SOME ASPECTS ON THE TAXONOMY, ECOLOGY, AND HISTOLOGY OF PYTHIUM PRINGSHEIM SPECIES ASSOCIATED WITH FUCUS DISTICHUS IN ESTUARIES AND MARINE HABITATS OF BRITISH COLUMBIA by TIMOTHY ALAN THOMPSON B.Sc., University Of Arizona, Tucson, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1981 Timothy Alan Thompson, 1981 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. BOTANY Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 rjate January 12, 1982 i i ABSTRACT Pythium un.dula.tum var. litorale Hohnk was found to infect Fucus distichus in the Squamish River estuary of southern British Columbia. This thesis adresses the questions of: 1.) whether this symbiosis can be found outside the Squamish River estuary, 2.) relationship of the infection within the estuary to the distribution of P. undulatum var. litorale in estuarine sediments, 3.) taxonomically defining those species associated with Fucus and/or in estuarine sediments, and 4.) the host parasite relationship as determined by means of histochemical and light microscope observations. Results indicated that outside the Squamish River estuary, associations between pythiaceous fungi and Fucus are uncommon in British Columbia coastal areas. Sampling of live and decaying Fucus plants from 10 field stations in British Columbia and Washington yielded only 4 species, the most common isolate being Phytophthora vesicula. Within the Squamish estuary, an association was found to exist between the distribution of P. undulatum var. 1itorale in the sediments and the distribution of infected Fucus plants. Sediment sampling from the Fraser River estuary, where Fucus does not occur, yielded P. undulatum var. litorale, suggesting that the fungus is probably indigenous to estuarine sediments. Numerous other species of Pythium were recovered from estuarine sediments, including P. butler i, P. carolinianum, P. catenulatum, P. gracile, P. torulosum , and P. volutum . Two taxa are described in detail. Pythium undulatum var. litorale was originally described by Hohnk (1953), but the varietal status was rejected by Waterhouse (1967). Arguments are presented for retention of the variety. Pythiogeten utriforme Minden is transferred to the genus Pythium and P. hohnkii is proposed as the nomen nova of this taxon. A discussion of the generic characteristics of the genus Pythiogeten is presented. In order to facilitate an understanding of the infection process by Pythium species, the anatomy and histochemistry of Fucus distichus were examined. Anatomically, F'. distichus agrees with earlier reports of other species of Fucus. The internal structure of cells was found to agree with descriptions in earlier publications, although higher physode content was noted in F. distichus. Histochemical staining suggested that cell walls of Fucus are three layered; having an outer fucan-rich layer, a middle layer composed principally of alginic acid, and an innermost layer of cellulose. Several phenolic-indicating reagents were tested on both fresh and fixed/embedded Fucus tissue, resulting in some interesting new observations of phenolics in the matrix. The host-parasite interface of P. undulatum var. 1itorale and F. distichus was also examined by use of histochemistry and the light microscope. Macroscopically, the infection of F. distichus occurs behind the most recent dichotomy, and lesions are necrotic, firm (flaccid with age), and are pink-to- red in color. Microscopically, fungal hyphae are confined to the cortical and medullary regions. Hyphae appear to i v penetrate host cell walls by means of an enzymatic dissolution of the alginic acid and cellulosic portions of the cell wall. Use of the Periodic Acid/Schiff's reagent shows a distinct non- staining halo at the point where hyphae cross the cell wall. Pit connections between cortical cells were observed to break down with hyphae present in only one cell, suggesting that the fungus is capable of parasitizing several cells via digestion of pits. Gemmae were observed to form in both cortical and medullary cells. The response by Fucus to infection is an active one; a hypersensitivity reaction analagous to that of higher plants is observed. Cells in advance of fungal hyphae are observed to autolyse. Normally metabolically quiescent medullary filaments are observed to have an increase in general protein levels and to have increased physode content. Physodes become polarized within the medullary cells, and coalesce to form larger units, which are then delimited from the producing cell by a cross wall. The fate of these 'giant' physodes was not observed, but it is believed that these cells autolyse and release their phenolic contents to the matrix, as levels of phenolic-reactive material were observed to increase in this region. Coupled with the buildup,of phenolics in the matrix is a decrease in the fucan component of the matrix. Stress and tear lines appear between cells, and eventually this region serves as an abscission zone by which the infected portions are dropped out of the plant. Behind the abscission zone, medullary filaments undergo transverse divisions to form V irregular, cuboidal cells which function as epidermis after abscission of the lesion occurs.. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES vii LIST OF FIGURES ix ACKNOWLEDGEMENTS xiii Introduction 1 Materials and methods 4 Recovery and Taxonomy of Pythium species 4 Histology of Healthy and Infected Fucus 10 Description of the principal study area 18 Results, Part 1 :Recovery of Pythiaceae from Fucus and from estuarine sediments in southern British Columbia and Washington 22 Effectiveness of Selective Media 22 Pythiaceous Fungi Associated With Fucus in British Columbia and Washington 24 Distribution of P. undulatum var. litorale in the Squamish and Fraser estuaries 24 Additional Oomycetes in Estuary Sediments 28 Discussion . 31 Part 2 : Pythium isolates recovered from Fucus and from estuarine sediments 34 Pythium undulatum var. litorale Hohnk 34 Pythium hohnki i nom nov 42 Brief Descriptions of Pythiacious fungi from Fraser and vii Squamish Estuaries 51 Part 3 : Observations on the infection of Fucus distichus by Pythium undulatum var. litorale 54 General Morpholgy and Histology of Uninfected Fucus distichus 54 Host-parasite interface: Natural infections 64 Laboratory Infection Studies 72 Discussion 73 Conclusion 87 References 90 Appendix-1: Fungal and Algal media employed 102 Appendix-2: Staining Procedures Employed 107 vi i i LIST OF TABLES Table 1: Histololgical techniques and reaction spec i £ ic it ies 11 Table 2: Laboratory tests on TTSM and GAM as selective media for the recovery of oomycetous fungi 23 Table 3: Oomycetes recovered from Fucus in British Columbia and Washington 25 Table 4: Pythium species recovered from sediments of the Squamish and Fraser river estuaries 25 Table 5: Reactivity of cell wall layers and matrix material with histological stains 57 Table 6: Reactions of Fucus matrix and cellular elements with phenolic-indicating reagents 62 LIST OF FIGURES Figure 1: Sampling sites in British Columbia and northern Washington 5 Figure 2: Diagram of culture apparatus 14 Figure 3: Fucus distichus growing on a log in central Squamish delta 20 Figure 4: Fucus distichus growing on sediments in central Squamish delta 20' Figure 5: Sites sampled and recovery of Pythium undulatum var. litorale from Fucus distichus in the Squamish River estuary 26 Figure 6: Sites sampled and recovery of Pythium undulatum var. litorale from the Fraser River estuary 29 Plate I: Pythium undulatum var. litorale 37 Figure 7: Lesions of F. distichus infected with P. undulatum var. litorale 37 Figure 8: Lobate sporangium 37 Figure 9; Inflated-filamentous sporangium 37 Figure 10: Toruloid sporangium 37 Figure 11: Mature sporangium showing refractile tip ... 37 Figure 12: Growth of hyphae through discharged sporangium 37 Figure 13: Zoospore formation 37 Figure 14: Zoospore germinating to reform sporangium .. 37 Figure 15: Chlamydospores formed in cortical cells of X Fucus 37 Plate II. Pythium hohnkii . 45 Figure 16: Highly branched hyphae 45 Figure 17: Hyphae digesting aborted oogonium 45 Figure 18: Bursiform sporangium 45 Figure 19: Bilobate sporangium 45 Figure 20: Spherical sporangium 45 Figure 21: Elongate emission tube 45 Figure 22: Refractile globules in mature zoosporangium 45 Figure 23: Zoosporogenesis, initiation 45 Figure 24: Zoosporogenesis, completion 45 Figure 25: Oogonium with multi-lobed antheridium 45 Figure 26: Mature oospore 45 Figure 27: Principle vegetative regions of F. distichus .. 55 Plate III. Anatomy of F. distichus '.. 58 Figure 28: Primary and secondary filaments showing outermost ring of fucans 58 Figure 29: Primary and seconday filaments showing inner ring of alginic acid 58 Figure 30: Epidermal cells stained with TBO pH=4.4 .... 58 Figure 31: Epidermal cells stained with TBO pH=1.0 .... 58 Figure 32: Phenolic-reactive material within medulla matrix 58 Figure 33: Erlichs reagent-reactive material in the epidermal and cortical regions 58 Figure 34: Cytoplasmic features of Fucus cells 58 Plate IV: Parasitism of F. distichus by P. undulatum xi var. 1 itorale 67 Figure 35: General parasitism of epidermal and cortical cells 67 Figure 36: Hyphae growing through former pit connection 67 Figure 37: PAS non-staining region at point where hyphae cross cell wall 67 Figure 38: PAS halo in medullary filament 67 Figure 39: Collapsed pit connections between cortical cells 67 Figure 40: Bacteria invading necrotic tissue 67 Plate V: Defense reaction of F.