Zoospore Production Research Articles

The pathogenicity of two isolates of Pythium spp. to mosquito larvae in the laboratory

Motile zoospores, the only infective stage of Pythium species, attached, encysted and infected 100% second instar larvae of Aedes aegypti, Aedes africanus, Aedes simpsoni , and Anopheles gambiae . Motile zoospores variably attached and infected Culex spp. and Eretmopodites but did not attack field collected Toxorhynchites sp. We observed mycelial development within the exposed larvae after exposure indicating that mortality was due to the fungus. The fungus infected 75 ± 10% (SD) second instar larvae of Aedes aegypti , under aseptically reared conditions. It infected 65 ± 12.8% (SD) second instar larvae of Culex quinquefasciatus in aseptic conditions in the laboratory. All field collected larvae were heavily infested with vorticella but after treatment with 5% sodium hypochlorite, control mortalities ranged from 0 to 8.6% (mean = 4.3%). The fungus was most infective between 20–27.5°C; at 30°C many zoospores were attached to the cuticle but did not infect. Mortality rates were as low as 51.5% at 30°C, with no infection at all at 35°C. pH range from 6–8 was optimum for infection, but pH above 8 inhibited infection. Infection level of 100% was obtained in aseptically reared larvae at higher densities of 1600 larvae of second instar larvae of Aedes aegypti and 97% infection for 1600 second instar larvae of C. quinquefasciatus when exposed to fungal infection. Phosphate, borate and acetate buffers inhibited zoospore production.

Factors affecting the production of sporophylls in the giant kelp Macrocystis pyrifera (L.) C.Ag.

The effects of variation in vegetative biomass, stand density, and stand location on sporophyll production in the giant kelp Macrocystis pyrifera (L.) C.Ag. were examined in a multifactorial experiment off the coast of Santa Barbara, California. Results indicate that vegetative biomass greatly influences zoospore production in this kelp, since the removal of 75% of vegetative fronds led to a drastic decrease in sporophyll production. Sporophyll biomass was shown to be closely correlated to zoospore production ( r = 0.995). Plants growing offshore produced significantly less sporophyll biomass per plant than those inshore. Site differences in sporophyll production may have been caused by differential shading resulting from the persistence of the surface canopy offshore and the senescence of it inshore. As a possible result, increased plant density significantly decreased sporophyll biomass offshore and had no effect inshore. The timing of events such as storms which remove vegetative biomass could affect the local population dynamics of this kelp by decreasing zoospore production.

Plectospira Myriandra, A Rediscovered Water Mold in Japanese Soil

One isolate of water mold from field soil in Shikoku Island, Japan, was Plectospira myriandra, rediscovered 59 years after its original description by Drechsler in 1927. During a survey of pythiaceous fungi in Japanese soils, an interesting fungus was isolated from a field in which bamboo had been grown in Nangoku City of Shikoku District (Island), Japan. This fungus was very similar to Aphanomcyes, but unlike Aphanomyces, it featured lobulate sporangia with uniseriate production of zoospores inside long evacuation tubes. Also, there were numerous antheridial elements per oogonium, and oospores and parthenospores borne in oogonia with and without antheridia, respectively. The morphology and taxonomy of the fungus identified as Plectospira myriandra Drechsler is described in this report. A preliminary report has been published (Watanabe, 1986).

Soil environment in relation to the incidence of Aphanomyces root rot of spinach. (II). Effect of nitrate on zoospore infection and its mechanisms.

Experiments were undertaken to elucidate the cause of reduction of spinach root rot by nitrate nitrogen application. It was confirmed in vitro that nitrate as Ca(NO3)2⋅4H2O has an effect on the inoculum potential of the pathogen rather than on susceptibility of the host.Accumulation of secondary zoospores of Aphanomyces cochlioides and infection of hypocotyls of host plants were observed in nitrate solutions at nitrogen levels of 0-200ppm, but not at levels of more than 300ppm. This phenomenon at high concentration of nitrate was the result of encystment and death of secondary zoospores. Inhibition of zoospore production from mycelia was also observed at high concentration of nitrate. Encystment and death at 0.75bar (N: ca. 300ppm) and 1.00bar (N: ca. 400ppm), respectively, were observed in nitrate solutions, but not in controls of sugar solutions, suggesting that nitrate toxicity is more closely responsible for the motility of secondary zoospores than increased osmotic pressure.

The susceptibility of barley cultivars to barley yellow mosaic virus (BaYMV) and its fungal vector, Polymyxa graminis

SUMMARYIn glasshouse experiments, barley seedlings were inoculated with barley yellow mosaic virus (BaYMV) either mechanically or by using zoospores or cystosori of a viruliferous isolate of the vector, Polymyxa graminis, maintained on barley in sand culture.Experiments using mechanical inoculation showed that seedlings became more resistant with age. Consistent cultivar differences were obtained: cvs Maris Otter and Halcyon were very susceptible and cv. Athene seemed immune. Symptoms developed more rapidly at 23 than at 17 or 11 oC. After vector inoculation, symptoms developed more slowly than after mechanical inoculation but cultivar ranking was similar. Cultivars did not differ in susceptibility to the vector, as measured by zoospore production on their roots. Spring barley cultivars supported the growth of the vector which remained viruliferous and some showed symptoms although, in the field, symptoms do not appear on spring‐sown crops.

Induction of sporulation in Blastocladiella emersonii: Influence of nutritional variables

We have studied the induction of zoosporangial sporulation in Blastocladiella emersonii by determining the influence of nutritional variables on the formation of the basal septum. This is an easy-to-monitor structure that has proved to be a useful marker of initiation of this cellular differentiation process. At any time before formation of the septum, the cells returned to the growth phase when incubated in the presence of 2% casamino acids. However, after septum formation has occurred, the cells were committed to sporulation. Certain of the amino acid components of the defined growth medium, DM 2, either prevent (tyrosine, phenylalanine, tryptophan, histidine, and threonine) or delay (valine, serine, arginine, and methionine) formation of the septum when added to the cultures together with the sporulation solution (buffered 1 m M CaCl 2). Sugars do not prevent the formation of the septum, although they can interrupt sporulation at later developmental phases. Glucosamine and acetylglucosamine can prevent formation of the discharge papilla and glucose can block completion of zoospore production. Glutamine, adenosine, and guanosine also caused long delays in septum formation. Taken together, these data favor (a) the hypothesis that amino acid starvation is one of the most important variables with respect to the induction of sporulation, and (b) the use of certain environmental chemical conditions that block sporulation at different steps as developmental tools for the study of this cellular differentiation process.

IRRADIANCE, DAYLENGTH AND TEMPERATURE EFFECTS ON ZOOSPOROGENESIS IN COLEOCHAETES CUTATA (CHAROPHYCEAE) 1

ABSTRACTUsing a factorial design, we investigated the effects of 150 different combinations of irradiance, daylength and temperature on zoosporogenesis in Coleochaete scutata. Analysis of variance (ANOVA) revealed that irradiance and daylength did not .significantly influence the response, out that temperature was highly significant. Exposure of thalli to 20°C for one to several days is sufficient to induce zoospore production in C. scutata and several other northern temperature species of Coleochaete. Results of the factorial experiment correlate well with field observations on the seasonal occurrence of asexual reproduction in several Coleochaete species. A technique based on results of this factorial study is described for using zoospores to obtain morphologically normal, unialgal cultures of Coleochaete .sp. It was concluded that the factorial approach to investigation of environmental control of zoosporogenesis can be a powerful tool for understanding natural algal population dynamics, as well as controlling growth and reproduction of algae in the laboratory.

Evidence that Blastocladiella emersonii zoospore maintenance factor is a sulfhydryl group-containing cyclic ribotide.

Blastocladiella emersonii zoospore maintenance factor (ZMF), released into the medium during zoospore production, mediates a reversible developmental block to zoospore encystment (Gottschalk, W. K., and Sonneborn, D. R. (1981) Exp. Mycol. 5, 1-14 and (1982) Dev. Biol. 93, 165-180). Crude ZMF and purified ZMF display indistinguishable sensitivities/insensitivities to inactivations by several different chemical or enzymatic treatments. Such data have provided additional support for the conclusion (Gottschalk, W. K., and Sonneborn, D. R. (1985) J. Biol. Chem. 260, 6588-6591) that ZMF biological activity resides in a single molecular species. The inactivation analyses have provided substantial evidence that ZMF is a newly discovered SH-containing cyclic ribotide. At least one SH-containing side group and at least one free amino group linked to an imidazole, as well as a ribosyl moiety containing a cyclic 3',5'-phosphate, a 2'-free hydroxyl, and a 1'-linkage to the imidazole, appear to be essential structural requirements for ZMF-mediated encystment blockage. The proposed structure of biologically functional ZMF is similar to that of a key intermediate in the de novo pathway of purine nucleotide biosynthesis (5'-phosphoribosyl-5-aminoimidazole-4-N-succino-carboxamide), except that ZMF, and not 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide, contains a cyclic phosphate and at least one reduced SH group.

LIGHT, TEMPERATURE AND PHOTOPERIOD AS FACTORS CONTROLLING REPRODUCTION IN ULOTHRIX ZONATA (ULVOPHYCEAE)1

ABSTRACTIn many lakes in the northern United States and Canada the filamentous green alga Ulothrix zonata (Weber and Mohr) Kütz grows abundantly in early spring in shallow waters. Asexual reproduction occurs by formation of quadriflagellate zoospores which disrupt, the integrity of the cells upon release causing the filament to disintegrate. Study of the effects of 100 different combinations of irradiance, temperature and photoperiod revealed that zoospore formation is favored by high temperatures near 20°C, high light levels of 520 μE·m−2·s−1 and photoperiods of either short day (8:16 h light‐dark) or long day cycles (16:8 h light‐dark). Zoospore formation is minimal under conditions of low temperature (5°C), low irradiance (32.5 μE·m−2·s−1) and neutral day‐lengths (12:12 h light‐dark). These observations explain the decline in U. zonata biomass when water temperatures rise above 10° C. The combined effect of rising water temperatures and increasing daylengths causes progressively more filaments to switch from vegetable growth to zoospore production resulting in an increasing loss of biomass.

Effects of salinity, pH, organic solutes, anaerobic conditions, and the presence of other microbes on production and survival of Lagenidium giganteum (Oomycetes: Lagenidiales) zoospores

The effects of several environmental factors on the production of viable zoospores by Lagenidium giganteum were determined by counts of germlings produced after induction of zoosporogenesis by suspension of mycelium in various substances. NaCl at a concentration of 0.6 g/liter virtually eliminated zoosporogenesis. At 0.2 g/liter NaCl there was a significant reduction in four of the five fungal isolates tested. Transmission of fungus between mosquito larvae at 0.8 g/liter NaCl suggests that estimates of solute effects from in vitro studies are exaggerated. Zoosporogenesis took place from pH 4.5 to 8.4 by three isolates and from 4.5 to 8 by two other isolates (±0.2). Anaerobic conditions halted zoosporogenesis. Reintroduction of oxygen within 7 days allowed the process to resume, but with reduced production for each day under anaerobic conditions. Five species of bacteria inhibited zoosporogenesis at concentrations of 10 7–10 8 cells/ml. The yeast, Saccharomyces cerevisiae, was inhibitory at 10 6 cells/ml. Three sugars, a sugar alcohol, three amino acids, and peptone all reduced zoosporogenesis at levels that generally correlated positively with their nutritional values for the fungus. Peptone, which can support lush vegetative growth of L. giganteum in the absence of other nutrients, was far more effective in inhibiting zoosporogenesis than the other solutes.

Zoospore induction and release in a Pythium causing equine phycomycosis

Zoospore induction and release were investigated in a Pythium causing equine phycomycosis. Zoospore formation and release were favoured in 24-h-old colonies. Induction in distilled water was maximized when this was preceded by four washes each of greater than 8 min duration. A reduction in nutrient levels rather than removal of staling products best explains zoospore induction. Induction occurred in media containing glucose, sucrose (10 m M ), and maltose and xylose (0.1 m M ). The most inhibitory of medium salts were CaSO 4 , NH 4 C1 and mono- and di-potassium phosphates when present singly. Large number of vesicles formed in the presence of NH 4 C1 (0.5 m M ) but few spores differentiated. At certain concentrations, the presence of Ca 2+ , Mg 2+ and K + ions appeared to stimulate sporulation but at higher concentrations were associated with the non-release of zoospores from vesicles. The non-released spores germinated in situ . Sitosterol also stimulated zoospore production when present in the induction medium.

An improved method for the synchronous production of zoospores from Phytophthora palmivora

A modification of the method described by J. Tokunaga and S. Bartnicki-Garcia (1971, Arch. Mikrobiol. 79: 283–292) allows the production of motile zoospores from Phytophthora palmivora at concentrations exceeding 2 × 10 6 ml −1. The zoospores are released within a period of 5 min, under conditions which allow greater control of the chemical environment than flooding of agar plates. The method depends on brushing the deciduous sporangia into distilled water at 24°C following a temperature shock.

In vitro production of zoospores by the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) on solid media

Reliable, large-scale production of Lagenidium giganteum zoospores was obtained on solid media. The fungus was grown for 7 days in a liquid medium of wheat germ, hemp seed, yeast extract, and glucose, then placed onto hemp-seed agar. Zoosporogenesis was induced on agar by immersing the fungal cultures into water. Zoospore production began 10 hr postimmersion, peaked at 18 hr, and ceased by 36 hr. A single, 10-cm Petri dish of fungus on hemp-seed agar produced 1.7−3.8 × 10 7 zoospores during the 26 hr of zoosporogenesis. Optimal zoospore production occurred with 4- to 7-day-old cultures; cultures older than 10 days produced few zoospores. The temperature range for zoosporogenesis was 15–35°C. The extent of zoosporogenesis was directly related to the volume of water used to induce zoospore formation and inversely proportional to agar thickness. Bioassay of zoospores against second instar Culex quinquefasciatus larvae yielded an LD 50 of 400 zoospores/ml.

Relative Toxicity of Certain Pesticides to Lagenidium giganteum (Oomycetes: Lagenidiales), a Fungal Pathogen of Mosquito Larvae1

Laboratory experiments were conducted to determine the relative toxicity of certain insecticides, herbicides, and fungicides to the vegetative growth and zoospore production in North Carolina (NC) and Louisiana (LA) isolates of Lagenidium giganteum. Zoospore production in both isolates was less tolerant than mycelial growth to most of the pesticides tested and, thus, the IC50 values (inhibition concentration) for mycelial growth rate on agar underestimated toxicity. The most toxic pesticides to one or both isolates of L. giganteum were captan, gamma-BHC. DDT. toxaphene. chlorpyrifos, and fenthion. These pesticides produced at least 10% inhibition of mycelial growth rate or completely inhibited zoospore production at concentrations below 5 parts per million (ppm). The least toxic pesticides caused a 10% inhibition in mycelial growth rate and failed to inhibit zoospore production at concentrations greater that 50 ppm. These were diflubenzuron. permethrin. temephos. and propoxur (LA isolate only). Pesticides of intermediate toxicity were: malathion, carbaryl, methoprene, alachlor, and atrazine. The two isolates differed substantially in their tolerance of the following pesticides: malathion, chlorpyrifos, toxaphene, carbaryl. propoxur. permethrin. and methoprene. At their recommended rates of application for control of mosquito larvae, methoprene, fenthion, malathion, and temephos would probably be compatible with both isolates of L. giganteum.

Quantitative assay of sporangia and zoospore production by Pythium aphanidermatum with a soaking plain water agar culture method.

A method was devised to assay sporangia and zoospore production of Pythium aphanidermatum quantitatively. One-day old 4-mm mycelial disc from PDA culture was placed centrally on a 2% water agar plate in a Petri dish, and flooded with 4ml of an incubation solution. With this method, sporangium formation and zoospore discharge were more stimulated by Petri's salt solution than Wills's salt solution, soil extract or distilled water as incubation solution. Sporangia and zoospore discharge were more abundant at 30C than at 25 or 20C.

Preliminary laboratory infection trials with a Fiji isolate of the mosquito pathogenic fungus Lagenidium

A mosquito-pathogenic oomycete, Lagenidium, has been isolated into pure culture from a dead larva of Aedes pseudoscutellaris in Fiji. Growth media prepared with soy flour was shown to be effective in obtaining sporulation in fresh water. Zoospore production was inhibited in 0.5% salt water. Using one or two soy agar disks as the inoculum produced the same infectivity as when between one and six infected larval cadavers were used. Seven species of mosquitoes occurring in Fiji were tested and all were found to be susceptible, although infection rates varied greatly from species to species. Aedes polynesiensis was found to be the most susceptible. The first two instars were more susceptible than the last two. The characteristics of this Lagenidium isolate are discussed.

Production and survival of Phytophthora cinnamomi zoospores in suppressive and conducive soils

The effect of selected Western Australian soils on sporulation, hyphal lysis and zoospore survival of P. cinnamomi was studied. The soils chosen represented extremes of disease incidence in the forest. They included a red earth, suppressive to P. cinnamomi; a yellow podsol and a red podsol, both conducive soils to disease development initiated by P. cinnamomi. The red earth soil extracts stimulated sporangial production and microbially induced hyphal lysis more than the other soils tested. A positive correlation between sporangial production and hyphal lysis was observed. This relationship could be explained if sporangial production is regarded as a survival response to hyphal lysis. Seven percent of encysted zoospores survived 43 days in all soil extracts. This survival was not influenced by soil sterility, temperature or soil type, suggesting that cysts are more resistant survival structures than hyphae. Motile zoospore survival is reduced by soil microbial factors; this effect is more pronounced in the red earth than in the two podsolic soils. Increased soil extract temperatures also reduced motile zoospore survival. These results show that survival of P. cinnamomi hyphae and zoospores is reduced in the suppressive red earth relative to conducive podsolic soils.

Assignment of the rumen anaerobe Neocallimastix frontalis to the Spizellomycetales (Chytridiomycetes) on the basis of its polyflagellate zoospore ultrastructure

Neocallimastix frontalis is an anaerobic herbivore gut inhabitant which occupies an uncertain taxonomic position, probably among zoospore fungi. It produces uninucleate zoospores which are bipartite with an organelle-free anterior portion and a posterior half bearing about 10 flagella and all organelles. The flagella are arranged primarily in two rows and beat as a unit. The flagellar roots are composed of two arrays of single microtubules, all of which emanate, in groups of about 14, from single kinetosome-associated spurs. The main array forms a divergent cone with which the microbodies (hydrogenosomes?) are intimately associated. The second group (the posterior fan) forms a fan-shaped array lying close to the posterior plasmalemma. Zoospores encyst, develop rhizoids, and can produce new zoospores at any stage in their subsequent development from a uninucleate to a multinucleate eucarpic thallus. Zoospore production involves intracytoplasmic flagellar vesicles, organelle clustering, and loss of the sporangium plasmalemma. Zoospore release follows nonlocalized dissolution of the entire sporangium wall. On these data, we assign the genus to a new family, the Neocallimasticaceae, in the Spizellomycetales of the Chytridiomycetes and thereby redefine these taxa to accommodate organisms producing polyflagellate zoospores.

Physiology of growth and asexual reproduction of a Pythium causing equine phycomycosis

The growth and asexual reproduction of a Pythium causing equine phycomycosis was examined on various media. The organism possesses a restricted ability to utilize carbohydrates. Glucose, maltose and starch gave clear support of growth and asexual reproduction. Galactose, glycerol and fructose supported growth but with the possible exception of galactose gave no evidence of stimulating subsequent zoosporogenesis. Ammonium nitrogen but not nitrite or nitrate nitrogen were utilized for growth and asexual reproduction. Various amino acids and urea also supported growth but only the latter gave clear evidence of supporting subsequent zoosporogenesis. A formulation providing around ten atoms of carbon for every atom of ammonium nitrogen was close to optimum for both growth and asexual reproduction. All forms of sulphur tested supported growth including sulphate. Asexual reproduction was achieved on colonies grown on cystine, thiosulphate, and some but not all sulphates. The calcium ion and sterols stimulated growth and the latter also stimulated asexual reproduction. Phosphate ions at concentrations in excess of 0.02 M deleteriously affected growth whereas zoospore production declined when colonies were grown on concentrations greater than 0.01 M.

Factors Influencing Zoospore Production in Liquid Cultures ofLagenidium Giganteum(Oomycetes, Lagenidiales)

A procedure is described for producing large numbers of zoospores in liquid cultures of mosquito-parasitizing fungus Lagenidium giganteum. The fungus was grown 4-6 da on a peptone, yeast extract and glucose medium, transferred to a medium of hempseed extract, wheat germ, yeast extract and glucose, propagated for 4-6 da, then washed and transferred to water. Zoospore production at 25 ? 1 C began at 18 h, peaked between 25-28 h, and ceased at 36 h. Fungus grown in the peptone medium for 4-6 da, and then transferred to hempseed medium and grown for 6 da, produced 13,700-21,000 zoospores per ml, whereas fungus grown 7 da in peptone medium and 6 da in hempseed medium produced 6100 zoospores per ml. Zoospore production occurred at a temperature range of 17-28 C with the optimum for production at 25-28 C. Effects of pH and buffers on zoospore production were studied. An LD50 of 1400 zoospores per ml was obtained with second instar larvae of Culex quinquefasciatus.