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.     

Primary simple assays of cellulose-degrading fungi

Some 25 fungi, including at least 14 basidiomycetes, one ascomycete, and five anamorphic fungi were evaluated for their cellulose-degrading abilities in Difco potato dextrose broth or Difco malt extract broth cultures with cellulosic substrates (e.g., filter paper) in plastic Petri dishes. Among them, Peniophora sp. 06-13 and Phlebia sp. 99-335 reduced the dry weights of the whole cultures with these substrates more than the dry weights of the respective original substrates after 30 days of culture, showing definite cellulose degradation. In the cultures with more than 10 test fungi including Pycnoporus coccineus 84-117, such weight losses did not occur. This assay technique for the primary screening for cellulose degrading fungi is simple, inexpensive, reproducible and accurate.     

Early breeding protects anuran eggs from Saprolegnia infection

Here, we studied the ecological significance of Saprolegnia infections (‘saprolegniasis’) on the survival and development of two populations of the endemic Patagonian anuran Pleurodema thaul (Anura, Leiuperidae). We found that four different Saprolegnia species infected eggs and embryos of P. thaul, indicating that the infection by these ‘zoosporic fungi’ was different in each anuran population and among different cohorts. Late anuran cohorts generally showed a higher incidence of infection than early cohorts, but we observed no clear overall pattern between populations. In addition, in laboratory experiments, we determined that some of the Saprolegnia species induce early hatching, and that hatching timing was variable between populations. In summary, we found that early breeding (by underlying priority effects) could improve the survival of the earliest cohorts of P. thaul by allowing them to survive the stress imposed by epidemic events of Saprolegnia.     

Effects of sodium chloride, formalin and iodine on the hatching success of common carp, Cyprinus carpio, eggs

Saprolegniales are ubiquitous in natural water supplies of fish hatcheries, and often cause serious disease problems. Sodium chloride, formalin and iodine, administered twice a day as a flush at different concentrations, were tested on infected eggs of common carp, Cyprinus carpio, to evaluate their antifungal activity and effect on hatching rates. Sodium chloride at 35 000 mg L^?1 and formalin at 400 mg L^?1 were found to be most effective in controlling Saprolegnia sp., with 85.4 and 91.8% hatching rates, respectively. Iodine increased the hatching rate by 27% at 200 mg L^?1 (P < 0.05). There were infections on eggs exposed to all levels of iodine, but not on eggs treated with sodium chloride and formalin. Sodium chloride and formalin were more effective than iodine in controlling Saprolegnia. Sodium chloride is a safe, efficacious and economical treatment of Saprolegniosis and is therefore recommended for treating common carp eggs.     

Detection of ‘long-haired’ Saprolegnia (S. parasitica) isolates using monoclonal antibodies

The ability of five monoclonal antibodies (Mabs) raised against a pathogenic Saprolegnia parasitica isolate from brown trout to detect and differentiate between isolates with bundles of long hairs (S. parasitica) and other Saprolegnia species was determined by means of an indirect immunofluorescence assay. Four of the Mabs used recognized some of the long-haired S. parasitica isolates but also cross-reacted with other Saprolegnia species without bundles of hairs and with Achlya sp. The other Mab (named 18A6) was able to differentiate between the asexual and most of the sexual isolates in the group of long-haired S. parasitica isolates, but did not recognize Achlya sp. or the Saprolegnia species without bundles of hairs, with the exception of S. hypogyna. These results indicate that isolates with bundles of long hairs are closely related with other members of genus Saprolegnia and share several antigens. However, Mab 18A6 seems to recognize an epitope that is expressed mainly in the asexual isolates in the long-haired S. parasitica isolates.     

Saprolegnia australis R. F. Elliott 1968 infection in Prussian carp Carassius gibelio (Bloch, 1782) eggs and its control with herb extracts

In order to control saprolegniosis in Prussian carp (Carassius gibelio (Bloch, 1782) eggs, it is important to screen herb extracts as potential anti‐Saprolegnia drugs in Prussian carp hatcheries. For this purpose, an oomycete water mould (strain SC) isolated from Prussian carp [Carassius gibelio (Bloch, 1782)] eggs suffering from saprolegniosis was characterised morphologically as well as from ITS rDNA sequence data. Initially identified as a Saprolegnia sp. based on its morphological features, the constructed phylogenetic tree using the neighbour joining method further indicated that the SC strain was closely related to Saprolegnia australis R. F. Elliott 1968 strain VI05733 (GenBank accession no. HE798564 ), and which could form biofilm communities as virulence factors. In addition, aqueous extracts from forty Chinese herbs were screened as possible anti‐Saprolegnia agents. Among them, a 1 g ml^?1 extract from Radix sanguisorbae was the most efficacious anti‐Saprolegnia agent, indicated by the minimum inhibitory concentration that was as low as 256 mg L^?1. Relative survival of 73 and 88% was obtained against the SC strain in fish eggs at concentrations of 256 and 1280 mg L^?1, respectively. This is the first known report of Saprolegnia australis R. F. Elliott 1968 infection in C. gibelio (Bloch, 1782) eggs involving the screening of R. sanguisorbae extracts as potential anti‐Saprolegnia agents.     

The behaviour of kinetochore microtubules during meiosis in the fungusSaprolegnia

Summary InSaprolegnia, kinetochore microtubules persist throughout the mitotic nuclear cycle but, whilst present at leptotene, they disappear coincidently with the formation of synaptonemal complexes at pachytene and reform at metaphase I. In some other fungi chromosomal segregation is random in meiosis and non-random in mitosis. The attachment of chromosomes to persistent kinetochore microtubules in mitosis, but not meiosis, inSaprolegnia provides a plausible explanation for such behaviour. At metaphase I each bivalent is connected to the spindle by 2 laterally paired kinetochore microtubules whereas at metaphase II (as in mitosis) each univalent bears only one kinetochore microtubule, thus showing that all kinetochores are fully active at all stages of meiosis.     

Decomposition of cellulose strips in relation to climate,litterfall nitrogen,phosphorus and C/N ratio in natural boreal forests

Isolates of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Penicillium sp., Rhizoctonia solani, Stemphylium sp., Thielaviopsis basicola, and Verticillium dahliae were cultured on potato–dextrose agar (PDA), barley-sand and alfalfa-sand substrates in petri-dish or in column microcosms. N-mineralization by fungi and fungal-feeding nematodes in combination or fungi alone was assessed. Numbers of Aphelenchus avenae or Aphelenchoides composticola supported by the fungi were measured every 7 days. Times for full colonization of the substrates by fungi ranged from 5 to 15 days. Rhizoctonia solani and B. cinerea on PDA supported the largest A. avenae and A. composticola populations, respectively. Penicillium sp. was a nonhost for A. composticola and A. avenae. Rhizoctonia solani, B. cinerea, V. dahliae, and F. oxysporum supported significantly more nematodes than the other four fungal species. The ranked order of fungi based on the amount of N mineralized in columns free of nematodes was A. alternata (with a rate of 0.052 μg N/g-sand per day), Stemphylium sp., V. dahliae, T. basicola, B. cinerea, F. oxysporum, R. solani, and Penicillium sp. (with a rate of 0.0045 μg N/g-sand perday). The presence of A. avenae resulted in significant increases in mineral N, compared to nematode-free columns colonized by F. oxysporum, R. solani, and T. basicola alone. The presence of A. composticola resulted in significant increases in mineral N, compared to nematode-free columns colonized by A. alternata, B. cinerea, F. oxysporum, and R. solani alone. There was more mineral N incolumns in the presence of A. composticola than A. avenae in most cases. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chitinolytic, Cellulolytic, and Pectinolytic Activity in vitro of Some Parasitic and Saprophytic Oomycctes

Seven lower phycomycetes, Afhanomyees astaci, A. euteiches, A. cladogamus, A. cochlioides, A. laevis. Pythium sp and Saprolegnia sp., have been investigated in respect of chitinolytic and cellulolytic activity. The five Aphanomyces species were also tested for pectinolytic activity. Different ages of stationary and shaken cultures, 2 or 3 different substrates, and 2 test pH were used for each enzyme to be tested. Chitinase and cellulase were assayed with 2 different methods. All the fungi showed chitinase activity, although in varying degree. In liquid cultures the strains of A. astaci gave the by far highest ac tivity. Differenl results were obtained with different growth media aud lest methods. Chitinase of some of the fungi was readily adsorbed on chilin. Adsorbed chitinase seemed to withstand heating more than unadsorbed enzyme. Only the three plant parasites, A. euteiches, A. cladogamus and A. codilioides, showed appreciable cellulase and pectinase activities which in some cases were stimulated by the presence in the medium of the enzyme substrate. Different assay methods and growth conditions showed difterent pattern regarding the difference in activity between the tested fungi and between used media. The conceivable roles in vivo of found activities were discussed.     

Effects of Nitrogen, Plant Density, Moisture Stress and Artificial Inoculation with Macrophomina phaseolina on Charcoal Rot Incidence in Grain Sorghum

The occurrence of pythiaceous fungi in pot plant cultures grown in ebb and flow bench systems was investigated monthly from May to December, Phytophthora, Pythium and Saprolegnia (in all 351) were isolated from water samples and identified. Nearly all the isolates of Pythium produced zoospores in water. A pathogenicity test involving 15 isolates of Pythium“group P”, and 7 of Pythium“group F” showed that 73 % were pathogenic on cucumber, 66 % on Gerbera, 59 % on lettuce, 50 % on tomato, and 32 % on cress. Control of Pythium and Phytophthora is important in order to improve the health of plants grown in ebb and flow systems.     

Fungi on submerged wood in the Koito River,Japan

 Woody substrates were collected from the Koito River in Japan, and the biodiversity of fungi on these substrates was investigated. Twenty-eight species were identified, comprising 12 ascomycete and 16 anamorphic taxa. The common fungi included Chaetosphaeria sp., Ophioceras commune, Pseudohalonectria lignicola, and Savoryella lignicola. The occurrence of fungi on submerged wood is discussed, and three interesting taxa – Pseudohalonectria lignicola, Bactrodesmium arnaudii, and B. pallidum – are described and illustrated. Received: August 19, 2002 / Accepted: November 25, 2002 Acknowledgments We are grateful to the financial support of the Special Coordination Fund for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Profs. K. Nishimura and M. Miyaji are thanked for scientific invitation and assistance in Chiba, Japan. Dr. Norio Hayashi from the Natural History Museum and Institute, Chiba, is thanked for providing physicochemical data for the Koito River. Y.M. Leung and M.H. Ng are thanked for technical assistance. Correspondence to:C.K.M. Tsui     

Ammonia fungi of iriomote Island in the southern Ryukyus,Japan and a new ammonia fungus,Hebeloma luchuense

Species composition and fruiting season of ammonia fungi were investigated in Iriomote Island of the southern Ryukyus, Okinawa Prefecture, Japan.Castanopsis andPinus forests were surveyed and 10 species of ammonia fungi were collected, including one new agaric species,Hebeloma luchuense sp. nov. This new fungus is characterized by having a rooting, squamulose-scaly stipe and cortinate veil and forms ectomycorrhizae withCastanopsis cuspidata var.sieboldii. Although the general mushroom season in theCastanopsis forest in Iriomote island was very short and restricted to summer, ammonia fungi were observed to fruit throughout the year in urea-treated plots.     

Observations on vertical distribution of fungi associated with standing senescent Acanthus ilicifolius stems at Mai Po Mangrove,Hong Kong

The present study was carried out to investigate the higher fungi colonizing the herbaceous mangrove associate Acanthus ilicifolius. This paper reports part of an investigation to determine if there is vertical distribution of fungi on the standing plant.The Mai Po Mangrove, Hong Kong is estuarine with great variations in salinity mainly due to the influence of the Pearl River. Senescent and dead stems of standing Acanthus ilicifolius were collected from mangroves in Mai Po from April to December 1992. The maximum tidal range observed was 2.6 m. A stratified sampling strategy was employed to assess the vertical distribution on the standing plant. A total of 44 fungi were collected: 32 Deuteromycotina, 11 Ascomycotina and 1 Basidiomycotina. Very frequent species were Acremonium sp.(55%), Colletotrichum gloesporioides cf.(42.5%), Phoma sp. (42.5%) Fusarium sp.,(25%), Tubercularia sp. (24.2%) and Phialophora sp. cf. (19.2%). Agerita sp., Corynespora cassiicola, Stachybotrys chartarum, Trichoderma sp. and D82 were frequent, while the remaining species were recorded at less than 10%. Vertical zonation of fungi colonizing the standing stems was observed. The apical portions were colonized by typical terrestrial fungi and the basal portions by marine species. This can be attributed to both the nature of the substratum and the degree of exposure due to tidal inundation.     

Further studies on Egyptian soil fungi: Succession of sugar and osmophilic fungi in soil amended with five organic substrates

The sugar and osmophilic fungal composition of soils amended with five organic substrates (newspaper, orange peel, bromegrass leaves, wheat straw and wood sawdust) was estimated after 2, 4, 6, 8 and 10 weeks using the dilution plate method on glucose and 50% sucrose Czapek's agar media. Wheat straw was the best substrate for total counts of both sugar and osmophilic fungi followed by newspaper, bromegrass leaves, wood sawdust and orange peel. Wood sawdust supported the highest average counts of total sugar fungi, Fusarium, Mucor, Scopulariopsis, Trichoderma and Trimmatostroma spp.; Newspaper, of Aspergillus (8 spp.), Penicillium (4 spp.) and Chaetomium sp. bromegrass leaves of Cladosporium sp., Humicola sp. and Sporotrichum sp.; orange peel, ofAlternaria sp., Circinella sp. and Stachybotrys sp.; and wheat straw, of Botryotrichum sp. and Myrothecium sp. Bromegrass leaves and orange peel supported the highest average counts of total osmophilic fungi, Aspergillus (10 spp.), Cladosporium sp. Paecillomyces sp. and Rhizopus sp.; and of Stemphylium sp., Trichoderma sp., Humicola sp. and Circinella sp. respectively; wheat straw, of Epicoccum sp., Scopulariopsis sp. and Trichothecium sp.; newspaper, of Penicillium (4 spp.) and Alternaria sp.; and wood sawdust of Curvularia sp. and Fusarium (3 spp.). The best colonizers throughout the experimental periods were Aspergilus and Penicillium spp.     

Contrasting responses to ectomycorrhizal inoculation in seedlings of six tropical African tree species

Five caesalpinioid legumes, Afzelia africana, Afzelia bella, Anthonotha macrophylla, Cryptosepalum tetraphylum and Paramacrolobium coeruleum, and one Euphorbiaceae species, Uapaca somon, with a considerable range in seed sizes, exhibited different responses to inoculation by four species of ectomycorrhizal (ECM) fungi, Scleroderma dictyosporum, S. verrucosum, Pisolithus sp. and one thelephoroid sp. in greenhouse conditions. Thelephoroid sp. efficiently colonized seedlings of all of the five caesalpinioid legumes except U. somon, but provided no more growth benefit than the other fungi. Thelephoroid sp. and S. dictyosporum colonized seedlings of U. somon poorly, but stimulated plant growth more than the other fungi. The relative mycorrhizal dependency (RMD) values of the caesalpinioid legumes were never higher than 50%, whilst U. somon had RMD values ranging from 84.6 to 88.6%, irrespective of the fungal species. The RMD values were negatively related to seed mass for all plant species. Potassium concentrations in leaves were more closely related than phosphorus to the stimulation of seedling biomass production by the ECM fungi. Our data support the hypothesis that African caesalpinioid legumes and euphorbe tree species with smaller seeds show higher RMD values than those with the larger seeds.     

Vesicular-arbuscular mycorrhizal fungi in coastal dune plant communities I. Spore formation ofGlomus spp. predominates under a patch ofElymus mollis

Vesicular-arbuscular (VA) mycorrhizal fungi in pure patches of coastal dune plantsElymus mollis, Wedelia prostrata andZoysia macrostachya were examined for frequency of occurrence and number of spores of VA mycorrhizal fungi over one year. Six species in three genera of VA mycorrhizal fungi were recovered. Under a patch ofE. mollis, spores ofAcaulospora sp. 1,Glomus tortuosum, Glomus sp. 1,Glomus sp. 2 andScutellospora gregaria were recovered. Spores ofGlomus spp. were most common. In patches ofW. prostrata andZ. macrostachya spores ofAcaulospora sp. 1,G. tortuosum, Glomus sp. 1,Glomus sp. 2,S. gregaria andScutellospora sp. 1 were found.Contribution No. 112, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Lignocellulose Decomposition and Production of Ligninolytic Enzymes During Interaction of White Rot Fungi with Soil Microorganisms

Abstract Four strains of white rot fungi, including two strains of Pleurotus sp., one Dichomitus squalens, and one Ganoderma applanatum, were grown on milled straw. After colonization of the straw by the fungi, sterile or nonsterile plugs of soil were added to the fungal substrates. The influence of the sterile soil and the indigenous soil microbiota on fungal growth, overall respiration, and production of ligninolytic exoenzymes was assessed. A method for extraction of laccase from soil samples was developed. Lignocellulose decomposition, and enzyme production of D. squalens were enhanced by the presence of sterile soil. The availability of inorganic compounds such as manganese may be a trigger for this stimulation. Neither growth nor the production of laccase and manganese peroxidase (MnP) of the Pleurotus strains was markedly affected by the soil microbiota. These fungi were highly competitive with the soil microbiota. It was demonstrated for the first time that the exoenzymes of such fungi are active in nonsterile soil. Enzyme activity in the aqueous phase of soil was high as in the aqueous phase of the straw substrate. D. squalens and G. applanatum did not withstand the competition with the soil microbiota, but the mycelia associated with straw were overgrown by soil microorganisms. Correspondingly, the fungi did not penetrate the soil, decomposition of lignocellulose was impeded, and the activities of laccase and MnP decreased dramatically. Received: 2 April 1996; Accepted: 7 June 1996     

Mycorrhizal inoculum potentials of pure reclamation materials and revegetated tailing sands from the Canadian oil sand industry

Recent improvements in the management of oil sand tailings used by the Canadian oil sand industry have resulted in the production of composite tailing sands (CT): a new challenging material for reclamation work. Jack pine (Pinus banksiana Lamb.), hybrid poplar (Populus deltoides Bartr. ex Marsh. ×Populus nigra L.) and red clover (Trifolium pratense L.) plants were used in an 8-week greenhouse bioassay to evaluate the mycorrhizal inoculum potential of CT. This inoculum potential was compared with that of three other reclamation materials [common tailing sands (TS), deep overburden (OB) and muskeg peat (MK)], and with three sites reclaimed in 1982 (R82), 1988 (R88) and 1999 (R99). CT was devoid of active mycorrhizal propagules while all other materials showed some level of inoculum potential. Arbuscular mycorrhizal fungi were observed on roots of clover or poplar grown in TS, OB, and all substrates containing peat (MK, R82, R88 and R99). Pine roots were also colonized by vesicle-forming hyphae of an unidentified fine endophyte and by dark septate fungi. Ectomycorrhizas (ECM) were observed on pine and poplar grown in OB, MK, and in soils from the two older reclaimed sites (R82 and R88). Using morpho- and molecular typing, six ECM fungi were identified to the genus or species level: Laccaria sp., Thelephora americana, Wilcoxina sp. (E-strain), Tuber sp. (I-type), a Sebacinoid, and a Pezizales species. Laccaria sp. and Wilcoxina sp. were the most frequently observed ECM species.     

Reprint of: Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.