Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.     

Der Einfluss des Wassergehaltes auf Inaktivierung und Mutationsrate von r?ntgen- und gammabestrahltenNeurospora Crassa-Conidien

Summary InNeurospora crassa X- and gamma-ray induced inactivation- and reversion-rates for conidia of HNO₂ induced mutants were measured. With constant radiation dose inactivation rate is lowest for conidia of medium moisture content. It increases for higher and also for very low moisture content. The semilogarithmic plot of inactivation data for conidia irradiated in suspension or at medium moisture content results in a shouldered curve. A formal analysis using the number of nuclei of multinucleated conidia as numerical reference indicates that ca. 0.5 hits per nucleus are needed to inactivate such a conidium. The data for extremely dry conidia plotted from 0–210 kr likewise closely approximate a single hit curve with a steeper slope than that of the corresponding curve for conidia of medium moisture content.Reversion rates of 5 among 7 mutants checked run parallel with inactivation with low figures for conidia of medium and increasing figures for higher as well as for very low moisture contents (class I). The reversion rate of conidia of very low moisture content of the other 2 mutants is equal or nearly equal to that of conidia of medium moisture content (class II).Possible explanations for the data obtained are discussed.

---

---

Mit 12 Textabbildungen     

Factors affecting sporulation by Phomopsis phaeolorum

Light and nutrition are the important factors in the production of pycnidia and conidia by cowpea isolates of the Phomopsis state of Diaporthe phaseolorum. The highest number of pycnidia and conidia were produced on plant tissue exposed to cool-white fluorescent light. In semisynthetic media more pycnidia were formed at high glucose concentrations, but they matured more slowly than those formed at lower glucose concentrations. Both the level of conidiation and the percentage of pycnidia that formed conidia were higher at lower glucose concentrations. The best artificial medium for inducing a high number of pycnidia containing abundant conidia was one that contained 0.4% glucose and 0.4% NaNO₃. A number of carbon sources could replace glucose in this medium.     

Development of a submerged-liquid sporulation medium for the potential smartweed bioherbicide Septoria polygonorum

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Septoria polygonorum. In phase I, 47 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, pea brine (5–10% v/v) provided best conidiation. In phase II, a fractional factorial design was utilized to screen 38 different medium adjuncts in combination with pea brine for improved sporulation. MgSO₄ was the only factor that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 8.88% v/v pea brine+0.1 molar MgSO₄ with an expected titer of 1.78×10⁸ conidia/ml. Actual mean titer attained with the model-derived medium was 1.15×10⁸ conidia/ml. No significant difference was observed in virulence of conidia produced on agar vs. the model-derived (liquid) medium.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

On the mechanism of mitotic recombination in Aspergillus nidulans. I. Intragenic recombination and DNA replication

Summary Adenine or pABA starvation induce mitotic recombination within the ad9 and paba1 cistrons respectively. Adenine concentrations in the plating medium as low as 1×10^-8 M increase recombination frequency; the concentration optimal in respect to induced recombination frequency is 5×10^-7 M.Recombination within the paba1 cistron is stimulated by low pABA concentrations, or caseine hydrolysate, or methionine.Aminopterin applied for one or two hours before conidia of pABA-requiring diploid are plated on proper selective media, induces recombination within the pro1, ad9 and paba1 cistrons. Conclusion is drawn that it is adenine or thymine starvation which induce mitotic recombination.The implications of this and other similar evidence are discussed.     

Medium Selection and Effect of Higher Oxygen Concentration Pulses on <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var<Emphasis Type="Italic">. lepidiotum</Emphasis> Conidial Production and Quality

Rice and oat flours were analyzed as media for the production of conidia by M. anisopliae var. lepidiotum. The presence of peptone increased conidia yield regardless of the substrate used; however, the highest yield was achieved on oat flour media. The effect of oxygen on conidia production using oat-peptone medium was also studied at two levels: Normal atmosphere (21% O₂) and Oxygen-rich pulses (26% O₂). Maximum conidia production (4.25 × 10⁷ conidia cm⁻²) was achieved using 26% O₂ pulses after 156 h of culture, which was higher than 100% relative to conidial levels under normal atmosphere. Conidia yield per gram of biomass was 2.6 times higher with 26% O₂ (1.12 × 10⁷ conidia mg⁻¹). Conidia quality parameters, such as germination and hydrophobicity, did not show significant differences (P < 0.05) between those treatments. Bioassays parameters, using Tenebrio molitor adults, were analyzed for conidia obtained in both atmospheres and data were fitted to an exponential model. The specific mortality rates were 2.22 and 1.26 days⁻¹, whereas lethal times for 50% mortality were 3.90 and 4.31 days, for 26% O₂ pulses and 21% O₂ atmosphere, respectively. These results are relevant for production processes since an oxygen increase allowed superior levels of conidia by M. anisopliae without altering quality parameters and virulence toward Tenebrio molitor adults.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

Effects of temperature,light, carbon and nitrogen nutrition on reproduction in Phoma medicaginis

Reproduction by three isolates ofPhoma medicaginis growing on potato dextrose agar was studied. The formation of pycnidia was optimum at 30°C whereas the optimum for the formation of conidia was 20°C. Light consistently increased the numbers of pycnidia and conidia over those formed in darkness and more conidia were produced in light at 23°C than at 30°C. The effects of carbon and nitrogen sources on reproduction were studied using modified Richard's medium as the basal medium. Fourteen carbohydrates, 11 amino acids, 9 inorganic nitrogen sources and urea were evaluated by replacing the carbohydrate or nitrogen in Richard's medium with the test substance. Generally, the monosaccharides and disaccharides were about alike and superior to polysaccharides for the production of pycnidia. The carbon sources were about equally useful for production of conidia, but the polysaccharides were superior to the other two classes of carbon sources when the number of conidia/pycnidium was calculated. Generally, the formation of pycnidia and conidia was favored by nitrate more than by ammonium nitrogen sources. The average number of conidia/pycnidium was greatest, however, when the nitrogen source was NH₄NO₃. All amino acids tested appeared to be useful nitrogen sources for production of pycnidia but none were especially good for conidia production. L-isoleucine was superior to the other amino acids tested. Of three isolates used, Illinois 23 consistently produced more pycnidia and conidia that did isolates Minnesota 2 and Missouri 5. Usually the significant interactions between isolates and other treatments were due to a greater response by isolate Ill. 23. It was concluded that the reproduction ofP. medicaginis varies significantly with the isolates of the fungus, the environment, and nutrients as well as with interactions among these factors, and conclusions about the influence of a particular factor will depend on whether the formation of pycnidia, conidia or conidia/pycnidium is being studied.Paper No. 7425, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Mitotic gene conversion, reciprocal recombination and gene replacement at the benA, beta-tubulin, locus of Aspergillus nidulans

Summary We have developed a procedure for determining the rates of mitotic recombination of an interrupted duplication created by integration of transforming plasmid sequences at the benA, beta-tubulin, locus of Aspergillus nidulans. Transformation of a strain carrying a benomyl-resistant benA allele with plasmid AIpGM4, which carries the wild-type benA allele and the pyr4 (orotidine-5-phosphate decarboxylase) gene of Neurospora crassa, creates an interrupted duplication with plasmid sequences flanked by two benA alleles, one wild type and one benomyl resdistant. Such transformants will not grow in the presence of high levels of benomyl. Mitotic recombination causes the loss of the wild-type benA allele or conversion of the wild-type to the mutant allele resulting in nuclei carrying only the benomylresistant allele. Conidia containing such nuclei can be selected on media with high benomyl allowing easy quantitation of mitotic recombination. We found that the rate of recombination giving rise to benomyl-resistant conidia was 4.6×10^-4. Reciprocal recombination leading to benomyl-resistant conidia lacking plasmid sequences occurred at a rate of 2.0×10^-4 and gene conversion leading to benomylresistant conidia occurred at a rate of 2.6×10^-4. We selected for reciprocal recombination leading to loss of pyr4 sequences on 5-fluoro-orotic acid and used this selection for two-step gene replacement of a mutant benA allele with the wild-type allele.     

A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata

Summary A diffusible self-inhibitor of germination of conidia of Glomerella cingulata appears to act as a regulator of protein synthesis. Both uptake of labeled amino acids and their incorporation into protein are reduced by the inhibitor or by crowding. Compared to conidia incubated without self-inhibitor, conidia incubated with self-inhibitor incorporated no labeled amino acids into protein in the first hour and 80% less in 6h. Thoroughly washed conidia were more permeable to amino acids and incorporated 6 times more precursor into proteins than unwashed conidia. At high density in nutrient medium, conidia of G. cingulata preferentially form secondary conidia instead of germ tubes and a mycelium. This inhibition of germination of conidia and regulation of development is mimicked by exposing them to an auto-inhibitor extracted from used culture medium and conidial washings. Germination of conidia of G. cingulata involves two steps, an initial step of 5 h duration which continues unaffected by crowing (1.7×10⁸/ml) and a subsequent 2 h step which conidia do not take unless they are sufficiently diluted. It is this step for which protein synthesis may be required.Non-Standard Abbreviations CHM cyloheximide - NM Neurospora minimal medium - psi pound per square inch - RPH reconstituted algal protein hydrolysate - TCA trichloroacetic acid     

THE VIRULENCE OF THE ENTOMOPHTHORALEAN FUNGUS PANDORA DELPHACZS TO THE BROWN PLANTHOPPER NILAPARVATA LUGENS1)

Abstract The entomophthoralean fungus, Pandwa delphacis (Hori) Humber, is a pathogen frequently causing epizootics of the brown planthopper, Nilaparvata lugens Stal, in rice growing areas of China. Two isolates (F95127 and F95129) of the fungus obtained from mycosis-killed planthoppers collected in Zhejiang Province were bioassayed for their virulence to N. lugens. For inoculation, botches of 4– or 5-in-star-old nymphs from a laboratory population were exposed to time-varying spore shower from sporulating plates of fungal mass produced in Sabouraud liquid medium, resulting in 8 and 6 dosages for F95129 and F95127, respectively. The nymphs treated at each dosage were maintained on rice plants at 25°C and a photophase of 12L: 12D in growth chamber (nearly 100% RH) and were examined daily for mortality. The resulting data were well fitted to time-dose-mortality model, generating parameters of time and dose effects for estimation of virulence indices. The LD₅₀ values 6–8 d after exposure were 327, 122 and 46 conidia/mm² for F95129 and 409, 133 and 74 conidia/mm² for F95127, respectively. The LT₅₀ estimated for F95127 was 7. 6 d at 95 conidia/mm² whereas that for F95129 decreased from 6. 6 d at 172 conidia/mm² to 5. 6 d at 525 conidia/mm². Based on these virulence indices and comparison of the slopes for dose effects from the model, the two isolates of P. delphacis had moderate virulence to N. lugens with insignificant difference to each other.     

Infectivity of two members of theEntomophthora muscae complex [Zygomycetes: Entomophthorales] forMusca domestica [Dipt.: Muscidae]

Dose-mortality studies were conducted with 2 members of theEntomophthora muscae (Cohn) Fresenius complex from southern California (CA) and Denmark (DA) infecting house flies,Musca domestica L., from southern California. Primary conidia of the DA form were significantly more infective (LC₅₀=34 conidia/mm²) than primary conidia of the CA form (LC₅₀=67 conidia/mm²) for 2–7 days old (‘young’) flies. Infectivity (single experimental trial) for 10–14 day old (‘old’) flies of primary conidia of the CA form (LC₅₀=34 conidia/mm²) was similar to that of the DA form for young flies. Secondary conidia of the CA form were markedly more infective (LC₅₀=0.36 conidia/mm²) than primary conidia of either form. Dose had no significant effect on incubation period for primary conidia of either form, but increased doses resulted in significantly shorter incubation periods for flies exposed to secondary conidia of the CA form.      

Germination of the entomopathogenic fungus Verticillium lecanii on scales of the glasshouse whitefly Trialeurodes vaporariorum

In a series of laboratory experiments, tobacco leaf discs infested with scales of the glasshouse whitefly Trialeurodes vaporariorum, were immersed in suspensions of conidia of three strains of the entomopathogenic fungus Verticillium lecanii. The germination of conidia on the insect was recorded and compared with the germination of conidia applied to Czapek—Dox Complete Medium. Laboratory bioassays were performed to record the mortality of whitefly scales exposed to conidia of all three fungal strains. The rate of germination of conidia on the whitefly scales was much reduced compared to that oh complete medium. Differences were recorded in the rate of germination of the three strains on complete medium, but not on the whitefly scales. There was no correlation between the pathogenicity of the strains to whitefly scales in laboratory bioassays and the rate of germination on the host.     

Production of the fungal biocontrol agent Epicoccum nigrum by solid substrate fermentation: effect of water activity on accumulation of compatible solutes

Epicoccum nigrum conidia were produced by solid fermentation on wheat grains (cv. Rendeveaux and Brigadier) at different water activities (a_w). Conidial production was highest at high a_w(0.996) than at reduced a_w (0.98). However, conidial production at reduced a_w was improved when the a_w of the substrate was adjusted with a mixture of glycerol and water. Maximum levels ofconidiation were 7–11 × 10⁶ conidia g⁻¹ grain. The a_w of the solid substrate affected the pattern of accumulation of compatible solutes in the conidia. Mannitol was the main polyol in all conidialtypes. However, the amounts of mannitol were higher in conidia produced at high a_w. At reduced a_w the conidia of E. nigrum accumulated moreglycerol, which is more efficient in the osmorregulation proccess than mannitol. Arabitol accumulated in low amounts, specifically in conidia produced at the lower a_w, on cv. Rendeveaux but not on cv. Brigadier. Trehalose was detected in higher amounts in cv. Rendeveaux than in cv. Brigadier, andthe amounts were higher in conidia produced at high a_w. A significant amount of endogenous solutes was detected in the washing liquid used for the separation of the conidia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Penicillium roqueforti conidia induced by L-amino acids can germinate without detectable swelling

Penicillium roqueforti is used for the production of blue-veined cheeses but is a spoilage fungus as well. It reproduces asexually by forming conidia. Germination of these spores can start the spoilage process of food. Germination is typically characterized by the processes of activation, swelling and germ tube formation. Here, we studied nutrient requirements for germination of P. roqueforti conidia. To this end,?>?300 conidia per condition were monitored in time using an oCelloScope imager and an asymmetric model was used to describe the germination process. Spores were incubated for 72 h in NaNO₃, Na₂HPO₄/NaH₂PO₄, MgSO₄ and KCl with 10 mM glucose or 10 mM of 1 out of the 20 proteogenic amino acids. In the case of glucose, the maximum number of spores (P_max) that had formed germ tubes was 12.7%, while time needed to reach 0.5 P_max (τ) was about 14 h. Arginine and alanine were the most inducing amino acids with a P_max of germ tube formation of 21% and 13%, respectively, and a τ of up to 33.5 h. Contrary to the typical stages of germination of fungal conidia, data show that P. roqueforti conidia can start forming germ tubes without a detectable swelling stage.

     

Effects of successivein vitro andIn vivo passages on the virulence of the entomopathogenic fungus,Nomuraea rileyi

There was no significant decrease or increase in the activity of conidia ofNomuraea rileyi after 12_{in vivo} serial passages in larvae ofTrichoplusia ni (Hübner) or after 12in vitro serial passages on a semi-synthetic medium. The LC-50 at the start of the serial passage was 16.8±4.0 conidia/mm²; after 12 serial passages the LC-50 for thein vitro- andin vivo-produced conidia was 16.2±2.5 conidia/mm² and 12.0±1.9 conidia/mm², respectively.

---

Résumé Il n'y a pas d'augmentation ou de réduction significative dans l'activité des conidies deNomuraea rileyi après 12 passages en sériein vivo chez les larves deTrichoplusia ni (Hübner) ou après 12 passages en sériein vitro sur un milieu semi-synthétique. La concentration léthale 50 au début de ces passages était de 16,8±4,0 conidies/mm², après 12 passages la CL 50 des conidies produitesin vitro etin vivo fut respectivement de 16,2±2,5 conidies/mm² et de 12,0±1,9 conidies/mm².

---

---

Mention of a proprietary product in this paper does not constitute a recommendation for use by the USDA.     

Attachment of Blastomyces dermatitidis conidia to murine bronchoalveolar macrophages: Characterization of binding the elicitation of respiratory burst

We studied the mechanisms of adherence of Blastomyces dermatitidis conidia to murine bronchoalveolar macrophages and the ability of the conidia to elicit an increase in macrophage O _inf2 ^sup- production, using an avirulent fungal strain. The number of cell associated conidia was counted by visual inspection of 2 hour macrophage monolayers incubated with conidia and O _inf2 ^sup- was measured by reduction of ferricytochrome c. Adherence of conidia to bronchoalveolar macrophages was time dependent and reached a plateau after 30 min (36±5%, 51±22%, and 36±17% macrophages with adherent conidia after 15, 30, and 60 min, respectively). Both Ca⁺² and Mg⁺² were required. The carbohydrates mannose, mannan, fucose, alpha-methylmannoside, beta-glucan, galactose, N-acetylglucosamine and chitotriose (100–1000 g/ml) did not inhibit adherence of conidia to macrophages. Trypsin treatment of macrophages or conidia did not affect binding. Conidia did not stimulate bronchoalveolar macrophage production of O _inf2 ^sup- above baseline concentrations (2.0±0.9 vs 0.8±0.5 nmol O _inf2 ^sup- , p>0.05). We conclude that murine bronchoalveolar macrophage-B. dermatitidis conidia interactions occur primarily by a non-lectin-like attachment and do not result in the production of macrophage derived O _inf2 ^sup- .     

Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

Growth and glucoamylase production by the thermophilic fungusThermomyces lanuginosus in a synthetic medium

Summary The production of glucogenic amylase from the thermophilic fungus Thermomyces lanuginosus was studied in shake flasks and laboratory fermentors. As conidia were not able to germinate in media without yeast extract, pregerminated conidia were applied as inoculum. By this procedure it was possible to use different NH _inf4 ^sup+ salts as the sole source of nitrogen for growth and amylase formation in a synthetic medium. In pH-controlled fermentors a fourfold increase in the extracellular glucogenic amylase activity was obtained with (NH₄)H₂PO₄ as the nitrogen source as compared with yeast extract. However, by fractionation of these activities, comparable yields of partially purified glucoamylases were obtained. The glucoamylase preparation from fermentations with either of the nitrogen sources had a temperature optimum at 70° C and showed similar thermal stability. By incubation without substrate at 60° C. 90% of the activity was still present after 5 h. At 70° C, 50% of the activity was retained after 30 min incubation. Offprint requests to: I. Hassum