annual crop loss of more than 10 %, amounting to billions of US dollars (Sasser 1989;

Shurtleff and Averre III 2000; Ferraz and Brown 2002) and root-knot nematodes (RKN) are responsible for major share of this damage. Sandy soil is usually preferred by PPN since this allows the larvae to move freely from their hatching site to new roots. The optimum temperature of 30 - 32 °C is necessary for reproduction which completes in about 15 to 18 days (Ferraz and Brown 2002; Weischer and Brown 2000).

Of more than 2,000 different species of plant parasitic nematodes, the most economically important groups are the root knot (Meloidogyne spp.), cyst (Heterodera spp.), root lesion

(Pratylenchus spp.), reniform (Rotylenchulus spp.) and sting (Belonolaimus spp.) nematodes (Shurtleff and AverreIII 2000; Siddiqi 2000; Ferraz and Brown 2002). The nematodes of interest for bioassays in this investigation are the root knot nematodes

(RKN), the group of greatest agronomic importance. RKN (Meloidogyne spp.) are widely distributed and have an extensive host range. They form galls on roots that block water and nutrient flow to the plant, therefore stunting growth, impairing fruit production and

15 Potential Application of Kava (Piper methysticum F.) in Nematode Control

causing foliage to yellow and wilt. This causes the roots to become rough and pimpled

and susceptible to cracking. RKN parasitism adversely affects the yield quantity and

quality. They also leave open wounds thus providing an entry to plant pathogenic fungi

and bacteria.

Root knot nematodes affect crops in the tropical and subtropical regions more severely as

climatic conditions favor growth and reproduction of the damaging species. This genus

comprises of more than 80 different species of which four are unquestionably the most

important plant parasitic nematodes on the planet (Sasser and Carter 1985; Taylor and

Sasser 1978). These four species are M. arenaria, M. hapla, M. incognita and M. javanica and are distributed widely in agricultural areas around the world. Meloidogyne

incognita (Kofoid and White) Chitwood is the most widespread and damaging of the

RKN species and can parasitize a wide range of hosts (Yepsen 1984; Sasser and Carter

1985; Caswell and Bugg 1991; Siddiqi 2000). The awareness of RKN occurrence in the

Pacific Island countries that are highly dependent on primary production provides a good

reason for this investigation.

1.6 Effect of Root Knot Nematodes on Plants

Sexual dimorphism is quite clear in this genus. Females are obese and saccate. They are

sedentary and are embedded in the roots where they lay eggs while males are typically

vermiform. The first stage juveniles (J1) from embryogenesis moult within the egg and

hatch as second stage juveniles (J2). The motile and infective J2, migrate through the soil

16 Potential Application of Kava (Piper methysticum F.) in Nematode Control in search of roots of suitable host plants (Croll 1970). The movement of juveniles in the soil may last for months if environmental conditions are not favorable. The energy they require at this time comes from fatty reserves accumulated in their intestines.

The second stage juveniles usually penetrate the roots just above the root cap. They move between undifferentiated root cells and come to rest with their heads in the developing stele near the region of cell elongation and bodies in the cortex. The cell walls are pierced with the stylets and the secretions from the esophageal glands are injected. These secretions lead to the enlargement of cells leading to the formation of giant cells, possible dissolution of cell walls, enlargement of nuclei and changes in composition of the cell contents (Ferraz and Brown 2002). The intense multiplication of cells around the larval head lead to the formation of the distinct galls that are known as knots.

The formation of galls suppresses root elongation and the infected roots appear darkened, frequently contain longitudinal cracks and many of the fine feeder roots are destroyed.

Nematode infection is also known to disrupt carbohydrate partitioning and affect the phenol oxidase activity. This makes the plant more susceptible to cold injury and bacterial infections, which have direct killing effect on the plants (Weischer and Brown

2000; Ferraz and Brown 2002). The overall growth of the plants is greatly reduced especially in young plants growing in nurseries.

17 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Shoot symptoms observed in infected plants are stunting, wilting and leaf chlorosis.

These observed symptoms indicate the inefficiency of the shortened root system to uptake water and nutrients from the soil. Root and plant weight reductions up to 50 % have been noted in several infected crops while mortality in forest nurseries was more than 50 % (Ferraz and Brown 2002; Sasser 1989).

1.7 Nematode Management

Various nematode management options include use of chemicals, resistant crop varieties, crop rotation, using organic matter and green manures, growing nematode suppressive crops, flooding or leaving the soil fallow, solarizing soil, and employing methods of biocontrol (Oka et al, 2000; Taylor and Sasser 1978). Chemical control has been the preferred method of nematode control in many countries. Many of the chemicals used to kill or suppress the pests persist in the environment for long periods. Those having bioaccumulative properties end up in the human body through the food chain where at high levels they have detrimental effects (Harris 2000).

1.7.1 Nematicides for Nematode Control

Use of chemicals for nematode control has been reported since 1911 when carbon bisulfide was used to kill soil nematodes and the left over chloropicrin from the First

World War was used by pineapple growers to control root knot and reniform nematodes

(Sasser and Carter 1985). Later in the 1940s, several halogenated hydrocarbons such as

18 Potential Application of Kava (Piper methysticum F.) in Nematode Control methyl bromide, a mixture of dichloropropane and dichloropropene, and ethylene dibromide, was being marketed as nematicides.

These chemicals were efficient fumigant nematicides and had high vapor pressure. When injected in soil they could disperse by gaseous diffusion through the soil pore spaces and kill the nematodes by interfering with their cell metabolism (Sasser and Carter 1985;

Ferraz and Brown 2002). Many of these chemicals were banned when information on the danger of these chemicals was found as soil residues, groundwater contamination and residues in edible plant produces. Since no alternatives for these chemicals were available, research was initiated to explore biological control methods which are environmentally friendly. Natural products from plant and microorganism populations have been explored and are desired since they are easily degraded in the soil (Prakash and

Rao 1996; Taylor and Sasser 1978).

1.7.2 Natural products for Nematode Control

Many plants have been used as organic amendments in soils for the control of root knot nematodes. The chemical properties of the plants used as amendments contribute to their efficacy towards the control of nematodes (Rodriguez-Kabana 1986). Compounds with nematicidal properties are released when organic matter is decomposed or are synthesized by microorganisms during the decomposing process. These compounds include organic acids, hydrogen sulfide, nitrogenous ammonia, phenols, and tannins

(Chavarria-Carvajal et al., 2001). Organic matter in soil also stimulates microbial

19 Potential Application of Kava (Piper methysticum F.) in Nematode Control

populations of fungi and bacteria, which may be foe to nematodes (Morgan-Jones and

Rodriguez-Kabana 1987).

Aqueous extracts of Conyza canadensis, Blumea oblique, Amaranthus viridis, Eclipta prostrate, Azadirachta indica, Chromolaena odorata, Strychnos nuxvomica and Pimenta

dioica were found to inhibit egg hatching and cause juvenile mortality of RKN in

laboratory experiments (Begum et al., 2003; Eapen and Kumar 2005). Soil amendments

with powdered root material of C. canadensis, B. oblique, A. viridis, E. prostrate in pots

of brinjal plants reduced nematode population and root knot development (Begum et al.,

2003). Incorporation of green leaves of S. nuxvomica in basins of black pepper reduced

the foliage from yellowing due to nematode infections (Eapen and Kumar 2005).

Ethanolic extracts of rhizomes of Artemisia vulgaris inhibited egg hatching upto 50 % at

2.35 mg/ml and caused 50 % mortality among second stage juveniles within 12 hrs of

exposure at 55 mg/ml. The ethanolic extract when applied to soil reduced root galling on

a susceptible host. The extract did not seem to lose activity upon dilution with water

when stored in the dark at 25 °C for up to 15 days (Costa et al., 2003). Hexane extract of

the leaves and stem of Cleome viscose L., was seen to have high nematicidal activity

against M. incognita (Williams et al., 2003).

20 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The introduction of castor oil plant which is postulated to have naturally occurring nematicides, reduced soil nematode population by 90 % while increasing the yield of cassava and cocoyam by 29 % and 28 % respectively (Ugbaja 1997). The degree of infection by nematodes on the tubers and corms were also reduced.

A number of plant species documented as Chinese traditional medicine have shown nematicidal properties (Ferris and Zheng 1999). The plant parts tested included roots, rhizomes and bulbs, stems, leaves, seeds, bark as well as whole plants. Some of the compounds identified in many of these plant extracts include alkaloids, glucosides, glycosides, organic acids and essential oils (Hsu et al, 1985; Huang 1993).

Application of molasses to soil by sprinkler irrigation system and by overhead boom sprayer, lowered soil populations of reniform nematodes and caused a marked improvement in plant growth and fruiting of papaya (Schenck 2001). Molasses supplys carbohydrate therefore alters the carbon-nitrogen ratio, which affects the soil microbial ecology causing a decrease in populations of plant parasitic nematodes, on the other hand having favorable effects on plant growth. The efficacy of molasses for the control of root knot nematodes was comparable to that of the chemical nematicide fenamiphos.

Soil amendments with raw and burnt rice husks in combination with solarization had shown to decrease nematode populations in nurseries. Tea waste, coconut husks and by- products from sugar cane when used as surface mulch or incorporated into soil managed to decrease nematode populations in nurseries as well (UNDP, n.d.). Organic

21 Potential Application of Kava (Piper methysticum F.) in Nematode Control amendments with pine bark, velvet bean and kudzu at the rate of 30 g/kg effectively decreased nematode population (Chavarria-Cavajal et al., 2001).

Crop residues of maize, panicum and velvet bean were found to decrease the population of plant parasitic nematodes as well (McSorley and Frederick 1999). Fresh neem leaf, neem cake, tobacco waste, fresh plant material of wild sunflower, Adathoda vesica and

Vetiveria zizaniodes when used as mulches effectively reduced nematode populations

(Ngigi and Ndalut 2000).

1.7.3 Compounds Isolated from Plants with Nematicidal Activity.

A number compounds have been isolated from plants with antagonistic effects towards plant parasitic nematodes. Phytochemicals with nematicidal and insecticidal properties include polythienyls, isothiocyanates, glucosinolates, cyanogenic, glycosides, polyacetylenes, alkaloids, lipids, terpenoids, sesquiterpenoids, diterpenoids, quassinoids, steroids, triternoids, simple and complex phenolics and several other classes (Chitwood

1993, 2002).

Furfural (2-furfuraldehyde) (21) is a liquid found in many essential oils from plants and in fruit juices, alcoholic beverages and bread. It is used in food industries as flavor compositions. Insecticidal properties of furfural have already been established (Flor

1926; Raeder et al, 1925). Fungicidal properties of furfural were first reported in 1926

(Rodriguez - Kabana et al., 1993). Soil amendments with furfural have been known to

22 Potential Application of Kava (Piper methysticum F.) in Nematode Control control southern blight in lentil. When furfural was added to soil at 0.1 - 1.0 ml/kg, populations of Meloidogyne arenaria and Pratylenchus brachyurus were suppressed

(Rodriguez - Kabana et al., 1993). Fewer galls on the roots and an increase in yields were noted.

Figure 1.6: Structure of 2-furfuraldehyde

Furostanol glycosides extracted from cell cultures of Dioscorea deltoidea decreased the susceptibility of infection by root knot nemtodes (M. incognita) in tomato and cucumber plants (Zinovieva et al., 1997). These plants when treated with furostanol glycoside showed a five-fold decrease in overall nematode population. The female nematodes present were smaller and there was a shift in the sex ratio towards more males.

Phytoecdy- steroid (20-hydroxyecdysone) (22), present in plants, caused fatalities to plant parasitic nematodes on direct exposure. This phytoecdy- steroid is in fact the moulting hormone of invertebrates and probably nematodes as well. Abnormal molting, immobility, reduced invasion, impaired development as well as death were some of the effects of this compound observed on plant parasitic nematodes (Soriano et al., 2002).

23 Potential Application of Kava (Piper methysticum F.) in Nematode Control

HO

Figure 1.7: Structure of 20-hydroxyecdysone

CHO

23 3 24

HC

CH OH HC 3 25

Figure 1.8: Structures of benzaldehyde, citral, furfural, menthol and α-terpineol

Other naturally occurring botanical compounds that have been tested for nematicidal properties are benzaldehyde (23), citral (24), furfural (21), menthol (25) and α-terpineol

(26). These compounds are used commercially for perfume and flavor production. When these compounds were mixed with soil at rates ranging from 0.1 to 0.5 mL/kg, populations of M. incognita decreased in both the roots and the soil. The health of the plants was not affected in any way by these compounds. Application of citral and menthol caused significant increase in plant heights as well (Bauske et al., 1994).

24 Potential Application of Kava (Piper methysticum F.) in Nematode Control

1.8 Aims and Objectives of Present Study

This study focused to obtain freeze-dried polar extracts of Fiji and Vanuatu kava (Piper methysticum), exhaustively remove ethanol solubles from this extract to attain highly polar extract and to separate this extract into fractions using semi-prep HPLC.

Additionally, to culture RKN on tomato plants to obtain populations of juveniles and eggs for bioassays, test the extracts for nematicidal activity in vitro,to conduct soil amendment experiments with powdered root material of kava in pot experiments and to compare the difference in bioactivity of Fiji and Vanuatu Kava.

25 Potential Application of Kava (Piper methysticum F.) in Nematode Control

CHAPTER 2

2.0 KAVA EXTRACTIONS AND SEPARATION

2.1 INTRODUCTION

The well known active ingredients in kava, the kava lactones are documented for their antibacterial, antifungal and amoebicidal properties in addition to various effects on the central nervous system of humans (Singh 1986; Sonfi et al., 1983; Sotheeswaran 1987;

Xuan et al., 20031; Walji 1998; Whitton et al., 2003). No reported work has been found on the nematicidal properties of kava.

In this study various extraction and subsequent separation procedures conducted for the preparation of samples for testing the nematicidal properties of kava are described. The first extract of kava was the water extract, which would be deemed to contain water solubles. This was one of the kava samples tested in this study and has been referred to as

Sample X. Sample X was further purified by removing the ethanol solubles in preparation for the highly polar testing material, referred to as Sample Y. Semi- preparative HPLC techniques, as described under section 2.2.4 were involved to further fractionate sample Y into its subsequent fractions that is Samples Y1, Y2, Y3 and Y4.

The above samples were subjected to various bioassays for evaluating the nematicidal properties of kava.

26 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2.2 METHODOLOGY

2.2.1 General Methodology

Commercial samples of dried (powdered) kava root material from Fiji (FJ) and Vanuatu

(VA) cultivars were bought from Suva Market and from a local importer respectively.

The exact cultivar of these kava samples were not known but the FJ kava used in this research was the one which is locally referred to as Kadavu kava while VA kava was known as Vanuatu kava only in the local market.

Solvents used for extraction were of high purity at purchase and were redistilled before use. Laboratory research grade water was used. Solvents and samples for use in HPLC were membrane filtered (0.45 μm cellulose acetate filter) and ultrasonically degassed prior to analysis. Samples were freeze dried (where necessary) using Dynavac

Engineering FD3 freeze drier and BUCHI Rotavapor R-114 equipped with BUCHI

Waterbath B-480 was used for rotor-evaporation.

Sections 2.2.2 to 2.2.4 outline the extraction procedure. (Refer to Appendix 1 for a flow diagram summary).

27 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2.2.2 Aqueous Extraction

About 500 g of powdered kava root material was soaked in approximately 1.5 L of water and left at ambient temperature. After 24 hrs it was filtered using cheesecloth to obtain a muddy colored suspension, which was first frozen followed by freeze-drying to get a solid sample of mass of about 62 g. The residue (brown in color) was stored at 4 °C in the refrigerator for use in bioassays. This extraction was carried out for both FJ and VA kava and the samples obtained are referred to as sample X-FJ and X-VA hereafter. For FJ kava, two samples X, i.e. X-FJ (stored) and X-FJ (fresh) were tested for juvenile mortality abilities.

2.2.3 Removal of Ethanol Solubles

A 20 g sample of the freeze-dried residue, sample X was subjected to exhaustive soxhlet extraction with 200 mL of absolute ethanol, for a period of 5 hrs. This was repeated four to five times until the extraction solvent ran clear, that is the original color of the solvent

(ethanol). The remaining residue (totally devoid of ethanol solubles) was air dried in a fume cupboard to get rid of any traces of ethanol from it. This residue (for both FJ and

VA kava) has been referred to as sample Y-FJ and Y-VA, and was stored at 4 °C in the refrigerator for use in various bioassays and for semi-prep HPLC fractionation. Sample

Y-FJ was obtained from sample X-FJ (fresh).

28 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2.2.3.1 HPLC analysis of Sample Y to verify the removal of Ethanol Solubles

The final ethanol extract from soxhlet was dried in vacuo and redissolved in a known volume of dichloromethane (DCM) to make a standard of approximately 1880 ppm. This sample was analysed by a Waters (c) 515 HPLC equipped with a 2487 Dual Wavelength

UV absorbance detector set at 254 nm. Econosil Silica 10μ column of size 250 mm x 4.6 mm (length and internal diameter), mobile phase composition of 70 % n- hexane and 30

% ethylacetate and attenuation of 256 was used for analysis.

2.2.4 Separation of Sample Y

Organic analysis of kava has been highly restricted to the isolation and characterization of kava lactones. Very little research has been done on the aqueous extract or the water soluble components of kava and definitely none in which attempts have been made to fractionate and isolate water-soluble components. Hence there was no available information on the HPLC parameters to be used to achieve separation of the compounds in sample Y for fractionation and isolation. Therefore method development for identifying the best chromatographic parameters was a major part of this section.

29 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2.2.4.1 Identification of λ max

Sample Y was dissolved in distilled water and placed in a quartz cuvette. Using Perkin-

Elmer Lambda 16 UV- Vis Spectrometer, the UV- Vis spectrum of this sample was obtained in the range of 200 and 800 nm.

2.2.4.2 Mobile Phase Composition Determination for separation of Sample Y using

Thin Layer Chromatography

Various mobile phase compositions were tested to identify the composition, which gave the best separation of the peaks. The three commonly used solvents (water, acetronitrile and methanol) in reverse phase HPLC were tested for the best possible separation of the compounds in Sample Y.

TLC of Sample Y was performed to identify the best condition for semi - preparative

HPLC fractionation. TLC of this sample was carried out using silica gel GF254 glass plates. Acetonitrile, methanol and water were the solvents used in various compositions as the mobile phase for TLC analysis (comprising of components of high polarity). A UV lamp was used to visualize the plates.

The best separation in TLC analysis of Sample Y was observed with a solvent system of acetonitrile and water in a ratio of 9 : 1. It was not possible to obtain distinct spots, however, long streaks were seen. Other solvent systems tested that did show some

30 Potential Application of Kava (Piper methysticum F.) in Nematode Control separation were various compositions of acetonitrile: methanol, methanol: water and acetonitrile: water: methanol.

2.2.4.3 Gradient HPLC Analysis of Sample Y

A Waters HPLC system comprising of 1525 binary HPLC pump, 717 plus autosampler,

2996 photodiode array detector aided with Empower software was used for gradient

HPLC analysis. A C18 Econosil column (250 x 4.6 mm, 10 μm particle size) was used for this analysis. A two solvent system and a three solvent system comprising of acetonitrile, methanol and water in various compositions were used as the mobile phase.

Sample Y was dissolved in the appropriate mobile phase and in water for injection.

Analysis was carried out at different flow rates along various gradients for each mobile phase. Chromatograms were scanned over a range of wavelengths (200 - 400 nm) to determine a specific wavelength at which maximum chromatographically distinguishable compounds could be detected.

The best separation was achieved at a flow rate of 0.5 mL/min. UV detection between the range from 200 - 400nm gave the best detection at 210 nm. The use of acetonitrile: methanol (80: 20 and 90: 10) as mobile phase gave good separation but column blocking resulting in build up of high pump pressure was noted. This solvent composition was considered not appropriate for analysis since column damage and pump damage could have occurred.

31 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The solvent system of acetonitrile: water (90: 10, 85: 15, 80: 20 and 75: 25) produced similar problem. A three solvent system of acetonitrile: water: methanol in various ratios was used next. Some separation was achieved initially but high pump pressure was observed afterwards. Therefore, this solvent system was not considered suitable for analysis as well. Since sample Y is mainly composed of highly polar components, complete elution was a problem (column blockage and excess build up of pump pressure). Prior purification using column chromatography could have eliminated this problem however, it was not engaged in this study. A highly polar solvent system was thus regarded necessary for good separation.

The water composition in the mobile phase with either acetonitrile or methanol was thus enhanced. It was observed that acetonitrile and water in a ratio of 1: 3 at flow rate of 0.5 mL/min and detection at 210 nm gave a relatively better separation without much pressure problems. These parameters were then used for semi-prep analysis.

2.2.4.4. HPLC Separation of Potential Nematicidal Compounds from Sample Y

The suitably identified HPLC analytical parameters (mobile phase; 1: 3, acetonitrile: water, flow rate of 0.5mL/min, UV detection at 210nm and an attenuation of 256) were used for fractionation of sample Y using semi- prep HPLC. Chromatogram obtained using these parameters is shown in Figure 2.3. The first fraction was collected between

12.3 to 17 mins, second from 17 to 21 mins, third between 21 to 23 mins and fourth,

32 Potential Application of Kava (Piper methysticum F.) in Nematode Control which was a single, slightly broad peak around 31 mins. The volume of sample injected was 200 μL and concentration was 0.01 g/mL.

These fractions were collected in vials and transferred in bottles which were kept at 4 °C in the refrigerator. Once the bottles were full, the solutions were transferred to 500 mL plastic beakers and frozen before freeze-drying. The dry residue obtained were weighed and transferred to vials. The four fractions collected from samples Y-FJ and Y-VA were labeled as Y1-FJ, Y1-VA,Y2-FJ, Y2-VA, Y3-FJ, Y3-VA, Y4-FJ and Y4-VA and kept in the refrigerator at 4 ºC for use in bioassays and for NMR analysis.

2.2.5 H-NMR Analysis of Fractions of Sample Y

analysis was conducted on the four fractions obtained. The H-NMR was conducted at University of New England. Bruker Avance 300 Spectrometer and a 5 mm inverse H/BB probe with z-gradient were used. Spectra were run in D2O at 30 °C and

128 scans were collected for the individual fractions.

33 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2.3 RESULTS AND DISCUSSION

2.3.1 HPLC analysis of Sample Y to verify the removal of Ethanol Solubles

The final ethanol extract obtained from soxhlet was analyzed using normal phase HPLC for the presence of any ethanol solubles. The peaks on the chromatogram were used as a guide to verify that the ethanol solubles have been removed from sample X. The following figures (2.1 and 2.2) show the chromatograms used for comparison.

I IT ! I I j /\ / i :=; / \^.--'

Figure 2.1: HPLC chromatogram showing peaks of the ethanol solubles

Figure 2.2: HPLC chromatogram of the final ethanol extract from soxhlet

34 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The compounds which were detected in the first ethanol extract were not detected in the final ethanol extract, as can be observed by the absence of the peaks in figure 2.2. It can be stipulated that exhaustive soxhlet extraction removed ethanol solubles, leaving behind the highly polar compounds that would be present in sample Y.

2.3.2 H-NMR Analysis of Fractions of Samples Yl, Y2, Y3 and Y4

The chromatogram in figure 2.3 shows the peaks which were used as a guideline to isolate fractions Y1, Y2, Y3 and Y1, Y2, Y3 and Y4 from sample Y.

Figure 2.3: HPLC chromatogram for sample Y showing the groups of peaks which were isolated

The four fractions were subjected to H-NMR analysis. The H-NMR spectra (Appendix

1) of these fractions as expected, were not very conclusive towards identification of the structure of the compounds but some functionalities could be deduced. The presence of anomeric protons was indicated by the peaks at 4.94 ppm and 5.34 ppm in fractions Y1

35 Potential Application of Kava (Piper methysticum F.) in Nematode Control and Y2, and at 4.94 ppm and 5.1 ppm fractions Y3 and Y4. The absorbance around 3.0 ppm - 4.0 ppm in all the fractions indicates resonance resulting from the presence of sugars around. The singlets and triplet around 1.1 ppm-1.3 ppm in all the fractions indicate the presence of methyl group. The large peak at 4.66 ppm is most likely that of water resulting from the exchange of the sugar protons with deuterium (D2O), as well as residual H20 in D2O and possibly in the sample. Peak at 5.41 ppm could be that of DCM, which was used to clean the tubes. Structure of the compounds present in the four fractions could not be identified since individual compounds were not isolated. Presence of large water and DCM peak also made structural elucidation difficult.

These results when taken together with the fact that fractions were highly polar, presence of glucosidic compounds in addition to carbohydrates can be suggested in fractions Y1,

Y2, Y3 and Y4. Many plants produce glycosylated compounds as secondary metabolites.

Glycosylated compounds are known to be better stored within plant vacuoles and are less reactive toward other cellular components (Pridham 1965).

36 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2.4 CONCLUSION

The procedure outlined in this chapter made possible the separation of various extracts of kava which were evaluated for nematicidal activity as will be reported in chapter 3.

Sample Y was isolated into sub-fractions Y1, Y2, Y3 and Y4 using RP semi-prep HPLC however individual compounds could not be isolated using the procedure identified in this research. Since the sample was not purified prior to subjecting to fractionation, build up of column pressure was noted making it impossible to further isolate the fractions.

The H-NMR data of the fractions suggests the probability of the presence of glucosides as postulated by Naiker and Prasad (2005) and Naiker et al. (2006) in addition to carbohydrates (Sotheeswaran et al., 1998). Actual structures of the compounds could not be deduced due to the complexity of the fractions. Each fraction consists of components of slightly different polarities. This was seen by the clustering of groups of peaks in the chromatogram.

The following chapter reports the bioactivity tests of various extracts of kava obtained.

37 Potential Application of Kava (Piper methysticum F.) in Nematode Control

CHAPTER 3

3.0 BIO ASSAYS

3.1 INTRODUCTION

The activity of kava and its subsequent extracts and fractions on nematodes can be demonstrated through bioassays. Testing of the isolated fractions will narrow down the chemistry of nematicidal compounds found in kava. Nematicidal activities of chemicals are commonly evaluated through in vitro testing, where nematodes are in direct contact with test chemicals or through amendments in soil. In soil amendment experiments chemicals are mixed in soil in which susceptible plants are grown and inoculated with nematodes.

The effect of different extracts of kava obtained (as described in chapter 2) was tested in vitro for mortality of juveniles and suppression of egg hatching of root knot nematodes.

Samples X and Y for both FJ and VA kava were tested on juveniles as well as eggs at different concentrations while samples Y1, Y2, Y3 and Y4 were only tested on juveniles at one concentration due to the small amount of samples obtained. Sample X is the aqueous extract, sample Y is the highly polar extract i.e. sample X less ethanol solubles and samples Y1, Y2, Y3 and Y4 are fractions isolated from sample Y using semi-prep

HPLC.

38 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Furthermore, dried kava root powder of Fiji and Vanuatu kava was used as the testing material for the soil amendment experiments. Kava powder at different concentrations and after degrading for various periods were used in pots for these experiments.

Subsequently, tomato seedlings were planted and inoculated with juvenile nematodes in these experimental pots. The efficacy of FJ and VA kava varieties towards the control of root knot nematodes i.e. its nematicidal activity and their phytotoxic effect (any detrimental effect) on the tomato plants was evaluated by observing the degree of root infection.

39 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.2 METHODOLOGY

3.2.1 General Methodology

Nematode cultures had been set up for use in the various bioassays in this research. Visits were made to vegetable farms and kitchen gardens along the Suva - Nausori corridor to identify plants infected with root knot nematodes (RKN). Infected plants were identified by randomly pulling out plants which looked sickly and looking for knots similar to those in Figure 3.1. An infected farm at Nailuva road was identified and samples for raising cultures were obtained from there.

'

Figure 3.1: Tomato roots infected by RKN as viewed with naked eyes

40 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.2.2 Nematode Culturing and Species Identification

Infected roots collected were washed in distilled water in a beaker, cut in pieces and transferred to a glass Petri dish with clean distilled water. The knots were observed under a stereoscopic microscope in the Petri dish to identify protruding egg sacs (masses) attached to females. Each egg sac was transferred to separate cavity blocks and the respective female was transferred to 0.9 % KCl (saline) solution before transferring to lactic acid solution. The saline solution maintains equilibrium of the fluid inside and outside the body of the female nematode and the lactic acid helps in clearing the body fluid so that it is easier to identify the perineal pattern on the posterior portion.

Female nematodes were cut in half and the anterior portion was discarded. The posterior portion was further trimmed around the perineal parts to obtain a small piece of the body tissue with the perineal pattern. The perineal portion was transferred on to a drop of glycerine on a slide and covered with a cover slip. The slide was sealed and left to dry before labeling and observing under compound microscope to identify the species of the root knot nematode. Perineal portions are like fingerprints which are different for different nematode species. Observation of the adult (females) and juvenile stage revealed that the nematodes infecting the plants were from Meloidogyne genus. Based on the observation of the morphology of perineal patterns (Taylor and Sasser, 1978) the nematodes infecting the plants were tentatively identified to be from the Meloidogyne incognita group.

41 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The egg sacs attached to females identified tentatively as M. incognita were transferred to clean distilled water in a 3.5 inches, disposable laboratory Petri dish and left in a BOD

(biological oxygen demand) incubator at 28 °C until the juveniles had hatched.

Suspensions of hatched juveniles were concentrated by allowing it to settle for few hours in a beaker before excess water was removed from the top gently, using a Pasteur pipette, being careful not to disturb the sediment at the bottom of the beaker.

Tomato seedlings of small fry variety were inoculated by planting them in sterilized soil and sand mixture (5:1 ratio) to which this concentrated juvenile suspension had been added. The tomato plants used were three weeks old and had been raised in sterilized soil.

These plants were allowed to grow undisturbed for about two months in the plot land with frequent watering to allow the nematode population to establish. Juvenile population and egg masses obtained from these culture plants were used for bioassays described in the research.

3.2.3 Sample Preparation

The samples to be tested for nematicidal properties were dissolved in water at different w/v (g/mL) concentrations. FJ kava had two X samples that is X-FJ (stored) and X-FJ

(fresh). Sample X-FJ (stored) had been stored in the refrigerator at 4 °C for six months while sample X-FJ (fresh) had been freshly extracted.

42 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Samples X and Y of both FJ and VA kava were tested at 1 % and 2 % concentrations while in some cases as indicated in the results, 0.5 % concentration was also tested. The activities of 2 % concentration of these samples were compared with 0.5 % concentration of gallic acid (standard). The fractions of Y samples i.e. Y1, Y2, Y3 and Y4 of FJ and

VA kava were tested at 0.1 % concentration and compared with gallic acid at 0.1 % w/v concentration.

Samples were added to distilled water in conical flasks and shaken for 20 minutes using

Stuart Scientific Flask Shaker SF 1 to assist in dissolving the samples faster.

3.2.4 Laboratory Assays of Kava Extracts

The in vitro experiments were conducted for juvenile mortality and suppression of egg hatching. The second stage juvenile (J2) populations and the egg masses needed for these experiments were obtained from the maintained Meloidogyne sp. cultures. The experiments for samples X and Y were performed in 3.5 inches disposable laboratory

Petri dishes, in which grids of 5 x 5 mm had been made using a needle while for samples

Y1, Y2, Y3 and Y4 were performed in 2 inches disposable Petri dishes with grids, 2.5 x

2.5 mm. The grids were marked to aid in the counting process.

43 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.2.4.1 Juvenile Mortality Tests

Activity of samples X-FJ (stored), X-FJ (fresh), X-VA, Y-FJ, Y-VA, Y1-FJ, Y1-VA, Y2-

FJ, Y2-VA, Y3-FJ, Y3-VA, Y4-FJ and Y4-VA was evaluated on second stage juveniles

(J2). All the bioassays were conducted at 28 ºC in BOD incubator while X-FJ (stored) and

X-FJ (fresh) were also tested at room temperature. Juveniles were obtained by allowing

the egg masses to hatch in distilled water in a BOD incubator for 7 to 10 days. J2

suspension was standardized to 100 juveniles/mL by either concentrating or diluting the

suspension.

Experiments for samples X and Y of FJ and VA were set using 10 mL of the respective

concentrations (2 %, 1 % and 0.5 %) of the test solutions and 1 mL juvenile suspension

per Petri dish. For samples Y1, Y2, Y3 and Y4, 3 mL of 0.1 % test solution and 1 mL of juvenile suspension was used. The 0 % concentration (distilled water) served as the

control for comparison.

Quantitative observation of the treated juveniles was made after 24 hrs and 48 hrs of

treatment after which the juveniles were transferred to distilled water. This was done by

transferring the contents of the Petri dish (juveniles and test solutions) in a beaker and

diluting it with distilled water. The beakers were left for two to three hours before

removing excess water using a Pasteur pipette. This was repeated three to four times to

further dilute the samples. The remaining mixture was transferred back to the respective

Petri dishes and left in the incubator for another 24 hrs before re-observation under the

44 Potential Application of Kava (Piper methysticum F.) in Nematode Control

microscope. This was to verify whether the effect of the samples tested was permanent or

not. All tests were conducted in seven replicates.

The percentage mortality of the juveniles was determined by counting the number of

dead and live juveniles at each observation using manual tally counters. As the Petri

dishes were scanned from one grid to another, the tally counters, one for dead and one for

live juveniles was clicked when a juvenile was spotted. Mortality of the juveniles was

indicated by immobilization and dead (slightly curved to almost straight) posture of the juveniles. The results obtained after revival in distilled water was subjected to analysis of

variance using regression with SPSS version 12.0.1 for windows software.

Figure 3.2: Live juveniles in motion in water as viewed under 40 x magnification

45 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Figure 3.3: Dead juveniles viewed under 40 x magnification (note the slightly curved to almost straight posture)

3.2.4.2 Egg Hatching Experiments

Egg hatching experiments were conducted for samples X-FJ (fresh), X-VA, Y-FJ and Y-

VA at 1 % and 2 % concentrations in seven replicates. The activity of 2 % concentration was compared with 0.5 % concentration of gallic acid. Experiments were set using 15 mL of the test solutions and two approximately equal sized egg masses per Petri dish at

28 °C in BOD incubator. These Petri dishes were replenished with about 8 mL of test solutions on the third and sixth day to prevent them from drying.

46 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Quantitative observation under the microscope was made on the third, sixth and eighth

day for sample X-FJ (fresh), since majority of the eggs hatch within 7 days. Since the

results of the third sixth and eight day did not differ much, observations for the other

experiments were recorded on the third and seventh day only. Total number of hatched juveniles as well as the number of dead and live juveniles was counted with a manual

tally counter and recorded. Results obtained were analyzed between subjects for

multivariate analysis using general linear model with SPSS software.

3.2.4.3 Comparison with the standard (Gallic Acid)

The activity of the kava extracts towards juvenile mortality and inhibition of egg hatching

was compared with the standard, gallic acid. These were separate experiments conducted

at 2 % concentration of the test solutions with 0.5 % concentration of standard. Gallic

acid was chosen as the standard since it is one of the phenolics present in methanolic

extracts of kava.

For comparing the activity of samples Y, a more branched or maybe a glycosidically

bound polyphenol should have been used since such compounds have been postulated to

be present in sample Y (Naiker and Prasad 2005). This is because extraction with ethanol

in preparation of sample Y would remove low molecular weight compounds, chemically

similar to gallic acid since these would be soluble in ethanol.

47 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.2.5 Pot Experiments (Soil Amendment Experiments)

The soil amendment experiments were conducted to evaluate the effect of kava on nematodes growing on plants and its effect on plants. These experiments were carried out using dried kava root material as soil amendment before planting and inoculating tomato seedlings with juvenile nematodes.

3.2.5.1 Preparation of Pots and Soil for Experiments

The soil amendment experiments were carried out in 9 x 9 cm (diameter and height) plastic pots, which were washed and dried prior to use. The soil to be used for the experiment was mixed with sand in a 5: 1 (soil and sand) ratio and sterilized in an autoclave at 80 °C and 20 psi for 1 hr to terminate all the pests in the soil including any nematodes that may have been present. The sand and soil mixture used per pot was 400 g.

3.2.5.2 Experimental Setup

Powdered kava root material was used for amendment of soil in these experiments. Four different experiments (where kava was degraded in the soil for different time periods) were set up for each kava sample. Each of these experiments had four different treatments and seven replicates.

48 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The different treatments were different kava concentrations in the soil which were 5 %, 2

%, 1 % and 0 % (control) weight to weight (w/w). These concentrations were obtained by manually mixing 20, 8, and 4 g of kava powder respectively to 400 g soil for each pot and only sterilized soil as control. A few (8 to 10) grains of urea, a commercial fertilizer was also added to the soil on the day of mixing kava and every 4 weeks thereafter. The pots with the different kava concentrations were placed randomly on the benches in the plot land.

The four different experiments were for 0, 2, 4, and 6 weeks of degradation of kava in soil. This was achieved by planting seedlings on the day of adding kava powder to soil, and 2, 4, and 6 weeks after adding kava to soil respectively. The pots were watered frequently to assist in the degradation process.

3.2.5.3 Planting of Seedlings and Inoculating

Tomato seedlings were raised in sterilized soil at atmospheric temperature and pressure.

Experiments were set using three weeks old seedlings of small fry variety of tomato that had been raised in sterilized soil. After the respective periods of degradation, a pit was made in each pot and 2 mL of standardized (100 juveniles/mL) J2 suspension was added.

One seedling per pot was planted and allowed to grow without much interference except for watering every second day. These experiments were terminated after 52 days as this gives sufficient time for RKN to complete at least one life cycle and gall formation by the next generation .

49 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Figure 3.4: Kava powder mixed in soil and left for degrading in the plot land

50 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Figure 3.5: The author planting the tomato plants in the pots after inoculating with juvenile nematodes

3.2.5.4 Plant Observations

On the 52nd day the plants were carefully removed from the pots avoiding breaking off

of any roots. These were then washed in water gently, preventing washing away of the

egg masses and were left on blotting paper for a few minutes before observing the degree

of nematode infection. Using magnifying lenses the number of galls were observed and

recorded. The plant heights and root lengths were also recorded. The results obtained

were compared to see the effect of different kava concentrations and the different

degradation periods on root galling by the nematodes on the plants.

51 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.3 RESULTS AND DISCUSSION

3.3.1 Laboratory Assays of Kava Extracts

3.3.1.1 Juvenile Mortality Experiments

Juvenile mortality experiments were carried out for samples X-FJ (stored), X-FJ (fresh),

X-VA, Y-FJ, Y-VA, Y1-FJ, Y1-VA, Y2-FJ, Y2-VA, Y3-FJ, Y3-VA, Y4-FJ and Y4-VA.

Samples X-FJ (stored), X-FJ (fresh) and Y-FJ were tested at room temperature as well as at controlled temperature (28 °C).

a) FJ Kava Extracts Tested at Room Temperature

Sample X (stored), which was stored in the refrigerator for about six months, was effective within 24 hrs of treatment time at both 2 and 1 % concentrations (Table 3.1).

Results summarized in Table 3.1, shows 100 % mortality at the 2 % concentration and 34

% mortality at 1 % concentration. Percentage mortality at both concentrations remained almost same even after 48 hrs of treatment. Observation after 24 hrs of revival in distilled water illustrated 30 % (at 2 % concentration) and 14 % (at 1 % concentration) revival of the juveniles. That is, % mortality after revival decreased to 70 and 21 % in 2 and 1 % treatments respectively. This indicates that the effect of sample X (stored) reversible to some degree even after 48 hrs of treatment. Analysis of variance gave P <

52 Potential Application of Kava (Piper methysticum F.) in Nematode Control

0.001 for both time and concentration effects thus indicating the significant effect of these on observed mortality.

Sample X (fresh) produced juvenile mortality after 24 hrs of treatment at both concentrations. Observed mortality increased after 48 hrs of treatment for the two concentrations. Percentage mortality was lower than sample X (stored) after 24 and 48 hrs of treatments. After 24 hrs of exposure to distilled water, juvenile mortality at 2 % concentration increased from that observed after 48 hrs of treatment, while it remained same for the 1 % concentration. This indicates that the toxic effect had been initiated after

48 hrs of treatment and was permanent since transferring the juveniles to distilled water did not help them to recover. The observed was P < 0.001 indicating a significant effect of concentration and treatment time on juvenile mortality.

The final mortality (after revival in distilled water) caused by sample X (fresh) was higher than sample X (stored). The 2 % concentration showed more significant results than the 1 % concentration. The variation in the efficacy of samples X (stored) and X

(fresh) could be due to some chemical changes which may have occurred over the storage period. However, this can not be concluded with certainty since these were single trials only.

Sample Y was tested at 2 %, 1 % as well as 0.5 % concentrations. Juvenile mortality was noted in all tested concentrations which increased with increasing treatment time.

Treatment time is one of the important factors contributing towards the efficacy of

53 Potential Application of Kava (Piper methysticum F.) in Nematode Control extracts since higher mortality was noted with longer periods of exposure to the chemicals. When the juveniles were transferred to distilled water for revival, a significant rise in mortality was observed for the 2 % test solution. This indicates that the activity of sample Y (especially at 2 % concentration), on the juveniles was slow but permanent.

Relatively high mortality rates were observed for the control as well. The temperature

(room temperature) at which these treatments were kept was not constant, thus it could possibly be contributing towards the variations in the results.

Table 3.1: FJ kava extracts tested for juvenile mortality at room temperature

Treatment Sample X-FJ (stored) Sample X-FJ (fresh) Sample Y Time 2 1 0.5* 2 1 0.5* 2 1 0.5 % % % Control % % % Control % % % Control 24hrs 11 100 34 X 3 67 26 X 3 20 14 21 48hrs 10 100 35 X 5 78 29 X 4 27 22 30 24 hrs revival in distilled 70 21 X 5 83 29 X 6 99 21 28 11 water * - this concentration was not tested.

Table 3.2: Analysis of Variance Results for FJ Kava Samples tested at Room

Temperature

Sample Tested Degrees of Observed F Observed P Freedom value value X-FJ (stored) at room 2, 15 16.2 0.0000 temperature X-FJ (fresh) at room temperature 2,15 15.2 0.0000

54 Potential Application of Kava (Piper methysticum F.) in Nematode Control

b) FJ Kava Extracts Tested at 28 °C

All three samples of FJ kava (X (stored), X (fresh) and Y) were tested for juvenile

mortality under controlled temperature. This was done to eliminate the effect of

fluctuating temperature, if any on observed mortality. Effect of sample X (stored) on juveniles was almost similar to that observed at room temperature. Concentration of test

solution as well as treatment times significantly affected juvenile mortality since P <

0.0001. Mortality increased when transferred to distilled water, which was more

significant for the 2 % concentration. This indicated that the effect of the extract on the juvenile was permanent and could not be reversed by isolating the juveniles from the

contact of chemicals.

Sample X (fresh) was tested at 2 %, 1 % and 0.5 % concentrations. The 1 % and 0.5 %

solutions presented very low mortality after 24 and 48 hrs of treatment but 2%

concentration was quite significant. When subjected to revival in distilled water, an

increase in mortality was noted for 2 % test solution, whilst 1 % and 0.5 % did not show

much variation. Percentage mortality observed after revival for both samples X (stored)

and X (fresh) was comparable for 2 % concentration. This indicates that storing the

sample at 4 °C did not cause any major chemical changes.

55 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The difference in activity observed in the experiments conducted at room temperature

(3.3.1.1), could have been more due to fluctuating atmospheric temperature than of any chemical changes over the storage period. Nematodes are quite sensitive to temperature and an optimum temperature between 25 to 32 °C is vital for their survival (Koenning et al., 2004). Temperatures higher than 35 °C and lower than 20 °C usually lead to mortality. Hence solarising, where soil temperatures are increased above 40 °C, has been adapted as one of the mechanisms for nematode control. Statistical analysis gave P <

0.001, indicating significance of concentration and treatment times on mortality.

Sample Y showed low mortality at all concentrations at 24 hrs but values increased after

48 hrs. This indicates that sample Y had a slower effect on juveniles compared to sample

X. Significant effect of concentration and treatment times were noted on observed mortality since P < 0.001. Some recovery of juveniles was noted when transferred to distilled water. Therefore the ethanol solubles present in sample X that were removed for the preparation of sample Y, can be said to be having a stronger and more permanent effect on the juveniles than the compounds remaining in sample Y. Furthermore, mortality was not only due to ethanol solubles but also the non-solubles or highly polar compounds present in kava, since juvenile mortality was observed in treatments with sample Y.

56 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Table 3.3: FJ kava extracts tested for juvenile mortality at 28°C

Treatment Sample X-FJ (stored) Sample X-FJ (fresh) Sample Y Time

2 1% 0.5*% Control 2% 1% 0.5% Control 2% 1% 0.5% Control % 24hrs 1 64 6 X 3 70 4 3 1 13 2 1 48hrs 2 64 12 X 3 70 7 5 1 75 14 4 24hrs revival in 2 distilled 81 16 X 3 82 8 5 1 74 4 1 water

* Concentration not tested

Table 3.4: Analysis of Variance Results for FJ Kava Samples tested at 28 °C

Sample Tested Degrees of Observed F value Observed P value Freedom X-FJ (stored) at 28°C 2,15 11.2 0.0001 X-FJ (fresh) at 28°C 3,20 13.4 0.0000

c) VA Kava Extracts Tested at 28 °C

Two samples of VA kava (X-VA and Y-VA) were tested. Sample X was effective at both

2 % and 1 % concentrations. Significance of concentration and treatment times were obvious since P < 0.0001. Observations after 48 hrs of treatment presented higher mortality for the higher concentrations. Mortality increased in both concentrations with increasing treatment times (Table 3.5). When transferred to distilled water after 48 hrs of

57 Potential Application of Kava (Piper methysticum F.) in Nematode Control exposure, juvenile recovery was noted in both concentrations. This indicated that the effect of sample X could be reversed to some extent. The mortality observed may be due to some other chemicals than that found in FJ kava since results for the two kava extracts were not comparable. On the other hand, it could also be possible that more than one compound is responsible for this activity which may be present in different concentrations in FJ and VA kava.

Sample Y was tested at 2 %, 1 % as well as 0.5 % concentrations. All of the three concentrations tested did not show much activity after 24 hrs of treatment but increased slightly after 48 hrs. The observed P < 0.0001, indicating the significance of concentration and treatment times. When transferred to distilled water for revival, mortality rate increased in 2 % concentration while it decreased in 1 % and 0.5 % concentrations. This indicated that 2 % concentration was the most effective concentration for sample Y of VA kava. The 1 % concentration of sample X showed a higher effect compared to the 1 % concentration of sample Y. Slightly higher mortality was observed at the 2 % concentration of sample X than sample Y. Therefore it could be said that ethanol solubles which were not present in sample Y had significant effect towards mortality.

58 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Table 3.5: VA kava extracts tested for juvenile mortality at 28°C

Treatment Time Sample X-VA Sample Y-VA

Control 2% 1 % 0.5* % Control 2% 1 % 0.5% 1 24 hrs 68 23 X 2 4 3 2 2 48 hrs 92 69 X 2 12 8 8 24 hrs revival in

70 37 X 2 62 4 4 2 distilled water

* - this concentration was not tested

Table 3.6: Analysis of Variance Results for VA Kava Samples tested at 28 °C

Sample Tested Degrees of Observed F value Observed P value Freedom X-VA at 28°C 2,15 19.6 0.0000 Y-VA at 28°C 3,20 107.1 0.0000

d) Comparison of sample X of FJ and VA Kava with the standard

The minimum concentration at which Gallic acid showed effective mortality was found to be 0.5 % (w/v). This concentration was as effective as those for the 2 % concentrations of sample X-FJ and X-VA on the juveniles. Mortality observed after 24 hrs of treatment was higher for sample X-VA compared to that of sample X-FJ and the standard (Table

3.7). An increase in mortality was noted in all treatments after 48 hrs. When exposed to distilled water for 24 hrs, mortality increased for X-FJ and gallic acid while for sample

X-VA some revival was noted. The mortality noted for the standard was in the range for

59 Potential Application of Kava (Piper methysticum F.) in Nematode Control that of FJ and VA kava. Results obtained as mentioned above were similar to those for the two kava extracts conducted separately (3.2.1.2 and 3.2.1.3).

Table 3.7: Comparison of the samples X of FJ and VA kava with gallic acid

2 % Sample X-FJ Treatment Time 2 % Sample X-VA 0.5 % Gallic Acid Control (fresh) 76

24 hrs 54 58 2 81

48 hrs 65 80 2

24 hrs revival in 95 80 84 2 distilled water

e) Comparison of sample Y of FJ and VA Kava with the Standard

The 2 % concentrations of samples Y of FJ and VA kava were compared with 0.5 %

Gallic acid. Juvenile mortality after 24 hrs of treatment was similar for FJ kava extract and standard while VA kava extract recorded lower mortality (Table 3.8). Subjecting treated juveniles to distilled water showed some recovery of juveniles in the FJ kava extract as noted in earlier experiment while mortality increased in VA kava extract and the standard. The results in this experiment were similar to those observed for previous experiments on sample Y (3.2.1.2 and 3.2.1.3).

Mortality results of samples X and Y are comparable with the results obtained for Gallic acid. Gallic acid together with other phenolics has been reported in 80 % methanol extract of kava and was observed to have allelopathic properties (Xuan et al., 20032).

60 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Table 3.8: Comparison of samples Y of FJ and VA kava with gallic acid

Treatment Time 2 % Sample Y-FJ 2 % Sample Y-VA 0.5% Gallic Acid Control 1 24hrs 75 50 76 2 48hrs 86 51 87 24 hrs revival in 83 65 89 2 distilled water

3.3.1.2 Juvenile Mortality Experiments of the Fractions

The four fractions isolated from sample Y-FJ were labeled as Y1-FJ, Y2-FJ, Y3-FJ and

Y4-FJ, while those isolated from Y-VA were labeled Y1-VA, Y2-VA, Y3-VA and Y4-

VA, in the order in which they eluted from the column. The column used had a non-polar stationary phase; therefore the fractions which eluted later were less polar compared to the earlier eluting ones. The activity of these fractions on juveniles were tested in vitro experiments and compared with control and the standard. The fractions and gallic acid

(standard) were tested at 0.1 % (w/v) concentration.

Juvenile mortality increased with increasing times in all treatments. Statistical analysis of both FJ and VA kava fractions gave P < 0.0001. Observations after 24 hrs of revival in distilled water showed reversible effect of the two highly polar fractions (Y1 and Y2) for both FJ and VA kava while the less polar fractions (Y3 and Y4) showed continued mortality that is increase in mortality from that observed after 48 hrs of treatment. Gallic acid showed similar activity to fractions Y3 and Y4 and was consistent with the effect observed in earlier experiments.

61 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The general trend in the activity of the four fractions isolated from FJ and VA kava samples showed similar activity thus indicating that compounds with similar chemistry were present in both FJ and VA kava. However, overall mortality was higher in sample

Y4-VA than Y4-FJ but was comparable for samples Y1-Y3 for the two kava samples.

Table 3.9: Analysis of Variance Results for FJ and VA Kava fractions tested at

28 °C

Sample Tested Degrees of Observed F value Observed P value Freedom Y1-FJ, Y2-FJ, Y3-FJ, Y4-FJ at 1,5 24.5 0.0000 28°C Y1-VA, Y2-VA, Y3-VA, Y4-VA at 1,5 12.1 0.0000 28°C

62 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Observed juvenile mortality over the treatment period for FJ Kava Fractions

30 25 i" 20 5 15

0 .m-, Y1 Y2 Y3 Y4 Gallic acid Control

• 24 hr s 48 hr s 24 hrs of revival in distilled water

Figure 3.6: Juvenile Mortality for FJ kava fractions

Observed juvenile mortality over the treatment 35 n period for VA Kava Fractions 30 25 20 15 10 5 0 Ilk Y1 Y2 Y3 Y4 Gallic acid Control • 24 hr s 48 hr s 24 hrs of revival in distilled water

Figure 3.7: Juvenile Mortality for VA kava fraction

63 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.3.1.3 Egg Hatch Experiments

The efficacy of samples X and Y at 2 % and 1 % concentrations for FJ and VA kava on suppressing egg hatch was investigated in vitro at 28 °C. The total number of juveniles hatched was counted, taking into account the number of dead and alive. Percentage hatching was calculated by taking the number of hatched juveniles in control as 100 % and percentage mortality was calculated based on total number hatched.

A problem encountered during these experiments was that the Petri dishes started drying over the observation period. This was overcome by adding about 8 to 10 ml of distilled water on the 3rd and 6th day for samples X and Y of both FJ and VA kava. The experiments where comparison was being made with standard, 8 to 10 ml of test solutions were added instead.

a) Activity of samples X and Y of FJ and VA kava

The results as seen in Figure 3.8 and 3.9 shows that both 2 % and 1 % concentrations of sample X-FJ (fresh) and X-VA suppressed egg hatching. Results for the activity of sample X-FJ (fresh) was recorded on third, sixth and eighth day while for sample X-VA, it was recorded on the third and seventh day only. The total number of hatched juveniles was found to be increasing in 2 %, 1 % as well as the control over the counting period, but the 2 % had the lowest increase while control had the highest. At 1 % treatment with sample X-FJ (fresh), the number of juveniles on the 8th day was almost half that of

64 Potential Application of Kava (Piper methysticum F.) in Nematode Control

control while in the 2 % concentration it was less than one fifth. Mortality of the juveniles was also noted in both the 2 % and 1 % concentrations and was seen that almost

50 % of the hatched juveniles were dead by the 8th day of observation. While for X-VA,

on the 7th day, 2 % concentration showed only about 7 % egg hatching and 1 % showed

around 19 % compared to the control, where a total number of 206 juveniles had hatched.

Moreover, mortality of the hatched juveniles was also noted in the 2 % and 1 %

concentration.

Sample Y-FJ also showed that both the concentrations tested were effective in

suppressing egg hatch (Figure 3.10). There was an increase seen for the total number of

hatched juveniles in the two test concentrations as well as the control over the two

observation days but was very slight in the 2 % concentration. Percentage hatching was

around 3 % in the 2 % concentration and 16 % for the 1 %. Mortality was also noted at

the higher concentrations.

Similar observations were made for sample Y-VA as seen in Figure 3.11. Both the

concentrations tested were effective in suppressing egg hatching. Observations on the 7th

day showed that there was no increase in the number of juveniles in 2 % concentration

but some increase was seen in the 1 % concentration.

Activity of the sample X when compared with sample Y for both FJ and VA kava

showed similar effect on egg hatching. For both FJ and VA kava, it was noted that the

number of hatched juveniles increased slightly over the experimental period for sample

65 Potential Application of Kava (Piper methysticum F.) in Nematode Control

X, while for samples Y there was hardly any increase. Hence sample Y could be said to be more effective in suppressing hatching of root knot nematode compared to samples X.

Table 3.10: Results for Sample X-FJ (fresh)

2% 1% Control

No. of No. of No. of % % % % % % Juveniles Juveniles Juveniles Hatching Dead Hatching Dead Hatching Hatched Hatched Dead Hatched

3rd Day 5 8 4 21 1 18 100 0 88

6th Day 15 32 30 38 7 75 100 2 199

8th Day 18 43 41 53 20 125 100 3 235

Table 3.11: Results for Sample X-VA

2% 1% Control

No. of No. of No. of % % % % % % Juveniles Juveniles Juveniles Hatching Dead Hatching Dead Hatching Dead Hatched Hatched Hatched 3rd 59 3 100 2 7 58 4 100 0 Day

7th 206 8 28 15 19 15 39 100 1 Day

66 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Treatment of Egg Masses with Sample X-FJ (fresh)

120 100 I 80 Ts 60 O) O) 40 LJJ o5 20 0 2% 1% Control

Thir d D a y Sixth Day —*— Eighth day

Figure 3.8: Treatment of egg masses with sample X-FJ (fresh)

Treatment of Egg Masses with Sample X-VA

120 100

3 80 re 60 O) 40 20 0 2% 1% Control

Third Day • Seventh Day

Figure 3.9: Treatment of egg masses with sample X-VA

67 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Table 3.12: Results for Sample Y-FJ

2% 1% Control

No. of No. of No. of % % % % % % Juveniles Juveniles Juveniles Hatching Dead Hatching Dead Hatching Hatched Hatched Dead Hatched

3rd 6 12 11 25 1 42 100 0 168 Day

7th 3 89 13 16 13 68 100 5 425 Day

Table 3.13: Results for Sample Y-VA

2% 1% Control

No. of No. of No. of % % % % % % Juveniles Juveniles Juveniles Hatching Dead Hatching Dead Hatching Dead Hatched Hatched Hatched

3rd 23 55 28 29 3 36 100 0 125 Day

7th 6 93 28 9 67 44 100 4 474 Day

68 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Treatment of Egg Masses with Sample Y-FJ

120 100 80 60

Eg g Hatchin 40 20

0 2% 1 % Control

Third Day -ySeventh Day

Figure 3.10: Treatment of egg masses with sample Y-FJ

Treatment of Egg Masses with Sample Y-VA

120 100

80 tch i n * 60

Ol UJ 40 S? 20 0 2% 1% Control

Third Day —•—Seventh Day

Figure 3.11: Treatment of egg masses with sample Y-VA

69 Potential Application of Kava (Piper methysticum F.) in Nematode Control

b) Comparison of Activity of Samples X and Y of FJ and VA Kava with Standard

Gallic acid was used for comparison of the activity of the two samples. Two separate

experiments were conducted to compare the activity of the X and Y samples with gallic

acid. Samples X-FJ (fresh) and X-VA were compared with standard in one experiment

while samples Y-FJ and Y-VA were compared in another. Both, samples X-FJ (fresh)

and X-VA, showed almost nil egg hatching on the 3rd day, while gallic acid showed about

5 % egg hatching compared to the control which had a total number of 155 hatched juveniles (Figure 3.12). The 7th day results were still quite low for the two test solutions.

The standard as well did not show any significant increase in the number of hatched juveniles hence lowering the percentage hatching for the 7th day with respect to that of

the control in which the numbers had almost doubled.

The results for samples X-FJ (fresh) and X-VA were similar to the separate experiments

carried out thus showing reproducibility and competence of the kava extracts in

suppressing egg hatch.

Samples Y-FJ and Y-VA as well as the standard showed some egg hatching on the 3rd

day. The number of hatched juveniles were almost same for the samples Y-FJ and Y-VA

but was little higher for the standard as per figure 3.13. The number of hatched juveniles

on the 7th day remained the same in all the experiments except the control, which showed

a three-fold increase.

70 Potential Application of Kava (Piper methysticum F.) in Nematode Control

It was noticed that the experiments in which test solutions were used to replenish the drying of solutions, the number of hatched juveniles did not increase from that recorded on day 3. While the experiments where water was added, allowed a slight increase in the number of juveniles after the 3rd day. Hence it could be concluded that addition of water diluted the effect of the chemicals thereby allowing some more eggs to hatch.

The observed nematicidal activity of the X samples can be due to any of the water- soluble components including suspended kava lactones which are consumed as kava drink. The kava lactones have been reported to have anesthetic effect on animals including humans (Cambie and Ash 1994; Cordell 1998; Davis and Brown 1999 and

Walji 1998). Since nematodes are very small organisms with simple body systems, the kava lactones could have led to irreversible paralyses and hence death of the nematodes.

The effect could not be on the digestive system of the nematodes since the J2 stage is the infective stage and the nematodes do not feed at this stage, instead they use the stored energy (Ferraz and Brown 2002; Weischer and Brown 2000). These lactones are also known to have antifungal, antibacterial and amoebicidal activities (Singh 1986;

Sotheeswaran 1987; Walji 1998; Xuan et al, 20031).

Other water soluble compounds reported in kava and which may be responsible for this nematicidal activity are glutathione, pigment molecules, bornyl esters and phenolic compounds (Basko 2002; O'hara et al, 1965; Singh 1986; Smith 1979, 1983;

Sotheeswaran et al., 1998; Whitton et al, 2003; Wu et al, 2002; Xuan et al, 20032).

These compounds could possibly be present in samples X, Y and the fractions of both FJ

71 Potential Application of Kava (Piper methysticum F.) in Nematode Control and VA kava. The phenolic compounds have been reported to be allelopathic activities

(Xuan et al., 20031). The nematicidal activity therefore can not be accredited to any one of the compound present in kava. It could be due to any of the compounds mentioned above or could be the result of the combined effect of a number of compounds.

72 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Comparision of Activity of Samples X of FJ and VA Kava with Gallic Acid for Egg Hatching 120

O) 100 80 60 U) UJ 40 20 0 2% Sample X-FJ 2% Sample X-VA 0.5% Galic Acid Control (fresh) • Third Day • Seventh Day

Figure 3.12: Comparison of activity of samples X of FJ and VA kava with Gallic acid for egg hatching

Comparison of Activivty of Sample Y of FJ and VA Kava with Gallic Acid for Egg Hatching 120

100 WWWT 80 I 60

*+++ UJ 40 *+++ *+++ *+++ 20 *+++ *+++ *+++ *+++ *+++ *+++ 0 2% Sample Y-FJ 2% Sample Y-VA 0.5% Gallic Acid IIControl • Third Day • Seventh Day

Figure 3.13: Comparison of activity of samples Y of FJ and VA kava with Gallic

Acid for egg hatching

73 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.3.2 Pot Experiments (Soil Amendment Experiments) for FJ and VA Kava

3.3.2.1 No Degradation Allowed (0 weeks)

The average number of root galls formed by RKN decreased as the concentration of kava was increased in the soil. Number of root galls shown for the 5 % concentration was from one plant only since plants in the other replicates had died. Some plant mortality was also noticed in the other treatments. Mortality increased with increasing concentration of kava in soil. This indicates that in addition to being effective in reducing root galls, kava also has some detrimental effect on plants especially at 5 % concentration. The plant height measurements showed a decreasing trend with increasing kava concentrations but the root length values were similar to control plants (Table 3.14 and 3.15). The average number of galls in the control was 119 and all the plants were healthy in these pots.

Results indicate that when seedlings are planted in the soil on the day of addition of kava, plant health is affected leading to death. The effect was more prominent as the concentrations increased. Results for VA kava are comparable to that FJ kava. The average of total number of galls in the control pots of FJ kava was 119 while for VA kava was 62. In both of these experiments it was noticed that though nematode population decreased by adding kava, there was some phytotoxic effects on the plants. The compounds present in kava responsible for these activities can not be determined at this stage.

74 Potential Application of Kava (Piper methysticum F.) in Nematode Control

0 Weeks Degradation FJ Kava

12 80 10 - 70 £ TO 60 re c 8 50 E re \ ^^ (D 6 \ \ 40 >. 30 ea l ve . 4 - < \ ?n i 2 ^B ^> 10 r\ U 1 2 5 % Kava in Soil

—•— % healthy plants (live plants) —•—Average of total number of Galls

Figure 3.14: Zero weeks degradation FJ kava

0 Weeks Degradation VA Kava

25 i • 60 20 50 TO

- —• 40 Plan t 30 10 20 ve . ga l Health y 5 10 r\ U i 1 2 5 % Kava in Soil

—•— % healthy plants (live plants) —•—Average of total number of Galls

Figure 3.15: Zero weeks degradation VA kava

75 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.3.2.2 Two Weeks of Degradation

The effect of degrading kava in soil for two weeks before planting and inoculating the seedlings was observed. All three treatments showed a decrease in root galls. The control of FJ kava recorded 34 as the average root galls while VA kava had 55. As in previous experiments nematode population was dependant on the concentration of kava in the soil.

Plant mortality was not noticed for treatments by FJ kava while treatments by VA kava caused some mortality at the higher concentrations. An increase in plant height was noticed in all the treatments compared to control for FJ kava, while root length decreased slightly. Treatments for VA kava led to a decrease in plant height as well as root lengths when compared to the control.

3.3.2.3 Four Weeks of Degradation

Four weeks of degradation of kava in the soil showed positive results towards control of nematode population. Root galls made by nematodes were quite less in treatment plants compared to control. The control for FJ kava showed an average of 97 root galls while

VA kava showed 66. As observed in other experiments, higher concentrations were more effective compared to lower ones. In regards to mortality of plants for FJ kava, 1 % treatment did not show any prominent effect but in 2 % and 5 % treatments, one and two plants respectively had died. For VA kava plant mortality remained same for 2 % concentration but was also noted at 1 % concentration. The plant heights increased with

76 Potential Application of Kava (Piper methysticum F.) in Nematode Control increasing concentrations for FJ kava but decreased with increasing concentration for VA kava. Root length showed a slight decreasing trend for all experiments.

3.3.2.4 Six Weeks of Degradation

In this experiment, kava had been allowed to degrade in the soil for six weeks before the seedlings were planted and inoculated. All of the three treatments were found to be effective in controlling root knot nematodes. The number of root galls were inversely related to the amount of kava added to soil. The control for FJ kava had an average number of 17 galls while VA kava had 67 galls. Two plants in 2 % and three plants in 5

% concentration had died in FJ kava treatment while for VA kava treatment, one plant each in 2 % and 5 % concentration had died. The general trend is similar in both FJ and

VA kava although in VA kava experiment, one plant had died in the control pots.

The plant height increased in 2 % and 1 % concentrations of FJ kava but a slight decrease compared to control was seen in the 5 % treatment. All the treatment concentrations on

VA kava showed slight decreased plant heights. Root length measurements were lower than control in all FJ kava treatments but for VA kava treatments lower values were recorded for 1 % and 2 % concentrations while 5 % concentration showed slightly higher value.

77 Potential Application of Kava (Piper methysticum F.) in Nematode Control

2 Weeks Degradation FJ Kava

120 OT +->

4 100 J5 Q. 3 3 80 > 6 2 60 to 1 40 0 + 20 0 0

% Kava in Soil

• % healthy plants (live plants) - Average of total number of Galls

Figure 3.16: Two weeks degradation FJ Kava

2 Weeks Degradation VA Kava

16 120 14 100 « 12 lin g 80 f

e . ga l 8 60 | < 6 40 S 4 2 20 $S 0 0

% Kava in Soil

% healthy plants (live plants) . Average of total number of Galls

Figure 3.17: Two weeks degradation VA kava

78 Potential Application of Kava (Piper methysticum F.) in Nematode Control

4 Weeks Degradation FJ Kava

10 -i r 120 9 100 8 7 - 80 6 60 Gallin g

5 th y Plant s 4 40 3 Ave . 2 x^ : 20 % Hea l 1 n n 1 2 5 % Kava in Soil

—•— % healthy plants (live plants )Average of total number of Galls

Figure 3.18: Four weeks degradation FJ kava

4 Weeks Degradation VA Kava

20 100

15 80 I 60 S 40 | 20 ^ 0 0

% Kava in Soil • % healthy plants (live plants) Average of total number of Galls

Figure 3.19: Four weeks degradation VA kava

79 Potential Application of Kava (Piper methysticum F.) in Nematode Control

6 Weeks Degradation FJ Kava

8 i j 120 7 - 6 o 5 C D O - ^ o

gallin g 4

" • th y Plant s 3 40 | Ave . 2 1 20 S? r\ U ~r 1 2 5 % Kava in Soil

—•— % healthy plants (live plants) —•—Average of total number of Galls

Figure 3.20: Six weeks degradation FJ kava

6 Weeks Degradation VA Kava

35 n 105 30 10 0 g> 25 c I 20 95 £ ° 15 90 I" < 10 85 I 5 80 S? 0 75 % Kava in Soil

% healthy plants (live plants ) Average of total number of Galls

Figure 3.21: Six weeks degradation VA Kava

80 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Table 3.14: Observed plant heights (cm) for the various treatment experiments of

FJ and VA kava

Concentration of FJ kava VA kava kava in soil (%)

0 wks 2 wks 4 wks 6 wks 0 wks 2 wks 4 wks 6 wks

0 30.2 35.3 37.0 26.5 28.1 27.5 32.3 35.2

1 28.1 43.3 35.6 31.3 22.6 24.7 30.5 31.3

2 13.4 40.4 35.5 33.8 20.2 24.7 27.1 27.9

5 9.5 36.9 31.8 24.6 20.0 25.8 25.6 30.9

Table 3.15: Observed root lengths (cm) for the various treatment experiments of FJ and VA kava

Concentration of kava in soil FJ kava VA kava (%) 0 wks 2 wks 4 wks 6 wks 0 wks 2 wks 4 wks 6 wks

0 16.6 22.5 21.3 17.2 22.9 23.3 22.1 17.4

1 29.7 18.0 17.4 11.7 7.6 17.8 19.1 16.5

2 16.4 19.5 19.5 17.0 12.6 18.8 17.5 15.2

5 14.5 18.1 19.6 16.7 10.2 16.8 18.8 18.7

81 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.3.2.5 Comparison of Observed Results at Different Concentrations over Various

Degradation Periods

Figures 3.22 and 3.23 summarize the results of the different degradation experiments of

FJ and VA kava at various concentrations tested. The number of galls observed at different concentrations did not differ much from each other since the trend line increases very slightly as the concentration is decreased. Statistical analysis shows that P < 0.001

(FFJ = 173.0, PFJ = 0.000, dfFJ = 1, 79; FVA = 152.2, PVA = 0.000, dfVA = 3, 73), which indicates that concentration of kava was significant in the experiment. Comparison of the different degradation periods over the concentration range does not show a very distinct variation for FJ kava but is distinct for VA kava.

The trend lines for the different degradation periods of FJ kava get closer to each other as the concentration is increased. There was significant interaction, P< 0.001 (FFJ = 27.4,

PFJ = 0.000, dfFJ = 9, 79) between the different concentrations and different degradation periods. The 0 and 4 weeks degradation experiments gave similar results at the 1 % concentration while the 2 and 6 weeks degradation showed slightly lower root gall numbers but were close to each other. For VA kava the 2 weeks degradation experiment was most effective in decreasing root gall numbers followed by 4, 0 and 6 weeks. The observed P > 0.1 (FVA = 0.6, PVA = 0.74, dfVA = 9, 73) which indicates that there was no significant interaction between the kava concentrations and the different degradation periods for VA kava.

82 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Root Gall Numbers against Kava Concentration

1 2 Kava Concentration in Soil (%) 0 weeks 2 2 weeks 4 weeks 6 weeks

Figure 3.22: Root gall numbers against kava concentration for FJ kava

Figure 3.23: Root gall numbers against kava concentration for VA kava

83 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.3.2.6 General Discussion

Kava when used as soil amendment was effective in controlling root- knot nematode population in all experiments. However some phytotoxic effect of kava was also noted when it was used as soil amendment. In the 0 week degradation experiment, a number of plants had died at the higher concentrations. Some plant mortality was also noted in the other experiments at higher concentrations but was not very significant. Since whole kava root material (powder) was used, any of the various compounds present in kava can be responsible for these two (nematicidal and phytotoxic) activities. Some phenolic compounds have already being isolated from kava which are also present in other plants with allelopathic properties (Xuan et al., 20031).

Allelopathic properties are the ability to suppress plant growth, especially the growth of troublesome weeds. Other chemicals identified in kava as reported by various authors elsewhere could be responsible for these nematicidal effects (Cambie et al, 1997;Lebot et al, 1992, 1997; O'hara et al. 1965; Singh and Blumenthal 1992, Singh 1986; Waiji

1998; Whitton et al, 2003; Xuan, et al, 20031).

Allowing kava to degrade in the soil before planting was seen to have lesser phytotoxic effect compared to instant application such as at 0 week degradation. It was noted that two weeks of degradation of kava in the soil was the most effective in suppressing root galling while having lesser phytotoxic effect. No plant mortality was noted in the 2 weeks degradation of FJ kava whilst some plant mortality was noted in VA kava. The difference

84 Potential Application of Kava (Piper methysticum F.) in Nematode Control in the results for phytotoxicity for the 0 and 2 weeks degradation experiment indicates that the compounds in kava responsible for this activity may have been destroyed

(degraded) or converted to harmless compounds over this period. In the case of VA kava, it may not have been totally degraded in this time period.

The difference in properties of the two kava types (FJ and VA) could be due to either different compounds or different amounts of the same compounds. If this activity is due to different compound(s), then it can be said that these compounds were not broken down during the degradation period or some other new compounds with phytotoxic effect may have been synthesized from these chemicals. Alternatively it can also be possible that higher concentration of the same compounds as that of FJ kava are present in VA kava and it could not be totally degraded by the available microorganism population. Since microorganism population was not determined in this study, these are only postulations for such an activity. Microorganisms can degrade harmful substances to harmless ones and can also produce new compounds from the existing compounds through in vitro biochemical conversion through processes such as cyclisation and esterification. These new compounds can be even more harmful than the initial compounds from which they are synthesized (Begum et al, 2003; Halbrendt 1996; Rodriguez-Kabana 1986).

For the six weeks degradation experiment, it was noted that for VA kava the number of plants dead in 2 % and 5 % concentrations were same as that in the control. The death of a plant in the control would definitely be due to reasons other than the effect of kava. If the plant mortality noted in the treatment pots are due to some other causes that is the

85 Potential Application of Kava (Piper methysticum F.) in Nematode Control same cause as that for control, it can be said that VA kava after six weeks of degradation does not have any phytotoxic effect. For FJ kava, plant mortality was noted in the six weeks degradation experiment, while at 2 weeks of degradation no plant mortality was seen. Hence it can be said that during this period new chemical compounds with phytotoxic activity may have been generated from FJ kava by the microorganisms. The nematicidal activity of kava was still prominent after six weeks of degradation. Thus kava can keep nematodes in control for quite some time after it has been applied to soil.

The compounds in kava responsible for these activities can not be identified as yet since chemical analysis of kava after various degradation periods was not carried out.

Generally all the experiments significantly decreased root gall numbers compared to the controls but in some, plant growth or health was affected as mentioned above.

An important observation made during the degradation period of kava was that, the pots in which kava had been added were usually dry. The dryness increased with increasing concentration and required daily watering. Thus the dying of plants in the experiment, in which no degradation was allowed, could be due to unavailability of water since water added may have been absorbed by certain component(s) of kava. Allowing kava to degrade in soil minimized this effect leading to healthier plants and decreased root galls.

This could possibly be due to degradation of the component(s) of kava which caused the soil to dry. Chemical analysis of kava after different degradation period will only allow the determination of the cause for this.

86 Potential Application of Kava (Piper methysticum F.) in Nematode Control

3.4 CONCLUSION

The observations have revealed that kava does have the active ingredients to suppress the root knot nematodes. Samples X as well as Y has shown positive results for nematode control. These samples had initiated juvenile mortality within 24 hrs of exposure which increased with increasing treatment times. The activity was permanent since none of the test samples showed more than 50 % revival when exposed to distilled water after being treated with the samples. Effective suppression of egg hatching was also noted for these extracts. All the extracts tested showed less than 50 % hatching over the observation period.

The results are also significant as the dosage found to be effective against these nematodes is quite low. Concentration as low as 2 % (w/v) showed significant juvenile mortality and effective suppression of egg hatch. The isolated fractions of samples Y-FJ and Y-VA were also effective towards juvenile mortality but recorded percentage mortality was lower than samples X and Y. It could have been possibly due to the low concentration (0.1 % w/v) at which these samples were tested. Since both sample X as well as Y (sample X less ethanol solubles) showed nematicidal activity, it can be concluded that the nematicidal activity of kava was not entirely due to groups of compounds with similar chemistry but the effect of the various compounds together. That is the result of the group of highly polar compounds present in sample Y as well as that of the ethanol solubles.

87 Potential Application of Kava (Piper methysticum F.) in Nematode Control

The pot experiments have shown that mixing kava powder in soil before planting can help in reducing infection by the root knot nematodes. These experiments have indicated that kava should be allowed to degrade in the soil for at least two weeks before planting to eliminate the phytotoxic effect. It was noticed that when seedlings were planted in the soil on the day of addition of kava powder, a high number of plants were dead. While the two, four and six weeks of degradation periods for both FJ and VA kava showed lesser affect on the health of the plant. It was also noted that the active ingredients in kava responsible for minimizing root gall numbers were effective even after 6 weeks since root galls were suppressed in the 6 weeks degradation experiments as well. Therefore using kava as soil amendment for controlling RKN would not require frequent re-application.

Kava has been found to be quite effective in these experiments. The extracts tested can be further fractionated and re-tested to identify the compound(s) with nematicidal activity.

Once the active compound has been identified, large scale isolations can been done to use kava as a nematicide. Moreover, with these initial positive results of kava, further exploration can be carried out in unused portions of kava plant such as the peelings and leaves as well as in related and wild kava species. Chemical analysis of kava after different degradation periods would permit determination of the different compounds present after the various degradation periods. This will assist in identifying the phytotoxic compounds in kava. Therefore these compounds which can be removed from kava when incorporating kava extracts for nematode control at large scale.

88 Potential Application of Kava (Piper methysticum F.) in Nematode Control

CHAPTER 4

4.0 GENERAL CONCLUSIONS

• The water soluble components as in the samples X of FJ and VA kava can be said

to contain different compounds or different amounts of the same compound. This

is because samples X of the two kava varieties showed different effect on the

root-knot nematode juveniles. While FJ kava showed continued mortality, VA

kava demonstrated some reversible of the initial effect noted when the juveniles

were transferred to distilled water.

• Samples Y of both kava types and their corresponding fractions could possibly

contain similar chemicals since they showed similar activity on juveniles but

further analysis needs to be done to confirm this.

• Samples X showed stronger nematicidal activity compared to sample Y for both

FJ and VA kava. This indicates that the ethanol solubles which had been removed

while preparing sample Y had significant effect on the root-knot nematodes.

• Both samples X and Y of FJ as well as VA kava were equally effective in

suppressing egg hatching. It can be said that the juvenile mortality and

suppression of egg hatching was due to different compounds in kava. However, it

89 Potential Application of Kava (Piper methysticum F.) in Nematode Control

could also be due to the same compounds since the effect of some chemicals on

different stages of an organisms life cycle are different.

Treatments that were replenished with distilled water on the 3rd day of observation

seemed to record slightly more hatched juveniles on the 7th day compared to the

treatments where the test solutions were added. It was noticed that addition of

water dilutes the effect of the test solutions thereby allowing more hatching.

• The pot experiments showed that a minimum of 2 weeks of degradation

eliminates/minimizes the phytotoxic effect of kava.

• Glucosides (in addition to carbohydrates) have been postulated to be present in

samples Y from the H-NMR data. A number of glucosides are known to have

nematicidal properties. Hence the glucosides in kava could be responsible for the

nematicidal activity. Moreover phenolic compounds present in kava have been

isolated from other plants and been tested positive for nematicidal properties as

well.

90 Potential Application of Kava (Piper methysticum F.) in Nematode Control

CHAPTER 5

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APPENDICES

Appendix 1

Flow Diagram outlining the extraction procedure for Kava

Powdered Kava Root Material

H2O

Residue Aqueous Extract

Freeze Dry

Residue (Sample X)

Extraction with Ethanol

Residue Ethanol Extract (Sample Y)

Semi-preparative HPLC fractionation

Sample Sample Sample Sample Y1 Y2 Y3 Y4

105 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Appendix 2

^ Spectrum of Sample Yl

i I—^

106 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Spectrum of Sample Y2

107 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Spectrum of Sample Y3

108 Potential Application of Kava (Piper methysticum F.) in Nematode Control

Spectrum of Sample Y4

109