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Some aspects on the taxonomy, ecology and histology of Pythium Pringsheim species associated with Fucus… Thompson, Timothy Alan 1982

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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 t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or pu b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. BOTANY Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 r j a t e January 12, 1982 i i ABSTRACT P y t h i u m un.dula.tum v a r . l i t o r a l e Hohnk was f o u n d t o i n f e c t F u c u s d i s t i c h u s i n t h e Squamish R i v e r e s t u a r y of s o u t h e r n B r i t i s h C o l u m b i a . T h i s t h e s i s a d r e s s e s t h e q u e s t i o n s o f : 1 . ) whether t h i s s y m b i o s i s c a n be f o u n d o u t s i d e t h e Squamish R i v e r e s t u a r y , 2 . ) r e l a t i o n s h i p of t h e i n f e c t i o n w i t h i n t h e e s t u a r y t o t h e d i s t r i b u t i o n of P. u n d u l a t u m v a r . l i t o r a l e i n e s t u a r i n e s e d i m e n t s , 3 . ) t a x o n o m i c a l l y d e f i n i n g t h o s e s p e c i e s a s s o c i a t e d w i t h F u c u s a n d / o r i n e s t u a r i n e s e d i m e n t s , and 4 . ) t h e h o s t p a r a s i t e r e l a t i o n s h i p a s d e t e r m i n e d by means of h i s t o c h e m i c a l and l i g h t m i c r o s c o p e o b s e r v a t i o n s . R e s u l t s i n d i c a t e d t h a t o u t s i d e t h e Squamish R i v e r e s t u a r y , a s s o c i a t i o n s between p y t h i a c e o u s f u n g i and F u c u s a r e uncommon i n B r i t i s h C o l u m b i a c o a s t a l a r e a s . S a m p l i n g of l i v e and d e c a y i n g F u c u s p l a n t s f r o m 10 f i e l d s t a t i o n s i n B r i t i s h C o l u m b i a and W a s h i n g t o n y i e l d e d o n l y 4 s p e c i e s , t h e most common i s o l a t e b e i n g P h y t o p h t h o r a v e s i c u l a . W i t h i n t h e Squamish e s t u a r y , an a s s o c i a t i o n was f o u n d t o e x i s t between t h e d i s t r i b u t i o n o f P. u n d u l a t u m v a r . 1 i t o r a l e i n t h e s e d i m e n t s and t h e d i s t r i b u t i o n o f i n f e c t e d F u c u s p l a n t s . S e d i m e n t s a m p l i n g f r o m t h e F r a s e r R i v e r e s t u a r y , where F u c u s d o e s n o t o c c u r , y i e l d e d P. u n d u l a t u m v a r . l i t o r a l e , s u g g e s t i n g t h a t t h e f u n g u s i s p r o b a b l y i n d i g e n o u s t o e s t u a r i n e s e d i m e n t s . Numerous o t h e r s p e c i e s o f P y t h i u m were r e c o v e r e d from e s t u a r i n e s e d i m e n t s , i n c l u d i n g P. b u t l e r i , P. c a r o l i n i a n u m , P. c a t e n u l a t u m , P. g r a c i l e , P. t o r u l o s u m , and P. v o l u t u m . Two t a x a a r e d e s c r i b e d i n d e t a i l . P y t h i u m u n d u l a t u m var. l i t o r a l e was o r i g i n a l l y described by Hohnk (1953), but the v a r i e t a l 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 i s proposed as the nomen nova of this taxon. A discussion of the generic c h a r a c t e r i s t i c s of the genus Pythiogeten is presented. In order to f a c i l i t a t e an understanding of the infection process by Pythium species, the anatomy and histochemistry of Fucus distichus were examined. Anatomically, F'. distichus agrees with e a r l i e r reports of other species of Fucus. The internal structure of c e l l s was found to agree with descriptions in e a r l i e r publications, although higher physode content was noted in F. d i s t i c h u s . Histochemical staining suggested that c e l l walls of Fucus are three layered; having an outer fucan-rich layer, a middle layer composed p r i n c i p a l l y of a l g i n i c acid, and an innermost layer of c e l l u l o s e . Several phenolic-indicating reagents were tested on both fresh and fixed/embedded Fucus tissue, r e s u l t i n g 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 l i g h t microscope. Macroscopically, the infection of F. distichus occurs behind the most recent dichotomy, and lesions are necrotic, firm ( f l a c c i d with age), and are pink-to-red in color. Microscopically, fungal hyphae are confined to the c o r t i c a l and medullary regions. Hyphae appear to i v penetrate host c e l l walls by means of an enzymatic dissolution of the a l g i n i c acid and c e l l u l o s i c portions of the c e l l wall. Use of the Periodic Acid/Schiff's reagent shows a d i s t i n c t non-staining halo at the point where hyphae cross the c e l l wall. Pit connections between c o r t i c a l c e l l s were observed to break down with hyphae present in only one c e l l , suggesting that the fungus i s capable of p a r a s i t i z i n g several c e l l s via digestion of p i t s . Gemmae were observed to form in both c o r t i c a l and medullary c e l l s . The response by Fucus to infection i s an active one; a hypersensitivity reaction analagous to that of higher plants is observed. C e l l s in advance of fungal hyphae are observed to autolyse. Normally metabolically quiescent medullary filaments are observed to have an increase in general protein lev e l s and to have increased physode content. Physodes become polarized within the medullary c e l l s , and coalesce to form larger units, which are then delimited from the producing c e l l by a cross wall. The fate of these 'giant' physodes was not observed, but i t i s believed that these c e l l s autolyse and release their phenolic contents to the matrix, as levels of phenolic-reactive material were observed to increase in t h i s region. Coupled with the buildup,of phenolics in the matrix i s a decrease in the fucan component of the matrix. Stress and tear l i n e s appear between c e l l s , and eventually t h i s 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 d i v i s i o n s to form V i r r e g u l a r , cuboidal c e l l s which function as epidermis after abscission of the lesion occurs.. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES ix ACKNOWLEDGEMENTS x i i i Introduction 1 Materials and methods 4 Recovery and Taxonomy of Pythium species 4 Histology of Healthy and Infected Fucus 10 Description of the p r i n c i p a l study area 18 Results, Part 1 :Recovery of Pythiaceae from Fucus and from estuarine sediments in southern B r i t i s h Columbia and Washington 22 Effectiveness of Selective Media 22 Pythiaceous Fungi Associated With Fucus in B r i t i s h Columbia and Washington 24 Dis t r i b u t i o n of P. undulatum var. l i t o r a l e 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. l i t o r a l e Hohnk 34 Pythium hohnki i nom nov 42 Brief Descriptions of Pythiacious fungi from Fraser and v i i Squamish Estuaries 51 Part 3 : Observations on the infection of Fucus distichus by Pythium undulatum var. l i t o r a l e 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 v i i i LIST OF TABLES Table 1: H i s t o l o l g i c a l techniques and reaction spec i £ ic i t 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 B r i t i s h Columbia and Washington 25 Table 4: Pythium species recovered from sediments of the Squamish and Fraser rive r estuaries 25 Table 5: Reactivity of c e l l wall layers and matrix material with h i s t o l o g i c a l stains 57 Table 6: Reactions of Fucus matrix and c e l l u l a r elements with phenolic-indicating reagents 62 LIST OF FIGURES Figure 1: Sampling s i t e s in B r i t i s h 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. l i t o r a l e from Fucus distichus in the Squamish River estuary 26 Figure 6: Sites sampled and recovery of Pythium undulatum var. l i t o r a l e from the Fraser River estuary 29 Plate I: Pythium undulatum var. l i t o r a l e 37 Figure 7: Lesions of F. distichus infected with P. undulatum var. l i t o r a l e 37 Figure 8: Lobate sporangium 37 Figure 9; Inflated-filamentous sporangium 37 Figure 10: Toruloid sporangium 37 Figure 11: Mature sporangium showing r e f r a c t i l e t i p ... 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 c o r t i c a l c e l l s of X Fucus 37 Plate I I . 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: R e f r a c t i l e globules in mature zoosporangium 45 Figure 23: Zoosporogenesis, i n i t i a t i o n 45 Figure 24: Zoosporogenesis, completion 45 Figure 25: Oogonium with multi-lobed antheridium 45 Figure 26: Mature oospore 45 Figure 27: Pr i n c i p l e vegetative regions of F. distichus .. 55 Plate I I I . 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 a l g i n i c acid 58 Figure 30: Epidermal c e l l s stained with TBO pH=4.4 .... 58 Figure 31: Epidermal c e l l s stained with TBO pH=1.0 .... 58 Figure 32: Phenolic-reactive material within medulla matrix 58 Figure 33: E r l i c h s reagent-reactive material in the epidermal and c o r t i c a l regions 58 Figure 34: Cytoplasmic features of Fucus c e l l s 58 Plate IV: Parasitism of F. distichus by P. undulatum xi var. 1 i t o r a l e 67 Figure 35: General parasitism of epidermal and c o r t i c a l c e l l s 67 Figure 36: Hyphae growing through former p i t connection 67 Figure 37: PAS non-staining region at point where hyphae cross c e l l wall 67 Figure 38: PAS halo in medullary filament 67 Figure 39: Collapsed p i t connections between c o r t i c a l c e l l s 67 Figure 40: Bacteria invading necrotic tissue 67 Plate V: Defense reaction of F. distichus to infec t i o n ... 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 c e l l s d i f f e r e n t i a t e d 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 x i i Figure 50: Giant physode in lab infection 74 Figure 51: Diagramatic summation of events during pathogenesis 79 ACKNOWLEDGEMENTS This work has been car r i e d out under the supervision of Dr. G.C. Hughes. I thank him for support, advice, editing of this thesis, but most of a l l for the education received here at UBC under his d i r e c t i o n . Dr. R.J. Bandoni encouraged, advised, and made si g n i f i c a n t 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 f a c i l i t i e s , and was equally generous in both technical and interpretive advice. Mr. Alan Burns contributed his time and talents to a s s i s t me in thi s study. I am indebted to both for the-i-r kindness. Many individuals at UBC contributed advice, discussion and technical assistance. I would especially l i k e 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. J o l l y H i b b i t t s of the Invertebrate Pathology Laboratory, NOAA, M u l k i t i l l o , Washington, is thanked for use of her laboratory and materials for GMS staining of plant tissues. F i n a l l y , my wife Suzanne has been the most important xiv source of help during the course of t h i s thesis. To my partner and friend go my most sincere thanks. 1 INTRODUCTION Since the c l a s s i c 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 l a s t 65 years, only recently have, fungi in the oceans been examined seriously for their disease-causing c a p a b i l i t i e s - - a s potential problems in developing a l g a l -aquaculture systems (Andrews, 1976,1979). Fungi, as important pathogens of cu l t i v a t e d seaweeds, have been recognized in Japanese 'nori' farms since the 1940's (Arasaki, 1947). Outside of Japan , l i t t l e 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 F u l l e r (1970,1977), and Walker, et a l . (1979), knowledge of the processes by which fungi attack and degrade algae i s 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 a l g i n i c acid ( Laminaria, Macrocystis, Nereocystis ). Of the fungi described as symbionts of the orders Laminariales and Fucales, a l l 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. d i c k s o n i i is also reported from the brown alg a l genera Feldmania, Punctaria, P y l a i e l l a , S t r i a r i a , and Stictyosiphen (see Andrews, 1976). In June,1980 , Dr. R.J. Bandoni c o l l e c t e d specimens of Fucus distichus L. from the Squamish River estuary which displayed pink-to-red, f l a c c i d lesions in the apical wings. Subsequent examination revealed the presence of Pythium  undulatum var. l i t o r a l e 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 p a r a s i t i c on a marine plant. The recovery of infected Fucus plants within the Squamish estuary raised interesting questions regarding the d i s t r i b u t i o n of P. undulatum var. l i t o r a l e within the estuary, as well as what other pythiaceous fungi might be present. Both the d i s t r i b u t i o n 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 d e t r i t a l food chains (Anastasiou and Churchland, 1969; F e l l and Master, 1975) or in seed set in mangrove plants (Newell, 1973,1976). This investigation, then, has two objectives: (1) to id e n t i f y species of Pythium and Phytophthora that occur in marine and estuarine l o c a l i t i e s in southern B r i t i s h 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. l i t o r a l e / F . distichus symbiosis. The former study was i n i t i a t e d (a) to determine i f t h i s 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 l o c a l estuaries. 4 MATERIALS AND METHODS Recovery and Taxonomy of P y t h i u m spec ies Fucus plants were sampled from June, 1980 to June,1981 from the following locations in B r i t i s h 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 s i t e , plants were co l l e c t e d for examination i f they displayed lesions similar to those observed at the Squamish River estuary. Healthy t h a l l i were co l l e c t e d 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 a l g a l tissue were placed in s t e r i l e glass p e t r i plates, covered with either s t e r i l e d i s t i l l e d water (SDW) or s t e r i l e seawater (SSW; 5 Figure 1: Sampling s i t e s in B r i t i s h Columbia and northern Washington . Boxes denote Squamish and Fraser River estuaries. »=site of sediment sampling; »=site of Fucus c o l l e c t i o n . Adpated from A.H. Hutchinson, 1928. Transactions of the Royal Society of Canada. Third Series, Volume 22, Section 5, Page 294. 7 a r t i f i c i a l seawater with an approximate s a l i n i t y of 27 o/oo), and allowed to incubate ' at 20 C u n t i l the appearance of oomycetous reproductive structures. Portions of healthy t h a l l i were processed in a similar manner to record the presence of any additional fungi. Usually, t h i s method allowed for ready and easy i s o l a t i o n of the pythiaceous fungi, but occasionally,severe contamination by various diatoms, bacteria, labyrinthulids, yeasts, and p a r t i c u l a r l y Deuteromycetes ( Penicillium , Cephalosporium, and Fusarium ), was encountered. A selective medium against the p r i n c i p a l 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 d i r e c t l y with a commonly used selective medium for pythiaceous fungi, G a l l i c Acid Medium (Flowers and Hendrix, 1969). Di s t r i b u t i o n of P. undulatum var. 1itorale in the Squamish River estuary was studied to determine i f t h i s species i s a regular member of the sediment mycoflora and , i f so, i f i t s presence in the sediment can be correlated with the incidence of infected Fucus. Additionally, i t 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 p a l u s t r i s (L.) Roemer and Schultes zone (see the Vegetation Zone Map, Squamish 8 River Estuary Habitat Work Group F i n a l Report ,1981), sediment sampling was r e s t r i c t e d to thi s area. To determine i f P. undulatum var. l i t o r a l e might occur in the sediments of other major estuaries of southern B r i t i s h Columbia, a limited sample was col l e c t e d from the Fraser River near the R e i f e l Island Game Preserve. Although Fucus was not observed to grow there, t h i s s i t e was chosen as i t too has a training dike, a l t e r i n g the flow of fresh water into the marsh. Sites sampled on the Squamish and Fraser r i v e r s are shown in Figures 5-6. Sediment samples were a s e p t i c a l l y c o l l e c t e d from Carex rhizomes into s t e r i l e "Whorl-Pac" bags for transportation to the lab. Some plant debris was coll e c t e d for hemp-seed ba i t i n g . At Squamish, the presence of infected plants within a 1/2 m2 from the sample s i t e was noted. Sediment samples were processed by direc t plating of small portions (ca.0.25 gm wt.) to PSM medium, 4 plates per sample s i t e . Incubation of plates was at 20 C. Plates were monitored d a i l y for the appearance of hyphae. When growth was observed, hyphal-tip transfers were made to a second plate of PSM, and i f the fungus was found to be free of contaminants, transfered to a tube of corn meal agar stored at 5 C u n t i l further study. A l l cultures were made axenic by the successive use of hyphal-tip transfers. For i d e n t i f i c a t i o n of recovered fungi, zoosporangial observations were made on s t e r i l e grass blades, hemp seeds, sesame seeds, or portions of Fucus t h a l l i , incubated in both SDW and SSW at 20 C. To induce sexual reproduction, iso l a t e s 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. l i t o r a l e and P. hohnki i were examined as to reproductive physiology in various d i l u t i o n s of seawater. Zoosporangia were again observed on the above substrata, and were compared for spore production in 0, 15, and 27 o/oo a r t i f i c i a l seawater (Instant Ocean, Aquarium Systems) for 3 to 10 days at 20 C. Zoosporogenesis was i n i t i a t e d by washing the seed or blade in several changes of fresh medium at appropriate s a l i n i t y . For production of sexual structures, 4 media, 3 s a l i n i t i e s and 3 temperatures were employed: Emerson's YpSs (Emerson, 1941), CMA, SchmA, and F u j i t a ' s agar (see Appendix 1), each made up using 0, 15, and 27 o/oo a r t i f i c i a l seawater. Both species were innoculated to a l l media, and then incubated at 5, 10, and 20 C for a period of up to 30 days. Plates were examined p e r i o d i c a l l y for presence of reproductive structures and, when found, measurements made d i r e c t l y on the agar surface. Diagnostic observations and measurments used in the descriptions are a combination of a l l the data acquired from the previously described treatments. Reported measurment data are from at least 100 observations. Placement of a l l species was checked against the keys of Middleton (1943), Waterhouse (1967) and Robertson (1980). 10 Histology of Healthy and Infected F u c u s Infected F. distichus plants were obtained from the Squamish River estuary. Infected portions were excised and transported to the lab for f i x a t i o n . Healthy plants were likewise processed for comparative purposes. A small sample was col l e c t e d 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 b r i e f l y in running tap water to remove any v i s i b l e 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 li g h t microscope, this f i x a t i o n procedure was found to y i e l d as high a quality f i x a t i o n 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 sl i d e s with d i s t i l l e d water, and dried on a warming plate. Cytochemical d e t a i l s , the stains used, color reactions of the stains, and histochemical s p e c i f i c i t i e s are l i s t e d in Table 1. I n i t i a l l y , there was some d i f f i c u l t y in d i f f e r e n t i a l l y staining fungal hyphae from al g a l c e l l s . Eventually the general protein s t a i n , Analine Blue-Black in combination with either TABLE-1: H i s t o l o g i c a l techniques and reaction s p e c i f i c i t i e s . H i s t o l o g i c a l Test S p e c i f i c i t y Reported Color Reaction Reference Acridine Orange Alc i a n Blue (AB) pH-0.5 Anallne Blue Black (ABB) Calcofluor White IK 2I/H 2S0 4 Periodic A c i d / S c h i f f ' s (PAS) Safranin-0 (Saf-O) Toluidine Blue-0 (TBO) pH-4.4 pH-1.0 Grocott's S i l v e r Methenamine Na^CO^ extraction C a C l 2 extraction Bordeaux Reagent E r l l c h ' s Reagent F e C l 3 Vannilin-HCl Sulfated polysaccharides Sulfated polysaccharides General Protein Cellulose Cellulose A l g i n l c Acid Cellulose Sulfated polysaccharides Fluorescence under UV l i g h t Cooke, 1977 Blue Blue to Black Fluorescence under UV l i g h t Blue Pink to red Red to orange Sulfated polysaccharides Pink Carboxylated polysaccharides Red to blue Physodes Sulfated polysaccharides Fungal hyphae Extracts a l g i n l c acid Extracts fucoldin Phenols Green Pink Black PAS or Calcofluor staining Stain with TBO ph 4.4 Brown Pink Grey-Black Reddish-Orange Parker and D i b o l l , 1966 Fisher, 1968 Heslop-Harrison and Hesslop-Harrison, 1980 Herth and Schnepf, 1980 Jensen, 1962 Feder and O'Brien,1968 McCully, 1966 Jensen, 1962 Splcer, 1960 McCully, 1970 II McCully, 1966 Evans and Holligan, 1972b McCully, 1970 Hib b l t t s , pers. comm. Parker and D i b o l l , 1966 Whyte, et a l . , 1981 Cragie and McLachlan, 1964 i b i d i b i d i b i d Evans and Holligan, 1972b OsO. Black Ragan, 1976 12 the Periodic Acid-Schiff's reaction or Safranin-O, was found to be e f f e c t i v e probably owing to the dense nature of the fungal protoplasm r e l a t i v e to that of Fucus c e l l s . Fast Green with Safranin-0 also adequately d i f f e r e n t i a t e d fungal and a l g a l tissues, as did Grocott's silver-methenamine stain. However, high cost of Grocott's prohibited i t s general use. Two additional stains useful in determining polysaccharides in Fucus were Toluidine Blue-0 (TBO) and Calcofluor-White: s p e c i f i c i t y of these two stains i s l i s t e d in Table 1. As certain cytoplasmic v e s i c l e s in brown algae, the so-c a l l e d "physodes", are thought to be important in biochemical defense of Fucus against epiphytic f l o r a and fauna, grazers and pathogens (reviewed by Ragan,l976), an attempt was made to locate these vesicles cytochemically, to see i f 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 i d e n t i f i c a t i o n were employed (Table 1). Due to possible interference of staining by the f i x a t i o n and embedding procedure, chemical tests were performed on both fresh (un-fixed) materials and p l a s t i c 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. A l l reagents employed were f i r s t 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. l i t o r a l e 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. I n i t i a l l y , an attempt was made to grow adult Fucus t h a l l i from f e r t i l i z e d zygotes by inducing the zygotes to s e t t l e on oyster s h e l l s , and then culturing them under the conditions described by McLachlan et a l . (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 t h a l l i , attached to loose rock substrate, were transported from Squamish to the lab, to an a r t i f i c i a l tide tank similar in construction to the one used by Fulcher and McCully (1969b). The culture apparatus (Figure 2) consisted of two v e r t i c a l l y arranged tanks i n s t a l l e d in a c i r c u l a t i n g water-bath table maintained at 13 C. The upper culture tank (55 l i t e r 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 l i t e r capacity) was designed to hold a l l the medium at low tide, and was equipped with 6-8 charcoal-glasswool corner f i l t e r s (Hartz Canada, Inc.) which supplied vigorous aeration and e f f e c t i v e l y f i l t e r e d the brown-colored exudates of Fucus plants. An additional corner f i l t e r was placed in the upper tank. Light was supplied by two V i t a - l i t e Duro-test fluorescent F i g u r e 2: D i a g r a m of c u l t u r e a p p a r a t u s . A = c o r n e r f i l t e r s ; B = c i r c u l a t i n g w a ter b a t h ; HT = h i g h t i d e l e v e l ; LT = low t i d e l e v e l ; P = p l e x i g l a s s p l a t f o r m ; T = t i m e r s ; a r r o w s i n d i c a t e d i r e c t i o n of water f l o w . 15 LIGHTS \ H T / PUMP C U L T U R E TANK / \ / 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 t i d a l cycles (two highs, two lows). Water temperature was maintained at 11-12 C, while a i r temperatures during 'low tide' fluctuated between 19-25 C in the l i g h t cycle, and 17-19 C during the dark cycle. The medium employed was a modification of the a r t i f i c i a l 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 a r t i f i c i a l seawater base. Composition of the medium i s given in Appendix 1. To simulate summer water conditions at the Squamish estuary, the a r t i f i c i a l seawater was made up to 8 o/oo t o t a l s a l t s (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 f e l l 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 car r i e d 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 l i t e r 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, c o l l e c t i n g the resultant zoospore suspension and pipetting the suspension d i r e c t l y onto the alg a l surfaces during a 'low tide' sequence. Plants were monitered da i l y 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 d i r e c t l y 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 fjo r d extending northeast from the southern S t r a i t of Georgia (Figure 1). The delta i s about 2 km wide at i t s seaward edge, and i s constrained by mountains on both sides of the fjord.. In 1972, as part of a port development plan, channelization of the Squamish River into i t s most westerly arm was accomplished by the construction of a river training dike. U n t i l completion of the dike in June,1972, the Squamish flowed into Howe Sound through two channels. The east arm of the river i s now blocked from dire c t freshwater input, except for two culverts i n s t a l l e d through the dike in May,1972. Since 1972, l i t t l e change in the p r i n c i p a l 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 l a u s t r i s are the p r i n c i p l e vascular plants in the zone in which this study was carried out. Although the primary vascular f l o r a 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, r e l a t i n g 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 i s widely d i s t r i b u t e d throughout the central d e l t a . Fucus i s 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 i s found not only on log substrata (Figure 3), but i s frequently d i r e c t l y associated with the sedge community; i t s holdfasts penetrating into the sediments near rhizomes of Carex (Figure 4). . ^ The Squamish River and i t s t r i b u t a r i e s are important with regards to salmon f i s h e r i e s . Numerous studies have been conducted by Federal and P r o v i n c i a l governments concerning the effects of i n d u s t r i a l development and rive r 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 Fi n a l  Report (1981) , and the Air and Water Quality Work Group Fi n a l  Report (1981), co-published by the Government of Canada and the Province of B r i t i s h Columbia. 20 F i g u r e 3: F u c u s d i s t i c h u s g r o w i n g on a l o g i n c e n t r a l Squamish d e l t a . Note s u r r o u n d i n g C a r e x . F i g u r e 4: F u c u s d i s t i c h u s g r o w i n g on s e d i m e n t s i n c e n t r a l S q uamish d e l t a 22 RESULTS, PART 1 :RECOVERY OF PYTHIACEAE FROM FUCUS AND FROM ESTUARINE SEDIMENTS IN SOUTHERN BRITISH COLUMBIA AND, WASHINGTON Effectiveness of Selective Media A l l oomycetous fungi tested to PSM medium grew, including the obligately marine species A t k i n s i e l l a 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 i n i t i a l l y 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 e f f e c t i v e in blocking the growth of Deuteromycetes, but i t also proved- to be i n h i b i t o r y to Oomycetes from marine habitats. This may not be due to the d i r e c t i n h i b i t i o n of these fungi by g a l l i c acid in GAM, but may result from the extremely low pH of GAM (pH<4.0). Throughout the course of this study, infected a l g a l 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 i s o l a t e of 0idium recovered from Squamish sediments. 23 T a b l e - 2 : L a b o r a t o r y t e s t s on TTSM and GAM as s e l e c t i v e media f o r t h e r e c o v e r y o f Oomycetous f u n g i , e x c l u s i v e o f D e u t e r o m y c e t e s and L a b y r i n t h u l o i d s . P o s i t i v e growth i n d i c a t e d by "+"; n e g a t i v e g r o w t h , n o t t e s t e d , Fungus TTSM GAM I . Oomycetes P y t h i u m a p h a n i dermatum + O P. h o h n k i i + P. m a r i num + P. monospermum + + P. p o r p h y r a e + P. s y l v a t i cum + o P. u l t i m u m + O P. u n d u l a t u m v a r . l i t o r a l e + P y t h i u m s p . + P h y t o p h t h o r a v e s i c u l a + A t k i n s i e l l a d u b i a + I I . D e u t e r o m y c e t e s C e p h a l o s p o r i u m s p . D e n d r y p h i e l l a s a l i n a F u s a r i u m s p . 1 - -s p . 2 - -P e n i c i l l i u m s p . 1 - -sp. 2 - -Z a l a r i o n m a r i t i m u m • -I I I . L a b y r i n t h u l o i d s L a b y r i n t h u l a s p . 1 s p . 2 T h r a u s t o c h y t r i u m s p . 24 Pythiaceous Fungi Associated With F u c u s in B r i t i s h Columbia and Washington Sampling indicated that pythiaceous fungi are not regular associates of l i v i n g or decaying Fucus fronds outside of the Squamish estuary. Of the 10 s i t e s sampled, only 6 yielded a t o t a l of 7 Oomycetes (Table 3). Three of the recovered fungi were Phytophthora vesicula Anastasiou and Churchland, which means that the t o t a l number of species found was only 4. Pythium undulatum var. l i t o r a l e was recovered from Fucus in the Squamish River estuary, Pythium hohnkii from the Skookumchuk Rapid s i t e , and an unidentified species of Pythium from Egmont. Dis t r i b u t i o n of P . u n d u l a t u m var. l i t o r a l e in  the Squamish and Fraser estuaries. Results of sampling in the Squamish delta are presented in Figure 5. P. undulatum var. l i t o r a l e i s widely di s t r i b u t e d 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. 1 i t o r a l e 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 i n B r i t i s h Columbia and Washington. Station Friday Harbor, Washington Bamfield, B r i t i s h Columbia Spanish Banks, B.C. Squamish, B.C. Skookumchuk Rapids, B.C. Egmont, B.C. Species Phytophthora v e s i c u l a Anst. et Church. P_. v e s i c u l a P_. v e s i c u l a Pythium undulatum var. l i t o r a l e Hohnk Pythium hohnkii nom. nov. Pythium sp. Table 4: Pythium species recovered from sediments of the Squamish and Fraser River estuaries, and the growth of these species i n fresh and marine water i n v i t r o . Growth indicated by "+"; no growth by "-". Species Pythium Carolinianum Matthews P_. catenulatum Matthews P_. b u t l e r i Subramanian P. g r a c i l e Schenk P_. torulosum Coker and Paterson P_. volutum Vanterpool and Truscott Pythium species I. Recovery Squamish Squamish, Fraser Squamish, Fraser Squamish, Fraser Squamish, Fraser Squamish, Fraser Squamish, Fraser Growth i n DW + + + + + + SW + + + + + 26 Figure 5: Sites sampled and recovery of Pythium undulatum var. l i t o r a l e 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 P l a n , Habitat Work Group F i n a l Report, 1981. Figure 18, Page 68. 28 fact that in the upper estuary, Fucus i s confined to submerged logs within the river channels, and i s not in dir e c t association with the s a l t marsh vegetation. P. undulatum var. l i t o r a l e was also recovered once from sediments at the Pelly Point s i t e in the Fraser River estuary (Figure 6). Additional Oomycetes in Estuary Sediments In addition to P. undulatum var. l i t o r a l e , 20 additional oomycetous fungi were recovered from sediments or plant debris plated on PSM. Because of d i f f i c u l t i e s involved in ide n t i f y i n g Pythium spp., and the time that would be required to complete a thorough investigation of a l l i s o l a t e s , only the most commonly occurring species were i d e n t i f i e d (Table 4 ) . A brief description of the i d e n t i f i e d fungi is presented below. A l l isol a t e s were of Pythium spp.; no Phytophthora species were recovered.. Pythium g r a c l i e and Pythium sp. 1 were the most commonly isolated species after P. undulatum var. l i t o r a l e 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 i t s 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. b u t l e r i which grew much better in SSW than in SDW. 29 Figure 6: Sites sampled and recovery of Pythium undulatum var. l i t o r a l e from the Fraser River estuary. o= sediment sample s i t e not yi e l d i n g P. undulatum var. 1 i t o r a l e ; • = s i t e of recovery of P. undulatum var. 1 i t o r a l e . 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 f i r s t 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 f l a t , and bottom sediments. What is evident from their studies, and other more recent reports by Scott (1962), Chowdery and Rai (1980), and th i s report, is that Pythium species are conspicuous members of the sediment mycoflora. The majority of the Pythium species reported from marine l o c a l i t i e s are not endemic, being frequently recovered from t e r r e s t r i a l s o i l s and fresh water. Hohnk, who had previously noted th i s 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 o r i g i n i s probably other than marine. Of the species recovered in this study, 3 have been previously reported from estuarine sediments (P. undulatum var. 1 i t o r a l e , P. catenulatum , P. gr a c i l e ; 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 e a r l i e r from several locations in adjacent Howe Sound (Churchland and McClaren, 1973) and i s reported here from decaying Fucus fronds in other locations in B r i t i s h Columbia. F e l l and Master (1975) demonstrated a unique assemblage of 32 Phytophthora species in mangrove swamps, but they employed di r e c t baiting and not sediment p l a t i n g . Schmit.thenne.r (1970) reported that Phytophthora species have not been successfully isolated from s o i l s by use of most selective media, although these same media were useful in i s o l a t i o n 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 a l l species of pythiaceous fungi (Hendrix and W.A. Campbell, l970;Hendrix and C.W. Campbell, 1973; Lumsden et al.,1975), and i t i s l i k e l y that p l a t i n g estuarine sediments on PSM did not result in a complete characterization of pythiaceous fungi in the estuary. It should be emphasized that t h i s represented only one sampling and thus can hardly be representitive of the t o t a l mycoflora of the estuary. Although the data on d i s t r i b u t i o n of P. undulatum var. 1itorale in sediments r e l a t i v e to infected Fucus plants are interesting, they do not imply a d i s t i n c t c o r r e l a t i o n as the c o l l e c t i n g techniques used do not allow for s t a t i s t i c a l analysis. It is possible that the high incidence of P. undulatum var. l i t o r a l e may have been due to production of zoospores from nearby infected plants. In p a r t i a l support of th i s idea is the observation that P. undulatum var. l i t o r a l e 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 l e v e l s . 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 s t a t i s t i c a l analysis of year-round sampling i s required before any d e f i n i t e correlations can be made. 34 PART 2 : PYTHIUM ISOLATES RECOVERED FROM FUCUS AND FROM ESTUARINE SEDIMENTS P y t h i u m u n d u l a t u m var. l i t o r a l e Hohnk Isolation : From necrotic lesions of F. distichus growing in the Squamish River estuary. Lesions occurring at the most advanced dichotomies on the l a t e r a l margins of the blades, or immediately in the crease of the most recent branching (Figure 7). Lesions are firm, becoming f l a c c i d 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 r a d i a l l y -advancing hyphae are r e s t r i c t e d to the sub-cortex and medullary regions of the a l g a l t h a l l u s . Beyond the i n t i t i a l point of penetration, rarely were epidermal c e l l s observed to host fungal hyphae. Death of Fucus duevto infection was not observed in f i e l d - c o l l e c t e d 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 B r i t i s h 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 l a t e r a l 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 r e f r a c t i l e globules in the zooplasm, with a d i s t i n c t r e f r a c t i l e cap (Figure 11). P r o l i f e r a t i o n of the hyphae through the old sporangia noted (Figure 12), although formation of new sporangia by these p r o l i f e r a t i o n s never observed. 36 Asexual Reproduction : Zoosporogenesis t y p i c a l for the genus (Figure 13), i n i t i a t e d 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 r e f r a c t i l e cap to form the external v e s i c l e , into which the undifferentiated zooplasm passes. F l a g e l l a are the f i r s t distinguishable features, sometimes v i s i b l e before the entire zooplasm has passed into the v e s i c l e . 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 vesi c l e occurs, releasing 40-60 l a t e r a l l y b i f l a g e l l a t e zoospores, 10.0-12.5 (x=l1.5) X 6.0-9.0 (x = 7.5)»». Zoospores encyst after a period of m o t i l i t y , 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 i n t e r n a l l y in Fucus c e l l s (Figure 15), 16-23 (X=20)M in diameter. Germination of chlamydospores not observed. 37 Plate I: Pythium undulatum var. l i t o r a l e , Figure 7: Lesions of F. distichus infected with P. undulatum var. l i t o r a l e . 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 r e f r a c t i l e t i p . 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 c o r t i c a l c e l l s 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 i d e n t i c a l sporangia were ever observed, the lobate form shown in Figure 8 was the type most c o n s i s t e n t l l y seen. The i n f l a t e d filamentous sporangia (Figure 9) occurred very early in the colonization of any substratum, but became rare as the culture aged. The t o r u l o i d 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 i s t y p i c a l l y pythiaceous, and proceeds i d e n t i c a l l y for a l l sporangial forms observed. In young, a c t i v e l y growing water-cultures, there appeared to be a high degree of synchrony with regards to zoosporogenesis. A single change of the culture water i n i t i a t e d 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 i n i t i a t e d the process. However, i t was observed that a few sporangia were induced to evacuate before maturity, as evidenced by their subsequent abortion, suggesting that some factor i s i n i t i a t i n g evacuation of a l l 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. l i t o r a l e as a v a l i d variety, stating that i t was not s u f f i c i e n t l y d i f f e r e n t from Petersen's (1909) description of the species to warrant v a r i e t a l 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. l i t o r a l e . P. undulatum has been defined as having narrow e l l i p s o i d a l sporangia with internal p r o l i f e r a t i o n of the sporangium, and/or sympodial branching with production of chlamydospores. Matthews (1931), Sparrow (1932) and Goldie-Smith (1952) a l l found that small ovoid or obpyriform sporangia were produced in culture. Dissman (1927) had previously carried out a number of experiments in solutions having d i f f e r e n t 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 l a t e r a l p r o l i f e r a t i o n s . Hohnk's contribution was to recognize that his isola t e showed s u f f i c i e n t s i m i l a r i t i e s to be c l a s s i f i e d with P. undulatum, but was unique in having the variable lobed 41 sporangial complexes. The v a r i a b l i 1 i t y in sporangial morphology in the B r i t i s h Columbia isolates highlights the d e s i r a b i l i t y of maintaining var. l i t o r a l e as a discrete entity within P. undulatum. As in Hohnk's is o l a t e (1953), the B r i t i s h Columbia fungus developed sporangia in a range of seawater d i l u t i o n s from 0 to 27 o/oo, indicating that the fungus i s well adapted to estuarine existance, where s a l i n i t i e s are usually in constant flux. However, the reduced a b i l i t y of this fungus to produce zoospores in the higher s a l i n i t i e s 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 i s not due to inadequate nutrient l e v e l s , growth temperatures, or s a l i n i t i e s . The p o s s i b i l i t y that t h i s fungus i s h e t e r o t h a l l i c , as in P. sylvaticum or P. heterothallicum (Hendrix and Campbell,1973), was not explored in t h i s study. 42 P y t h i u m h o h n k i i nom nov. = Pythiogeten u t r i 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, B r i t i s h 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 s a l i n i t y 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 b a l l 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 a l l three s a l i n i t e s , are dark, thin-walled, and p r i n c i p a l l y 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 l i e at right angles to, or may be p a r a l l e l 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 r e f r a c t i l e body (Figure 22) and a r e f r a c t i l e cap on the discharge tube. Asexual Reproduction : Zoosporogenesis is i n t i t i a t e d by the passage of undifferentiated zooplasm into an external v e s i c l e (Figure 23) that has formed from the r e f r a c t i l e cap of the discharge tube. This v e s i c l e is persistent throughout zoospore formation (Figure 24); at no time was the v e s i c l e observed to dissipate, nor was zoospore cleavage 'free in the water' observed. Individual zoospores are recognizable within 2 min. after passage into the v e s i c l e , but cleavage is not completed u n t i l 3-5 min. This i s followed by a period of slow, sluggish 44 swimming, gradually increasing in vigour u n t i l zoospore a c t i v i t y ruptures the v e s i c l e wall, allowing the escape, of 15-30 b i f l a g e l l a t e 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 p r o l i f e r a t i o n of sporangia not observed. Sexual Reproduction : Antheridia s t r i c t l y monoclinous, single with multi-lobes applied at the base of the oogonium (Figure 25). Two to fi v e a n t h e r i d i a l 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, p l e r o t i c , yellow-brown, having a thick outer wall 3.0-5.5 (x=4.5)»i, and a large central o i l 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 a l l 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 pla t e s ) , but low in the 5 and 10 C plates (less than 1%). Oogonia not forming at 27 o/oo. 45 Plate I I . Pythium hohnki i . Figure 16: Highly branched hyphae. Bar= 25M. Figure 17: Hyphae digesting aborted oogonium. Bar= 100*/. Figure 18: Bursiform sporangium t y p i c a l 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: R e f r a c t i l e globules in mature zoosporangium (arrows ) . Bar= 45M. Figure* 23:- Zoosporogenesis, ini t i a t i o n * . Note intact v e s i c l e (arrows) . Bar= 1 0M . Figure 24: Zoosporogenesis, completion. Note ves i c l e s t i l l 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 f i r s t recognized the importance of the Pythiaceae in estuaries, and who f i r s t reported the pythiaceous nature of zoospore formation in Minden's Pythiogeten utriforme. Holotype : University of B r i t i s h Columbia Herbarium, UBC, s l i d e s numbered TT71380. Cultures deposited at ATCC, CBS, and IFO. Discussion : Although t h i s i s the f i r s t published description of t h i s fungus from western Canadian coastal waters, Anastasiou (pers. comm.) recovered what he i d e n t i f i e d as Pythiogeten  utriforme growing on submerged leaves of Arbutus menziesii Pursh. Hohnk (1939,1953) isolated P. utriforme from sandy beach s o i l 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 F l o r i d a . Species placed in the genus Pythiogeten are generally considered to be saprobes (Johnson and Sparrow, 1961), and i t i s l i k e l y that the presence of this fungus on Fucus was incidental and not pathogenic. Lack of extant material of Minden's o r i g i n a l type, or of any subsequently c o l l e c t e d material of Pythiogeten, makes diagnosis and direct comparison d i f f i c u l t . Although the type of 48 zoosporangium, p l e r o t i c oospore, and monoclinous antheridium appear to a l l y the B r i t i s h Columbia isolate with Pythiogeten  utriforme, several c h a r a c t e r i s t i c s stand out in marked contrast at both the generic and s p e c i f i c l e v e l s . The o r i g i n 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 i s unique to t h i s i s o l a t e . Minden did allow for an antheridium bearing a short appendage, but his drawings, and Sparrow's subsequent drawings (1936), bear l i t t l e resemblance to the antheridium of the B r i t i s h Columbia i s o l a t e . Hohnk (1939,1953) did not observe sexual reproduction, but he only observed water cultures, and oospores were not produced by my i s o l a t e , save on agar culture. Lund (1934) observed sexual reproduction, but his i d e n t i f i c a t i o n of P. utriforme i s questionable as he described diclinous antheridia for his i s o l a t e , although the species as o r i g i n a l l y described is monoclinous. To my knowledge, th i s i s the f i r s t recorded i s o l a t i o n 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 i s o l a t e s to Pythiogeten based on sporangial morphology, and did not observe either zoospore or oospore formation. Sparrow (1960) described oospore germination, but t h i s process 49 was not observed in my i s o l a t e . The B r i t i s h Columbia iso l a t e lacks internal p r o l i f e r a t i o n of sporangia, but this feature was also found lacking by Sparrow (1960), Cantino (1949) or Hohnk (1939,1953), although i t was recorded by Drechsler (1932) for Pythiogeten autossytum and Lund (1934) for P. utriforme. The major difference between Minden's Pythiogeten and my iso l a t e i s the persistence of the v e s i c l e during zoosporogenesis. The character upon which Minden erected the genus Pythiogeten was a Pythium-like zoospore i n i t i a t i o n , but with a quickly evanescent v e s i c l e and maturation of the spores occurring 'free' in the medium . Hohnk noticed this same inconsistancy in his isola t e s of P. utriforme, but was of the opinion that the v e s i c l e in Pythium was conspicuously larger, and that the vesi c l e in Pythiogeten was ephemeral and e l a s t i c . The v e s i c l e in the B r i t i s h Columbia fungus does not appear to be d i f f e r e n t than that noted in general for Pythium species. The v e s i c l e i s semi-persistant after spore release, something which Hohnk also noticed. Drechsler (1932) descibed the formation of zoospores independent of a ve s i c l e in Pythiogeten autossytum , but he too noted frequent -completion of spore formation within an intact v e s i c l e . Cantino (1949) also describes "naked" maturation of zoospores in an isolate tentatively i d e n t i f i e d as P. uniforme . Wolf (1944) figured an elongated discharged zooplasm (see his Figure 51), but f a i l e d to discuss zoosporogenesis. Sparrow (1960) retained the genus in the second edition of Aquatic 50 Phycomycetes , but i t i s not clear from that edition or his e a r l i e r work (1936,1952.) i f he actually observed zoospore formation in this fungus. Therefore, whether the v e s i c l e membrane dissolves or not during zoosporogenesseems a questionable generic character, in view of the inconsistency of the published reports regarding that p a r t i c u l a r t r a i t . Much more work is required before one could consider transferring a l l 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 l a t e r 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 t h i s taxon. 51 Brief Descriptions of Pythiacious fungi from Fraser and Squamish Estuaries Pythium b u t l e r i Subramanian: Hyphae sparse in SDW, vigorous in SSW, branched, 2.5-6.0,/ wide. Sporangia consist of t o r u l o i d outgrowths, zoosporangia producing 10-20 zoospores. Oogonia terminal or intercalary, 8-36,/ in diameter. Antheridia s t r i c t l y d i c l i n o u s , 1-2 (usually one). Aplerotic, 8.0-24.0,/ in diameter. Isolated from three s i t e s 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 e l l i p s o i d a l , 18.0-30.0,/ in diameter. Sporangia frequently with discharge tube, although not consistent in t h i s 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 o i l 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, t o r u l o i d with communicating elements, 5.0-8.0 X 5.0-16.0M, mostly terminal, rarely intercalary. Oogonia mostly terminal, terminal on short l a t e r a l branches, rarely intercalary, 13.0-20.0* in diameter. Antheridia monoclinous, 1-3, a r i s i n g from the oogonial stalk. Oogonia p l e r o t i c . 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 h e l i c a l l y involved with the oogonial stalk. Oospores a p l e r o t i c , 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 F u c u s d i s t i c h u s The general anatomy of healthy F. distichus agrees with e a r l i e r descriptions of other. species in this, genus (McCully,1965,1966). A mature vegetative thallus exhibits three d i s t i n c t tissue regions (Figure 27): 1.) an epidermis composed of a single layer of columnar c e l l s , capped to the outside by a heavy, TBO-staining c u t i c l e , 2.) 2-4 layers of c o r t i c a l c e l l s , and 3.) a reticulum of filamentous c e l l s in the center of the thallus embedded in a mucilaginous matrix, the medulla. McCully (1966) distinguished the large, wide-lumen c e l l s of the medulla as primary filaments, and the smaller, narrow-lumen c e l l s as secondary filaments. Observations on the histochemistry of the c e l l walls shows three layers of d i s t i n c t r e a c t i v i t y . The outermost layer reacts to stains s p e c i f i c for sulfated polysaccharides; the wide, middle layer has a f f i n i t y for stains s p e c i f i c to molecules with 2- v i c i n a l hydroxyl groups; a thin, innermost layer that also demonstrates adjacent hydroxyl groups, but a d d i t i o n a l l y reacts with the fluorescent marker Calcofluor-White (Table 5; Figures 28-31). These results suggest that the three layers are 55 F i g u r e 27: P r i n c i p l e v e g e t a t i v e r e g i o n s o f F. d i s t i c h u s . N o m e n c l a t u r e of M c C u l l y ( 1 9 6 6 ) . TBO s t a i n i n g . 56 T a b l e - 5 : R e a c t i v i t y - o f c e l l w a l l l a y e r s and m a t r i x m a t e r i a l w i t h h i s t o l o g i c a l s t a i n s . P o s i t i v e r e a c t i o n , " +"; n e g a t i v e r e a c t i o n , "-" P i t c o n n e c t i o n s S t a i n O u t e r m o s t w a l l M i d d l e w a l l I n n e r w a l l and c r o s s w a l l s M a t r i x A c r i d i n e Orange + - - + A l c i a n B l u e (pH=0.5) + - - + C a l c o f l u o r - + + -I K 2 I - H 2 S O u - - -PAS + + -S a f - 0 + + - + TBO (pH = 1.0r + + + + TBO (pH=4.4) ' + + + + + Na^HCO^ e x t r a c t i o n - + + -( C a l c o f l u o r ) ^ Na^HCO^ e x t r a c t i o n (PAS ) - + + -C a C l ^ e x t r a c t i o n + + + -(TBO, pH=4.4) 1. I n d i c a t e s s t a i n u s e d a f t e r e x t r a c t i o n p r o c e d u r e . 58 Plate I I I . Anatomy of F. di s t i c h u s . 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 a l g i n i c acid. Contrast with Figure 28. PAS/ABB staining; Bar = 15K. Figure 30: Epidermal c e l l s stained with TBO pH=4.4. Note heavy staining of c e l l s walls at this pH (arrow). Bar = 25K. Figure 31: Epidermal c e l l s stained with TBO pH=1.0. Note at th i s pH, the c e l l walls do not stain (arrows). Bar = 20*.. Figure 32: Phenolic-reactive material, within, medulla- matrix. TBO staining; Bar = 30K. Figure 33: E r l i c h s reagent-reactive material in the epidermal and c o r t i c a l regions. Staining proceeded embedding. Bar = 100K. Figure 34: Cytoplasmic features of Fucus c e l l s . Note the basipetal location of nuclei in epidermal c e l l s (arrow), the c e n t r a l l y located nuclei (N), and the numerous physodes (P). ABB/Saf-0 staining; Bar = 15K. 60 composed of fucans 1, a l g i n i c acid, and c e l l u l o s e respectively. Calcofluor staining also demonstrated strong fluorescence in the pit-connections between epidermal c e l l s , a fluorescence that was contiguous with the thin, inner c e l l wall layer. Furthermore, cross walls between primary medullary c e l l s were found to be strongly PAS-positive and to fluoresce with Calcofluor. Secondary c e l l walls were found to be PAS-positive after a l k a l i n e extraction and to fluoresce with Calcofluor. Although these results strongly suggest the presence of c e l l u l o s e , some caution should be noted since the standard test for c e l l u l o s e , IK 2I with H 2SO„, f a i l e d to show any r e a c t i v i t y in methacrylate-embedded sections, and the same test applied to fresh material showed only very f a i n t r e a c t i v i t y in secondary c e l l s and cross walls, with, no r e a c t i v i t y in the p i t 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 r e a c t i v i t y only appeared in material fixed with 1% ca f f i n e in the f i x a t i v e . Fresh sections stained with phenolic-indicating 1. A chemically d i s t i n c t fucan sulfate (fucoidin) is not believed to occur in mature vegetative Phaeophytes. Rather, there i s a family of int e r - r e l a t e d glycans r i c h in sulfated L-fucose (McCandless and Cragie, 1979). For convenience, the term "fucans" in this thesis w i l l refer to th i s group of sulfated polysaccharides. 61 reagents revealed areas of positive r e a c t i v i t y that corresponded to the TBO-positive regions. (Table 6). V a n i l l i n HCl, E r l i c h ' s Reagent, and Bordeaux Salt were p a r t i c u l a r l y useful in demonstrating posi t i v e r e a c t i v i t y in the epidermal region (Figure 37), but V a n i l l i n HCl had limited use as i t 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. F i r s t , unlike results o r i g i n a l l y reported by McCully (1966), authentic phloroglucinol was not found to react metachromatically with TBO in v i t r o . Secondly, phenolic reagents did not show r e a c t i v i t y when applied to embedded-tissue; only freshly sectioned material was e f f e c t i v e l y stained. Internally, both epidermal and parenchyma c e l l s of Fucus are highly vacuolate. In the epidermis, the nuclei are basally located with cytoplasmic strands running from the nucleus to the p l a s t i d s , located on the l a t e r a l walls (Figure 38). In parenchyma and filament c e l l s , nuclei are located more-or-less c e n t r a l l y and are connected to the peripherally located p l a s t i d s by transvacuolar strands. Filament c e l l s are in t e r n a l l y similar to parenchyma c e l l s , except that they are largely devoid of p l a s t i d s and vacuoles. Epidermal nuclei f a i l e d to stain metachromatically with TBO, as had been reported for other species (McCully, 1966). However, nuclei and pla s t i d s did react with E r l i c h ' s reagent and F e C l 3 , indicating the possible presence of phenolic materials. Vacuoles were p r i n c i p a l l y of two types: small (less than Table-6: Reactions of Fucus matrix and c e l l u l a r elements with phenolic-indicating reagents. A l l reagents f i r s t tested against authentic p h l o r o g l u c i n o l , and then against both fresh and fixed-embedded Fucus material. Color reactions within plant that were similar to i n v i t r o reaction of phloroglucinol indicated by "++•"; les s e r 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 + - -FeCl 3 grey-black - - -black - - 0 0 TBO, ph U.U blue 1 - - - • • Vanillin HCl orange - -GMS not tested 0 0 0 - +++ o 2 1. McCully (1966, 1970) reported green metachromasia of TBO with phloroglucinol. Concentrations ranging from 0.05 to 1.0% i n aqueous or alchohol s o l u t i o n s , or i n phosphate or cacodylate b u f f e r s , f a i l e d to induce uietachromusiu In v i t r o . 2. Color development by physode3 with t h i s s t a i n varied with length of staining: less than 30 seconds, no s t a i n ; 1-2 minuteB, turquiose-blue; greater than 3 minutes, dark blue. 63 1 it) v e s i c l e s staining dark blue with TBO and red with Saf-O, and large (4-6^) vesic l e s that were col o r l e s s in l i v e 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 a l g a l l i t e r a t u r e (McCully, 1966,1970; Evans and Holligan, 1972b; Ragan, 1976; Ragan and Cragie, 1976). These were found to occupy most of the c e l l space in the epidermis and parenchyma. While the smaller bodies were found in a l l c e l l types, the physodes were rare in primary and secondary medullary filaments. Physodes are reported to contain phenolic materials, but h i s t o l o g i c a l tests employed in th i s study provided a confusing array of res u l t s . The standard stain test for phenolics in Fucus physodes, TBO (McCully,1970), stained these bodies d i f f e r e n t i a l l y , depending upon the length of staining time. If methacrylate sections were stained with just s u f f i c i e n t time to metachromatically d i f f e r e n t i a t e the sulfated matrix (about 30s) the physodes were unstained and retained their yellow-color. If l e f t 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 V a n i l l i n HCl, brown with Bordeaux Salt after 1 h., but f a i l e d to react with F e C l 3 or E r l i c h ' s reagent. As before, none of these reagents had any eff e c t on embedded tissue. An unexpected result occurred when using Grocott's Silver-methanamine stain 64 to d i f f e r e n t i a t e fungi i n Fucus t i s s u e s . Physodes s t a i n e d dark black with t h i s s t a i n , while no other c e l l u l a r components s t a i n e d at a l l . H o s t - p a r a s i t e i n t e r f a c e : N a t u r a l i n f e c t i o n s . M a c r o s c o p i c a l l y , i n i t i a l l e s i o n s on F. d i s t i c h u s appear i n i t i a l l y as pink to red n e c r o t i c r e g i o ns c o n f i n e d to the wings of d i s t a l t i p s , u s u a l l y at or near the p o i n t of the f i r s t dichotomy (Figure 7 ) . L e s i o n s were r a r e l y observed at the a p i c e s or on o l d e r m i d - r i b t i s s u e . With age of i n f e c t i o n , the n e c r o t i c r e gion becomes f l a c c i d , and a c l e a r e d t i s s u e band develops at the l e s i o n p e rimeter. T h i s zone a c t s as an a b s c i s s i o n l a y e r dropping the i n f e c t e d p o r t i o n from the p l a n t . L e s i o n s r a r e l y spread beyond t h i s zone; death of the p l a n t due to i n f e c t i o n c o u l d never be v e r i f i e d i n f i e l d m a t e r i a l . I n f e c t i o n was probably i n i t i a t e d by p e n e t r a t i o n of zoospore germ tube d i r e c t l y through the epidermal c e l l s , although t h i s process was never observed i n methacrylate s e c t i o n s . A p p r e s o r i a were not observed. R e l a t i v e l y few epidermal c e l l s were invaded; beyond the i n i t i a l p o i n t of i n f e c t i o n , r a r e l y were the hyphae ever observed i n epidermal c e l l s and never were hyphae seen to grow on the a l g a l s u r f a c e . I n f e c t i n g hyphae spread r a d i a l l y from the p o i n t of i n f e c t i o n and are c o n f i n e d to the parenchyma and medullary regions ( F i g u r e 35). 65 Parasitism of individual host c e l l s i s i n i t i t a t e d by what appears to be an enzyme-mediated digestion of the a l g i n i c acid portion of the host c e l l wall. In Fucus, the algin portion of the c e l l wall stains pink in the PAS reaction (Figure 29), but in infected c e l l s , at the point where the hyphae cross the c e l l wall, there i s a large discontinuity in the c e l l wall staining (Figure 37-38). There i s no evidence to suggest that the fucan components of the c e l l wall or matrix are also dissolved. However, there i s some indication that the c e l l u l o s e component of the wall i s digested. Hyphae are frequently observed to grow between two c e l l s through the former p i t connection, which now shows no staining a f f i n i t y for either PAS or Calcofluor (Figure 36). There i s also the suggestion that while physically occupying one c e l l the fungus may be capable of p a r a s i t i z i n g three or four adjacent c e l l s via digestion of the p i t connection. Figure 39 shows several c e l l s with both contiguous collapsed cytoplasm through the former p i t connection and areas in which the p i t connections show li g h t e r staining. Hyphal cons t r i c t i o n s or appressorial formation, commonly observed in fungi having mechanical (pressure) mediated penetration, were never observed. Haustoria are not formed in host c e l l s . The exact sequence of events in the digestion of host c e l l s i s s t i l l obscure, as intermediates of degradation were not observed. Occasionaly, a remnant nucleus i s observed in par a s i t i z e d c e l l s , suggesting that t h i s i s the last structure degraded. Infected c e l l s have collapsed cytoplasm and, h i s t o l o g i c a l l y , c e l l contents stain as an amorphous mixture of 66 protein, polysaccharides, and phenolic materials. In older infected regions, the structural i n t e g r i t y of the thallus is l o s t ; c e l l s of the epidermis begin to slough o f f , and p r i n c i p a l l y rod-shaped bacteria are evident (Figure 40). In material co l l e c t e d 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 c e l l s . Thick-walled chlamydospores are often formed in c o r t i c a l and medullary c e l l s (Figure 15). The response by Fucus to the invasion i s an active one: a hypersensitivity response (HR) occurs in advance of the fungal hyphae, and here the sequence of events is cle a r e r . The process i n i t i a t e s in the c o r t i c a l and medullary c e l l s , but extends outward to include epidermal c e l l s . P l astids and cytoplasm are the f i r s t features to be disrupted. The nucleus shows a decreasingly intensive reaction with ABB, becoming swollen and distended u n t i l i t is apparently disbanded. The physodes are the l a s t d i s t i n c t structures, but these too dissipate and the remnant contents of the c e l l stain intensely green with TBO. This green staining material i s not evident in more advanced infections, and in the same region a lack of matrix material i s noted; stress and tear l i n e begin to appear between c e l l s . C e l l walls have a d i s j o i n t e d , f i b r i l l a r appearance and h i s t o l o g i c a l l y stain only for a l g i n i c acid and c e l l u l o s e . Beyond the developing hypersensitivity region is an area of intense metabolic and meristematic a c t i v i t y . A s i g n i f i c a n t 67 Plate IV: Parasitism of F. distichus by P. undulatum var. l i t o r a l e . Figure 35: General parasitism of epidermal and c o r t i c a l c e l l s . PAS-ABB staining; Bar = 20M. Figure 36: Hyphae growing through former p i t connection. PAS-ABB staining; Bar = 5M . Figure 37: PAS non-staining region at point where hyphae cross c e l l wall (arrows). PAS-ABB staining; Bar = 10M. Figure ,38: PAS halo in medullary filament (arrows). PAS-ABB staining; Bar = 10M. Figure 39: Collapsed p i t connections between c o r t i c a l c e l l s . F = fungal hyphae; P = collapsed p i t connection. Note that the cytoplasm is now contiguous between c e l l s 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 l e v e l of proteinaceous material. Concurrent with this i s the appearance of numerous physodes, normally rare or absent in medullary elements (Figure 41). In some cases, these c e l l s become so packed with vesi c l e s that other c e l l elements are obscured. A d i s t i n c t p o l a r i z a t i o n of physodes occurs, followed by coalescence of individual v e s i c l e s to form one large unit (Figures 42-43). These 'giant' vesicles are subsequently delimited from the producing c e l l by an irregular PAS-positive wall (Figure 43). These c e l l s contribute to the developing HR; autolysis occurs and the c e l l s stain b r i l l i a n t green with TBO. A s i g n i f i c a n t l e v e l 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 l i n e s begin to appear between c e l l s . The sequence of events in t h i s region follows that of the early HR: disappearance of phenolic-staining material and of fucan matrix with a d i s j o i n t e d , f i b r i l l a r appearance of the c e l l walls. In the f i n a l stages of the infection process, c o r t i c a l and medullary filaments undergo transverse and l o n g i t u i d i n a l d i v i s i o n s to form a 1-2 c e l l protection layer (Figure 46), which functions as epidermis after abscission has occurred. These c e l l s 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 tha l l u s , terminating the 70 P l a t e V: D e f e n s e r e a c t i o n o f F. d i s t i c h u s t o i n f e c t i o n . F i g u r e 41: Low m a g n i f i c a t i o n of d e f e n s e r e a c t i o n i n m e d u l l a . Note i n c r e a s e d l e v e l s o f A B B - s t a i n i n g m a ' t e r i a l and f o r m a t i o n o f l a r g e , p o l a r i z e d p h y s o d e s ( a r r o w s ) . PAS-ABB s t a i n i n g ; Bar = 3 0 M . F i g u r e 42: P r i m a r y f i l a m e n t s h owing f o r m a t i o n o f g i a n t p h y s o d e s from c o a l e s c e n c e o f s m a l l e r v e s i c l e s ( a r r o w s ) . PAS-ABB s t a i n i n g ; Bar = 1 5M . F i g u r e 43: H i g h m a g n i f i c a t i o n of g i a n t p h y s o d e . Note PAS-p o s i t i v e w a l l s e p e r a t i n g ' g i a n t ' p h ysode from d e l i m i t i n g c e l l . PAS-ABB s t a i n i n g ; Bar = 1 0M . F i g u r e 44: I n c r e a s e i n p h e n o l i c m a t e r i a l s i n t h e m a t r i x o f t h e h y p e r s e n s i t i v e r e g i o n . R e a c t i v e a r e a s i n d i c a t e d by a r r o w s . TBO, pH=4.4, s t a i n i n g ; Bar = 2 5 M . F i g u r e 45: A b s c i s s i o n zone ( a r r o w ) . Note t h e a b s e n c e of t h e d a r k - s t a i n i n g f u c a n m a t r i x i n t h i s r e g i o n . TBO, pH=4.4; Bar = 2 0M . F i g u r e 46: I r r e g u l a r , c u b i o d a l c e l l s d i f f e r e n t i a t e d f r o m m e d u l l a r y f i l a m e n t s . T h e s e c e l l s f u n c t i o n as e p i d e r m i s a f t e r a b s c i s s i o n has o c c u r r e d . TBO, pH=4.4; Bar = 1 0M . 72 in f e c t i o n . 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 aft e r the 30 day incubation period. Within 2-3 d of application of innoculum, numerous small (1-2mm), brick-red lesions appeared on the al g a l fronds (Figure 48) p r i n c i p a l l y at the apices. By 5-7 days, a l l plants in the tank showed i n f e c t i o n . 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 f i e l d - i n f e c t i o n s , 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 l a t e r a l wings. A d i s t i n c t i v e 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. l i t o r a l e . In thin sections, a great reduction in the physode content i s 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 i s noted in the medulla (Figure 49), where the infection seems to be mainly confined. Occasionally, a 'giant' physode i s seen to form (Figure 50), but i t s occurrence i s sporadic and does not seem to be associated with any concerted defense reaction by the plant. Discussion While the findings of t h i s study p a r a l l e l previous findings of Fucus and brown algae in general, a few new observations were' made concerning- the morphology of healthy vegetative Fucus distichus in B r i t i s h Columbia. One addition i s the histochemical l o c a l i z a t i o n of c e l l u l o s e in the mature thallus by means of Calcofluor-white and the PAS reaction. Cellulose i s known to occur in small quantities in brown algae (Percival and McDowell, 1967) and has a demonstrated prominent role in c e l l wall development in several species of Fucus (Novotny and Forman,l975; Quatrano and Stevens,1976). McCully (1970) reported a weak reaction with IK 2I-H 2SO„ and a positive PAS reaction after a l k a l i n e extraction in the walls of 74 P l a t e V I : L a b o r a t o r y i n f e c t i o n . F i g u r e 47: R e d u c t i o n i n physode c o n t e n t a f t e r 30 day i n c u b a t i o n p e r i o d . ABB/Saf-0 s t a i n i n g ; Bar = 10M . F i g u r e 48: L a b o r a t o r y l e s i o n s on Fucus. ! p r i n t . l e ( . f 49) F i g u r e 49: Low m a g n i f i c a t i o n of l a b o r a t o r y i n f e c t i o n . F = f u n g a l hyphae. ABB/Saf-0 s t a i n i n g ; Bar = 30*.. F i g u r e 50: G i a n t physode i n l a b i n f e c t i o n . ABB/Saf-O s t a i n i n g ; Bar = 10*>. 75 76 secondary f i b r e s of Fucus. Positive r e a c t i v i t y with Calcofluor of the secondary filament walls before and after the alkaline extraction used in t h i s study provides additional evidence for c e l l u l o s e in these walls. Furthermore, similar r e a c t i v i t y in the cross walls of primary filaments and in the p i t connections between epidermal and c o r t i c a l c e l l s suggests a higher c e l l u l o s e content than previously considered for these regions. The p o s s i b i l i t y does exist that Calcofluor-White may be binding to a l g i n i c acid, as > t h i s stain i s thought to have a f f i n i t y for Beta 1-3* and Beta 1-4 linked mannuronic and guluronic units (Maeda and Ishida,l976; Takeuchi and Komamine,1978). In l i g h t of the ambiguous reaction with the IK 2I-H 2SO a s t a i n , t h i s factor must be considered. However, alkaline extraction removed the a l g i n , as indicated by PAS staining, but l e f t the Calcofluor-positive regions i n t a c t . The presence of phenolic materials in the matrix of Fucus has not been noted previously, and t h i s may in part be due to the apparent differences in stain a f f i n i t y between fresh and fixed-embedded material. Phluroglucinol and i t s derivatives are alcohol and water soluble (Cragie and McLachlan,1964; Ragan and Cragie,1976), and are probably removed during f i x a t i o n and dehydration procedures. Inclusion of 1% caffeine in the f i x a t i v e was found to be an e f f e c t i v e means of preserving the phenolics for histochemical d i f f e r e n t i a t i o n , although the technique was not completely e f f i c i e n t as the amount of material staining in the c o r t i c a l region with E r l i c h ' 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 f i x a t i o n . This is in contrast to e a r l i e r reports where physodes in mature epidermal and medullary c e l l s of Dictyota were not maintained after gluteraldehyde or acrolein f i x a t i o n (Evans and Holligan,1972b). This lack of physode preservation in a c r o l e i n -fixed tissue may account for the differences in physode content of epidermal c e l l s of F. distichus in t h i s report and those figured.by McCully for F. vesiculosus and F. edentatus , since she used acrolein f i x a t i o n for her material (1966, see McCully's Figures 1B and 5, and contrast with Figure 38 of t h i s report). The reason for the development of a yellow-color in the physodes in JB-4 methacrylate i s unknown, but presumably must be related to the embedding procedure as tissues examined immediately after f i x a t i o n 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 d i f f e r e n t levels or d i f f e r e n t types of phenolic compounds in physodes. Because of the f a i l u r e of TBO to react metachromatically with authentic phloroglucinol in_ v i t r o , green color developed by physodes in the present study i s 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. d i s t i c h u s . These differences may be due to differences in f i x a t i o n s , or may r e f l e c t genuine differences in sub-cellular bodies betwen these species, or between physical environments (Atlantic vs. P a c i f i c oceans). Despite the i r r e g u l a r i t i e s 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 E r l i c h ' s Reagents and F e C l 3 f a i l e d to react in physodes, while V a n i l l i n HCl, Fast Bordeaux Salt, and 0s0 4 did, i s not c l e a r . Presumably there are other cytoplasmic elements which interfere in color development with these reagents. Why Grocott's s i l v e r -methanamine stain also reacted with physodes in p l a s t i c section i s unknown, but i t i s suggested that i t has to do with the s i l v e r ion binding with the phenol, as s i l v e r n i t r a t e i s often used as a phenolic indicator in biochemical thin-layer chromatography (Cragie and McLachlan,1964). The f a i l u r e of the other reagents to react with physodes or matrix phenolics in methacrylate-embeded sections c l e a r l y demonstrates the need for caution when interpreting histochemistry solely from staining properties of embedded materials. Figure 51 summarizes the infection process. Zoospores i n i t i a t e the infection by penetation through the epidermal 79 Figure 51: Diagramatic summation of events during pathogenesis. A. Pathogenesis 1. B i f l a g e l l a t e zoospore as primary inoculum. 2. Zoospore encysts, and then penetrates the epidermis by means of dir e c t hyphal penetration. 3. Hyphae grow mainly in the c o r t i c a l and medullary regions. Penetration of individual c e l l s may be in part due to an enzymatic digestion of the a l g i n i c acid and c e l l u l o s e portions of the c e l l wall (3'). P i t connections are dissolved between c e l l s and the fungus appears to be capable of digesting the contents of several c e l l s while physically occupying only one (3"). 4. Lesions on Fucus become v i s i b l e , are pink-to-red in color, and are i n i t i a l l y firm, but become f l a c c i d with age. 5. Thick-walled chlamydospores formed inside c o r t i c a l and medullary filaments. 6. Sporangiophores push up between epidermal c e l l s , and form the lobate sporaangia of P. undulatum var. l i t o r a l e , reproducing the primary inoculum. B. Host Response 1. Hypersensitivity of host c e l l s in advance of fungal hyphae. Medullary filaments show an increase in general protein l e v e l s , coupled with the appearance- of numerous physodes, normally rare or absent in t h i s region. Physodes coalesce to form larger units, which are then believed to autolyse and release their phenolic materials into the matrix. Stress and tear l i n e s appear between c e l l s 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 c e l l s that function as epidermis once abscission has occurred. 80 81 c e l l s . Hyphae advance r a d i a l l y and a r e m a i n l y c o n f i n e d t o t h e c o r t i c a l and m e d u l l a r y c e l l s . P e n e t r a t i o n of i n d i v i d u a l c e l l s i s i n i t i a t e d by what a p e a r s t o be an e n z y m a t i c d i s s o l u t i o n o f t h e a l g i n i c a c i d and c e l l u l o s e components of t h e c e l l w a l l s . H a u s t o r i a o r m o d i f i e d a b s o r p t i o n s t r u c t u r e s a r e a b s e n t . By u t i l i z i n g i t s a b i l i t y t o d i g e s t c e l l w a l l s , t h e f u n g u s i s c a p a b l e o f p a r a s i t i z i n g s e v e r a l c e l l s v i a d i s s o l u t i o n of t h e p i t s , a l t h o u g h t h e f u n g a l f i l a m e n t may p h y s i c a l l y o c c u p y o n l y one c e l l . L o b a t e s p o r a n g i a , a r e formed o u t s i d e t h e t h a l l u s ; t h e s e r e l e a s e 30-60 z o o s p o r e s w h i c h r e c y c l e t h e i n f e c t o n . T h i c k - w a l l e d c h l a m y d o s p o r e s a r e formed i n c o r t i c a l and m e d u l l a r y c e l l s , but t h e f u n c t i o n of t h e s e i s unknown. A h y p e r s e n s i t i v i t y r e s p o n s e (HR) o c c u r s i n a d v a n c e o f t h e f u n g a l h yphae. In t h e f i r s t s t a g e , a l l c e l l t y p e s a r e i n v o l v e d and r a p i d l y n e c r o s e w i t h remnant c e l l c o n t e n t s s t a i n i n g f o r p h e n o l i c m a t e r i a l s , c o u p l e d w i t h a d i s a p p e a r a n c e o f m a t r i x m a t e r i a l . The s e c o n d s t a g e o f t h e HR i s c h a r a c t e r i z e d by an i n c r e a s e i n m e t a b o l i c a c t i v i t y , as e v i d e n c e d by i n c r e a s e d l e v e l o f g e n e r a l p r o t e i n s t a i n i n g and t h e a p p a r e n t p r o d u c t i o n of numerous p h y s o d e s i n t h e n o r m a l l y q u i e s c e n t m e d u l l a r y f i l a m e n t s . T h e s e p h y s o d e s c o a l e s c e t o form ' g i a n t ' p h y s o d e s , and a u t o l y s i s o c c u r s i n t h e s e c e l l s , w h i c h t h e n s t a i n h e a v i l y f o r amorphous p h e n o l i c m a t e r i a l s . Heavy d e p o s i t s of p h e n o l i c -r e a c t i v e m a t e r i a l a p p e a r i n t h e m a t r i x , w h i l e s t a i n i n g o f t h e m a t r i x f u c a n s i s l o s t . In t h e l a t e r s t a g e s o f HR, b o t h f u c a n m a t r i x a n d p h e n o l i c m a t e r i a l s - have d i s a p e a r e d , HR c e l l w a l l s have a f i b r o u s d i s t e n d e d a p p e a r a n c e and become s e p a r a t e d 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, c o r t i c a l and medullary filaments have undergone p r i n c i p a l l y transverse d i v i s i o n s to form i r r e g u l a r , cuboidal c e l l s that function as epidermal c e l l s once abscission has occurred. Pentration of Fucus epidermal c e l l s 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 F u l l e r , 1970) or of Phycomyces  blakesleeanus by Pythium acanthium (Hoch and F u l l e r , 1977) does not. The gap in PAS c e l l wall staining where hyphae cross c e l l walls has been interpretted in this study to be as a result of chemical dissolution of the c e l l wall during penetration. Similar non-staining c e l l wall 'halos' have been noted in penetration of barley epidermal c e l l s by Erysiphe graminis at both the l i g h t and electron microscope levels (McKeen e_t a l . , 1969; Edwards and Allen,1970). Kazama (1969) f e l t that penetration of Porphyra c e l l 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 i s accomplished by mechanical means. In the absence of correlated electron microscopic studies, t h i s report can only suggest that enzymatic digestion of Fucus c e l l walls i s occurring. Enzymes which participate in c e l l 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 c e l l u l o s i c f r a c t i o n s . 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 a c t i v i t y been adequately demonstrated in a fungus (Wainwright,1980). Based on the h i s t o l o g i c a l results, P. undulatum var. l i t o r a l e might be an ideal candidate for further examination of alginase a c t i v i t y in the fungi. The hypersensitivity and abscission response to infection seen in Fucus has not been reported previously for any marine alga. F u l l e r , et a_l. (1966) reported that in naturally infected Porphyra perforata , host c e l l s had altered pigmentation in advance of infecting hyphae of Pythium marinum . However, Kazama and F u l l e r (1970) found that in a r t i f i c i a l l y infected Porphyra plants t h i s reaction was never observed. Necrosis in advance of Pythium porphyrae in f e c t i n g other Porphyra species has never been observed (Fujita,pers. comm.). Muller (1959) defined the HR as encompassing a l l morphological and h i s t o l o g i c a l changes that, when produced by an infection agent, e l i c i t the premature dying off (necrosis) 84 of the infected tissue as well as inactivation and l o c a l i z a t i o n of the infectious agent. Certainly the necrosis observed d i s t a l to hyphal advance, the h i s t o l o g i c a l changes in phenol levels in both cortex and medulla, and the meristimatic a c t i v i t y producing a new epidermis by Fucus in response to inf e c t i o n , f a l l within t h i s d e f i n i t i o n . The HR response of Fucus i s similar to the reponse described as the 'shot-hole' syndrome in plant pathological l i t e r a t u r e . As exemplified by the response of Prunus spp. to infection with Cladosporium carpophilum, spread of the pathogen is halted r e l a t i v e l y l a t e , a hypersensitivity necrosis develops, and meristematic a c t i v i t y is induced d i s t a l to the necrosis bringing about the development of a concentric periderm for the seperation of necrotic tissue (Muller,1959). However, in t h i s HR, abscission i s 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 c e l l s and autodigestion of matrix fucans in the necrotic region, with the p o s s i b i l i t y of enzymatic degradation of c e l l walls. Alginate lyase a c t i v i t y has been demonstrated for several brown alg a l genera (Madgwick e_t al.,1973; Shiraiwa et al.,1975), and i t i s l i k e l y 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 f i b r i l l a r elements in the c e l l wall. Dissolution of middle lamellae is accepted as a c r u c i a l 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 c e l l s , 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 i n h i b i t o r s has been correlated with resistance to pathogenic organisms (Bateman and Basham, 1976). The role of polyphenolics in brown algae i s believed to be as a pre-formed biochemical defense agent against bacteria (Conover and Sieburth,1964), fungi (Khaleafa et al.,1975), e p i f l o r a (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 i t i s possible that the phenolic buildup in the HR of Fucus may serve to inactivate the fungal enzymes, checking the spread of hyphae. The f i n a l step in the hypersensitivity response is formation of the new epidermis. Divisions in the c o r t i c a l and medullary c e l l s 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 c e l l s 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 c e l l u l a r and e x t r a - c e l l u l a r materials. The Kohlmeyers (1979) have noted that d i f f i c u l t i e s 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. l i t o r a l e proved pathogenicity, f a i l u r e of the plants to develop an HR and abscission zone layer prevented unequivocal demonstration of Koch's Postulates. Physiological parameters ( l i g h t , temperature, mineral s a l t s , etc.) have an important influence on HR in higher plants (Addicott,1965), and i t i s l i k e l y that environmental physiology i s equally important for the HR in Fucus. While uninnoculated plants appeared healthy throughout the course of t h i s study, the devastating effect of infection in v i t r o indicates that conditions were probably less than optimal. In support of t h i s i s the reduction of physode content during the 30 day pre-infection incubation period. 87 CONCLUSION While any d e f i n i t e c o n c l u s i o n s , based on the l i m i t e d data of t h i s t h e s i s , are not j u s t i f i e d , a few o b s e r v a t i o n s r e g a r d i n g the work as a whole are warranted. The absence of Fucus i n the Squamish River estuary p r i o r to dike c o n s t r u c t i o n , combined with, the suggestion that Pythium  undulatum var. l i t o r a l e i s an indigenous member of the e s t u a r i n e sediment mycoflora, and with the absence of i n f e c t i o n o u t s i d e the e s t u a r y , suggest that a unique set of p h y s i o g r a p h i c a l parameters e x i s t w i t h i n the estuary that allow t h i s symbiosis to occur. The most probable p h y s i o l o g i c a l element to examine i s the change i n s a l i n i t y p a t t e r n s w i t h i n the estuary a f t e r dike c o n s t r u c t i o n . P e n e t r a t i o n of Fucus i n t o the e s t u a r y , up to the blocked channels of the r i v e r , demonstrates the. d r a s t i c a l t e r a t i o n s , i n s a l i n i t y that occurred with r i v e r t r a i n i n g . However, i t i s l i k e l y that the estuary i s a marginal h a b i t a t f o r Fucus, as s a l i n i t i e s are d i l u t e d by the freshwater outflow from the Squamish R i v e r . In the d i l u t e environment, metabolism in Fucus may be s u f f i c i e n t l y s t r e s s e d so as to cause a r e d u c t i o n i n l e v e l s of b i o c h e m i c a l defense agents capable of p r e v e n t i n g i n f e c t i o n . P. undulatum var. l i t o r a l e i s most l i k e l y s a l i n e -i n f l u e n c e d as to i t s d i s t r i b u t i o n . Both v e g e t a t i v e growth and asexual r e p r o d u c t i o n were i n h i b i t e d at higher s a l i n i t i e s i n 88 laboratory cultures. Not finding P. undulatum var. l i t o r a l e outside of the estuarine environment further suggests that the fungus is saline limited. Thus, within the Squamish River estuary, a c l a s s i c a l triangular interaction of host, parasite, and environment seems to e x i s t . If s a l i n i t y i s the factor to be examined, one could expect that during winter, when s a l i n i t i e s are higher in the estuary, the incidence of disease would be reduced, whereas during the spring freshet of the Squamish River s a l i n i t i e s would be reduced and disease incidence would increase. Monthly sampling of s a l i n i t i e s and infected plans could provide some interesting answers regarding the influence of s a l i n i t y on disease development. Laboratory examination of infection under standardized conditions ( l i g h t , temperature, nutrients, etc.), while varying s a l i n i t y , would also be useful. The observations made on the infection process are limited in the abscence of correlated electron microscope data. Additional data on c e l l wall dissolution by hyphae, formation of the 'giant' physodes, and a l l factors r e l a t i n g to development of the hypersensitive reaction are necessary to support the interpretations given in thi s t h e s i s . 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 lim i t a t i o n s 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 i n f e c t i o n . This study notes that the infection of Fucus i s the f i r s t 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 o r i g i n of these species is t e r r e s t r i a l . 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Mangrove fungi: the succession in the mycoflora of red mangrove ( Rhizophora mangle L.) seedlings, in "Recent Advances in Aquatic Mycology (ed E.B.G. JonesT. pp 51-91. Wiley. New York. 98 Nbvotny, A.M., and M. Forman. 1975. The composition and development of c e l l walls of Fucus embryos. Planta 122: 67-78. Parker, B.C., and A.G. D i b o l l . 1966. Alcian stains for histochemical l o c a l i z a t i o n of acid and sulfated polysaccharides in algae. Phycologia 6:37-46. Percival,E., and R.H. McDowell. 1967. "Chemistry and Enzymology of Marine Algal Polysaccharides". Academic Press, London and New York. 219 pp. Petersen, H.E. 1909. Studier over Ferskvands-Phycomyceter. Bot. Tiddskr. 29:1-440. . 1910. An account of Danish freshwater Phycomycetes, with b i o l o g i c a l and systematical remarks. Ann. Mycologici 8:494-560. Pomeroy, W.M., and J.G. Stockner. 1976. Effects of environmental disturbance on the d i s t r i b u t i o n and primary production of benthic algae in a B r i t i s h Columbia estuary. J. Fish. Res. Board Canada 33:1175-1187. Preis, J., and G. Ashwell. 1962. A l g i n i c acid metabolism in bacteria. J. B i o l . Chem. 237:309-316. Quantro, R.S., and P.T. Stevens. 1976. C e l l wall assembly in Fucus zygotes. I . Characterization of the- polysaccharide components. Plant Physiol. 58:224-231. Ragan, M.A. 1976. Physodes and the phenolic compounds of brown algae. Composition and significance of physodes _in vivo . Bot. Mar. 19:145-154. , and J.S. Cragie. 1976. Physodes and the phenolic compounds of brown algae. Can. J . Biochem. 54:66-73. Robertson, G.I. 1980. The genus Pythium in New Zeland. New Zeland J. Bot. 18:73-102. 99 Schmitthenner, A.F. 1970. Significance of populations of Pythium and Phytophthora in s o i l s , in "Root Diseases and Soil-Borne Pathogens" (eds., T.H. Toussoun, R.V. Bega, and P.E. Nelson). UC Berkely Press. 252 pp. Scott, W.W. 1962. The aquatic phycomycetous f l o r a of marine and brackish waters in the v i c i n i t y of Gloucester Point, V i r g i n i a . V i r g . Inst. Mar. S c i . Special Report No. 36. 16 pp. Shen, S.C. and W.N. Siang. 1948. Studies in the aquatic Phycomycetes of China. S c i . Repts. Nat. Tsing Hua Univ., Ser. B: B i o l . and Psychol S c i . 3:179-203. Shiraiwa, Y. , K. Abe, S..F. Sasaki, T. Ikawa, and K. N i s i z a . 197'5. Alginate lkyase a c t i v i t i e s in the extracts from several brown algae. Bot. Mar. 18:97-104. Sieburth, J.Mc, and J.T. Conover. 1965. Sargassum tannin, an a n t i b i o t i c which retards fouling. Nature (London) 208:52-53. Siepman, R. 1959. Ein Beitrag zur saprophytischen P i l z f l o r a . d e s Wattes der Wesermundung. I. Systematischer T e i l . Verhoff Inst. Meersforsch. Bremerhaven 6:213-281. Sparrow, F.K. 1932. Observations on the aquatic fungi of Cold Spring Harbor. Mycologia 24:268-303. . 1934. Observations on marine phycomycetes col l e c t e d in Denmark. Dansk. Bot. Ark. 8:1-24. . 1936. A contribution to our knowledge of the aquatic phycomycetes of Great Britan. J. Linn. Soc. London (Bot) 1v(50,417-478) . 1952. A contribution to our knowledge pf the phycomycetes of Cuba. I I I . Res. Soc. Cubana Bot. 9:104-108. 100 . 1960. "Aquatic Phycomycetes". University of Michigan Press. Ann Arbor. 1187 pp. Spencer, J.A., and W.E. Cooper. 1967. Pathogenisis of cotton ( Gossypium hirsutum ) by Pythium spp.: Zoospore and mycelium att r a c t i o n and i n f e c t i v i t y . Phytopath. 57:1332-1338. Spicer, S.S. 1960. A c o r r e l a t i v e study of the histochemical properties of rodent acid mucopolysaccharides. J. Histochem. and Cytochem. 8:18-35. Squamish River Estuary Management Plan. Air and Water Quality Work Group Final Report. 1981. Co-published by the Government of Canada and the Province of B r i t i s h Columbia. 261 pp. . Habitat Work Group F i n a l Report. 1981. Government of Canada and Province of B r i t i s h Columbia. 159 pp. Sutherland, G.K. 1915a. New marine fungi on P e l v i t i a . New Phytol. J_4:33-42. . 1915b. Additional notes on marine Pyrenomycetes. New Phytol. 14:183-193. . 1916. Marine Fungi Imperfecti. New Phytol. 15: 35-48. Takeuchi, Y., and A. Komamine. 1978. Composition of the c e l l wall formed by protoplasts isolated from c e l l suspension cultures of Vinca rosea . Planta 140:227-232. Wainwright, M. 1980. Alginate degredation by the marine fungus Dendryphiella salina . Mar. B i o l . Let. _1_:35l-354. Walker, D. 1980. "Sorus Abscission From Laminae of Nerocystis  lutkeana (Mert.) Post, and Rup.". PhD. thesis, Dept. of Botany, Univ. of B.C. 463 pp. 101 , G.C. Hughes, and T. Bisalputra. 1979. A new interpretation of the i n t e r f a c i a l zone between Spathulosp.ora (Ascomycetes) and Balli.a (Florideophyceae) . Trans. Br. mycol. Soc. 73:193-206. Waterhouse, G.M. 1967. "Key to Pythium Pringsheim". Mycol. Paper. CMI 109. 15 pp. Whyte, J.N.C., J.R. Engler, and P.E. Borgman. 1981. Compositional changes on freshwater leaching of the marine algae Nereocystis lutkeana and Macrocystis  i n t e g r i f o l i a . Can. J. Fish. Aquat. S c i . 38:193-198. Wolf, F.T. 1944. "The Aquatic Oomycetes of Wisconsin. Part 1". Univ. of Wisconsin Press, Madison. 64 pp. Yaphe, W., and K. Morgan. 1959. Enzymatic hydrolysis of fucoidin by Psuedomonas a t l a n t i c a and Pseudomonas  carrageenovora . Nature (Lond.) 183:761. 1 02 APPENDIX-1: FUNGAL AND ALGAL MEDIA EMPLOYED I . Fungal Media (Dr. Y. F u j i t a , pers. comm.) Fuj i t a ' s Seawater Agar Materials fresh corn kernels 20 gm d i s t i l l e d water 500' ml seawater 500 ml agar 20 gm Procedure B o i l corn in DW for 30 m. F i l t e r , add seawater. Bring f i n a l volume to 1 l i t e r with DW. . Adjust the pH to 7.3-7.5, add agar, autoclave. Comments Dr. F u j i t a has found t h i s 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 l e v e l of sexual reproduction as Schmitthener's agar. G a l l i c Ac id Medium (Flowers and Hendrix, 1969) Materials sucrose 30.0 gm 1 03 NaNO 3 2.0 MgSO, 7H20 0.5 KH2POa 1.0 yeast extract 0.5 g a l l i c acid 425 mg rose bengal 0.5 mg pentachloronitrobenzene (PCNB) 25.0 mg P e n i c i l l i n G 80,000 units nystatin 100,000 units agar 20 gm d i s t i l l e d water 1 1 Comments G a l l i c acid, rose bengal, PCNB, Pen-G and nystatin were added a s e p t i c a l l y to the autoclaved, cooled (58-60 C) nutrient agar. Schmitthener's Agar (Robertson, 1980) Mater i a l s sucrose 2.5 gm asparagine 0.27 KH 2PO„ 0.15 K 2HPO„ 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 i s 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 a s e p t i c a l l y to autoclaved, cooled (58-60 C) ShmA (minus the sucrose) or water agar medium made with 50 or 100% seawater. Comments The use of Ge0 2 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 i s o l a t i o n 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 K 2HPO„ 1.0 MgSO« 0.5 agar 20 II. Algal Media A r t i f i c i a l Seawater Medium (after Harrison et a l . , 1980) Nutrient and trace metals stock solution NaN03 4.7 g K 2HPO„ 0.1 Na2EDTA 0.55 Fe(NH») 2 (SO«) 2 6H20 0.23 FeSO„ 0.02 ZnSO, 7H20 0.008 MnSO„ 4H20 0.054 CoCL 2 6H20 0.002 d i s t i l l e d water 1 l i t e r Vitamin Stock solution Thiamine HCl 0.1 g B-12 0.002 Biot i n 0.001 d i s t i l l e d water 1 l i t e r To make medium, add 10 mis of nutrient solution and 1 ml of vitamin solution to 1 l i t e r of 'Instant Ocean'. 1 06 Comments Na 2Si0 3 9H,20 and H 3B0 3 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% g l a c i a l acetic acid 7% aqueous g l a c i a l acetic acid Procedure Stain 1.5-2.On sections at 55 C for 10 m. B r i e f l y dip s l i d e into 7% acetic acid to remove excess s t a i n . Wash in tap water, a i r dry, mount. Comments PAS i s an excellent co-stain with ABB,-»but must be done before staining with ABB. Saf-0 in 7% acetic acid i s also an excellent counter-stain for ABB (see procedure l i s t e d under Fast Green stain for Saf-0 sta i n i n g ) . Acridine Orange (Cooke, 1977) Materials 0.1% Acridine Orange in 1N HCl Procedure Adjust pH of stain to 0.5 i f necessary. Stain 1.5-2.On sections for 1.5 minutes. Dry, mount, and observe with a fluorescent microscope. Comments At t h i s pH only sulfate groups are ionized, making the 108 stain s p e c i f i c for fucans. Cooke (1977) reports that the staining s p e c i f i c i t y l a s t s only two hours after mounting. Alcian Blue (Parker and D i b o l l , 1966) Materials 0.5% alcian blue in 1N HCl Procedure Stain 1.5-2.On sections for 30-60 m. Wash b r i e f l y in DW with pH adjusted to 0.5". Wash well in DW. Comments At th i s pH, only sulfate groups are ionized, and this test may be in conjunction with Acridine Orange to iden t i f y fucans. Parker and DiB o l l report that i f sections are counterstained with Alcian Yellow (pH=2.5), carboxylated polysaccharides w i l l stain yellow and be eas i l y d i f f e r e n t i a t e d 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 a l . , 1981) Materials 1% CaCl 2 in aqueous solution Procedure Extract sections for 1 h at 60 C. Comments Caution must be exercised as the p l a s t i c sections become extremely soft at the elevated temperature. Allow sections to cool completely in the solution before removal for staining. E r l i c h ' 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 d i r e c t l y or embbed in p l a s t i c . Comments Tissue tends to become macerated i f l e f t 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 c a l l e d for an overspray with Na 2C0 3, 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 a c i d i f i e d 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 i s a good general protein stain that has the same s p e c i f i c i t y as ABB. Fungal hyphae, along with plant nuclei, p l a s t i d s , and cytoplasmic proteins are v i s i b l y demonstrated with t h i s s t a i n . Staining beyond the 1 m mark with Saf-0 results in removal of FG. FeCl 3 Procedure (Cragie and McLachlan, 1964) Materials 2% F e C l 3 in DW Procedure Place fresh (unembedded) thallus pieces in fresh F e C l 3 solution for 2-3" hours. Cut on microtome and observe. Comments 111 Specimens embedded to methacrylate after staining did not retain the nuclear or p l a s t i d coloration. Grocott's Silver-Methenamine Stain (Hibbitts, pers. comm.) Materials . 4% chromic acid or 1% periodic acid 5% aqueous s i l v e r n i t r a t e 3% aqueous hexamethylenetetramine 5% sodium borate 1% sodium b i s u l f i t e 1% sodium t h i o s u l f i t e 1% gold chloride Procedure 1. ) Oxidize specimens in chromic acid or periodic acid for 1 h. 2. ) Rinse, then place in sodium b i s u l f i t e solution for 1 m, followed by an additional rinse. 3. ) Stain in freshly mixed methenamine-silver nitr a t e solution (see below) at 58-60 C for up to 100 m. This part i s subjective and each group of s l i d e needs to be checked to see when they need to be taken out. 4. ) Allow solution and s l i d e to cool to 20 C before removing sections from s t a i n . Failure to do so may result in severe wrinkling of the soft, heated p l a s t i c 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 d i f f i c u l t . ABB, Fast Green, Saf-O, and TBO did not e f f e c t i v e l y penetrate the p l a s t i c to color tissue after application of GMS. Methenamine-Silver Nitrate solution Methenamine solution 100 ml Si l v e r n i t r a t e solution 5 ml ( s t i r u n t i l p r e c i p i t a t e dissolves) D i s t i l l e d Water 100 ml Borax solution 8 ml Na 2C0 3 extraction of Al g i n i c Acid (Parker and D i b o l l , 1966) Materials 1% aqueous Na 2C0 3 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 i a l s 1% Periodic Acid solution (freshly made) Sc h i f f ' s Reagent (see below) 0.5% Na 2S 20„ 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 S c h i f f ' s reagent for 10 minutes. Follow S c h i f f ' s stain by three quick dips in Na 2S 2Oii. Wash in cold tap water. Comments PAS staining must be proceded by blocking residual aldehyde groups remaining from aldehyde f i x a t i o n . See Feder and O'Brien (1968) for procedures of aldehyde blocking for methacrylate thin-sections. An excellent counter-stain for PAS i s ABB. However, the PAS precedure must procede protein staining. S c h i f f ' s Reagent Bring 100 ml DW to a b o i l . Remove from heat and immediately dissolve 1 g Basic Fuchsin. Allow t h i s solution to cool to 60 C, f i l t e r , and add 2 g sodium meta-bisulate and 20 ml 1N HCl. [ NOTE: This reaction release C l 2 gas; perform t h i s 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 f i l t e r i n g . 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 a c i d i f i e d DW. Comments These sections are best viewed immediately, as a i r drying tends to allow some loss of metachromasia. If the sections have been a i r 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% v a n i l i n 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 i s subjective, so i t s best to monitor the material c l o s e l y as too long an exposure to the acid macerates tissue beyond recognition. 

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