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. distichus to infection ... 70
Figure 41: Low magnification of defense reaction in
medulla 70
Figure 42: Primary filament showing formation of giant
physodes 70
Figure 43: High magnification of giant physode 70
Figure 44: Increase in phenolic materials in the matrix
of the hypersensitive region 70
Figure 45: Abscission zone 70
Figure 46: Irregular, cubiodal cells differentiated
from medullary filaments 70
Plate VI: Laboratory infection 74
Figure 47: Reduction in physode content after 30 day
incubation period 74
Figure 48: Laboratory lesions on Fucus 74
Figure 49: Low magnification of laboratory infection .. 74 xii
Figure 50: Giant physode in lab infection 74
Figure 51: Diagramatic summation of events during
pathogenesis 79 ACKNOWLEDGEMENTS
This work has been carried out under the supervision of
Dr. G.C. Hughes. I thank him for support, advice, editing of this thesis, but most of all for the education received here at
UBC under his direction.
Dr. R.J. Bandoni encouraged, advised, and made
significant contributions to my knowledge of the fungi as a
group. Dr. R. Copeman was always available with open, frank
advice, and also contributed to my education at UBC. To these
gentlemen go my thanks and appreciation.
Dr. T. Bisalputra generously allowed me to use his
laboratory facilities, and was equally generous in both
technical and interpretive advice. Mr. Alan Burns contributed
his time and talents to assist me in this study. I am indebted
to both for the-i-r kindness.
Many individuals at UBC contributed advice, discussion and
technical assistance. I would especially like to thank Drs.
B. Bohm, P.G. Harrison, L. Olivera, G. Rouse, G. Towers,
and I. Whyte, for their contributions. The kind assistance of
Dave Zitten, Bioscience Data Center is acknowledged. Ms.
Jolly Hibbitts of the Invertebrate Pathology Laboratory, NOAA,
Mulkitillo, Washington, is thanked for use of her laboratory
and materials for GMS staining of plant tissues.
Finally, my wife Suzanne has been the most important xiv
source of help during the course of this thesis. To my partner and friend go my most sincere thanks. 1
INTRODUCTION
Since the classic studies of Sutherland (1915a,b,1916), mycologists have expressed interest in the study of fungal symbionts of marine algae. Although the number of known species, and our knowledge of them, has increased considerably in the last 65 years, only recently have, fungi in the oceans been examined seriously for their disease-causing capabilities--as potential problems in developing algal- aquaculture systems (Andrews, 1976,1979). Fungi, as important pathogens of cultivated seaweeds, have been recognized in
Japanese 'nori' farms since the 1940's (Arasaki, 1947). Outside of Japan , little work has been done on the process of pathogenesis in marine algae. Beyond contributions by Kohlmeyer
(1968,1972), Kohlmeyer and Kohlmeyer (1973,1975), Kazama and
Fuller (1970,1977), and Walker, et al. (1979), knowledge of the processes by which fungi attack and degrade algae is scant. In
North America, members of the Phaeophyta (brown algae) are being considered for commercial use; as food condiments (eg.,
"Kombu" made from Laminaria spp.), or as a source of alginic acid ( Laminaria, Macrocystis, Nereocystis ). Of the fungi described as symbionts of the orders Laminariales and Fucales, all are in the Ascomycotina or Deuteromycotina. No zoosporic
fungi have been described from the parenchymatous brown algae.
Ectocarpus species have been reported to be infected by 2
Eurychasmidium dicksoni i (Wright) Magnus (Johnson and Sparrow,
1961), by Olpidiopsis andreii (Lagerheim) Karling (Sparrow,
1960), and by Anisolpidium ectocarpi i Karling (Karling, 1943).
E. dicksonii is also reported from the brown algal genera
Feldmania, Punctaria, Pylaiella, Striaria, and Stictyosiphen
(see Andrews, 1976).
In June,1980 , Dr. R.J. Bandoni collected specimens of
Fucus distichus L. from the Squamish River estuary which displayed pink-to-red, flaccid lesions in the apical wings.
Subsequent examination revealed the presence of Pythium undulatum var. litorale Hohnk in the infected tissues.
Pythiums, as pathogens of marine algae, had been previously known to occur only in the red algae ( Porphyra and Ceramium )
(Sparrow,1932; Arasaki,1947). Thus, a unique opportunity was presented to study the presence of a zoosporic fungus symbiotic with a parenchymatous brown alga, and to observe a second species of Pythium parasitic on a marine plant.
The recovery of infected Fucus plants within the Squamish estuary raised interesting questions regarding the distribution of P. undulatum var. litorale within the estuary, as well as what other pythiaceous fungi might be present. Both the distribution and role of the Pythiaceae in estuarine ecological systems have been largely ignored, despite the demonstrations by Hohnk (1939,1953,1956) and Siepman (1959) that these fungi were present in salt marsh and estuarine sediments. These workers, and later Harrison and Jones (1974),showed that
Pythium and Phytophthora spp. could adapt to the more stringent 3
physiological requirements of estuaries and oceans. Only more recently have workers turned their attention to the possible role of marine-associated Oomycetes in detrital food chains
(Anastasiou and Churchland, 1969; Fell and Master, 1975) or in seed set in mangrove plants (Newell, 1973,1976).
This investigation, then, has two objectives: (1) to identify species of Pythium and Phytophthora that occur in marine and estuarine localities in southern British Columbia and northern Washington state that may have an association with
Fucus, and (2) to describe the host-parasite interface of the
P. undulatum var. litorale/F. distichus symbiosis. The former study was initiated (a) to determine if this relationship only occurs in the Squamish estuary, (b) to taxonomically define those species associated with Fucus, and (c) to describe any other Oomycetes that might be present in local estuaries. 4
MATERIALS AND METHODS
Recovery and Taxonomy of Pythium spec ies
Fucus plants were sampled from June, 1980 to June,1981 from the following locations in British Columbia and Washington
(Figure 1): (1) Friday Harbor, Washington (48°33'N, 123°00'W),
(2) Cattle Point, San Juan Is., Washington (48°27.5'N,
123°00'W), (3) Limekiln Light, San Juan Is., Washington
(48°30'N, 123°9'W), (4) Sooke Harbor, B.C., (48°22'N,
123°43'W), (5) Point-no- Point, B.C. (48°45'N, 123°53'W), (6)
Bamfield, B.C. (48°50'N, 125°08'W), (7) Egmont, B.C. (49°45'N,
123°56'W), (8) Skookumchuck Narrows, B.C. (49°42'N, 123°54'W),
(9) Squamish River estuary, B.C. (49°42'N, 123°09'W), (10)
Spanish Banks, Burrard- Inlet, B.C. (49°06,N, 123°18'W).
At each site, plants were collected for examination if
they displayed lesions similar to those observed at the
Squamish River estuary. Healthy thalli were collected next to
infected plants. These plants were transported to the lab where
presence or absence of internal hyphae was confirmed by
freezing microtomy. Upon confirmation of a filamentous
pythiaceous fungus, portions of infected algal tissue were
placed in sterile glass petri plates, covered with either
sterile distilled water (SDW) or sterile seawater (SSW; 5
Figure 1: Sampling sites in British Columbia and northern Washington . Boxes denote Squamish and Fraser River estuaries. »=site of sediment sampling; »=site of Fucus collection. Adpated from A.H. Hutchinson, 1928. Transactions of the Royal Society of Canada. Third Series, Volume 22, Section 5, Page 294. 7
artificial seawater with an approximate salinity of 27 o/oo), and allowed to incubate ' at 20 C until the appearance of
oomycetous reproductive structures. Portions of healthy thalli
were processed in a similar manner to record the presence of
any additional fungi.
Usually, this method allowed for ready and easy isolation
of the pythiaceous fungi, but occasionally,severe contamination
by various diatoms, bacteria, labyrinthulids, yeasts, and
particularly Deuteromycetes ( Penicillium, Cephalosporium, and
Fusarium ), was encountered. A selective medium against the
principal fungal contaminants was developed and designated
Pythiaceous Selective Medium (PSM). The active factors of the
medium were pentachloronitrobenzene, benomyl, and mycostatin
(see Appendix-1). Before general application of PSM, the medium
was tested in laboratory with several species of the most
commonly encountered contaminants, and compared directly with a
commonly used selective medium for pythiaceous fungi, Gallic
Acid Medium (Flowers and Hendrix, 1969).
Distribution of P. undulatum var. 1itorale in the Squamish
River estuary was studied to determine if this species is a
regular member of the sediment mycoflora and , if so, if its
presence in the sediment can be correlated with the incidence
of infected Fucus. Additionally, it was of some interest to
determine what other Oomycetes might be present in the Squamish
and Fraser estuary sediments. As the infected Fucus occurred
only in the Carex lyngbyei Hornem - Eleocharis palustris (L.)
Roemer and Schultes zone (see the Vegetation Zone Map, Squamish 8
River Estuary Habitat Work Group Final Report ,1981), sediment sampling was restricted to this area. To determine if
P. undulatum var. litorale might occur in the sediments of other major estuaries of southern British Columbia, a limited
sample was collected from the Fraser River near the Reifel
Island Game Preserve. Although Fucus was not observed to grow
there, this site was chosen as it too has a training dike, altering the flow of fresh water into the marsh.
Sites sampled on the Squamish and Fraser rivers are shown
in Figures 5-6. Sediment samples were aseptically collected
from Carex rhizomes into sterile "Whorl-Pac" bags for
transportation to the lab. Some plant debris was collected for
hemp-seed baiting. At Squamish, the presence of infected plants
within a 1/2 m2 from the sample site was noted.
Sediment samples were processed by direct plating of small
portions (ca.0.25 gm wt.) to PSM medium, 4 plates per sample
site. Incubation of plates was at 20 C. Plates were monitored
daily for the appearance of hyphae. When growth was observed,
hyphal-tip transfers were made to a second plate of PSM, and if
the fungus was found to be free of contaminants, transfered to
a tube of corn meal agar stored at 5 C until further study. All
cultures were made axenic by the successive use of hyphal-tip
transfers.
For identification of recovered fungi, zoosporangial
observations were made on sterile grass blades, hemp seeds,
sesame seeds, or portions of Fucus thalli, incubated in both
SDW and SSW at 20 C. To induce sexual reproduction, isolates 9
were plated to both Corn Meal Agar (CMA, Difco) and to
Schmitthener's agar (SchmA; see Appendix 1), incubated at 20 C.
P. undulatum var. litorale and P. hohnki i were examined as to reproductive physiology in various dilutions of seawater.
Zoosporangia were again observed on the above substrata, and were compared for spore production in 0, 15, and 27 o/oo artificial seawater (Instant Ocean, Aquarium Systems) for 3 to
10 days at 20 C. Zoosporogenesis was initiated by washing the
seed or blade in several changes of fresh medium at appropriate
salinity. For production of sexual structures, 4 media, 3
salinities and 3 temperatures were employed: Emerson's YpSs
(Emerson, 1941), CMA, SchmA, and Fujita's agar (see Appendix
1), each made up using 0, 15, and 27 o/oo artificial seawater.
Both species were innoculated to all media, and then incubated
at 5, 10, and 20 C for a period of up to 30 days. Plates were
examined periodically for presence of reproductive structures
and, when found, measurements made directly on the agar
surface. Diagnostic observations and measurments used in the
descriptions are a combination of all the data acquired from
the previously described treatments. Reported measurment data
are from at least 100 observations. Placement of all species
was checked against the keys of Middleton (1943), Waterhouse
(1967) and Robertson (1980). 10
Histology of Healthy and Infected Fucus
Infected F. distichus plants were obtained from the
Squamish River estuary. Infected portions were excised and transported to the lab for fixation. Healthy plants were likewise processed for comparative purposes. A small sample was collected from Point-no-Point, B.C. to compare estuarine Fucus cytology with that of outer coast plants.
On return to the laboratory, infected and healthy material were washed briefly in running tap water to remove any visible foreign material or epiphytes. Fixation proceeded in 2% gluteraldehyde in 0.2M Hepes-seawater buffer (Sigma Chemicals), pH=7.3, with 1% caffeine added for phenolic preservation
(Bisalputra, pers. comm.), for 24-48 hours at 5 C. For the light microscope, this fixation procedure was found to yield as high a quality fixation as gluteraldehyde in the more toxic sodium cacodylate buffer. After a post-fixation rinse in buffer solution (for about 30 min.), material was dehydrated to 95%, in a methyl alcohol series and embedded in JB-4 methacrylate
(Polysciences, Inc.). Sections were cut at 1/2 to 2 1/2 y on a
Sorvall-DuPont microtome, floated onto acid-cleaned glass
slides with distilled water, and dried on a warming plate.
Cytochemical details, the stains used, color reactions of
the stains, and histochemical specificities are listed in Table
1. Initially, there was some difficulty in differentially
staining fungal hyphae from algal cells. Eventually the general protein stain, Analine Blue-Black in combination with either TABLE-1: Histological techniques and reaction specificities.
Histological Test Specificity Reported Color Reaction Reference
Acridine Orange Sulfated polysaccharides Fluorescence under UV light Cooke, 1977
Alcian Blue (AB) Sulfated polysaccharides Blue Parker and Diboll, pH-0.5 1966
Anallne Blue Black (ABB) General Protein Blue to Black Fisher, 1968
Calcofluor White Cellulose Heslop-Harrison and Hesslop-Harrison, 1980 Fluorescence under UV light Herth and Schnepf, 1980
IK2I/H2S04 Cellulose Jensen, 1962 Blue Periodic Acid/Schiff's Alginlc Acid Feder and O'Brien,1968 (PAS) Cellulose McCully, 1966 Jensen, 1962 Pink to red Safranin-0 (Saf-O) Sulfated polysaccharides Splcer, 1960
Toluidine Blue-0 (TBO) pH-4.4 Sulfated polysaccharides Pink McCully, 1970 Red to orange Carboxylated polysaccharides Red to blue II Physodes Green McCully, 1966 Evans and Holligan, 1972b
pH-1.0 Sulfated polysaccharides Pink McCully, 1970
Grocott's Silver Fungal hyphae Black Hibbltts, pers. comm. Methenamine
Na^CO^ extraction Extracts alginlc acid PAS or Calcofluor staining Parker and Diboll, 1966
CaCl2 extraction Extracts fucoldin Stain with TBO ph 4.4 Whyte, et al., 1981
Bordeaux Reagent Phenols Brown Cragie and McLachlan, 1964
Erllch's Reagent Pink ibid
Grey-Black ibid FeCl3
Vannilin-HCl Reddish-Orange ibid Evans and Holligan, 1972b
OsO. Black Ragan, 1976 12
the Periodic Acid-Schiff's reaction or Safranin-O, was found to be effective probably owing to the dense nature of the fungal protoplasm relative to that of Fucus cells. Fast Green with
Safranin-0 also adequately differentiated fungal and algal tissues, as did Grocott's silver-methenamine stain. However, high cost of Grocott's prohibited its general use. Two additional stains useful in determining polysaccharides in
Fucus were Toluidine Blue-0 (TBO) and Calcofluor-White: specificity of these two stains is listed in Table 1.
As certain cytoplasmic vesicles in brown algae, the so- called "physodes", are thought to be important in biochemical defense of Fucus against epiphytic flora and fauna, grazers and pathogens (reviewed by Ragan,l976), an attempt was made to
locate these vesicles cytochemically, to see if the production of these compounds could be associated with the infection process. As the physodes are thought to contain phloroglucinol,
or polymeric derivatives of phloroglucinol (Ragan, 1976),
reagents commonly used for phenolic identification were
employed (Table 1). Due to possible interference of staining by
the fixation and embedding procedure, chemical tests were
performed on both fresh (un-fixed) materials and plastic
embedded sections. Fresh materials were sectioned with a
freezing microtome and then stained, or were cut into small
portions, placed in the test reagent, and then embedded to JB-4
for sectioning. All reagents employed were first tested against
authentic phloroglucinol to record any deviations from the
expected color reactions. Details of the cytochemical staining 1 3
procedures used are given in Appendix 2.
To prove that P. undulatum var. litorale was actually pathogenic to Fucus, experiments were conducted to demonstrate
Koch's Postulates. It was also of some interest to observe development of the host parasite-interface over time.
Initially, an attempt was made to grow adult Fucus thalli from fertilized zygotes by inducing the zygotes to settle on oyster shells, and then culturing them under the conditions described by McLachlan et al. (1 971 ).• However, this method of obtaining experimental plants proved unsatisfactory as plants grew too slowly—obtaining a maximum height of only 5 mm after 6 months.
As an alternative, whole adult thalli, attached to loose rock substrate, were transported from Squamish to the lab, to an artificial tide tank similar in construction to the one used by Fulcher and McCully (1969b). The culture apparatus
(Figure 2) consisted of two vertically arranged tanks installed in a circulating water-bath table maintained at 13 C. The upper culture tank (55 liter capacity) was equipped with 3 perforated-plexiglass platforms, designed to maintain the plants above the low water mark during 'low-tide'. The lower holding tank (105 liter capacity) was designed to hold all the medium at low tide, and was equipped with 6-8 charcoal- glasswool corner filters (Hartz Canada, Inc.) which supplied vigorous aeration and effectively filtered the brown-colored exudates of Fucus plants. An additional corner filter was placed in the upper tank.
Light was supplied by two Vita-lite Duro-test fluorescent Figure 2: Diagram of culture apparatus. A = corner filters; B = circulating water bath; HT = high tide level; LT = low tide level; P = plexiglass platform; T = timers; arrows indicate direction of water flow. 15
LIGHTS
\ H T
CULTURE TANK / / \
PUMP
HOLDING TANK / 1 6
lamps, which provided 39 #/E at the water surface. Timers were
used to supply a 14:10 photoperiod, and 4 six-hour tidal cycles
(two highs, two lows). Water temperature was maintained at 11-
12 C, while air temperatures during 'low tide' fluctuated
between 19-25 C in the light cycle, and 17-19 C during the dark
cycle.
The medium employed was a modification of the artificial
seawater medium developed by Harrison et §_1. (1980). The only
major departure from their medium was to substitue "Instant
1 Ocean" (Aquarium Systems) for the artificial seawater base.
Composition of the medium is given in Appendix 1. To simulate
summer water conditions at the Squamish estuary, the artificial
seawater was made up to 8 o/oo total salts (based on the data
of Levings et §JL. , 1976) before addition of the nutrient and
vitamin solution. The pH of the medium was monitored weekly,
but never fell below 7.6. Nutrient and vitamin solutions were
added weekly to the culture vessels and de-ionized water was
used to maintain volume.
Prior to innoculation with the test fungus, Fucus plants
were held for 30 days to allow acclimation to the new
environment. Any necrosis that developed as a result of the
transfer, or the development of infection due to residual
innoculum carried from the Squamish estuary, resulted in those
plants being removed from the tank prior to innoculation. A set
of plants was removed from the remaining group, placed in a 100
liter continual-submergence tank, and watched for disease
and/or necrosis development without innoculation. Healthy 17
thallus portions were also embedded in methacrylate for comparative purposes.
Innoculation was achieved by flooding a single CMA culture plate with 10 mis SDW, allowing 48 hr incubation at 20 C, collecting the resultant zoospore suspension and pipetting the suspension directly onto the algal surfaces during a 'low tide' sequence.
Plants were monitered daily for symptom expression up to a period of 30 days beyond the innoculation date. Symptoms were noted, and portions of the lesions were processed to JB-4 methacrylate for sectioning. Recovery of the causal organism was effected by directly plating portions of necrotic lesions to PSM. 18
DESCRIPTION OF THE PRINCIPAL STUDY AREA
The estuary of the Squamish River is located at the head of Howe Sound,a fjord extending northeast from the southern
Strait of Georgia (Figure 1). The delta is about 2 km wide at its seaward edge, and is constrained by mountains on both sides of the fjord.. In 1972, as part of a port development plan, channelization of the Squamish River into its most westerly arm was accomplished by the construction of a river training dike.
Until completion of the dike in June,1972, the Squamish flowed into Howe Sound through two channels. The east arm of the river is now blocked from direct freshwater input, except for two culverts installed through the dike in May,1972.
Since 1972, little change in the principal vegetation of the central and eastern deltas has occurred, despite the deflection of freshwater from these areas, and the, subsequent penetration of a marine 'wedge' into the upper marsh (Levings,
1980). Carex lyngbyei and Eleocharis laustris are the principle vascular plants in the zone in which this study was carried out. Although the primary vascular flora appears to be unchanged in the 9 years since the dike construction (Levings, pers. comm.), the advance of F. distichus into the upper regions of the delta has been dramatic. In 1972, no Fucus occurred in the central delta (Pomeroy and Stockton, 1976); the only Fucus within the estuary at that time being confined to 19
low logs at the mouth of the estuary, relating to the penetration of the marine waters. By 1976-1977, Fucus began to appear on the seaward edge of the training dike (Levings, pers. comm.), and today is widely distributed throughout the central delta. Fucus is most luxuriant on the east side of the training dike and at the seaward edge of the central delta, but extends as far back as the blocked channel. Within the central delta, Fucus is found not only on log substrata (Figure 3), but is frequently directly associated with the sedge community; its holdfasts penetrating into the sediments near rhizomes of Carex
(Figure 4). . ^
The Squamish River and its tributaries are important with regards to salmon fisheries. Numerous studies have been conducted by Federal and Provincial governments concerning the effects of industrial development and river training on the estuary and adjacent regions of Howe Sound. The most recent and relevant physiographical data on the region can be found in the
Squamish Estuary Managment Plan, Habitat Work Group Final
Report (1981) , and the Air and Water Quality Work Group Final
Report (1981), co-published by the Government of Canada and the
Province of British Columbia. 20
Figure 3: Fucus distichus growing on a log in central Squamish delta . Note surrounding Carex .
Figure 4: Fucus distichus growing on sediments in central Squamish delta
22
RESULTS, PART 1 :RECOVERY OF PYTHIACEAE FROM FUCUS AND FROM
ESTUARINE SEDIMENTS IN SOUTHERN BRITISH COLUMBIA AND, WASHINGTON
Effectiveness of Selective Media
All oomycetous fungi tested to PSM medium grew, including the obligately marine species Atkinsiella dubia Vishniac. None of the frequently encountered Deuteromycete contaminants were able to grow on PSM during the regular period of incubation
(15-20 days) (Table 2). The labyrinthulids tested initially
showed no growth, but during subsequent sediment platings a
Labyrinthula sp. was recovered on PSM that did not seem to be affected by the presence of the fungistats.
GAM was effective in blocking the growth of
Deuteromycetes, but it also proved- to be inhibitory to
Oomycetes from marine habitats. This may not be due to the
direct inhibition of these fungi by gallic acid in GAM, but may
result from the extremely low pH of GAM (pH<4.0).
Throughout the course of this study, infected algal
portions and sediment plating to PSM consistently yielded
pythiaceous fungi without contamination by other fungi or
diatoms, except for the above-mentioned species of Labyrinthula
, and a single isolate of 0idium recovered from Squamish
sediments. 23
Table-2: Laboratory tests on TTSM and GAM as selective media for the recovery of Oomycetous fungi, exclusive of Deuteromycetes and Labyrinthuloids. Positive growth indicated by "+"; negative growth, not tested,
Fungus TTSM GAM
I. Oomycetes Pythium aphani dermatum + O
P. hohnkii + P. mari num + P. monospermum + + P. porphyrae + P. sylvati cum + o P. ultimum + O P. undulatum var. litorale + Pythium sp. + Phytophthora vesicula + Atkinsiella dubia +
II. Deuteromycetes Cephalosporium sp. Dendryphiella salina Fusarium sp. 1 - - sp. 2 - - Penicillium sp. 1 - - sp. 2 - - Zalarion maritimum • -
III. Labyrinthuloids Labyrinthula sp. 1 sp. 2 Thraustochytrium sp. 24
Pythiaceous Fungi Associated With Fucus in British
Columbia and Washington
Sampling indicated that pythiaceous fungi are not regular associates of living or decaying Fucus fronds outside of the
Squamish estuary. Of the 10 sites sampled, only 6 yielded a total of 7 Oomycetes (Table 3). Three of the recovered fungi were Phytophthora vesicula Anastasiou and Churchland, which means that the total number of species found was only 4.
Pythium undulatum var. litorale was recovered from Fucus in the
Squamish River estuary, Pythium hohnkii from the Skookumchuk
Rapid site, and an unidentified species of Pythium from Egmont.
Distribution of P . undulatum var. litorale in
the Squamish and Fraser estuaries.
Results of sampling in the Squamish delta are presented in
Figure 5. P. undulatum var. litorale is widely distributed throughout the estuary, being routinely recovered from both
sediments and plant debris in the central and eastern deltas.
Where Fucus plants were found to be infected, P. undulatum var. 1itorale was recovered from nearby sediments or plant debris. However, in the upper estuary near the blocked channels, the fungus was recovered from sediments, but infected
Fucus was not observed. This may, in part, be explained by the Table 3: Oomycetes recovered from Fucus in British Columbia and Washington.
Station Species
Friday Harbor, Washington Phytophthora vesicula Anst. et Church. Bamfield, British Columbia P_. vesicula Spanish Banks, B.C. P_. vesicula Squamish, B.C. Pythium undulatum var. litorale Hohnk Skookumchuk Rapids, B.C. Pythium hohnkii nom. nov. Egmont, B.C. Pythium sp.
Table 4: Pythium species recovered from sediments of the Squamish and Fraser River estuaries, and the growth of these species in fresh and marine water in vitro. Growth indicated by "+"; no growth by "-".
Growth in Species Recovery DW SW
Pythium Carolinianum Matthews Squamish + P_. catenulatum Matthews Squamish, Fraser + P_. butleri Subramanian Squamish, Fraser + P. gracile Schenk Squamish, Fraser + + P_. torulosum Coker and Paterson Squamish, Fraser + + P_. volutum Vanterpool and Truscott Squamish, Fraser + + Pythium species I. Squamish, Fraser + + 26
Figure 5: Sites sampled and recovery of Pythium undulatum var. litorale from Fucus distichus in the Squamish River estuary, o=non-infected Fucus; • =infected Fucus; A= recovery of P. undulatum var. 1itorale from sediment. Bar = 2 km. 27
Adapted from the Squamish Estuary Management Plan, Habitat Work Group Final Report, 1981. Figure 18, Page 68. 28
fact that in the upper estuary, Fucus is confined to submerged logs within the river channels, and is not in direct association with the salt marsh vegetation. P. undulatum var. litorale was also recovered once from sediments at the
Pelly Point site in the Fraser River estuary (Figure 6).
Additional Oomycetes in Estuary Sediments
In addition to P. undulatum var. litorale, 20 additional oomycetous fungi were recovered from sediments or plant debris plated on PSM. Because of difficulties involved in identifying
Pythium spp., and the time that would be required to complete a thorough investigation of all isolates, only the most commonly occurring species were identified (Table 4). A brief description of the identified fungi is presented below. All
isolates were of Pythium spp.; no Phytophthora species were
recovered.. Pythium graclie and Pythium sp. 1 were the most commonly isolated species after P. undulatum var. litorale in
the Squamish estuary, but both were more frequently isolated in
the Fraser delta. Pythium sp. 1 strongly resembles P. monospermum , but lacks the sexual structures necessary for determining its taxonomy with certainty. Most species showed
equally good growth in both SDW and SSW (Table 4). Exceptions
were P. carolinianum and P. catenulatum which did not grow in
SSW, and P. butleri which grew much better in SSW than in SDW. 29
Figure 6: Sites sampled and recovery of Pythium undulatum var. litorale from the Fraser River estuary. o= sediment sample site not yielding P. undulatum var. 1itorale; • = site of recovery of P. undulatum var. 1itorale. Bar = 1/2 km. Adapted from Chart 3488 of the Canadian Hydrographic Service, Marine Sciences Branch, Dept. of the Environment, Ottawa. 31
Di scussion
This is not the first study to demonstrate Pythium spp. in brackish and seawater sediments. Over 18 species of Pythium were isolated by Hohnk (1939, 1953, 1956) and Siepman (1959) from salt marsh, mud flat, and bottom sediments. What is evident from their studies, and other more recent reports by
Scott (1962), Chowdery and Rai (1980), and this report, is that
Pythium species are conspicuous members of the sediment mycoflora. The majority of the Pythium species reported from marine localities are not endemic, being frequently recovered from terrestrial soils and fresh water. Hohnk, who had previously noted this in his study of the Weser estuary (1956), suggested that at least some species of Pythium are inhabitants of brackish waters, but concluded that their origin is probably other than marine. Of the species recovered in this study, 3 have been previously reported from estuarine sediments
(P. undulatum var. 1itorale, P. catenulatum , P. gracile ;
Hohnk, 1953, 1956; Scott,1962), while the other 4 are new reports of these fungi in estuarine sediments.
Phytophthora species were absent in samples from the
Squamish and Fraser estuaries, and were rarely reported by previous workers (Hohnk, 1956; Siepman, 1959). P. vesicula was
isolated earlier from several locations in adjacent Howe Sound
(Churchland and McClaren, 1973) and is reported here from decaying Fucus fronds in other locations in British Columbia.
Fell and Master (1975) demonstrated a unique assemblage of 32
Phytophthora species in mangrove swamps, but they employed direct baiting and not sediment plating. Schmit.thenne.r (1970) reported that Phytophthora species have not been successfully isolated from soils by use of most selective media, although these same media were useful in isolation from infected tissue.
PSM was useful in recovering Phytophthora from decaying Fucus fronds, but was probably inadequate for sediment isolation.Previous studies have shown that none of, the described selective media can be used to isolate all species of pythiaceous fungi (Hendrix and W.A. Campbell, l970;Hendrix and
C.W. Campbell, 1973; Lumsden et al.,1975), and it is likely that plating estuarine sediments on PSM did not result in a complete characterization of pythiaceous fungi in the estuary.
It should be emphasized that this represented only one sampling and thus can hardly be representitive of the total mycoflora of the estuary.
Although the data on distribution of P. undulatum var. 1itorale in sediments relative to infected Fucus plants are interesting, they do not imply a distinct correlation as the collecting techniques used do not allow for statistical analysis. It is possible that the high incidence of
P. undulatum var. litorale may have been due to production of zoospores from nearby infected plants. In partial support of
this idea is the observation that P. undulatum var. litorale was infrequently recovered (only once) from the Fraser delta,
suggesting that the presence of a suitable host plant in the
Squamish delta, may have helped increase the pathogen population 33
levels. On the other hand, recovery of the fungus high in the central delta, where Fucus. is confined to low logs, suggests that the fungus may be indigenous to the sediments in the Carex vegetation zone. Obviously a careful study involving
statistical analysis of year-round sampling is required before any definite correlations can be made. 34
PART 2 : PYTHIUM ISOLATES RECOVERED FROM FUCUS AND FROM
ESTUARINE SEDIMENTS
P y t h i u m undulatum var. litorale Hohnk
Isolation : From necrotic lesions of F. distichus growing in
the Squamish River estuary. Lesions occurring at the
most advanced dichotomies on the lateral margins of the
blades, or immediately in the crease of the most recent
branching (Figure 7). Lesions are firm, becoming
flaccid with age, pinkish-to-red in color, becoming
brown with drying of host plants, ranging in diameter
from a few mm to 1 1/2 cm. Methacrylate-embedded
sections of the infected tissue show that the radially-
advancing hyphae are restricted to the sub-cortex and
medullary regions of the algal thallus. Beyond the
intitial point of penetration, rarely were epidermal
cells observed to host fungal hyphae. Death of Fucus
duevto infection was not observed in field-collected
material; a hypersensitivity reaction appears to take
place, resulting in the abscission of the parasitized
portions. Herbarium specimens of infected plants are
deposited in the University of British Columbia
Herbarium, UBC. 35
Hyphae : Hyaline, smooth-walled, aseptate, becoming septate
with age, infrequently branched, 3.0-7.5 (x=4.5)n. In
water culture on hemp seed or grass blade, few
vegetative hyphae produced; most hyphae terminated by a
sporangium. When grown on Fucus thallus pieces or
sesame seed, abundant vegetative hyphae evident.
Zoosporangia : Numerous in water or agar culture, forming at 0,
15, and 27 o/oo seawater. Terminal, less frequently
terminal on a short lateral branch, rarely intercalary,
separated from sporangiophore by a septum.
Sporangiophore may be indistinguishable from vegetative
hyphae, or may be short and swollen. Sporangial form
highly variable: commonly multilobate, less frequenly
inflated-filamentous, rarely forming highly lobate
(toruloid) complexes (Figures 8-10), 84-150 (X=170.5) X
9.0-24.0 (x=20.0)/i, with lobes 9.0-31.5*/ in diameter.
Mature sporangia marked by 1-3 (usually 1) large
refractile globules in the zooplasm, with a distinct
refractile cap (Figure 11). Proliferation of the hyphae
through the old sporangia noted (Figure 12), although
formation of new sporangia by these proliferations
never observed. 36
Asexual Reproduction : Zoosporogenesis typical for the genus
(Figure 13), initiated by rinsing sporangia in a fresh
change of 0 or 15 o/oo culture water, rarely occurring
in 27 o/oo. Within a few minutes, evacuation of the
zooplasm begins with expansion of the refractile cap to
form the external vesicle, into which the
undifferentiated zooplasm passes. Flagella are the
first distinguishable features, sometimes visible
before the entire zooplasm has passed into the vesicle.
These begin to beat, setting the mass into a slow,
rocking motion. Cleavage furrows appear within one
min. after evacuation; individual zoospores are
recognizable by 5-6 min., and these undergo a period of
slow, sluggish movement, gradually increasing in
vigour. By 9-10 min., rupture of the vesicle occurs,
releasing 40-60 laterally biflagellate zoospores, 10.0-
12.5 (x=l1.5) X 6.0-9.0 (x = 7.5)»». Zoospores encyst
after a period of motility, and subsequently germinate
via germ tube. These may then form vegetative mycelia,
or may immediately produce zoosporangia (Figure 14).
Internal and intercalary chlamydospores formed both in
culture and internally in Fucus cells (Figure 15), 16-
23 (X=20)M in diameter. Germination of chlamydospores
not observed. 37
Plate I: Pythium undulatum var. litorale,
Figure 7: Lesions of F. distichus infected with P. undulatum var. litorale. Bar=15»/.
Figure 8: Lobate sporangium. Bar=15»i.
Figure 9; Inflated-filamentous sporangium. Bar=15p
Figure 10: Toruloid sporangium. Bar=15»i.
Figure 11: Mature sporangium showing refractile tip. Bar=15n.
Figure 12: Growth of hyphae through discharged sporangium. Bar = 20,1.
Figure 13: Zoospore formation. Bar = 20»».
Figure 14: Zoospore germinating to reform sporangium. Bar=20^.
Figure 15: Chlamydospores formed in cortical cells of Fucus, Bar=10M.
39
Sexual Reproduction : The formation of sexual organs was not
observed to occur under these culture conditions.
Discussion : When lesions of Fucus were excised and placed in
SDW or SSW, numerous lobate sporangia were produced within 48 h. While no two morphologically identical sporangia were ever observed, the lobate form shown in Figure 8 was the type most consistentlly seen. The inflated filamentous sporangia (Figure
9) occurred very early in the colonization of any substratum, but became rare as the culture aged. The toruloid forms (Figure
10) were rare under any circumstance, but formed most
frequently in sesame seed cultures after long periods of
incubation at 20 C.
Zoosporogenesis is typically pythiaceous, and proceeds
identically for all sporangial forms observed. In young, actively growing water-cultures, there appeared to be a high degree of synchrony with regards to zoosporogenesis. A single
change of the culture water initiated evacuation of the
zooplasm in many sporangia. This may not represent synchrony in
zoospores spore formation; rather a number of sporangia may
have existed in a state of physiological 'readiness', and the
change in medium simply initiated the process. However, it was
observed that a few sporangia were induced to evacuate before
maturity, as evidenced by their subsequent abortion, suggesting
that some factor is initiating evacuation of all zoosporangia
in the state of, or near the state of, reproductive maturity.
In any case, the numerous sporangia, and the copious quantities 40
of zoospores produced, make this species an ideal candidate for ultrast.ructural studies of zoosporogenesis.
Johnson and Sparrow (1961) questioned, and Waterhouse
(1967) rejected, Hohnk's acceptance of P. undulatum var. litorale as a valid variety, stating that it was not sufficiently different from Petersen's (1909) description of the species to warrant varietal status. However, careful comparison of Petersen's studies (1909,1910), and later reports of the species (Dissman,1927; Matthews,1931; Sparrow,1932;
Middleton,1943; Drechsler,1946; Goldie-Smith,1952) withHohnk's
(1953) description of the variety argue for the retention of var. litorale. P. undulatum has been defined as having narrow ellipsoidal sporangia with internal proliferation of the sporangium, and/or sympodial branching with production of chlamydospores. Matthews (1931), Sparrow (1932) and Goldie-
Smith (1952) all found that small ovoid or obpyriform sporangia were produced in culture. Dissman (1927) had previously carried out a number of experiments in solutions having different composition and concentrations of nutrients, and concluded that while the size of the sporangia may vary, the shape was constant. Sparrow (1932) noted that under 'foul' conditions the
sporangia assumed a variety of shapes, but he declined to define either the conditions or the resultant aberrant shapes.
No allowance had been made for either constrictions or lateral proliferations. Hohnk's contribution was to recognize that his
isolate showed sufficient similarities to be classified with
P. undulatum, but was unique in having the variable lobed 41
sporangial complexes. The variabli1ity in sporangial morphology in the British Columbia isolates highlights the desirability of maintaining var. litorale as a discrete entity within P. undulatum.
As in Hohnk's isolate (1953), the British Columbia fungus developed sporangia in a range of seawater dilutions from 0 to
27 o/oo, indicating that the fungus is well adapted to estuarine existance, where salinities are usually in constant
flux. However, the reduced ability of this fungus to produce zoospores in the higher salinities probably indicates that occurrence of the fungus would be rare or non-existent outside of estuaries. Sexual reproduction has not been reported in
P.. undulatum , and the findings of Dissman (1927) and this
study suggest that lack of sexual reproduction is not due to
inadequate nutrient levels, growth temperatures, or salinities.
The possibility that this fungus is heterothallic, as in
P. sylvaticum or P. heterothallicum (Hendrix and
Campbell,1973), was not explored in this study. 42
Pythium h o h n k i i nom nov.
= Pythiogeten utri forme Minden. Falk. Mykolog. Untersuch.
Berichter, 2(2):242, 1916
Isolation : From Fucus distichus L. growing in a small cove
facing the Sechelt Rapids , Skookumchuk Provincial
Park, British Columbia. Fucus tissue healthy.
Hyphae : Hyaline, smooth-walled, aseptate, becoming septate
with age, 1.5-5.0 (X=4.0M). Highly branched in water
cultures (Figure 16); as observed in agar
culture,hyphal growth form varied depending upon the
salinity of the agar medium. On SDW plates, highly
branched with frequent bulbous swellings and tortuous
hyphae; this condition rarely observed in the 15 o/oo
plates and never observed in the 27 o/oo plates. On SDW
and 15 o/oo plates, hyphae near aborted oogonia
observed to grow toward and wrap around the aborted
structure, resembling a ball of yarn (Figure 17) at
later stages. These hyphae appeared to be digesting the
aborted oogonium. 43
Zoosporangia : Always terminal, separated from the unbranched
sporangiophore by a septum. Sporangia, which form
readily in water culture at all three salinites, are
dark, thin-walled, and principally bursiform (Figure
18), but may be spherical, lobate or bilobate (Figures
19-20). Sporangia having a more-or less prolonged
discharge tube, the long axis of which may lie at right
angles to, or may be parallel to, the sporangiophore.
Main body of the sporangia 66.6-104.0 (x=81.5) X 35.0-
65.5 (x=48.5)n at the widest point of the lobe.
Emission tube 40.5-283.0 (x=55.5) X 11.5-21.5 (x=16.0)n
(Figure 21), hyaline, may be branched, but with only a
single functional exit pore. Mature sporangium marked
by a large refractile body (Figure 22) and a refractile
cap on the discharge tube.
Asexual Reproduction : Zoosporogenesis is intitiated by the
passage of undifferentiated zooplasm into an external
vesicle (Figure 23) that has formed from the refractile
cap of the discharge tube. This vesicle is persistent
throughout zoospore formation (Figure 24); at no time
was the vesicle observed to dissipate, nor was zoospore
cleavage 'free in the water' observed. Individual
zoospores are recognizable within 2 min. after passage
into the vesicle, but cleavage is not completed until
3-5 min. This is followed by a period of slow, sluggish 44
swimming, gradually increasing in vigour until zoospore
activity ruptures the vesicle wall, allowing the escape,
of 15-30 biflagellate zoospores, 10.5-14.0 (x=12.5) X
7.0-8.0 (x=7.5) f. Zoosporogenesis infrequent in SDW,
frequent in 15 o/oo, but not occurring in 27 o/oo SSW.
Sporangia are semi-persistant, internal proliferation
of sporangia not observed.
Sexual Reproduction : Antheridia strictly monoclinous, single
with multi-lobes applied at the base of the oogonium
(Figure 25). Two to five antheridial lobes (most
commonly 3-4), 8.0-36.0 (x=14.5) X .5.5-6.5 (x=6.0) »,
antheridiophore highly tortuous. Oogonia terminal,
smooth, hyaline (Figure 26). Oospore single, spherical,
plerotic, yellow-brown, having a thick outer wall 3.0-
5.5 (x=4.5)»i, and a large central oil vacuole. Oospores
31-67 (x=50.0) um in diameter. Sexual structures never
observed in water culture, but form readily on SchmA of
0 and 15 o/oo at all three test temperatures, although
the oogonial abortion frequency at 20 C was high (100%
in the 0 o/oo and 64% in the 15 o/oo plates), but low
in the 5 and 10 C plates (less than 1%). Oogonia not
forming at 27 o/oo. 45
Plate II. Pythium hohnki i.
Figure 16: Highly branched hyphae. Bar= 25M.
Figure 17: Hyphae digesting aborted oogonium. Bar= 100*/.
Figure 18: Bursiform sporangium typical of genus. Bar= 15//.
Figure 19: Bilobate sporangium. Bar= 10**.
Figure. 20: Spherical sporangium. Bar= 30M.
Figure 21: Elongate emission tube. This emission tube unusually long, but none-the-less functional. Bar= 50M.
Figure 22: Refractile globules in mature zoosporangium (arrows ) . Bar= 45M.
Figure* 23:- Zoosporogenesis, initiation*. Note intact vesicle (arrows) . Bar= 1 0M.
Figure 24: Zoosporogenesis, completion. Note vesicle still intact (arrows). Bar= 10M.
Figure 25: Oogonium with multi-lobed antheridium (arrows). Note the tortuous antheridiophore. Bar= 25M.
Figure 26: Mature oospore. Bar = 10M. 46 47
Etiology : Named for Dr. W. Hohnk, Inst, fur Meeresforschung,
Bremerhaven, BRD, who first recognized the importance
of the Pythiaceae in estuaries, and who first reported
the pythiaceous nature of zoospore formation in
Minden's Pythiogeten utriforme.
Holotype : University of British Columbia Herbarium, UBC,
slides numbered TT71380. Cultures deposited at ATCC,
CBS, and IFO.
Discussion : Although this is the first published description
of this fungus from western Canadian coastal waters, Anastasiou
(pers. comm.) recovered what he identified as Pythiogeten
utriforme growing on submerged leaves of Arbutus menziesii
Pursh. Hohnk (1939,1953) isolated P. utriforme from sandy beach
soil adjacent to brackish water, as well as from an open ocea'n
water sample. Newell (1973,1976) reports the recovery of
P. utriforme from mangrove seedlings in Florida. Species placed
in the genus Pythiogeten are generally considered to be
saprobes (Johnson and Sparrow, 1961), and it is likely that the
presence of this fungus on Fucus was incidental and not
pathogenic.
Lack of extant material of Minden's original type, or of
any subsequently collected material of Pythiogeten, makes
diagnosis and direct comparison difficult. Although the type of 48
zoosporangium, plerotic oospore, and monoclinous antheridium appear to ally the British Columbia isolate with Pythiogeten utriforme, several characteristics stand out in marked contrast at both the generic and specific levels.
The origin of the antheridia and their mode of application to the oogonia are in agreement with Minden (1916) and Sparrow
(1960), but the multi-lobed nature of the antheridium is unique to this isolate. Minden did allow for an antheridium bearing a short appendage, but his drawings, and Sparrow's subsequent drawings (1936), bear little resemblance to the antheridium of the British Columbia isolate. Hohnk (1939,1953) did not observe sexual reproduction, but he only observed water cultures, and oospores were not produced by my isolate, save on agar culture.
Lund (1934) observed sexual reproduction, but his
identification of P. utriforme is questionable as he described diclinous antheridia for his isolate, although the species as originally described is monoclinous.
To my knowledge, this is the first recorded isolation of a
Pythiogeten-like organism which reproduces sexually in pure culture. Pythiogeten spp. have been cultured only twice before
(Drechsler, 1932; Cantino,1949), although numerous authors have
recovered fungi they ascribed to the genus (Gaertner, 1954; Ito
and Nagai,l931; Shen and Siang, 1948; Sparrow,1932,1934,1952;
Wolf,1944; Newell,1973,1976). Most of these authors assigned
their isolates to Pythiogeten based on sporangial morphology,
and did not observe either zoospore or oospore formation.
Sparrow (1960) described oospore germination, but this process 49
was not observed in my isolate.
The British Columbia isolate lacks internal proliferation of sporangia, but this feature was also found lacking by
Sparrow (1960), Cantino (1949) or Hohnk (1939,1953), although
it was recorded by Drechsler (1932) for Pythiogeten autossytum and Lund (1934) for P. utriforme.
The major difference between Minden's Pythiogeten and my
isolate is the persistence of the vesicle during zoosporogenesis. The character upon which Minden erected the
genus Pythiogeten was a Pythium-like zoospore initiation, but with a quickly evanescent vesicle and maturation of the spores
occurring 'free' in the medium . Hohnk noticed this same
inconsistancy in his isolates of P. utriforme, but was of the
opinion that the vesicle in Pythium was conspicuously larger,
and that the vesicle in Pythiogeten was ephemeral and elastic.
The vesicle in the British Columbia fungus does not appear to
be different than that noted in general for Pythium species.
The vesicle is semi-persistant after spore release, something
which Hohnk also noticed.
Drechsler (1932) descibed the formation of zoospores
independent of a vesicle in Pythiogeten autossytum , but he too
noted frequent -completion of spore formation within an intact
vesicle. Cantino (1949) also describes "naked" maturation of
zoospores in an isolate tentatively identified as P. uniforme .
Wolf (1944) figured an elongated discharged zooplasm (see his
Figure 51), but failed to discuss zoosporogenesis. Sparrow
(1960) retained the genus in the second edition of Aquatic 50
Phycomycetes , but it is not clear from that edition or his earlier work (1936,1952.) if he actually observed zoospore
formation in this fungus.
Therefore, whether the vesicle membrane dissolves or not
during zoosporogenesseems a questionable generic character, in
view of the inconsistency of the published reports regarding
that particular trait. Much more work is required before one
could consider transferring all species of Pythiogeten to
Pythium, but I believe that the extant evidence warrants
transfer of Pythiogeten utriforme, to the genus Pythium.
Since transfer of Pythiogeten utriforme to Pythium
utriforme would create a later homonym of Pythium utriforme
Cornu (1872), despite the fact that Cornu's name was rejected
by Waterhouse (1967), a new epithet must be chosen on
transferring Pythiogeten utriforme to Pythium. I have chosen
Pythium hohnkii as the nomen nova for this taxon. 51
Brief Descriptions of Pythiacious fungi from Fraser and
Squamish Estuaries
Pythium butleri Subramanian: Hyphae sparse in SDW, vigorous in
SSW, branched, 2.5-6.0,/ wide. Sporangia consist of
toruloid outgrowths, zoosporangia producing 10-20
zoospores. Oogonia terminal or intercalary, 8-36,/ in
diameter. Antheridia strictly diclinous, 1-2 (usually
one). Aplerotic, 8.0-24.0,/ in diameter. Isolated from
three sites on central Squamish delta and once from
South Jetty, Fraser River.
Pythium carolinianum Matthews: Hyphae delicate, sparsely
branched with some septation, 2.0-3.0,/ in diameter.
Sporangia single or ocasionally forming a catenulate
chain, globose, rarely ellipsoidal, 18.0-30.0,/ in
diameter. Sporangia frequently with discharge tube,
although not consistent in this character, 2.0-5.0,/
wide by 15.0-21.On long. Sporangia frequently
germinating by germ tube to form mycelia. Sexual
reproduction not observed. Isolated from Squamish River
estuary only. 52
Pythium catenulatum Matthews: Mycelia highly branched, septate with age, 2.5-6.0*/. Sporangia composed of highly catenulate spherical elements, 10.0-15.0*/ in diameter, 20.0-60.0*/ long. Oogonia aplerotic, smooth, terminal, or less often intercalary, 18.0-30.0*/ in diameter. Antheridia monoclinous or diclinous, 4-10 per oogonium, antheridiophore sometimes helically involved with the oogonial stalk. Oospores 16.0-25.0*/ in diameter. Isolated from central Squamish delta and from Pelly Point, Fraser River.
Pythium gracile Schenk: Hyphae much branched, 2.0-4.0*/ in diameter, producing toruloid appressoria, 2.0-8.0*/ in diameter. Sporangia filamentous, indistinguishable from vegetative hyphae, producing 2-15 biflagellate spores. Oogonia terminal or intercalary, occasionally 3-4 catenulate oogonia formed on same branch, smooth- walled, 13.0-30.0*/. Antheridia single, rarely double, diclinous. Oospores aplerotic, single, smooth-walled, 9.0-26.0*/, having a thick outer wall (2.0*>), and a large centric to sub-centric oil vacuole. Isolated frequently from Squamish River estuary and Fraser estuary. An additional isolate was recovered from Carex debris at the Fraser River, that differed only in commonly having oospores formed in catenulate chains, and having mono- or diclinous antheridia. 53
Pythium torulosum Coker and Patterson: Hyphae sparsely
branched, 2.5-4.1% in diameter. Sporangia few, toruloid
with communicating elements, 5.0-8.0 X 5.0-16.0M,
mostly terminal, rarely intercalary. Oogonia mostly
terminal, terminal on short lateral branches, rarely
intercalary, 13.0-20.0* in diameter. Antheridia
monoclinous, 1-3, arising from the oogonial stalk.
Oogonia plerotic. Isolated from Fraser River, Pelly
Point, and South Jetty, and from the upper Squamish
Central delta.
Pythium volutum Vanterpool and Truscott: Hyphae sparsely
branched, 3.0-6.0M. Sporangiophore may be branched or
unbranched, bearing ovoid to lobate sporangia. Oogonia
terminal, rarely intercalary, smooth walled, 27.0-36.On
in diameter. Antheridia 1-7, diclinous, rarely
monoclinous, antheridiophore frequently helically
involved with the oogonial stalk. Oospores aplerotic,
25.0-30.0M. Isolated from Pelly Point and South Jetty
sediments, and from Carex debris at the Fraser River. 54
PART 3 : OBSERVATIONS ON THE INFECTION OF FUCUS DISTICHUS BY
PYTHIUM UNDULATUM VAR. LITORALE
General Morpholgy and Histology of Uninfected Fucus
distichus
The general anatomy of healthy F. distichus agrees with earlier descriptions of other. species in this, genus
(McCully,1965,1966). A mature vegetative thallus exhibits three distinct tissue regions (Figure 27): 1.) an epidermis composed of a single layer of columnar cells, capped to the outside by a heavy, TBO-staining cuticle, 2.) 2-4 layers of cortical cells, and 3.) a reticulum of filamentous cells in the center of the thallus embedded in a mucilaginous matrix, the medulla. McCully
(1966) distinguished the large, wide-lumen cells of the medulla as primary filaments, and the smaller, narrow-lumen cells as secondary filaments.
Observations on the histochemistry of the cell walls shows three layers of distinct reactivity. The outermost layer reacts to stains specific for sulfated polysaccharides; the wide, middle layer has affinity for stains specific to molecules with
2-vicinal hydroxyl groups; a thin, innermost layer that also demonstrates adjacent hydroxyl groups, but additionally reacts with the fluorescent marker Calcofluor-White (Table 5; Figures
28-31). These results suggest that the three layers are 55
Figure 27: Principle vegetative regions of F. distichus. Nomenclature of McCully (1966). TBO staining. 56 Table-5: Reactivity- of cell wall layers and matrix material with histological stains. Positive reaction, " +"; negative reaction, "-"
Pit connections Stain Outermost wall Middle wall Inner wall and cross walls Matrix
Acridine Orange + - - + Alcian Blue (pH=0.5) + - - + Calcofluor - + + -
IK2I-H2SOu - - -
+ + - PAS + + - + Saf-0 TBO (pH = 1.0r + + + + TBO (pH=4.4)' + + + + + Na^HCO^ extraction - + + - (Calcofluor)^ Na^HCO^ extraction - + + - (PAS ) CaCl^ extraction + + + - (TBO, pH=4.4)
1. Indicates stain used after extraction procedure. 58
Plate III. Anatomy of F. distichus.
Figure 28: Primary and secondary filaments showing outermost ring of fucans. ABB/Saf-0 staining; Bar = 5K.
Figure 29: Primar.y and. seconday filaments showing inner ring of alginic acid. Contrast with Figure 28. PAS/ABB staining; Bar = 15K.
Figure 30: Epidermal cells stained with TBO pH=4.4. Note heavy staining of cells walls at this pH (arrow). Bar = 25K.
Figure 31: Epidermal cells stained with TBO pH=1.0. Note at this pH, the cell walls do not stain (arrows). Bar = 20*..
Figure 32: Phenolic-reactive material, within, medulla- matrix. TBO staining; Bar = 30K.
Figure 33: Erlichs reagent-reactive material in the epidermal and cortical regions. Staining proceeded embedding. Bar = 100K.
Figure 34: Cytoplasmic features of Fucus cells . Note the basipetal location of nuclei in epidermal cells (arrow), the centrally located nuclei (N), and the numerous physodes (P). ABB/Saf-0 staining; Bar = 15K.
60
composed of fucans1, alginic acid, and cellulose respectively.
Calcofluor staining also demonstrated strong fluorescence in
the pit-connections between epidermal cells, a fluorescence
that was contiguous with the thin, inner cell wall layer.
Furthermore, cross walls between primary medullary cells were
found to be strongly PAS-positive and to fluoresce with
Calcofluor. Secondary cell walls were found to be PAS-positive
after alkaline extraction and to fluoresce with Calcofluor.
Although these results strongly suggest the presence of
cellulose, some caution should be noted since the standard test
for cellulose, IK2I with H2SO„, failed to show any reactivity
in methacrylate-embedded sections, and the same test applied to
fresh material showed only very faint reactivity in secondary
cells and cross walls, with, no reactivity in the pit
connection.
Staining with TBO revealed the matrix material to be
largely fucans, as revealed by the red-purple metachromasia,
but also demonstrated the presence of large areas of a
turquoise-blue staining material (Figure 36). This reactivity
only appeared in material fixed with 1% caffine in the
fixative. Fresh sections stained with phenolic-indicating
1. A chemically distinct fucan sulfate (fucoidin) is not believed to occur in mature vegetative Phaeophytes. Rather, there is a family of inter-related glycans rich in sulfated L- fucose (McCandless and Cragie, 1979). For convenience, the term "fucans" in this thesis will refer to this group of sulfated polysaccharides. 61
reagents revealed areas of positive reactivity that
corresponded to the TBO-positive regions. (Table 6). Vanillin
HCl, Erlich's Reagent, and Bordeaux Salt were particularly
useful in demonstrating positive reactivity in the epidermal
region (Figure 37), but Vanillin HCl had limited use as it
quickly macerated tissue beyond recognition. Results of these
tests are taken to indicate the presence of phenolics in the
matrix, but some anomalies were noted. First, unlike results
originally reported by McCully (1966), authentic phloroglucinol
was not found to react metachromatically with TBO in vitro.
Secondly, phenolic reagents did not show reactivity when
applied to embedded-tissue; only freshly sectioned material was
effectively stained.
Internally, both epidermal and parenchyma cells of Fucus
are highly vacuolate. In the epidermis, the nuclei are basally
located with cytoplasmic strands running from the nucleus to
the plastids, located on the lateral walls (Figure 38). In
parenchyma and filament cells, nuclei are located more-or-less
centrally and are connected to the peripherally located
plastids by transvacuolar strands. Filament cells are
internally similar to parenchyma cells, except that they are
largely devoid of plastids and vacuoles. Epidermal nuclei
failed to stain metachromatically with TBO, as had been
reported for other species (McCully, 1966). However, nuclei and
plastids did react with Erlich's reagent and FeCl3, indicating
the possible presence of phenolic materials.
Vacuoles were principally of two types: small (less than Table-6: Reactions of Fucus matrix and cellular elements with phenolic-indicating reagents. All reagents first tested against authentic phloroglucinol,
and then against both fresh and fixed-embedded Fucus material. Color reactions within plant that were similar to in vitro reaction of
phloroglucinol indicated by "++•"; lesser reactions by "++" or no reaction by not tested by "o".
FKKSH FIXED-EMBEDDED
TEST REAGENT PHLOROGLUCINOL MATRIX PHYSODES NUCLEI-PLASTIDS MATRIX PHYSODES MJCLEI-PLASTIDS
Bordeaux rgnt. orange-brown —
Erlich a rgnt. pink + - -
FeCl3 grey-black - - - black - - 0 0 o 2 TBO, ph U.U blue 1 - - - • •
Vanillin HCl orange - - +++ GMS not tested 0 0 0 -
1. McCully (1966, 1970) reported green metachromasia of TBO with phloroglucinol. Concentrations ranging from 0.05 to 1.0% in aqueous or alchohol solutions, or in phosphate or cacodylate buffers, failed to induce uietachromusiu In vitro.
2. Color development by physode3 with this stain varied with length of staining: less than 30 seconds, no stain; 1-2 minuteB, turquiose-blue;
greater than 3 minutes, dark blue. 63
1 it) vesicles staining dark blue with TBO and red with Saf-O, and large (4-6^) vesicles that were colorless in live material, but yellow in fixed and embedded material (Figure 38). These larger vesicles correspond with the so-called "physodes", or
"fucosan-bodies", so frequently mentioned in the brown algal literature (McCully, 1966,1970; Evans and Holligan, 1972b;
Ragan, 1976; Ragan and Cragie, 1976). These were found to occupy most of the cell space in the epidermis and parenchyma.
While the smaller bodies were found in all cell types, the physodes were rare in primary and secondary medullary filaments.
Physodes are reported to contain phenolic materials, but histological tests employed in this study provided a confusing array of results. The standard stain test for phenolics in
Fucus physodes, TBO (McCully,1970), stained these bodies differentially, depending upon the length of staining time. If methacrylate sections were stained with just sufficient time to metachromatically differentiate the sulfated matrix (about 30s) the physodes were unstained and retained their yellow-color. If left for a period of 1-2 min., they began to take on a turquoise-green color, and beyond 3 min., were stained dark blue. Physodes in fresh material were unstained by TBO after 24 h., stained reddish-orange after 1 h. exposure to Vanillin
HCl, brown with Bordeaux Salt after 1 h., but failed to react
with FeCl3 or Erlich's reagent. As before, none of these reagents had any effect on embedded tissue. An unexpected result occurred when using Grocott's Silver-methanamine stain 64
to differentiate fungi in Fucus tissues. Physodes stained dark black with this stain, while no other cellular components stained at all.
Host-parasite interface : Natural infect ions.
Macroscopically, initial lesions on F. distichus appear
initially as pink to red necrotic regions confined to the wings of distal tips, usually at or near the point of the first dichotomy (Figure 7). Lesions were rarely observed at the apices or on older mid-rib tissue. With age of infection, the necrotic region becomes flaccid, and a cleared tissue band develops at the lesion perimeter. This zone acts as an abscission layer dropping the infected portion from the plant.
Lesions rarely spread beyond this zone; death of the plant due
to infection could never be verified in field material.
Infection was probably initiated by penetration of
zoospore germ tube directly through the epidermal cells,
although this process was never observed in methacrylate
sections. Appresoria were not observed. Relatively few
epidermal cells were invaded; beyond the initial point of
infection, rarely were the hyphae ever observed in epidermal
cells and never were hyphae seen to grow on the algal surface.
Infecting hyphae spread radially from the point of infection
and are confined to the parenchyma and medullary regions
(Figure 35). 65
Parasitism of individual host cells is inititated by what appears to be an enzyme-mediated digestion of the alginic acid portion of the host cell wall. In Fucus, the algin portion of the cell wall stains pink in the PAS reaction (Figure 29), but in infected cells, at the point where the hyphae cross the cell wall, there is a large discontinuity in the cell wall staining
(Figure 37-38). There is no evidence to suggest that the fucan components of the cell wall or matrix are also dissolved.
However, there is some indication that the cellulose component of the wall is digested. Hyphae are frequently observed to grow between two cells through the former pit connection, which now shows no staining affinity for either PAS or Calcofluor (Figure
36). There is also the suggestion that while physically occupying one cell the fungus may be capable of parasitizing three or four adjacent cells via digestion of the pit connection. Figure 39 shows several cells with both contiguous collapsed cytoplasm through the former pit connection and areas in which the pit connections show lighter staining. Hyphal constrictions or appressorial formation, commonly observed in fungi having mechanical (pressure) mediated penetration, were never observed. Haustoria are not formed in host cells.
The exact sequence of events in the digestion of host cells is still obscure, as intermediates of degradation were not observed. Occasionaly, a remnant nucleus is observed in parasitized cells, suggesting that this is the last structure degraded. Infected cells have collapsed cytoplasm and, histologically, cell contents stain as an amorphous mixture of 66
protein, polysaccharides, and phenolic materials.
In older infected regions, the structural integrity of the thallus is lost; cells of the epidermis begin to slough off, and principally rod-shaped bacteria are evident (Figure 40). In material collected and immediately fixed, no zoosporangia were ever observed. If allowed to incubate 24-48 h. in seawater, numerous sporangia are formed from the medulla by the pushing up of sporangiophores between epidermal cells. Thick-walled chlamydospores are often formed in cortical and medullary cells
(Figure 15).
The response by Fucus to the invasion is an active one: a hypersensitivity response (HR) occurs in advance of the fungal hyphae, and here the sequence of events is clearer. The process initiates in the cortical and medullary cells, but extends outward to include epidermal cells. Plastids and cytoplasm are the first features to be disrupted. The nucleus shows a decreasingly intensive reaction with ABB, becoming swollen and distended until it is apparently disbanded. The physodes are the last distinct structures, but these too dissipate and the remnant contents of the cell stain intensely green with TBO.
This green staining material is not evident in more advanced infections, and in the same region a lack of matrix material is noted; stress and tear line begin to appear between cells. Cell walls have a disjointed, fibrillar appearance and histologically stain only for alginic acid and cellulose.
Beyond the developing hypersensitivity region is an area of intense metabolic and meristematic activity. A significant 67
Plate IV: Parasitism of F. distichus by P. undulatum var. litorale.
Figure 35: General parasitism of epidermal and cortical cells. PAS-ABB staining; Bar = 20M.
Figure 36: Hyphae growing through former pit connection. PAS-ABB staining; Bar = 5M.
Figure 37: PAS non-staining region at point where hyphae cross cell wall (arrows). PAS-ABB staining; Bar = 10M.
Figure ,38: PAS halo in medullary filament (arrows). PAS-ABB staining; Bar = 10M.
Figure 39: Collapsed pit connections between cortical cells. F = fungal hyphae; P = collapsed pit connection. Note that the cytoplasm is now contiguous between cells PAS- ABB staining; Bar = 10M.
Figure 40: Bacteria invading necrotic tissue in late stages of disease. PAS-ABB staining; Bar = 10M. 68 69
increase in ABB-reactive material in the normally quiescent primary and secondary filaments indicates a higher level of proteinaceous material. Concurrent with this is the appearance of numerous physodes, normally rare or absent in medullary elements (Figure 41). In some cases, these cells become so packed with vesicles that other cell elements are obscured. A distinct polarization of physodes occurs, followed by coalescence of individual vesicles to form one large unit
(Figures 42-43). These 'giant' vesicles are subsequently delimited from the producing cell by an irregular PAS-positive wall (Figure 43). These cells contribute to the developing HR; autolysis occurs and the cells stain brilliant green with TBO.
A significant level of phenolic-staining material appears in the matrix (Figure 44), but at the same time the matrix fucans show a decreasing metachromasia with TBO, and stress lines begin to appear between cells. The sequence of events in this region follows that of the early HR: disappearance of phenolic- staining material and of fucan matrix with a disjointed, fibrillar appearance of the cell walls.
In the final stages of the infection process, cortical and medullary filaments undergo transverse and longituidinal divisions to form a 1-2 cell protection layer (Figure 46), which functions as epidermis after abscission has occurred.
These cells have numerous physodes, but none of the 'giant' physodes are evident. The hypersensitive.region now marks a well-defined abscission zone (Figure 45) by which the infected portion is dropped out of the Fucus thallus, terminating the 70
Plate V: Defense reaction of F. distichus to infection .
Figure 41: Low magnification of defense reaction in medulla. Note increased levels of ABB-staining ma'terial and formation of large, polarized physodes (arrows). PAS- ABB staining; Bar = 30M.
Figure 42: Primary filament showing formation of giant physodes from coalescence of smaller vesicles (arrows). PAS-ABB staining; Bar = 15M.
Figure 43: High magnification of giant physode. Note PAS- positive wall seperating 'giant' physode from delimiting cell. PAS-ABB staining; Bar = 10M.
Figure 44: Increase in phenolic materials in the matrix of the hypersensitive region. Reactive areas indicated by arrows. TBO, pH=4.4, staining; Bar = 25M.
Figure 45: Abscission zone (arrow). Note the absence of the dark-staining fucan matrix in this region. TBO, pH=4.4; Bar = 20M.
Figure 46: Irregular, cubiodal cells differentiated from medullary filaments. These cells function as epidermis after abscission has occurred. TBO, pH=4.4; Bar = 10M.
72
infection.
Laboratory Infection Studies
Fucus plants transported to the laboratory from the
Squamish River estuary adapted well to lab conditions. Very few of the plants developed necrosis due to the new culture conditions, and in many plants new apical growth was evident after the 30 day incubation period.
Within 2-3 d of application of innoculum, numerous small
(1-2mm), brick-red lesions appeared on the algal fronds (Figure
48) principally at the apices. By 5-7 days, all plants in the tank showed infection. Healthy, uninnoculated plants in holding tanks did not develop lesions, indicating that the necrosis observed was due to infection., and not to culture conditions.
Unlike field-infections, lesions spread outward, coalesced and rapidly decayed the apical tips- of infected plants. Coupled- with decay, a yellow-brown substance was released into the culture vessel that, when concentrated by roto-evaporation, gave positive reactions with phenolic indicators. Brown exudates did not accumulate in tanks with healthy, un• innoculated plants. Infections spread rapidly into older tissues, and by 10-15 d most plants had additional lesions develop on the lateral wings. A distinctive abscission zone was not observed to develop under these culture conditions. By the end of the 30 d culture period, most plants were decayed beyond 73
recognition. Excision of infected zones and plating on PSM recovered P. undulatum var. litorale.
In thin sections, a great reduction in the physode content
is noted after the pre-innoculation incubation period (Figure
47). The host appears to have no resistance mechanism to the
fungal attack. A higher density of fungal filaments is noted in the medulla (Figure 49), where the infection seems to be mainly confined. Occasionally, a 'giant' physode is seen to form
(Figure 50), but its occurrence is sporadic and does not seem
to be associated with any concerted defense reaction by the plant.
Discussion
While the findings of this study parallel previous
findings of Fucus and brown algae in general, a few new
observations were' made concerning- the morphology of healthy
vegetative Fucus distichus in British Columbia. One addition is
the histochemical localization of cellulose in the mature
thallus by means of Calcofluor-white and the PAS reaction.
Cellulose is known to occur in small quantities in brown algae
(Percival and McDowell, 1967) and has a demonstrated prominent
role in cell wall development in several species of Fucus
(Novotny and Forman,l975; Quatrano and Stevens,1976). McCully
(1970) reported a weak reaction with IK2I-H2SO„ and a positive
PAS reaction after alkaline extraction in the walls of 74
Plate VI: Laboratory infection.
Figure 47: Reduction in physode content after 30 day incubation period. ABB/Saf-0 staining; Bar = 10M.
Figure 48: Laboratory lesions on Fucus. !print.le(.f 49) Figure 49: Low magnification of laboratory infection. F = fungal hyphae. ABB/Saf-0 staining; Bar = 30*..
Figure 50: Giant physode in lab infection. ABB/Saf-O staining; Bar = 10*>. 75 76
secondary fibres of Fucus. Positive reactivity with Calcofluor of the secondary filament walls before and after the alkaline extraction used in this study provides additional evidence for cellulose in these walls. Furthermore, similar reactivity in the cross walls of primary filaments and in the pit connections between epidermal and cortical cells suggests a higher cellulose content than previously considered for these regions.
The possibility does exist that Calcofluor-White may be binding to alginic acid, as > this stain is thought to have affinity for Beta 1-3* and Beta 1-4 linked mannuronic and guluronic units (Maeda and Ishida,l976; Takeuchi and
Komamine,1978). In light of the ambiguous reaction with the
IK2I-H2SOa stain, this factor must be considered. However, alkaline extraction removed the algin, as indicated by PAS staining, but left the Calcofluor-positive regions intact.
The presence of phenolic materials in the matrix of Fucus has not been noted previously, and this may in part be due to the apparent differences in stain affinity between fresh and fixed-embedded material. Phluroglucinol and its derivatives are alcohol and water soluble (Cragie and McLachlan,1964; Ragan and
Cragie,1976), and are probably removed during fixation and dehydration procedures. Inclusion of 1% caffeine in the fixative was found to be an effective means of preserving the phenolics for histochemical differentiation, although the technique was not completely efficient as the amount of material staining in the cortical region with Erlich's reagent applied to fresh material is not observed in TBO staining. 77
Excellent preservation of physodes in vegetative tissue was obtained with the gluteraldehyde-Hepes buffer seawater fixation. This is in contrast to earlier reports where physodes in mature epidermal and medullary cells of Dictyota were not maintained after gluteraldehyde or acrolein fixation (Evans and
Holligan,1972b). This lack of physode preservation in acrolein- fixed tissue may account for the differences in physode content of epidermal cells of F. distichus in this report and those figured.by McCully for F. vesiculosus and F. edentatus , since she used acrolein fixation for her material (1966, see
McCully's Figures 1B and 5, and contrast with Figure 38 of this report). The reason for the development of a yellow-color in the physodes in JB-4 methacrylate is unknown, but presumably must be related to the embedding procedure as tissues examined immediately after fixation were not so colored.
The physodes reported here correspond to the various green, TBO-staining bodies reported by McCully (1966), although the staining anomalies with TBO were noted. Evan and Holligan
(1972) noted that physodes in mature tissue of Dictyota stained dark blue with TBO, and suggested that differences in color reaction (green to blue) were due to either different levels or different types of phenolic compounds in physodes. Because of the failure of TBO to react metachromatically with authentic phloroglucinol in_ vitro , green color developed by physodes in the present study is believed to due to the yellow color imparted to the physodes during embedding in combination with the blue color of the TBO dye. Of the four other types of 78
vacuoles or inclusions distinguished with TBO staining in
McCully's report (1966), only the small, deep-staining granules were observed in F. distichus. These differences may be due to differences in fixations, or may reflect genuine differences in sub-cellular bodies betwen these species, or between physical environments (Atlantic vs. Pacific oceans).
Despite the irregularities of staining encountered, evidence presented here and elsewhere strongly argue for the presence of polyphenolics in physodes (Evans and
Holligan,1972b; Ragan,1976). As blue or green TBO-staining areas correspond to materials stained by the other phenolic reagents, these areas are believed to contain phenolics. Why
Erlich's Reagents and FeCl3 failed to react in physodes, while
Vanillin HCl, Fast Bordeaux Salt, and 0s04 did, is not clear.
Presumably there are other cytoplasmic elements which interfere in color development with these reagents. Why Grocott's silver- methanamine stain also reacted with physodes in plastic section is unknown, but it is suggested that it has to do with the silver ion binding with the phenol, as silver nitrate is often used as a phenolic indicator in biochemical thin-layer chromatography (Cragie and McLachlan,1964). The failure of the other reagents to react with physodes or matrix phenolics in methacrylate-embeded sections clearly demonstrates the need for caution when interpreting histochemistry solely from staining properties of embedded materials.
Figure 51 summarizes the infection process. Zoospores initiate the infection by penetation through the epidermal 79
Figure 51: Diagramatic summation of events during pathogenesis. A. Pathogenesis 1. Biflagellate zoospore as primary inoculum. 2. Zoospore encysts, and then penetrates the epidermis by means of direct hyphal penetration. 3. Hyphae grow mainly in the cortical and medullary regions. Penetration of individual cells may be in part due to an enzymatic digestion of the alginic acid and cellulose portions of the cell wall (3'). Pit connections are dissolved between cells and the fungus appears to be capable of digesting the contents of several cells while physically occupying only one (3"). 4. Lesions on Fucus become visible, are pink-to- red in color, and are initially firm, but become flaccid with age. 5. Thick-walled chlamydospores formed inside cortical and medullary filaments. 6. Sporangiophores push up between epidermal cells, and form the lobate sporaangia of P. undulatum var. litorale, reproducing the primary inoculum. B. Host Response 1. Hypersensitivity of host cells in advance of fungal hyphae. Medullary filaments show an increase in general protein levels, coupled with the appearance- of numerous physodes, normally rare or absent in this region. Physodes coalesce to form larger units, which are then believed to autolyse and release their phenolic materials into the matrix. Stress and tear lines appear between cells of this region due to the disapearance of fucan matrix. Eventually, this region acts as an abscission zone. 2. Medullary filaments divide transversely to form irregular, cuboidal cells that function as epidermis once abscission has occurred. 80 81
cells. Hyphae advance radially and are mainly confined to the cortical and medullary cells. Penetration of individual cells is initiated by what apears to be an enzymatic dissolution of the alginic acid and cellulose components of the cell walls.
Haustoria or modified absorption structures are absent. By utilizing its ability to digest cell walls, the fungus is capable of parasitizing several cells via dissolution of the pits, although the fungal filament may physically occupy only one cell. Lobate sporangia, are formed outside the thallus; these release 30-60 zoospores which recycle the infecton.
Thick-walled chlamydospores are formed in cortical and medullary cells, but the function of these is unknown.
A hypersensitivity response (HR) occurs in advance of the fungal hyphae. In the first stage, all cell types are involved and rapidly necrose with remnant cell contents staining for phenolic materials, coupled with a disappearance of matrix material. The second stage of the HR is characterized by an increase in metabolic activity, as evidenced by increased level of general protein staining and the apparent production of numerous physodes in the normally quiescent medullary filaments. These physodes coalesce to form 'giant' physodes, and autolysis occurs in these cells, which then stain heavily for amorphous phenolic materials. Heavy deposits of phenolic- reactive material appear in the matrix, while staining of the matrix fucans is lost. In the later stages of HR, both fucan matrix and phenolic materials- have disapeared, HR cell walls have a fibrous distended appearance and become separated from 82
each other. This region now marks a well-defined abscission zone by which the infected portions are isolated from healthy tissue and dropped from the plant.
Immediately behind the abscission zone, cortical and medullary filaments have undergone principally transverse divisions to form irregular, cuboidal cells that function as epidermal cells once abscission has occurred.
Pentration of Fucus epidermal cells takes place without the formation of either appressoria or penetration pegs. In
Pythium, hyphal penetration of host tissue may or may not involve appressorial formation (Aist,l976), depending on the species and host. Penetration of bentgrass by P.aphanidermaturn
(Kraft et a_l.,l967) and of Ceramium rubrum by P. marinum
(Sparrow,1934) involve appressoria; infection by P. marinum of
Porphyra perforata (Kazama and Fuller, 1970) or of Phycomyces blakesleeanus by Pythium acanthium (Hoch and Fuller, 1977) does not.
The gap in PAS cell wall staining where hyphae cross cell walls has been interpretted in this study to be as a result of chemical dissolution of the cell wall during penetration.
Similar non-staining cell wall 'halos' have been noted in penetration of barley epidermal cells by Erysiphe graminis at both the light and electron microscope levels (McKeen e_t al. ,
1969; Edwards and Allen,1970). Kazama (1969) felt that penetration of Porphyra cell walls by Pythium mar inum is by mechanical pressure and not by chemical breaching, while
Spencer and Cooper (1967) argue that penetration of cotton 83
roots by Pythium species is accomplished by mechanical means.
In the absence of correlated electron microscopic studies, this report can only suggest that enzymatic digestion of Fucus cell walls is occurring. Enzymes which participate in cell wall degradation are known to occur in a wide variety of plant pathogens, including Pythium species (Bateman and Basham,
1976), although most of the work to date has centered on degradation of the pectic and cellulosic fractions. Degradation of fucans and alginates has been studied in marine bacteria
(Chesters et a_l.,l954'; Yaphe and Morgan, 1959; Preis and
Ashwell,1962; Lynn et al.,1968), but only recently has alginate lyase activity been adequately demonstrated in a fungus
(Wainwright,1980). Based on the histological results,
P. undulatum var. litorale might be an ideal candidate for further examination of alginase activity in the fungi.
The hypersensitivity and abscission response to infection seen in Fucus has not been reported previously for any marine alga. Fuller, et a_l. (1966) reported that in naturally infected
Porphyra perforata , host cells had altered pigmentation in advance of infecting hyphae of Pythium marinum . However,
Kazama and Fuller (1970) found that in artificially infected
Porphyra plants this reaction was never observed. Necrosis in advance of Pythium porphyrae infecting other Porphyra species has never been observed (Fujita,pers. comm.).
Muller (1959) defined the HR as encompassing all morphological and histological changes that, when produced by an infection agent, elicit the premature dying off (necrosis) 84
of the infected tissue as well as inactivation and localization of the infectious agent. Certainly the necrosis observed distal to hyphal advance, the histological changes in phenol levels in both cortex and medulla, and the meristimatic activity producing a new epidermis by Fucus in response to infection, fall within this definition.
The HR response of Fucus is similar to the reponse described as the 'shot-hole' syndrome in plant pathological literature. As exemplified by the response of Prunus spp. to infection with Cladosporium carpophilum, spread of the pathogen is halted relatively late, a hypersensitivity necrosis develops, and meristematic activity is induced distal to the necrosis bringing about the development of a concentric periderm for the seperation of necrotic tissue (Muller,1959).
However, in this HR, abscission is induced by meristematic formation of an abscission layer (Akai,l959), while in Fucus the abscission appears to be brought about by autolysis of pre• existing cells and autodigestion of matrix fucans in the necrotic region, with the possibility of enzymatic degradation of cell walls. Alginate lyase activity has been demonstrated for several brown algal genera (Madgwick e_t al.,1973; Shiraiwa et al.,1975), and it is likely that the same kinds of enzymes are active in the Fucus HR. Walker (1980) found that sorus release in Nereocystis lutkeana involved dissolution of matrix material, middle lamellae, and a spreading of the fibrillar elements in the cell wall. Dissolution of middle lamellae is accepted as a crucial aspect of abscission in higher plants 85
(Addicott,1965; Abies,1969; Esau, 1977).
A further analogy between the Fucus HR and higher plant HR is the occurrence of phenolic-reactive compounds in the hypersensitized cells, and in the increased production of these phenyl compounds. Higher plants contain phenylated compounds that function as non-specific enzyme-inhibitors (Bateman and
Millar,1966) and the occurrence of enzyme inhibitors has been correlated with resistance to pathogenic organisms (Bateman and
Basham, 1976). The role of polyphenolics in brown algae is believed to be as a pre-formed biochemical defense agent against bacteria (Conover and Sieburth,1964), fungi (Khaleafa et al.,1975), epiflora (McLachlan and Cragie,1964,1966) and epifauna (Conover and Sieburth,1965; Sieburth and
Conover,1965). Phenols have been shown to inactivate secreted fungal enzymes (Mukherjee and Kundu,l973), and it is possible that the phenolic buildup in the HR of Fucus may serve to inactivate the fungal enzymes, checking the spread of hyphae.
The final step in the hypersensitivity response is formation of the new epidermis. Divisions in the cortical and medullary cells proceed in a similar fashion to that described for wound response in other species of Fucus (Fulcher and
McCully,1969) and other brown algae (Fagerberg and Dawes,1976;
Walker,1980), although higher levels of physodes are noted in the defense reaction cells of Fucus. The formation of this epidermal defense layer not only functions in preventing the injurious effect of a secondary infection of even weakly pathogenic fungi or bacteria, but also prevents further loss of 86
cellular and extra-cellular materials.
The Kohlmeyers (1979) have noted that difficulties
encountered in growing host plants in culture have limited data accumulation on pathogenicity of algicolous fungi. Certainly
their observations are applicable in the laboratory infection
studies reported here. Although infection of Fucus and
subsequent recovery of P. undulatum var. litorale proved
pathogenicity, failure of the plants to develop an HR and
abscission zone layer prevented unequivocal demonstration of
Koch's Postulates. Physiological parameters (light,
temperature, mineral salts, etc.) have an important influence
on HR in higher plants (Addicott,1965), and it is likely that
environmental physiology is equally important for the HR in
Fucus. While uninnoculated plants appeared healthy throughout
the course of this study, the devastating effect of infection
in vitro indicates that conditions were probably less than
optimal. In support of this is the reduction of physode content
during the 30 day pre-infection incubation period. 87
CONCLUSION
While any definite conclusions, based on the limited data of this thesis, are not justified, a few observations regarding the work as a whole are warranted.
The absence of Fucus in the Squamish River estuary prior to dike construction, combined with, the suggestion that Pythium undulatum var. litorale is an indigenous member of the estuarine sediment mycoflora, and with the absence of infection outside the estuary, suggest that a unique set of physiographical parameters exist within the estuary that allow this symbiosis to occur. The most probable physiological element to examine is the change in salinity patterns within the estuary after dike construction. Penetration of Fucus into the estuary, up to the blocked channels of the river, demonstrates the. drastic alterations, in salinity that occurred with river training. However, it is likely that the estuary is a marginal habitat for Fucus, as salinities are diluted by the
freshwater outflow from the Squamish River. In the dilute environment, metabolism in Fucus may be sufficiently stressed so as to cause a reduction in levels of biochemical defense agents capable of preventing infection.
P. undulatum var. litorale is most likely saline-
influenced as to its distribution. Both vegetative growth and asexual reproduction were inhibited at higher salinities in 88
laboratory cultures. Not finding P. undulatum var. litorale outside of the estuarine environment further suggests that the fungus is saline limited.
Thus, within the Squamish River estuary, a classical triangular interaction of host, parasite, and environment seems to exist. If salinity is the factor to be examined, one could expect that during winter, when salinities are higher in the estuary, the incidence of disease would be reduced, whereas during the spring freshet of the Squamish River salinities would be reduced and disease incidence would increase. Monthly sampling of salinities and infected plans could provide some interesting answers regarding the influence of salinity on disease development. Laboratory examination of infection under standardized conditions (light, temperature, nutrients, etc.), while varying salinity, would also be useful.
The observations made on the infection process are limited in the abscence of correlated electron microscope data.
Additional data on cell wall dissolution by hyphae, formation of the 'giant' physodes, and all factors relating to development of the hypersensitive reaction are necessary to support the interpretations given in this thesis. Finding a laboratory system that allows development of the hypersensitive reaction, so that the infection can be observed sequentially, is also necessary to gain a successful understanding of these processes.
Despite the limitations placed on the data- provided by this thesis, sone useful.contributions were made toward our 89
knowledge of pythiaceous fungi in marine habitats, and toward the methods in which algae react to infection. This study notes that the infection of Fucus is the first record of infection by a species of Pythium on a phaeophyte. It notes that members of the Pythiaceae are common in estuarine sediments and supports
Hohnk's (1956) contention that the origin of these species is terrestrial. Taxonomic interpretations are made regarding correct placement of P. undulatum var. litorale and transfer is made of Pythiogeten utri forme to Pythium hohnki i. The observation of the hypersensitive reaction of F. distichus is a new observation for this process in marine algae. Previously this process was known to occur only in higher plants. 90
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APPENDIX-1: FUNGAL AND ALGAL MEDIA EMPLOYED
I . Fungal Media (Dr. Y. Fujita, pers. comm.)
Fuj ita's Seawater Agar
Materials
fresh corn kernels 20 gm
distilled water 500' ml
seawater 500 ml
agar 20 gm
Procedure
Boil corn in DW for 30 m. Filter, add seawater. Bring
final volume to 1 liter with DW. . Adjust the pH to 7.3-
7.5, add agar, autoclave.
Comments
Dr. Fujita has found this medium to be ideal for the
production of sexual characters in Pythium porphyrae
Takahashi et Sasaki. I found the medium to be adequate
for vegetative growth of pythiaceous fungi in this study,
but did not support the same level of sexual reproduction
as Schmitthener's agar.
Gallic Ac id Medium (Flowers and Hendrix, 1969)
Materials
sucrose 30.0 gm 1 03
NaNO3 2.0
MgSO, 7H20 0.5
KH2POa 1.0
yeast extract 0.5
gallic acid 425 mg
rose bengal 0.5 mg
pentachloronitrobenzene (PCNB) 25.0 mg
Penicillin G 80,000 units
nystatin 100,000 units
agar 20 gm
distilled water 1 1
Comments
Gallic acid, rose bengal, PCNB, Pen-G and nystatin were
added aseptically to the autoclaved, cooled (58-60 C)
nutrient agar.
Schmitthener's Agar (Robertson, 1980)
Mater ials
sucrose 2.5 gm
asparagine 0.27
KH2PO„ 0.15
K2HPO„ 0.15
MgSC 7H20 0. 1
ergosterol 0.01
agar 15.0
water 1 1
pH adjusted to 7.3-7.5 104
Comments
Ergosterol is insoluble in water, but can be dissolved in
95% EtOH.
PSM
Materials
Benomyl (as Benlate, DuPont) 10 ppm
Pentachloronitrobenzene 100 ppm
Nystatin (as Mycostatin, Aeirst) 100,000 units
GeO2 5 mg/l
agar 15 gm
Procedure
Add above fungistats aseptically to autoclaved, cooled
(58-60 C) ShmA (minus the sucrose) or water agar medium
made with 50 or 100% seawater.
Comments
The use of Ge02 could be substitued for by use of a van
Tieghem ring, or by placing a portion of the diatom-
contaminated material underneath the agar. Allowing the
fungus to grow up through the agar, leaving the
contaminant behind. Other workers have included
bactericides in their isolation media, but this was
generally not necessary as the pythiacous fungi readily
out grew the bacteria.
YpSs (Emerson, 1941)
Materials 1 05
yeast extract (Difco) 4.0 g
soluable starch 15.0
K2HPO„ 1.0
MgSO« 0.5
agar 20
II. Algal Media
Artificial Seawater Medium (after Harrison et al., 1980)
Nutrient and trace metals stock solution
NaN03 4.7 g
K2HPO„ 0.1
Na2EDTA 0.55
Fe(NH»)2 (SO«)2 6H20 0.23
FeSO„ 0.02
ZnSO, 7H20 0.008
MnSO„ 4H20 0.054
CoCL2 6H20 0.002
distilled water 1 liter
Vitamin Stock solution
Thiamine HCl 0.1 g
B-12 0.002
Biotin 0.001
distilled water 1 liter
To make medium, add 10 mis of nutrient solution and 1 ml of vitamin solution to 1 liter of 'Instant Ocean'. 1 06
Comments
Na2Si03 9H,20 and H3B03 eliminated from, nutrient solution
as adequate amounts of these minerals were represented in
the 'Instant Ocean' mix. 1 07
APPENDIX-2: STAINING PROCEDURES EMPLOYED
Analine Blue Black (Fisher, 1968)
Materials
1% Analine Blue Black in 7% glacial acetic acid
7% aqueous glacial acetic acid
Procedure
Stain 1.5-2.On sections at 55 C for 10 m. Briefly dip
slide into 7% acetic acid to remove excess stain. Wash in tap water, air dry, mount.
Comments
PAS is an excellent co-stain with ABB,-»but must be done
before staining with ABB. Saf-0 in 7% acetic acid is
also an excellent counter-stain for ABB (see procedure
listed under Fast Green stain for Saf-0 staining).
Acridine Orange (Cooke, 1977)
Materials
0.1% Acridine Orange in 1N HCl
Procedure
Adjust pH of stain to 0.5 if necessary. Stain 1.5-2.On
sections for 1.5 minutes. Dry, mount, and observe with
a fluorescent microscope.
Comments
At this pH only sulfate groups are ionized, making the 108
stain specific for fucans. Cooke (1977) reports that
the staining specificity lasts only two hours after
mounting.
Alcian Blue (Parker and Diboll, 1966)
Materials
0.5% alcian blue in 1N HCl
Procedure
Stain 1.5-2.On sections for 30-60 m. Wash briefly in DW
with pH adjusted to 0.5". Wash well in DW.
Comments
At this pH, only sulfate groups are ionized, and this
test may be in conjunction with Acridine Orange to
identify fucans. Parker and DiBoll report that if
sections are counterstained with Alcian Yellow (pH=2.5),
carboxylated polysaccharides will stain yellow and be
easily differentiated from fucans.
Calcofluor White (Heslop-Harrison and Heslop-Harrison, 1981)
Materials
0.1% aqueous Calcofluor white
Procedure
Stain 1.5-2.0*/ sections for 1 m. Rinse in DW. Observe
sections with fluorescent microscope.
Comments
Attempts to counter stain with either Acridine Orange or
Saf-0 resulted in loss of UV fluorescence. 109
CaCl 2 extraction of Fucans (Whyte et al., 1981)
Materials
1% CaCl2 in aqueous solution
Procedure
Extract sections for 1 h at 60 C.
Comments
Caution must be exercised as the plastic sections become
extremely soft at the elevated temperature. Allow
sections to cool completely in the solution before
removal for staining.
Erlich's Reagent (Cragie and McLachlan, 1964)
Materials
0.5% p-dimethylaminobenzaldehyde in 95% EtOH + 2.5% HCl
Procedure
Stain fresh material (non-embedded) by immersing the
specimen for 1-2 hours. Rinse in EtOH, and then section
directly or embbed in plastic.
Comments
Tissue tends to become macerated if left in the reagent
much beyond 2 hours.
Fast Bordeaux Reagent (Cragie and McLachlan, 1964)
Materials
0.1% Fast Bordeaux Salt in DW
Procedure
This technique was employed by Cragie and McLachlan to 1 10
identify phenolic compounds in Fucus on thin-layer
chromatography. While their technique called for an
overspray with Na2C03, no difference was found in
reaction with or without use of base in tissue or
authentic phloroglucinol in my study.
Fast-Green/Safranin-0 (Spicer, 1960)
Materials
0.2% aqueous Fast-Green acidified to pH=2.0 with 1N HCl
0.1% Safranin-0 in 1% acetic acid (pH=2.0)
Procedure
Stain tissue in FG for 10m at 20 C. Rinse in DW and
then counterstain with Saf-0 for 1 m at 20 C.
Comments
FG is a good general protein stain that has the same
specificity as ABB. Fungal hyphae, along with plant
nuclei, plastids, and cytoplasmic proteins are visibly
demonstrated with this stain. Staining beyond the 1 m
mark with Saf-0 results in removal of FG.
FeCl 3 Procedure (Cragie and McLachlan, 1964)
Materials
2% FeCl3 in DW
Procedure
Place fresh (unembedded) thallus pieces in fresh FeCl3
solution for 2-3" hours. Cut on microtome and observe.
Comments 111
Specimens embedded to methacrylate after staining did not
retain the nuclear or plastid coloration.
Grocott's Silver-Methenamine Stain (Hibbitts, pers. comm.)
Materials .
4% chromic acid or 1% periodic acid
5% aqueous silver nitrate
3% aqueous hexamethylenetetramine
5% sodium borate
1% sodium bisulfite
1% sodium thiosulfite
1% gold chloride
Procedure
1. ) Oxidize specimens in chromic acid or periodic acid
for 1 h.
2. ) Rinse, then place in sodium bisulfite solution for 1
m, followed by an additional rinse.
3. ) Stain in freshly mixed methenamine-silver nitrate
solution (see below) at 58-60 C for up to 100 m. This
part is subjective and each group of slide needs to be
checked to see when they need to be taken out.
4. ) Allow solution and slide to cool to 20 C before
removing sections from stain. Failure to do so may
result in severe wrinkling of the soft, heated plastic
sections.
5. ) Rinse in DW, then tone in gold chloride solution for
5 minutes, and follow with another DW rinse. 1 1 2
Comments
Counter staining after GMS was found to be difficult.
ABB, Fast Green, Saf-O, and TBO did not effectively
penetrate the plastic to color tissue after application
of GMS.
Methenamine-Silver Nitrate solution
Methenamine solution 100 ml
Silver nitrate solution 5 ml (stir until precipitate
dissolves)
Distilled Water 100 ml
Borax solution 8 ml
Na2C03 extraction of Alginic Acid (Parker and Diboll, 1966)
Materials
1% aqueous Na2C03
Procedure
Extract 1.5-2.0M sections for 2 h at 20 C
Comments
none
Periodic Acid-Schiff's Reagent (Feder and O'Brien, 1968)
Mater ials
1% Periodic Acid solution (freshly made)
Schiff's Reagent (see below)
0.5% Na2S20„ solution (freshly made)
Procedure 1 1 3
Place 1.5-2.0»i sections in periodic acid for 10 minutes.
Rinse in tap water and then stain, in Schiff's reagent for
10 minutes. Follow Schiff's stain by three quick dips
in Na2S2Oii. Wash in cold tap water.
Comments
PAS staining must be proceded by blocking residual
aldehyde groups remaining from aldehyde fixation. See
Feder and O'Brien (1968) for procedures of aldehyde
blocking for methacrylate thin-sections. An excellent
counter-stain for PAS is ABB. However, the PAS
precedure must procede protein staining.
Schiff's Reagent
Bring 100 ml DW to a boil. Remove from heat and
immediately dissolve 1 g Basic Fuchsin. Allow this
solution to cool to 60 C, filter, and add 2 g sodium
meta-bisulate and 20 ml 1N HCl. [ NOTE: This reaction
release Cl2 gas; perform this function in fume hood.]
Stopper and store solution in dark for 18-24 h. After
storage, add 200 mg activated charcol, shake vigorously
for 1 m before filtering. Store at 0-5 C.
Toluidine Blue-Q (McCully, 1970)
Materials
0.01% Toluidine Blue-0 in phosphate buffer, pH=4.4
0.01% Toluidine Blue-0 in 1N HCl, pH=1.0
Procedure 1 1 4
Stain 1.5-2.0*/ sections for 30 seconds. Rinse pH=4.4
sections in DW, rinse pH=1.0 sections in acidified DW.
Comments
These sections are best viewed immediately, as air drying
tends to allow some loss of metachromasia. If the
sections have been air dried, a measure of moisture can
be added back to the specimens by gently breathing on
them just before mounting.
Vanilin-HCl (Evans and Holligan, 1972b)
Materials
0.14% vanilin in 10N HCl
Procedure
Place fresh material in fresh working solution for 1
hour. Fresh, freezing-microtomed sections may also be
stained, but the stain time should be reduced to 15m.
Comments
The timing of staining is subjective, so its best to
monitor the material closely as too long an exposure to
the acid macerates tissue beyond recognition.