Carbon Concentration and Carbon-to-Nitrogen Ratio Influence Submerged-Culture Conidiation by the Potential Bioherbicide Colletotrichum truncatum NRRL 13737

We assessed the influence of various carbon concentrations and carbon-to-nitrogen (C:N) ratios on Colletotrichum truncatum NRRL 13737 conidium formation in submerged cultures grown in a basal salts medium containing various amounts of glucose and Casamino Acids. Under the nutritional conditions tested, the highest conidium concentrations were produced in media with carbon concentrations of 4.0 to 15.3 g/liter. High carbon concentrations (20.4 to 40.8 g/liter) inhibited sporulation and enhanced the formation of microsclerotiumlike hyphal masses. At all the carbon concentrations tested, a culture grown in a medium with a C:N ratio of 15:1 produced more conidia than cultures grown in media with C:N ratios of 40:1 or 5:1. While glucose exhaustion was often coincident with conidium formation, cultures containing residual glucose sporulated and those with high carbon concentrations (>25 g/liter) exhausted glucose without sporulation. Nitrogen source studies showed that the levels of C. truncatum NRRL 13737 conidiation were similar for all protein hydrolysates tested. Reduced conidiation occurred when amino acid and inorganic nitrogen sources were used. Of the nine carbon sources evaluated, acetate as the sole carbon source resulted in the lowest level of sporulation.     

Enhancement of disease caused by Colletotrichum truncatum in Sesbania exaltata by coinoculating with epiphytic bacteria

Colletotrichum truncatum isolate NRRL 13737 (ARS patent culture No. 18434) is being evaluated for development as a mycoherbicide against the problematic weed Sesbania exaltata. Studies were conducted to determine whether selected phylloplane microorganisms, used as coinoculants, could increase the severity of disease incited by C. truncatum in S. exaltata. Hemp Sesbania seedlings were grown in a variety of soils and environments, sprayed with conidia of C. truncatum, and the formation of appressoria was examined on leaves using epifluorescence microscopy. From hemp sesbania plants that supported high levels of appressoria formation, over 200 phyllosphere microorganisms were isolated. Fifteen of 73 microbial isolates assayed stimulated appressoria formation in vitro on cellophane membranes. Five of 8 superior isolates from the in vitro assay also enhanced disease symptoms induced by C. truncatum on S. exaltata compared to seedlings treated with conidia only. Populations of three selected superior isolates remained high on leaves during plant exposure to dew. The superior isolates initiated no apparent symptoms and rarely decreased seedling growth parameters in the absence of C. truncatum. This is the first instance of utilizing phylloplane microorganisms to increase the level of disease incited by a mycoherbicide agent. Microbial facilitators may provide a means of improving the weed control efficacy of mycoherbicides.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Production ofPaecilomyces Fumosoroseus conidia in submerged culture

Mexican isolates ofPaecilomyces fumosoroseus (Wize) Brown & Smith virulent to nymphs and adults ofBemisia tabaci Gennadius (Homoptera: Aleyrodidae) were screened in terms of spore production in submerged culture. Effects of light, temperature stress and yeast extract on sporulation were studied. Cycles of 12 hours light/12 hours dark increased spore production as well as an incubation for 24 hours at 37°C prior to incubation at 30°C. In absence of organic nitrogen both fungal growth and sporulation were very low. Spore production in fermentors with a culture media of a C:N ratio of 25 was doubled as compared to a media with a C:N ratio of 11. Both conidia and blastospores were produced. Production of conidia directly from blastospores through microcyclic sporulation was observed. The proportion of conidia obtained under optimal conditions was 88.8%. Submerged culture ofP. fumosoroseus seemed advantageous compared to ricefilled plastic bags production method because of shorter fermentation times, higher spore yields and substantially higher volumetric spore productivity. Results indicated that careful manipulation of nutritional and environmental conditions allowed for production of conidia during submerged growth ofP. fumosoroseus, microcyclic sporulation being induced under a set of environmental conditions including temperature stress and nutrients limitation.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Biocontrol potential of five Burkholderia and Pseudomonas strains against Colletotrichum truncatum infecting chilli pepper

The effect of bacterial antagonism against the causal agent, Colletotrichum truncatum was assessed as a potential alternative in managing anthracnose in chilli pepper. Out of 104 contrasting bacterial colonies isolated from rhizosphere soil of a forest floor, five isolates caused the radial growth inhibition greater than 90% (Significant at p?<?0.05 level) of C. truncatum in dual cultures. Based on 16S rRNA analysis, these antagonistic bacterial isolates were identified as Pseudomonas aeruginosa, Burkholderia arboris, Burkholderia gladioli and Burkholderia rinojensis. The selected promising antagonists showed nearly 100% inhibition of the spore germination of C. truncatum in vitro. These antagonists produced antifungal compounds which are diffusible in nature. Microscopic studies of blackened fungal hyphae, which were subjected to antagonism showed many deformations such as thickening, swelling and malformation. In vivo study revealed that C. truncatum inoculated chilli pepper seeds treated with the five antagonists significantly inhibited the incidence of seed colonisation (p?<?0.05) by the pathogen. At the post emergence stage, the survival percentages and vigour indices of all the antagonists’ treatments, except B. rinojensis strain 1, were significantly higher compared to the untreated control. The efficacy of the selected antagonists in managing anthracnose fruit rot was 100% at the colour breaking stage of chilli pepper fruits. These bacterial antagonists had a negative effect on C. truncatum spore attachment and subsequent colonisation on chilli pepper leaves except in the treatment of B. arboris. The results of in-vitro and in-vivo studies, suggest that the screened antagonistic bacterial isolates are potential biocontrol agents and need to be further studied for the biochemical basis of their activity against C. truncatum.     

Effects of carbon concentration and carbon-to-nitrogen ratio on growth, conidiation, spore germination and efficacy of the potential bioherbicide Colletotrichum coccodes

The effect of carbon concentration and carbon-to-nitrogen ratio (C:N) as well as their interaction on Colletotrichum coccodes growth and sporulation in submerged flask culture were evaluated. When C:N ratios were held constant, both mycelial dry biomass and spore yield increased with increasing carbon concentration. The specific spore yields (spore yield g⁻¹ carbon), however, were not significantly different for the same C:N ratio in most cases. The highest spore yields (1.3 × 10⁸ spores per ml) were obtained from media containing 20 g per liter carbon with C:N ratios ranging from 5:1 to 10:1. When the C:N ratio was greater than 15:1, spore yields were significantly decreased with increasing C:N ratios. High carbon concentration (20 g L⁻¹) combined with high C:N ratios (above 15:1) reduced both mycelial growth and sporulation, and increased spore matrix production. Spores produced in medium containing 10 g L⁻¹ carbon with C:N ratios from 10:1 to 15:1 had 90% germination on potato dextrose agar after 12 h and caused extensive shoot dry weight reduction on the target weed, velvetleaf. These results suggest that C:N ratios from 10:1 to 15:1 are optimal for C. coccodes spore production. Received 9 December 1997/ Accepted in revised form 22 May 1998     

Development of pilot-scale fermentation and stabilisation processes for the production of microsclerotia of the entomopathogenic fungus Metarhizium brunneum strain F52

Using 100 L stirred-tank bioreactors, we evaluated the effect of fermentation parameters and drying protocols on the production and stabilisation of microsclerotia (MS) of the entomopathogenic fungus Metarhizium brunneum (formerly M. anisopliae F52). Results showed that stirred-tank bioreactors can be used to mass produce stable MS of Metarhizium and that culturing and drying protocols significantly affected MS yield and stability. Length of fermentation (4–7 days) for Metarhizium cultures had no significant impact on biomass accumulation, MS formation or the storage stability of the air-dried MS granules. Although cultures of Metarhizium grown on media with a carbon-to-nitrogen (C:N) ratio of 30:1 produced significantly more biomass when compared to cultures grown in media with a C:N ratio of 50:1, MS formation and desiccation tolerance following drying were similar. After storage for 1 year at 4°C, conidia production by air-dried MS granules from 50:1 media was significantly higher compared to MS granules from 30:1 media. The addition of diatomaceous earth (DE) to cultures of Metarhizium prior to drying at rates of 0–60 g L^?1 had no significant effect on MS desiccation tolerance but did impact conidia production. Air-dried MS granules without DE produced significantly more conidia g^?1 during the first 4 months of storage, but after 1 year, conidia production was similar regardless of DE content of the MS granule. Microsclerotial granules with higher moisture levels (2.6–5.0% w/w) produced significantly more conidia immediately after drying and MS granules with low moisture (0–2.5% w/w) produced more conidia after 12 months storage.     

Perturbation of alkaloid production by cadaverine in hairy root cultures of Nicotiana rustica

Cadaverine (1–10 mM) stimulated the production of anabasine by hairy root cultures of Nicotiana rustica transformed with Agrobacterium rhizogenes. In control cultures, nicotine accounted for at least 70–80% of the total alkaloid produced, whereas in cultures supplemented with 5 mM cadaverine about two-thirds of the alkaloid was anabasine and nicotine production was markedly diminished. Putrescine and agmatine caused some stimulation of alkaloid production, but the ratio of nicotine to anabasine was essentially unaffected. Lysine caused no substantial increase in anabasine formation.     

Sources of Variability in the Measurement of Fungal Spore Yields

Variability in the production of fungal spores and in the measurement of spore yields was investigated in four species of fungi: Colletotrichum gloeosporioides, Colletotrichum coccodes, Colletotrichum phomoides, and Acremonium strictum. When the fungi were grown on solid medium in microplates and spore yields were measured by counting the subsamples with a hemacytometer, the variability among hemacytometer squares was always the largest source of variation, accounting for 51 to 91% of the total variation. Variability among replicate cultures and results of repeat experiments were generally also significant. The effect of square-to-square variability on the precision of spore yield measurement was minimized by counting a moderate number (ca. 30) of squares per culture. Culture-to-culture variability limited the practical precision of spore production measurements to a 95% confidence interval of approximately the mean ± 25%. We provide guidelines for determining the number of replicate cultures required to attain this or other degrees of precision. Particle counter-derived spore counts and counts based on spore weights were much less variable than were hemacytometer counts, but they did not improve spore production estimates very much because of culture-to-culture variability. Results obtained by both of these methods differed from those obtained with a hemacytometer; particle counter measurements required a correction for spore pairs, while the relationship between spore weights and spore counts changed as the cultures aged.     

Partial Chemical and Physical Characterization of Two Extracellular Polysaccharides Produced by Marine, Periphytic Pseudomonas sp. Strain NCMB 2021

The marine bacterium Pseudomonas sp. strain NCMB 2021, which can attach to solid, and especially hydrophobic, surfaces, elaborates two different extracellular polysaccharides in batch cultures. One (polysaccharide A) was produced only during exponential growth and contained glucose, galactose, glucuronic acid, and galacturonic acid in a molar ratio of 1.00:0.81:0.42:0.32. It produced viscous solutions, formed gels at high concentrations, and precipitated with several multivalent cations. The other (polysaccharide B) was released at the end of the exponential phase and in the stationary phase. It contained equimolar amounts of N-acetylglucosamine, 2-keto-3-deoxyoctulosonic acid, an unidentified 6-deoxyhexose, and also O-acetyl groups. Despite its high molecular weight (10⁵ to 10⁶ as judged by gel filtration), the polysaccharide produced aqueous solutions with very low viscosities and was also soluble in 90% aqueous phenol, 80% methanol, and 80% ethanol.     

Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid’s pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.     

Oxygen delivery requirements of Colletotrichum truncatum during germination,vegetative growth,and sporulation

Optimization of O₂ delivery was the key to successful conidiation of Colletotrichum truncatum in submerged fermentor cultures supplied with 20 g carbon/l and C:N at the optimal 10:1 mass ratio for spore efficacy. Minimal mycelial fragmentation and maximal biomass and spore yields were provided by an O₂ transfer program that called for gradual increases in stirring rate to compensate for rising cell concentration and viscosity. The utility of an event-based O₂ transfer program was further supported by our observation of different O₂ requirements for each phase of the life cycle. Spore germination did not occur in cultures sparged with N₂. However, even low levels of O₂ [10% dissolved O₂ tension (DOT)] allowed 100% germination. The specific growth rate of the mycelia was a Monod-like function of DOT. The maximal growth rate was achieved when 15% DOT was provided via O₂ transfer at a specific rate of 5.4 × 10^–3 mol/g per hour. Sporulation had a strict O₂ requirement, and its rate and yield were optimized by providing 55% DOT following the cessation of growth. The specific O₂ demand of optimally sporulating mycelia was 4.9 × 10^–4 mol/g per hour, an order of magnitude less than that associated with growing mycelia. Behaving as a pseudoplastic fluid, the fermentation broth reached a maximum apparent viscosity of 70 P at the onset of sporulation when the O₂ demand was low. However, the maximum power requirement approx. 7.9 W/l occurred during the last 36 h of growth when the O₂ demand was highest. Correspondence to: P. J. Slininger     

Utilization of oil palm decanter cake for cellulase and polyoses production

The abundance of oil palm decanter cake (OPDC) is a problem in oil palm mills. However, this lignocellulosic biomass can be utilized for cellulase and polyoses production. The effectiveness of chemical and physical pretreatment in reducing the lignin content was studied by saccharification using a Celluclast 1.5 L and scanning electron microscope. Physicochemical pretreatment of OPDC with 1% (w/v) NaOH and autoclaving at 121°C for 20 min increased potential polyoses produced to 52.5% and removed 28.7% of the lignin content. The optimized conditions for cellulase production by a locally isolated fungus were a time of 120 h, a substrate of untreated OPDC, a spore concentration of 1 × 10⁷ spore/mL, a temperature of 30°C, and a pH between 7.0 and 7.5. Trichoderma asperellum UPM1 produced carboxymethylcellulase (CMCase), ??-glucosidase and filter paper activity (FPase) in the following concentrations: 17.35, 0.53, and 0.28 U/mL, respectively. Aspergillus fumigatus UPM2 produced the CMCase, ??-glucosidase and FPase in the following amounts: 10.93, 0.76, and 0.24 U/mL. The cellulases from T. asperellum UPM1 produced 2.33 g/L of polyoses and the cellulases from A. fumigatus UPM2 produced 4.37 g/L of polyoses.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

Reduction of the infectivity of baculovirus stocks frozen at ultra‐low temperature in serum‐free media: The role of lipid emulsions

The infectivity of stocks of baculoviruses produced in serum‐free media is sensitive to freezing at ultra‐low temperatures. The objective of this work was to elucidate the causes of such sensitivity, using as a model the freezing of stocks of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a baculovirus widely employed as biological insecticide. Titers of supernatants of cell cultures infected with AgMNPV in four different serum‐free media supplemented with lipid emulsions were reduced by 50 to 90% after six months freezing. By using a full factorial experiment, freezing and lipid emulsion, as well as the interaction between them, were identified as the main factors reducing the viral titer. The virucidal effect of the lipid emulsion was reproduced by one of their components, the surfactant Polysorbate 80. Damaged viral envelopes were observed by transmission electron microscopy in most particles frozen in a medium supplemented with lipid emulsion or Polysorbate 80. Additionally, Polysorbate 80 also affected the infectivity of AgMNPV stocks that were incubated at 27°C. The identification of the roles played by the lipid emulsion and Polysorbate 80 is not only a contribution to the understanding of the mechanisms underlying the inactivation of baculovirus stocks produced in serum‐free media during storage at ultra‐low temperature, but is also an input for the rational development of new procedures aimed at improving both the preservation of baculovirus stocks and the composition of culture media for the production of baculovirus‐based bioproducts in insect cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1559–1569, 2016     

Characteristics of the Vegetative Growth of Bacillus popilliae

Growth characteristics of the insect pathogen, Bacillus popilliae Dutky, were studied by propagation in shaken flasks and in 2-liter fermentors. Maximal populations between 5 × 10⁸ and 2 × 10⁹ viable cells per milliliter of culture medium routinely were obtained in incubation periods of 18 to 24 hr at 30 C in a medium composed of 1.5% yeast extract, 0.6% K₂HPO₄, and 0.2% glucose or trehalose. The carbohydrate required for growth in liquid media was fermented with the formation of 2 meq of acid per mmole of carbohydrate utilized; acid products ordinarily were not subsequently metabolized. B. popilliae is an aerobe, and the amount of growth obtained varied with aeration to an optimum at oxygen absorption rates of about 0.5. Maximal populations persist in a culture for periods of only 1 to 4 hr; cessation of growth was followed immediately by rapid death of cultures, so that less than 1% of the cells remained viable after 48 hr, and viability often was lost entirely by the end of 72 hr of incubation. No cytological evidence for spore formation was observed under any growth condition. Death was not associated with lysis of the cells, although extensive granulation ultimately occurred. Continuous neutralizaiton, augmented buffering, various techniques of dialysis, or slow feeding of the carbohydrate did not markedly alleviate the characteristic death of the cultures.     

Sporulation of the “Thermophilic Anaerobes.”

A reasonable degree of synchrony in the sporulation of Clostridium thermosaccharolyticum 3814 was obtained by using three 10% transfers of 8-hr cultures in a medium containing 0.5% L-arabinose, 0.5% peptone, 0.5% yeast extract, and Gc minerals. Sporulation was stimulated by L-arabinose and L-xylose, but was repressed by glucose, mannose, fructose, and D-pentoses. Sporulating cells were long and thin, whereas repressed cells were shorter and thicker. The optimal pH for sporulation was in the range of pH 5.0 to 5.5. As sporulation continued, the accumulated acetate decreased. Label studies indicated that a significant amount of acetate-2-C¹⁴ was incorporated into the spore lipid. The calcium, phosphorus, and dipicolinic acid (DPA) concentrations on a dry weight basis were 2.55, 2.60, and 7.25%, respectively. The molar ratio of Ca-DPA was 1.47.     

Aquastella gen. nov.: A new genus of saprolegniaceous oomycete rotifer parasites related to Aphanomyces, with unique sporangial outgrowths

The oomycete genus Aquastella is described to accommodate two new species of parasites of rotifers observed in Brooktrout Lake, New York State, USA. Three rotifer species – Keratella taurocephala, Polyarthra vulgaris, and Ploesoma truncatum – were infected, and this is the first report of oomycete infection in these species. Aquastella attenuata was specific to K. taurocephala and Aquastella acicularis was specific to P. vulgaris and P. truncatum. The occurrence of infections correlated with peak host population densities and rotifers were infected in the upper layers of the water column. Sequencing of 18S rRNA and phylogenetic analysis of both species placed them within the order Saprolegniales, in a clade closely related to Aphanomyces. The Aquastella species were morphologically distinct from other rotifer parasites as the developing sporangia penetrated out through the host body following its death to produce unique tapered outgrowths. Aquastella attenuata produced long, narrow, tapering, finger-like outgrowths, whilst A. acicularis produced shorter, spike-like outgrowths. We hypothesize that the outgrowths serve to deter predation and slow descent in the water column. Spore cleavage was intrasporangial with spore release through exit tubes. Aquastella attenuata produced primary zoospores, whereas A. acicularis released spherical primary aplanospores, more typical of other genera in the Aphanomyces clade.