Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence from a Phylogenetic Analysis of Internal Transcribed Spacer Sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon.Saprolegniaemerges as the most basal genus, sister toAchlya, Thraustotheca,andDictyuchus. AchlyaandThraustothecaare most closely related, whileDictyuchusappears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited bySaprolegniarepresents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall.     

Water stress and sporangial emptying in Achlya (Saprolegniaceae)

Time-course experimenls demonstrate that sporangial emptying in Achlya inlricata is inhibited by experimental depression of the external osmotic potential. The sporangial wall is a macromolecular sieve, impermeable to polyethylene glycols (PEGs) with molecular weight 3350. The process of emptying can be precisely manipulated using PEG-8000 which suggests that spore discharge in an aqueous environment is effected by a reduction in the osmotic potential of the sporangial lumen relative to the external solution.     

北京地区水霉科真菌季节性分布

本文报道北京地区水霉科(Saprolegniaceae)真菌季节性分布的调查、研究结果,发现影响水霉季节性分布的主要因素是水温的季节性变化。不同属、种具不同的季节性分布,而种的季节性分布与它们卵孢子的结构有关。     

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.     

Growth of Certain Aquatic Omycetes on Amino Acids I. Saprolegnia, Achlya, Leptolegnia, and Dictyuchus

Four fungi in the order Saprolegniales —Saprolegnia sp., Achlya ambisexualis, Leptolegnia eccentrica and Dictyuchus sterile– were tested for growth in synthetic media containing one of the following carbon sources: glucose, maltose, sucrose, alanine, proline, glutamate, leucine, arginine and phenylalanine. All of these compounds were effective substrates for one or more of the four fungi. The ability of Saprolegnia sp. to utilize other substrates was studied. Saprolegnia sp. can metabolize soluble starch, fructose, ornithine, aspartate, serine, and lysine but did not grow on galactose and eight additional amino acids.     

Effect of the fungicide chlorothalonil (Bravo) on some metabolic activities of aquatic fungi

Physiological responses ofAchlya proliferoides, Saprolegnia ferax andDictyuchus sterilis as affected by the fungicide chlorothalonil (Bravo) were determined. Glucose consumption differed in dependence on the organisms used. Ammonia and peptide nitrogen secretion were stimulated inS. ferax but inhibited in the other two organisms. All doses of the fungicide used decreased phosphorus absorption and increased acid phosphatase activity. The lowest concentrations (30 ppm) of the fungicide increased DNA, RNA and protein synthesis while inhibition was observed at moderate or high concentrations. Aspartate aminotransferase and alanine aminotransferase activities were inhibited inA. proliferoides, stimulated inS. ferax but remained similar to that of the control inD. sterilis.     

Comparative study of the effect of stress by the heavy metals Cd+2, Pb+2, and Zn+2 on morphological characteristics of Saprolegnia delica Coker and Dictyuchus carpophorus Zopf.

The effects of essential (Zn+2) and non-essential (Cd+2 and Pb+2) heavy metals on morphogenesis of two represantatives of informal group zoosporic fungi namely; Saprolegnia delica Coker and Dictyuchus carpophorus Zopf. were studied. These two species varied in their tolerance of each amended heavy metal. Lead had the most potent effect amongst the tested heavy metals in inhibiting the radial extension of the vegetative hyphae of the two tested species. The vegetative hyphae of S. delica and D. carpophorus assumed different morphological alterations compared with that at controls depending upon the applied heavy metal and the dose concentration. Both zoosporangial formation and discharges of the two tested fungi were greatly inhibited even at the low concentrations of Cd. Zoosporangia of D. carpophorus appeared curved at high concentrations of Cd. Zoosporangial formation and discharge of the two zoosporic fungi showed variable deformation when treated with Pb. The different applications of Zn nearly stimulated sporangial elongation in both zoosporic fungi. Sex organs varied in their numbers and morphogenesis at each treatment of the applied heavy metal. The gemmae of S. delica were greatly reduced or missed at the elevated toxic levels of Cd whereas they enhanced in numbers and size at most Pb treatments and little affected at Zn applications.     

Studies on species of fungi associated with mycotic infections of fish in a Nigerian freshwater fish pond

A survey was carried out on the species of fungi associated with mycotic infections of fish in a Nigerian freshwater fish pond. A total of 24 fungal species belonging to 6 genera of aquatic phycomycete were isolated from the infected fishes. Achlya racemosa, Aphanomyces laevis, Dictyuchus sterile, Saprolegnia ferax, S. litoralis and S. parasitica had 100% frequency of occurrence amongst the infected fishes. There were similarities in the species of fungi isolated from the infected fishes in the fish pond and those isolated from the hatchery. Of the infected fishes, Clarias lazera had the highest number of fungal isolates. It was followed by Tilapia zilli, then by Chanos chanos, Ethmalosa fimbriata and finally by Cyprinus carpio. The implications of the results obtained on the development of fish farming in Nigeria are discussed.     

Transcellular ion currents during sporangium development in the water mould Achlya bisexualis

Changes in the pattern of electric currents that accompany the transformation of growing hyphae of Achlya bisexualis into sporangia have been examined. When hyphae were transferred to a non-nutrient buffer, they continued to extend for several hours and then gave rise to sporangia. Throughout this process, current (positive charge) flowed into the apical region that corresponds approximately to the future sporangium. The current ceased after the crosswall appeared. The sporangium then remained electrically quiescent, except for a brief intense burst of outward current at the 'homogeneous' stage of spore cleavage. The inward current during sporangium formation largely represents an influx of protons. Addition of nitrate abolished the flow of electric current with little effect on sporulation. The late burst of outward current is most probably an artefact, generated by the discharge of salts from the sporangial vacuole. The transcellular electric current apparently plays no role in sporangium formation or in spore cleavage. Calcium ions, however, are required and may traverse the plasma membrane.     

A phylogenetic circumscription of Polytrichastrum (Polytrichaceae): Reassessment of sporophyte morphology supports molecular phylogeny

Mosses arguably possess the most structurally complex sporangia of any extant land plants, a consequence of being the monosporangiophyte lineage most strongly adapted to terrestrial environments. Morphological and functional variation in the mechanisms that regulate spore release in one of the major classes of mosses, the Polytrichopsida, is largely unexplored, while recent research indicates that the most distinctive structure, the peristome, has evolved independently in the Polytrichopsida and in other mosses. The genus Polytrichastrum was separated from Polytrichum on the basis of such sporangial characters, although the critical features had until recently only been examined using light microscopy, and strong evidence from molecular data indicated that Polytrichastrum as currently circumscribed is polyphyletic. Here we use Bayesian ancestral character state reconstruction in conjunction with extensive scanning electron micrographic studies to elucidate probable morphology at ancestral nodes and define natural taxa. As well as clarifying the structure, evolution, and aspects of development of the peristome-epiphragm complex in this highly prominent group of mosses, the results provide a basis for a revised phylogenetic taxonomy in which the species of Polytrichastrum sect. Aporotheca are recognized once more within Polytrichum.     

Fungi parasitising the eggs of certain fresh water fishes

By the present investigations the parasitic abilities of five isolates of Saprolegniaceous fungi have been established on the eggs of three fresh water fishesviz., Channa marulius Ham.,Channa gachua Ham., andNotoptenis notopterus Ham. These fungi areAchlya orion CokerAchlya prolifera (Nees) De Bary,Aphanomyces laevis Minden andSaprolegnia ferax (Gruith) Thuret.     

Basic nuclear proteins in the aquatic fungus Achlya ambisexualis.

The complement of basic chromosomal proteins in the aquatic fungus Achlya ambisexualis has been characterized. Achlya nuclei contain proteins with electrophoretic mobilities on acetic acid/urea and dodecyl sulphate polyacrylamide gels which are comparable to rabbit kidney histones H3, H4 and H2A. In contrast, the behavior of putative H2B and H1 proteins from Achlya showed greater analogy on acid/urea gels to higher plant histones. A closely related water fungus Saprolegnia ferax contained basic nuclear proteins which were very similar to those of Achlya.     

Wall formation in the saprolegniales

Summary The primary and secondary cysts of Saprolegnia ferax and the secondary cysts of Dictyuchus sterile have a two layered wall structure, the outer layer of which bears various types of spines. These spines, and the outer wall layer are derived from preformed structures (bars) found in the cytoplasm prior to encystment. Golgi derived vesicles appear to contribute to the inner layer of the primary cyst wall of S. ferax. The outer surface of the secondary cyst walls of this species has fibrils which are not embedded in matrix material.     

Re-evaluation of the enigmatic species complex Saprolegnia diclina-Saprolegnia parasitica based on morphological, physiological and molecular data

The phylogenetic relationships among isolates of the Saprolegnia diclina-Saprolegnia parasitica complex were investigated based on ITS rDNA sequences, and correlated with morphological and physiological characters. The isolates studied belong to five phylogenetically separate clades. The majority of presumed parasitic isolates, mostly isolated from fish lesions, fell within a clade that comprises isolates which has been variously named as S. diclina Type 1, S. parasitica, Saprolegnia salmonis or just as unnamed Saprolegnia sp. Presence of bundles of long-hooked hairs on secondary cysts, high frequency of retracted germination, and oogonia production at 7 degrees C (when occurring) were characteristic of this clade. A single isolate identified as S. diclina Type 2 clustered in a clade along with Saprolegnia ferax isolates. The isolates identified as S. diclina s. str. (S. diclina Type 3) distributed in two clades and appeared closely related to Saprolegnia multispora and to a number of Chilean isolates identified as Saprolegnia australis. The ITS sequences of clade I were almost identical even though the isolates were of diverse geographical origins and showed physiological and morphological differences and variations in their pathogenicity. This suggest these species reproduces clonally even in apparently sexually competent isolates. Adaptation to parasitism in Saprolegnia might have occurred at spore level by the development of long-hooked hairs to facilitate host attachment and selection of a retracting germination. The use of the name S. parasitica should be assigned to isolates of clade I that contained isolates forming cysts with bundles of long-hooked hairs.     

Synchytrium solstitiale: reclassification based on the function and role of resting spores

Studies were made about resting spores of Synchytrium solstitiale, a chytrid that causes false rust disease of yellow starthistle (YST). During evaluation of this fungus for biological control of YST, a protocol for resting spore germination was developed. Details of resting spore germination and study of long-term survival of the fungus were documented. Resting spores from dried leaves germinated after incubating them on water agar at least 7 d at 10-15 C. Resting spores were viable after storage in air-dried leaves more than 2 y at room temperature, suggesting they have a role in off-season and long-term survival of the fungus. Each resting spore produced a single sorus that contained a single sporangium, which on germination released zoospores through a pore. YST inoculated with germinated resting spores developed symptoms typical of false rust disease. All spore forms of S. solstitiale have been found to be functional, and the life cycle of S. solstitiale has been completed under controlled laboratory and greenhouse conditions. Resting spore galls differ from sporangial galls both morphologically and biologically, and in comparison, each sporangial gall cleaves into several sori and each sorus produces 5-25 sporangia that rupture during release of zoospores. For this reason S. solstitiale should be reclassified as diheterogallic sensu Karling (Am J Bot 42:540-545). Because resting spores function as prosori and produce an external sorus, S. solstitiale is best placed in into the subgenus Exosynchytrium.     

The developmental patterns of lysosomal enzyme activities during Ca2+-induced sporangium formation in Achlya bisexualis

The present paper describes intracellular changes in ribonuclease specific activity during Ca²⁺-induced sporangium formation in the water mold Achlya bisexualis. The enzymes undergo a decrease in activity prior to crosswall formation followed by an increase in activity during spore cleavage. As spore discharge occurs the RNase activity again decreases. A large percentage of the nuclease activity is associated with a lysosomal-like fraction of the cell, but there is also considerably activity associated with nuclear and microsomal fractions. Addition of cycloheximide or actinomycin D at various times during development prevents further decrease or increase in the enzyme activity. Mixing of cell extracts from different developmental stages provides evidence that inhibitors or activators of the enzyme activity are not responsible for the activity levels evident at the different stages. There is a change in the total levels of presumptive mRNA during Ca²⁺-induced sporangial formation which appears to be associated with the patterns of RNase activity. Utilizing total cellular RNA and Poly(A)⁺ RNA with the crude ribonuclease preparations, no substrate specificity could be ascertained.     

A mechanism of propagule release from unilocular reproductive structures in brown algae

Summary The structure of unilocular sporangia inP. littoralis was investigated along with several other species of brown algae in order to study the mechanism by which propagules are released from unilocular reproductive structures. Unilocular sporangia inP. littoralis are composed of a spherical cell wall of two distinct layers and contain a number of zoospores. The mass of spores is surrounded and permeated by mucilaginous carbohydrates. It is suggested that the production of these carbohydrates generates the necessary pressure to weaken the sporangial wall. In addition, ultrastructural observations indicate that further weakening seems to occur due to digestion of the inner wall layer. Walls of sporangia were mechanically broken just prior to normal spore release in order to investigate whether internal pressure exists, and if it can effect spore discharge. Results show that an internal pressure does exist prior to normal spore discharge and that this pressure is not generated by turgor pressure of the spores themselves or by a semi-permeable wall osmoticum system. The discharge of spores seems to occur when the carbohydrate around the spores swells. The adsorption of water when plants are immersed by the incoming tide thus seems a likely mechanism of spore discharge. The similarities of unilocular reproductive structures and spore release in several brown algal species suggests common mechanisms of propagule discharge for members of thePhaeophyta.     

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.     

Epidermal 'alarm substance' cells of fishes maintained by non-alarm functions: possible defence against pathogens, parasites and UVB radiation

Many fishes possess specialized epidermal cells that are ruptured by the teeth of predators, thus reliably indicating the presence of an actively foraging predator. Understanding the evolution of these cells has intrigued evolutionary ecologists because the release of these alarm chemicals is not voluntary. Here, we show that predation pressure does not influence alarm cell production in fishes. Alarm cell production is stimulated by exposure to skin-penetrating pathogens (water moulds: Saprolegnia ferax and Saprolegnia parasitica), skin-penetrating parasites (larval trematodes: Teleorchis sp. and Uvulifer sp.) and correlated with exposure to UV radiation. Suppression of the immune system with environmentally relevant levels of Cd inhibits alarm cell production of fishes challenged with Saprolegnia. These data are the first evidence that alarm substance cells have an immune function against ubiquitous environmental challenges to epidermal integrity. Our results indicate that these specialized cells arose and are maintained by natural selection owing to selfish benefits unrelated to predator-prey interactions. Cell contents released when these cells are damaged in predator attacks have secondarily acquired an ecological role as alarm cues because selection favours receivers to detect and respond adaptively to public information about predation.     

Some species of saprolegniaceae collected from Gorakhpur,India

Summary Eleven species of Saprolegniaceae viz., Saprolegnia diclina, S. ferax, Isoachlya anisospora, Achlya dubia, A. proliferoides, A. diffusa, A. orion, A. prolifera, A. conspicua, Aphanomyces helicoides, and Leptolegnia sp. (sterile) collected from waters of Gorakhpur have been described.This forms part of a thesis accepted for the degree of Ph. D. in the University of Gorakhpur, Gorakhpur.