The secondary zoospore of Aphanomyces astaci and A. laevis (Oomycetes, Saprolegniales)

The ultrastructure of the secondary zoospores of Aphanomyces astaci and A. laevis was compared. The general appearance of the organelles and their compartmental–ization is the same, but some subtle differences were found. A. laevis has a less distinct distribution of fuzzy vesicles around the border of the water expulsion apparatus than A. astaci. The content of the U–body of the A. astaci zoospore is paracrystalline but the A. laevis U–body has a tubular content. In the peripheral cytoplasm of both species are seen vesicles with a granular content. Flattened cisternae are found in a narrow zone just below the plasmalemma. These two structures are confined to the zoospore stage of the fungi. The lipid–microbody complex of A. laevis cysts is not present in the zoospore. The ultrastructural organization of uniflagellate and bi–flagellate zoospores is compared.     

The occurrence of Achlya recurva (Saprolegniales, Oomycetes) in hydrocarbon-polluted soil from Argentina

Saprolegniaceae are ubiquitous filamentous water molds. They occur as saprotrophs or parasitic on aquatic and terrestial plants and on aquatic animals, and a number of interesting fungi belonging to the Saprolegniaceae have been isolated from the soil. In the course of an investigation of the water molds in Argentina, an unusual species of Achlya was isolated once from soil collections. Brassica napus seeds were used as bait as well as for the maintenance of this fungus in gross water cultures. Achlya recurva Cornu (Saprolegniales, Oomycetes), was isolated from a hydrocarbon-polluted soil (crude oil) in the neighborhood of an oil tank from a distillery (Ensenada, Buenos Aires Province). This species is reported, described and illustrated for the first time from Argentina. Remarks on the habitat are also made because it was never found in polluted areas.     

Phylogenetic relationships among plant and animal parasites,and saprotrophs in Aphanomyces (Oomycetes)

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.     

Comparative Physiology of Respiration in Aquatic Fungi II. The Saprolegniales, Especially Aphanomyces astaci

The endogenous respiration of six members of Saprolegniaceae (Oomycetes), Saprotegnia sp., Thraustotheca sp., Achlya sp., Dichtyuchus sp., Aphanomyces euteiches, and A. astaci were studied in the presence and in the absence of exogenous substrate using conventional manometric techniques. Glucose stimulated the rate of oxygen uptake of unstarved mycelia to some extent in all the fungi. Attempts to increase the weak stimulation of respiration by glucose in A. astaci were not successful. The respiratory quotients of the fungi tested were usually in the range of 0.7 to 0.9 during endogenous respiration, and addition of glucose increased these values more than expected. L-leucine and L-glutamic acid stimulated respiration of A. astaci only when the fugus was starved, and acetic acid and butyric acid were inhibitory. Fructose and acetic acid increased respiration in starved mycelium of A. euteiches while L-leucine and L-glutamic acid had little effect. Antimycin A, HOQNO, HCN, and fluoroacetate strongly inhibited endogenous oxygen uptake by A. astaci. Amytal and azide were also markedly inhibitory while rotenone and CO had little effect. DNP and diphenylamine inhibited respiration at a high concentration but at a lower concentration DNP was stimulatory. In contrast the respiration of Saprolegnia sp. was resistant to cyanide, antimycin A, and HOQNO. Spectrophotometric observations on homogenized mycelia of Saprolegnia sp. and of A. astaci indicated the presence of cytochrome c (551 nm), two b-type cytochromes (557 and 564 nm) and cytochrome a-a₃ (605 nm) all in approximately equimolar concentrations. In both strains CO combined with cytochrome a₃ and an unidentified pigment. A remarkable similarity in the cytochrome system seems to exist between these two strains and some members of the Leptomitales.     

An ultrastructural study of the invasion of Culex quinquefasciatus larvae by Leptolegnia chapmanii (Oomycetes: Saprolegniales)

Transmission electron microscopy of the invasion of Culex quinquefasciatus by Leptolegnia chapmanii confirmed that it is a primary pathogen and revealed several differences between penetration via the gut and penetration via the integument. The latter often involved aggregations of zoospores, appressoria-like swellings of the invasive hyphae, and lateral growth of hyphae between the epicutical and endocuticle. These features were not detected in the case of gut invasion, but hyphal septa at the point of entry were apparently peculiar to this route. There was no evident tissue specificity, and death presumably resulted from generalized destruction of tissues.     

Differectial Induction of Zoospore Encystment and Germination in Aphanomyces astaci, Oomycetes

Genmination and encystment of zoosspores of Aphanomyces astaci, the very serious pathogen of European freshwater crayfish, Astacus astacus, were studied in ritro. Encystment of motile zoospores was achieved using mild heating, stirring, or addition of alkali metal chlorides or mannitol. The physical treatments were inhibitory to the subsequent germination, while encytment could be achieved with potassium chloride, indicating that the two processes are differently induced and can be studied separately. The effect of temperature on these processes was also somewhat different. Some ionic substances drastically influenced the final shape of the cyst. but without noticeably influencing the subsequent germination. Germination was induced by a short exposure to the stimulatory substances. A synergistic effect between ionic and non-ionic compounds was found. The receptivity of the zosspores to the triggering substances was low in newly formed spores but increased during the first 3 h of motility. The initiation of germination is suggested to be parltly to be partly due to an osmotic effects on the zoospore. The relevance of the experimental results to in vivo conditions in discuessd.     

Growth of Certain Aquatic Oomycetes on Amino Acids II. Apodachlya, Aphanomyces, and Pythium

Four aquatic fungi —Apodachlya brachynema and A. minima (Leptomitales), Aphanomyces laevis (Saprolegniales), and Pythium ultimum (Peronosporales) —were tested for growth in synthetic media containing one of a variety of carbon sources. Apodachlya brachynema readily utilized five amino acids — alanine, glutamate, aspartate, proline and leucine — as well as glucose and acetate. Growth on sucrose as a carbon source was slight. Apodachlya minima differed from A. brachynema in that it could not utilize proline and leucine. Aphanomyces laevis grew well on only three of the substrates tested — glucose, alanine and glutamate. Pythium ultimum utilized glucose, sucrose, maltose, cellobiose, alanine, glutamate, aspartate, proline, asparagine, ornithine, and serine, but not eight other amino acids. All of these fungi hydrolyzed gelatin. Radioactively labeled carbon dioxide was released during incubation of Aphanomyces laevis in media containing labeled leucine, proline, or phenylalanine. These data provide evidence of some catabolism of the three substrates although none of these substrates can support the growth of Aphanomyces laevis as a sole source of carbon and nitrogen.     

Evaluation of histological techniques for the detection of fungal infections caused byLeptolegnia chapmanii (Oomycetes: Saprolegniales) inAedes aegypti (Diptera: Culicidae) larvae

We evaluated which of the fixatives and stains most frequently used for observation of insect tissues were the most appropriate for histopathological visualization of entomopathogenic fungal infections with Leptolegnia chapmanii in larvae of Aedes aegypti. The best contrast between the host tissues and the fungal structures was obtained when using a combination of Camoy fixative with Grocott staining contrasted with light green. Masson trichromic stain combined with 10% formaldehyde-phosphate buffer also provided satisfactory results--a good contrast and clearly distinguishable host tissues and fungal structures.     

Chitinase and protease activities in germinating zoospore cysts of a parasitic fungus,Aphanomyces astaci,Oomycetes

In germinating spores of the parasitic fungus, Aphanomyces astaci, chitinase was first demonstrated shortly before the germ-tube began to branch, in contrast to protease which was present in both ungerminated and germinated spores. The time at which chitinase would be required when this fungus penetrates the crayfish cuticle is correlated with that of the in vitro production of chitinase.     

The effect of fungicides on survival of the crayfish plague fungus,Aphanomyces astaci,Oomycetes, growing on fish scales

An antifungal factor isolated from extracts of Cymbidium (Orchidaceae) roots and infected pseudobulbs was identified as monolinolein.     

The Scoliolegnia asterophora aggregate, formerly Saprolegnia asterophora de Bary (Oomycetes)

The taxonomy of isolates referred to Saprolegnia asterophora de Bary has been investigated. Two new species are described and Saprolegnia asterophora has been re-defined. These three species are regarded as segregate species within an aggregate and are placed in a new genus of the Saprolegniaceae, Scoliolegnia Dick.     

Life cycle and ultrastructure ofDucellieria chodati (Oomycetes)

Ducellieria chodati forms colourless, evidently plastid-lacking aggregates. For the first time, details of the reproduction cycle are reported: Biflagellate zoospores, released from the aggregates, infest coniferous pollen grains drifting on lake surfaces. The unicellular thallus growing inside the pollen grain develops into a sporangium. 10 to c. 60 spores are discharged, gather at the mouth of the discharge tube, and form new aggregates. After more than 30 cycles, the formation of aggregates ceases in favour of the direct production of zoospores which again infest pollen grains. If several zoospores infest the same grain, a resting spore can be produced, probably by a sexual process. It is evident from this complex life cycle thatDucellieria chodati is misplaced inChlorophyceae orXanthophyceae and needs to be grouped within theOomycetes. Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

A new genus of the Verrucalvaceae (Oomycetes)

DICK. M. W., CROFT, B. J., MAGARF.Y, R. C, de COCK, A. W. A. M. & CLARK, G., 1989. A new genus of the Verrucalvaceae (Oomycetes) . A fungus, isolated from sugar cane in Queensland, Australia and causing the Poor Root Syndrome disease, is described and shown to be related to Verrucalvus and possesses verrucate oogonia containing plerotic oospores; it is placed in the Verrucalvaceae (Sclerosporales). On the basis of morphological and DNA studies the Verrucalvaceae has to be placed in the Saprolegniomycetidae. It follows that the order Sclerosporales (and also the Leptomitales) must be removed from the Peronosporomycetidae and placed in the Saprolegniomycetidae.     

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. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents 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. Copyright 1998 Academic Press.     

A new species of Brevilegnia (Saprolegniales, Straminipila) from Buenos Aires Province, Argentina

Brevilegnia ensenadensis sp. nov. is described from litter (floating twigs, leaves, and roots) in a man-made, polluted channel near a petroleum refinery, in Buenos Aires Province, Argentina. The species is illustrated and compared with other species of the genus; it can be identified by the preponderance of smooth or papillate oogonia, with a distinctive irregular inner wall, borne in bent or coiled oogonial stalks, which are terminal, lateral or of the intercalary position. The oogonia often are irregular in shape, and the oospheres mostly are immature. The single oospore is eccentric inside the great oogonia, and the species develops mainly diclinous antheridial branches and has fewer monoclinous and androgynous ones.     

Ecological roles of saprotrophic Peronosporales (Oomycetes,Straminipila) in natural environments

The fungus-like Peronosporales are composed of several lineages of mainly biotrophic and hemibiotrophic representatives. Saprotrophic species of Peronosporales are limited to the genera Halophytophthora and Salisapilia, and to some species in Phytopythium and Phytophthora Clades 6 and 9, which inhabit terrestrial and all types of aquatic ecosystems. The recent discovery of species of Phytophthora in marine habitats and of Halophytophthora in freshwater indicated that these genera are not only morphologically but also ecologically poorly delineated. In addition, half of these genera are not monophyletic. They play key ecological roles by upgrading nutrients to higher trophic levels through colonization of plant debris, which makes substrata more palatable for detritivores or through zoospore grazing by zooplankton, although their role as saprotrophs is still largely neglected. Some species of Phytophthora can be aggressive opportunistic pathogens of riverine forests in the presence of susceptible hosts and favourable environmental conditions and, as a consequence, most studies have focused on their role as pathogens. Identification of species is challenging due to hybridization and species complexes that harbour multiple cryptic species and, therefore, is not reliable without DNA sequencing tools.     

Species boundaries within Saprolegnia (Saprolegniales, Oomycota) based on morphological and DNA sequence data

Saprolegnia is a common and widespread genus of Oomycetes, however species identifications are difficult and uncertain. To test whether keys based on morphological characters could identify species as determined by molecular characters we determined partial DNA sequences for the 28S rRNA gene and the complete internal transcribed spacer (ITS) region for 55 isolates belonging to Saprolegnia and one isolate of Protoachlya hypogyna that exhibited saprolegnoid zoospore discharge in water culture. Phylogenetic analyses of the combined sequence data yielded 10 robustly supported clades that probably represent separate species. Morphological analyses of all isolates revealed that each DNA-based clade could be delimited from others by autapomorphic or unique combinations of morphological character states but not without employing several features previously not used at the species level. Taxonomic implications of these results are discussed and recommendations for less equivocal characterization of new Saprolegnia species are made.     

Image analysis of hyphal morphogenesis in Saprolegniaceae (Oomycetes)

Because of their wide range of apical morphology, several members of saprolegniaceous fungi (Oomycetes) were chosen to examine concordance with the vesicle supply center (VSC) model of hyphal morphogenesis. Two computer routines were devised to measure diameter changes over long stretches of hyphae and to test compatibility with the theoretical hyphoid shape, y = xcot(xV/N). In all four genera examined, the apex followed closely the contour described by the hyphoid equation; divergences became evident in the subapex. The hyphae of Saprolegnia parasitica showed maximum concordance with the VSC model, i.e., their profile matched a hyphoid curve from the apex to the entire length of the mature hyphal tube. In Aphanomyces and Leptolegnia, growth in the subapical region subsided becoming less than that specified by the hyphoid equation. In Achlya bisexualis, the reverse was true, the subapical region expanded beyond that specified by the hyphoid equation. The two divergent subapical tendencies gave the hyphal tips a cylindroid or conoid appearance, respectively. Since the hyphal apex of all four species conformed to the curvature dictated by the hyphoid equation, we concluded that a basic VSC mechanism operates in all of these oomycetous fungi. Accordingly, we suggest that the shape of an oomycetous hypha is generated by a VSC-driven gradient of wall formation, which is subject to additional modification in the subapex to produce a range of hyphal tip morphologies. The mathematical basis for generating a conoid hyphal tip by elongating the VSC is described in Appendix A.