OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XI. A PHYSODERMA ON AGROPYRON REPENS

Sparrow , Frederick K. (U. Michigan, Ann Arbor), Joyce E. Griffin , and Robert M. Johns . Observations on chytridiaceous parasites of phanerogams. XI. A Physoderma on Agropyron repens. Amer. Jour. Bot. 48(9): 850–858. Illus. 1961.—A Physoderma has been found in Michigan on Agropyron repens, which produces dark-brown, non-erumpent streaks up to 20 mm (usually 4–6 mm) in length by 0.5 mm in width and very local discoloration of the leaf blades and sheaths. There is no systemic infection, and plants parasitized are normal in size and bear flowers. Resting spores upon germination produce zoospores which may develop either into the endobiotic, poly-centric, resting spore-bearing thallus or into monocentric, epibiotic, ephemeral sporangia. The latter bear either colorless or orange globuled zoospores. These zoospores may give rise to new epibiotic sporangia, or, apparently without fusing in pairs, to the endobiotic system. Comparison with other congeneric fungi reported on Agropyron repens reveals the Michigan fungus to be distinct from them. Whether it can be embraced within Physoderma graminis (Büsgen) de Wild. or other described taxa on grasses awaits the results of future cross-inoculation studies.     

Observations on chytridiaceous parasites of Phanerogams

Summary A reexamination of Physoderma maculare Wallroth the type species of the genus, on Alisma, confirms Clinton's (1902) account of the production of an epibiotic stage from resting spore zoospores. The latter on mature host tissue may also give rise to the endobiotic stage which bears resting spores. On seedlings, however, resting spore zoospores produce only epibiotic sporangia. The fungus could not successfully infect Sagittaria, a closely related host.Contribution No. 1152     

THE DEVELOPMENT AND CYTOLOGY OF THE EPIBIOTIC PHASE OF PHYSODERMA PULPOSUM

Lingappa , Yamuna . (U. Michigan, Ann Arbor.) The development and cytology of the epibiotic phase of Physoderma pulposum. Amer. Jour. Bot. 46(3) : 145-150. Illus. 1959.—Physoderma pulposum, a chytrid parasite on Chenopodium album L. and Atriplex patula L., has a zoosporangial epibiotic phase. The latter consists of extramatrical sporangia and intramatrical bushy rhizoids, both enclosed in large protruding galls. The sporangia are subspherical, up to 350μ in diameter, and may produce hundreds of planospores. If planospores settle on the host surface, they develop narrow germ tubes which penetrate the epidermal cells and develop into rhizoids. The planospore body, however, remains on the host surface and develops into a mature epibiotic sporangium in about 20-25 days at 16°C., 12-15 days at 20-25°C., or 6-8 days at 30°C. During development, its nucleus and daughter nuclei divide mitotically with intranuclear spindles until the sporangium contains several hundred nuclei. This is followed by progressive cleavage which delimits the planospore rudiments. When mature sporangia are placed in fresh water, the planospores are quickly formed within 1 hr. at 25°C. and begin to swarm within the sporangia. They escape in large numbers through an opening formed by the deliquescence of a papillum in the sporangial wall. The planospores are subspherical or elongate, 3-5 × 4-6 μ, and each has an eccentric orange-yellow refractive globule and a flagellum 18-22 μ in length. The electron micrographs of the flagella indicate that the flagella are absorbed from tip backward during encystment of the planospores. By periodic inoculation of the host plants with planospores from epibiotic sporangia, as well as from germinating resting sporangia, generation after generation of epibiotic sporangia have been obtained for 4 years. This proves the existence of a eucarpic, epibiotic, ephemeral zoosporangial phase in P. pulposum. Field observations on the duration and sequence of development of the fungus indicate that the endobiotic resting sporangial phase always follows the epibiotic phase. The results of infection experiments also indicate that the epi- and endobiotic phases belong to one and the same fungus, P. pulposum.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. VIII. UROPHLYCTIS (PHYSODERMA) PLURIANNULATUS AND U. MAJUS

Sparrow , Frederick K. and Yamunga Lingappa . (U. Michigan, Ann Arbor.) Observations on chytridiaceous parasites of phanerogams. VIII. Urophlyctis (Physoderma) pluriannnlatus and U. majus. Amer. Jour. Bot. 47(3): 202—209. Illus. 1960.—Urophlyctis pluriannulatus, an obligate parasite of Sanicula spp., has an endobiotic phase which is strongly polycentric and produces small crateriform galls on the petioles and blades of the host leaves. The agent accomplishing infection is not known but is probably a zygote. The first cell of the parasite established in the host is the so-called “primary turbinate organ.” This becomes multinucleate, is somewhat pyriform and becomes multicellular by 2 methods: (1) by cleavage into peripheral segments; or (2) by division into cells, each with its own cell wall. Replication of the thallus is accomplished by the production of nucleated outgrowths bearing haustoria which elongate, become ribbon-like, somewhat roughened and lumened, and produce distally turbinate organs of a second order. Tertiary, etc. turbinate organs are produced in like manner. Resting spores usually form at the tip of an extremely short outgrowth from the apex of a turbinate organ. These bear a supra-equatorial crown of 7—10 branched haustoria. Rarely, monocentric thalli are formed, in which a single turbinate organ becomes converted into a resting spore. All nuclear division figures were intranuclear. The fungus produced marked enlargement of infected host cells and their nuclei, and caused division of neighboring cells. As development continues, lysis of the surrounding host walls takes place and a large cavity bearing a dense symplast and numerous host nuclei is formed, within which is the thallus of the parasite. At maturity, all traces of symplast and of fungus, except for resting spores, disappear. Urophlyctis majus, a parasite on leaves of Rumex orbiculatus, hitherto known only from its resting spore stage, has a pattern of development strikingly similar to that of U. pluriannulatus. Here, however, turbinate cells only form peripheral segments. Furthermore, the “hyphae” are smooth and without a lumen. Aside from size differences, the mature thallus with resting spores, unbranched (not branched) haustorial tufts, etc. is like that of the Sanicula parasite. The galls produced were compartmentalized, dark red to black, usually surrounded by a reddish zone, and early dropped from the leaf. No undoubted evidence of the epibiotic gametangial phase was found in either species.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XXVII. A FURTHER STUDY OF PHYSODERMA HYDROCOTYLIDIS VIÉGAS AND TEIXEIRA

A further study of Physoderma hydrocotylidis from California on its host Hydrocotyle ranunculoides gives further details on its structure and development. EM micrographs reveal that the notably thin wall of the resting spore consists of an outer, an intermediate, and an inner layer. The complete wall thickness from measurements of these EM preparations is only 0.5 μm, the thinnest of any known Physoderma resting spore. Such resting spores germinate readily in 2–5 h by the dehiscence of a broad cap and formation of a protruding endosporganium. Zoospores from the latter infect epidermal host cells and produce either the endobiotic, polycentric rhizoidal system with tenuous filaments, turbinate cells and eventually resting spores, or a hitherto unknown epibiotic, monocentric, rhizidiaceous sporangial stage. Zoospores from the latter may all bear colorless lipid globules or all faintly orange-colored ones.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XIV. PHYSODERMA CALAMI

Physoderma calami, a chytrid (Phycomycetes), has not been studied or apparently even collected in Europe since 1895 and has never been recorded in this hemisphere. Material widely distributed in herbaria indicated it was a Physoderma but no details save color, shape, and size of resting spores were known. A study of living material from Vermont indicates resting spore germination occurs by dehiscence of a lid and production of a sporangium with zoospores. The latter may form either an epibiotic or “ephemeral” sporangial stage and zoospores, or an endobiotic one, with extensive polycentric thallus bearing turbinate cells along the rhizoids, and, as outgrowths of these turbinate cells, straw-colored, thick-walled resting spores. The latter stage produces dark-brown spots on infected parts of the host.     

MORPHOLOGICAL AND ECOLOGICAL STUDY OF PHYSODERMA DULICHII

A new species of aquatic Phycomycete, Physoderma dulichii Johns, parasitic on the aquatic sedge Dulichium arundinaceum (L.) Britt., is described from northern Michigan. This parasite infects and kills the upper epidermal cells of the host leaves. Macroscopically, infection by P. dulichii is indicated by striking brown bands with irregular margins, at intervals on the upper surfaces of the leaves. Like other species of Physoderma, this organism's development includes two distinct phases, an epibiotic monocentric phase producing asexual zoospores and an endobiotic polycentric phase bearing thick-walled resting spores that germinate after an extensive period of maturation at low temperature to form zoospores. The morphology and development of the two phases and of resting spore germination are reported in detail. Only the immature leaves of the host are susceptible to infection, which may be initiated by the introduction of mature resting spores, zoospores from germinated resting spores, or zoospores from epibiotic sporangia. Resting-spore zoospores may also produce the endobiotic stage directly. Initiation of infection in nature requires that the terminal cluster of immature leaves on the host plant be submerged, but infection of subsequently formed leaves of emergent culms can be accomplished through the agency of zoospores from epibiotic sporangia on older leaves. The relation of infected stands of hosts to their environment is discussed and the importance of standing water to infection noted. The geographical distribution of the parasite shows correlation with the drainage basins of the Great Lakes, the St. Lawrence River, and the northern Atlantic Coastal Plain     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XXV. PHYSODERMA JOHNSII SPARROW,A PARASITE OF CALTHA PALUSTRIS L.

The morphology of the endobiotic and epibiotic stages of Physoderma johnsii Sparrow on Caltha palustris is described. Highly characteristic of the endobiotic stage is the formation of numbers of large, narrowly pyriform cells with a tuft of rhizoids at the broader (distal) end. Early developmental features are not included since germination of the resting spore has not as yet been achieved. Reasons for maintaining this taxon distinct from older ones on Caltha palustris are given.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XX. RESTING SPORE GERMINATION AND EPIBIOTIC STAGE OF PHYSODERMA BUTOMI SCHROETER

The development and morphology of Physoderma butomi Schroeter on Butomus umbellalus L., found in Michigan, is followed from germination of the resting spore through formation of epibiotic and endobiotic stages, to resting spore. No evidence for sexuality was found.     

The endobiotic thallus ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The endobiotic thallus ofPhysoderma maydis is characterized by the presence of an extremely fine rhizomycelium which passes through the host cell wall, allowing the spread of the disease, and irregularly shaped turbinate cells, which may be septate or nonseptate and which are in close association with developing resting sporangia. The formation of the resting sporangium wall is first seen as localized depositions on the rounded surface of the sporangium and only later on the flattened surface of the sporangium which will form the operculum. The substructure of the resting sporangium wall is typical for members of theBlastocladiales. The resting sporangium is contiguous with the rhizomycelium during development and is finally sealed-off from the rhizomycelium by a further deposition of wall material. After the sealing-off of the resting sporangium from the rhizomycelium the content of the sporangium is compartmentalized and the two inner wall layers are deposited. The centre of the sporangium is filled with an electron dense accretion. At the periphery of the sporangium is a layer of lipid bodies. Between the lipid bodies and the central electron dense accretion is a thin layer of cytoplasm which contains the nuclei. The outer surface of the resting sporangium is smooth.     

Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

Early Devonian fungi: a blastocladalean fungus with sexual reproduction

In this paper we describe a fossil fungus–Paleoblastocladia milleri gen. et sp. nov.–from the 400 million-year-old Early Devonian Rhynie chert that shares numerous features with modern zoosporic fungi placed in the order Blastocladiales. The fungus occurs in tufts that arise from stomata or between the cuticle and epidermis of Aglaophyton major axes. Thallus development begins from an irregular bipolar basal cell that produces a system of intramatrical rhizoids and clavate-shaped extramatrical, nonseptate hyphae. These hyphae develop into two types of mature thalli. Sporothalli are characterized by several orders of dichotomous branching and the production of terminal, globose zoosporangia, as well as thick-walled, pitted resting sporangia. On separate dichotomously branched thalli (gametothalli) are terminal chains of two or three gametangia, in which the terminal one is slightly larger. Despite the fact that all of the reproductive organs contain either zoospores or gametes, none show evidence of discharge papillae. The fossil fungus is compared with extant members of the Blastocladiales, and the presence of sexual reproduction is discussed.     

OBSERVATIONS ON THE LIFE HISTORY OF PAPENFUSSIELLA CALLITRICHA (PHAEOPHYCEAE,CHORDARIALES) IN CULTURE1

Papenfussiella callitricha (Rosenv.) Kylin from eastern Canada was studied in culture. Zoids from unilocular sporangia develop into microscopic, filamentous, dioecious gametophytes which produce isogametes in filament cells and few-chambered plurilocular gametangia. Unfused gametes germinate to reproduce the gametophytes. Fusion takes place between a settled (“female”) and a motile (“male”) gamete. The zygote gives rise to a filamentous plethysmothallus that reproduces asexually by zoids formed in thallus cells and in few-chambered plurilocular zoidangia. Erect macrothalli are produced on the plethysmothallus, beginning with the formation of upright filaments. Later on, these filaments become the terminal assimilators of the macrothalli. Further assimilatory filaments, rhizoids, and unilocular sporangia are produced in a branching region at the base of the terminal assimilator. Zoids from unilocular sporangia formed in culture germinate to reestablish the gametophyte phase. Chromosome counts yielded n = 19 ± 3 for the gametophytes, and 32 ± 6 for the sporophyte, both plethysmothallus and macrothallus.     

MORPHOLOGY AND LIFE CYCLE OF A NEW CHYTRID WITH AERIAL SPORANGIA

An investigation of the life cycle of Caulochytrium gloeosporii Voos and Olive, a parasite of Gloeosporium, has confirmed the existence of an alternation of generations. Zoospores from sessile sporangia may develop vegetatively into similar sporangia or they may function as isogametes, fusing in pairs to produce cysts (zygotes) that give rise to slender-stalked aerial sporangia. Meiosis is believed to occur in the aerial sporangium, which at germination liberates eight asexual zoospores. The species is homothallic.     

Observations on chytridiaceous parasites of phanerogams

Summary Resting spores of the chytrid parasite of Claytonia virginica or Spring Beauty, Physoderma claytonianum var. sparrowii, were overwintered in plastic containers buried at the site of collection near Ann Arbor, Mich., USA. Germination took place in the field in early March. The process was followed in the laboratory and involved dehiscence of an operculum, protrusion of an endosporangium, zoospore formation and discharge. Resting spore zoospores were repeatedly placed in contact with young Claytonia plants but there was no evidence that they ever developed into epibiotic, ephemeral sporangia. Rather, in all instances within 36 hours the endobiotic, polycentric thallus was established inside the host. Both observational evidence and the speed with which the endobiotic thallus first appeared pointed to the complete lack of an epibiotic stage in our material.Paper No. 1131, Department of Botany, University of Michigan. Acknowledgement is made to the National Science Foundation for financial support of this project.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XIX. A PHYSODERMA ON EURASIAN WATER MILFOIL (MYRIOPYLLUM SPICATUM)

A Physoderma was found on Myriophyllum spicatum in Michigan. This is the first recorded on this host from this hemisphere. It differs from the Danish P. myriophylli Rostrup in not producing galls on the host and in several aspects of its endobiotic stage. It more closely resembles a Swedish fungus attributed to Physoderma hippuridis by Lagerheim occurring on the same species of host.     

Cytology of Thamnidium elegans link

The resting nuclei in hyphae, sporangiophores and sporangiospores of sporangia and sporangiola of Thamnidium elegans consist of a large centrals nucleolus and a shell of chromatin surrounding the nucleolus. Division of the nucleus in hyphae and sporangiospores is achieved by elongation and constriction.     

On Chytridium braun,Diplochytridium N. G., and Canteria N. G. (Chytridiales)

Summary The operculate genus Chytridium Braun is emended and restricted to approximately 25 known species and several incompletely known members which lack an apophysis and develop much like species of Rhizophydium, insofar as the zoosporangium and absorbing or rhizoidal system are concerned, and form endobiotic or intramatrical resting spores. This interpretation corresponds fairly closely to Braun's diagnosis of the type species.A new genus, Diplochytridium, is established to segregate the former Chytridium species with an endobiotic or intramatrical apophysis or prosporangium and resting spores, which may develop sexually or asexually. Some of these species have a well-defined endo-exogenous alternation of growth and development in which the apophysis appears to function as a prosporangium. As interpreted here, Diplochytridium includes approximately 20 of the known species.Another new genus, Canteria, is created for a parasite of Mougeotia which Canter first described as a species of Phlyctidium but later found to develop endobiotic resting spores or zygospores by the fusion of conjugation tubes.     

Observations on chytridiaceous parasites of phanerogams

Summary A study of the morphology and life cycle of two Physoderma taxa occurring on Eleocharis spp. in Michigan is presented in detail. Both fungi possess almost identical endobiotic and epibiotic stages. They differ significantly with respect to the ease of germination of the resting spores, those on E. compressa being difficult to germinate whereas those from Eleocharis sp. from the vicinity of Ann Arbor do so readily.Resting spore stages of other collections, American, Polynesian and European, including the type of Physoderma heleocharidis (Fuckel) Schroeter, when compared, do not differ significantly from one another. P. schroeteri Krieger described from Scirpus is hardly distinguishable from P. heleocharidis on resting spore stage alone. Furthermore, we have successfully produced mature thalli and resting spores of the Physoderma on Eleocharis from Ann Arbor on Scirpus actus. Other such successful cross incculations will be needed before we will say with certainty P. schroeteri is not distinct from P. heleocharidis.Enough similarity is to be found in all the fungi on Eleocharis to place them for now, at least, in a single taxon, Physoderma heleocharidis. Future work with living material will no doubt necessitate a different treatment. An expanded technical diagnosis of P. heleocharidis based on our studies is included.Contribution No. 1143 from the Botany Department, and from the Biological Station, and Botanical Gardens, University of Michigan.Deceased, 1963.     

DEVELOPMENT OF ENDOPHYTIC FRANKIA SPORANGIA IN FIELD- AND LABORATORY-GROWN NODULES OF COMPTONIA PEREGRINA AND MYRICA GALE

Field-collected nodules of Comptonia peregrina (L.) Coult. and Myrica gale L. (Myricaceae), infected by the nitrogen-fixing actinomycete Frankia sp., were of two types: those that lacked sporangia entirely, designated spore(-), and those that showed extensive sporangial development, designated spore(+). In spore(+) nodules of C. peregrina, sporangia began to develop after the differentiation of endophytic vesicles and the concomitant onset of nitrogenase activity. At the onset of sporangial differentiation, infected host cells appeared healthy. However, endophytic vesicles and host cell cytoplasm and nuclei began to senesce rapidly as sporangia developed. Staining of sectioned material with the fluorescent stain Calcofluor White suggested that vesicles, hyphae and young sporangia were enclosed within a host-derived encapsulation layer, but mature sporangia were no longer encapsulated. In both C. peregrina and M. gale, vesicles were more short-lived in spore(+) than in spore(-) nodules. Field-collected spore(+) M. gale nodules exhibited a pronounced seasonality of sporangial formation. Sporangia began to differentiate in June, after the formation of vesicles and became more prominent in late summer. Inter- and intraspecific cross-inoculations suggest that the ability to form sporangia in the symbiotic state is controlled by endophytic strain type rather than host genotype or host/endophyte combination. The host may, however, influence the number and seasonal appearance of sporangia formed.