A STUDY ON THE DISEASE RESISTANCE IN HEVEA BRASILIENSIS

Transcription

1 . - A STUDY ON THE DISEASE RESISTANCE IN HEVEA BRASILIENSIS TO COLLETOTRICHUM GLOEOSPORIOIDES A thesis submitted to the Faculty of Applied Science, University of Sri Jayawardenapura, Sri Lanka for the Degree of Master of Science by PALLEPITIYA KANKANAMALAGE SAMARADEEWA B.Sc.(Sri Lanka) October, Department of Biological Science, Sri Jayawardenapura University, Nugegoda, Sri Lanka.

2 VII A B S T R ACT The factors responsible for disease resistance of Hevea clones to ~. gloeosporioides were st~died. First, field observations showed that the s~sceptibility of Hevea to the disease varied in different clones and an interaction was seen between clones and locations. When the infection was low incidence and severity of the disease were linearly related; a c~rvilinear relationship was evident when the level of infection was high. Isolates of. gloeosporioides varied in their growth characteristics, morphology and spore prod~ction. They showed differential s~sceptibility to vario~s clones. Leachate of resistant clones suppressed the growth of the pathogen and favo~ed appressorium formation. 1 vitro st~dies indicated that growth promoting s~bstances were not cond~cive for formation of appressoria. There was no difference either q~antitatively or q~alitatively in s~gars and amino acids in the leachate of different clones. Waxes and s~bstances associated with it are inhibitory on the pathogen at high concentrations. These substances separated by TLC showed two inhibitory zones. Phenolic and acidic s~bstances are associated with the cr~de wax extract. Leaves of resistant clones s~pported less germt~be growth than s~sceptible leaves, b~t favoured appressori~m formation. On penetration of resistant tiss~es a rapid disorganization of the cell content was observed, arresting hyphal growth. In s~sceptible clones hyphae invaded deeper in to the tissues and colonized the mesophyll cells both inter and intracell~lerly;

3 VIII aoervuli were formed 12 h after inoculation. The migration of nuolei towards the site of penetration was observed in resistant olones. There was no relationship between either total or orthodihydroxy phenol level and field resistance of Hevea olones. But the infection was accompanied by a change in the orthodihydroxy phenol level, particularly in resistant clones. In Hevea,peroxidase activity was higher than polyphenoloxidase and inorease in peroxidase level with the infection was more pronounced in resistant clones. Infection also resulted in the increase of soluble protein content.

4 II CONTENTS MEMORANDUM I CON TEN T S II A C K NOW LED G E 1'1E N T S VI A B S T R ACT VII A B B REV I A T ION S IX 1.INTRODUCTION 1 2. LIT ERA T U R ERE V I E W 9 3. MAT E R I A L SAN D MET HOD S 21 I. Isolation and maintenance of cultures 21 II. preparation of inoculum 21 III. Selection of plant materials Disease incidence and severity of Hevea clones Assessment of incidence and severity in different environments 22 3~1.2 Relationship between disease incidence and severity 24 i. Linear relationship 24 ii. Comparison between different functions Pathogenicity of different isolates Pre-penetration behaviour Growth characteristics of the isolates Leaf exudate (leachate) Preparation of leaf exudate and examination of its effect on the pathogen Chromatographic analysis of the exudate 21 i. Sugars 27 ii. Amino acids In vitro studies with sugars and amino acids 28,

5 III Determination of phenolic substances present in the leachate Leaf surface waxes Determination of surface waxes Bioassay with the crude wax Ether extraction of acidic substances 31 3&4.4 Direct bioassay using thin-layer chromatography Detection of phenolic compounds co-extracted with wax Histopathology Leaf clearing and growth measurements on the leaf surface 3~ Fixation of tissues and staining for microscopic observations Phenols Extraction and determination of phenols Quantitative assay for total and orthodihydroxy phenols Qualitative variation of phenols Identification of phenolic compounds in leaf extract Bioassay with the chromatography elute Effect of leaf extract on spore germination Enzyme assay Preparation of acetone powder Enzyme extraction and purification Measurement of enzyme activity 40,

6 IV 3.8 Protein determination 4. RES U L T S 4.1 Disease incidence and severity of Hevea clones Influence of environment on disease incidence and severity Relationship between incidence and severity 53 i. Linear relationship 53 ii. Comparison between functions Pathogenicity of different isolates Pre-penetration behaviour Growth characteristics of the isolates Leaf exudate (leachate) Effect of leaf exudate on development of the pathogen 68 4.,.2 Nutritive components in the leaf exudate Effect of sugars and amino acids on the pathogen Phenolic compounds in the leaf exudate Waxes and associated substances Content of leaf surface waxes Effect of crude chloroform extract on the pathogen Effect of different extracts Bioassay on thin-layer chromatography Chromatographic separation of waxy materials Histopathology 90 \ Pre-penetration studies of the pathogen Post-penetration studies of the pathogen 98

7 v 4.6 Role of phenols in resistant Hevea clones Phenols in healthy Hevea leaf tissues Effect of infection on the phenol level ) Qualitative estimation of phenols Bioassays with the elutes of naturally occuring phenols 11) Identification of phenols 11) Toxicity of the leaf extract Peroxidase and polyphenoloxidase activity Enzyme activity in healthy leaves Variation of peroxidase and polyphenoloxidase activity with the infection Effect of infection on the protein content D I S C U S S ION REF ERE NeE S A P PEN D I X 116