Conidial Density Research Articles

Surface Colonization, Penetration, and Lesion Formation on Grapes Inoculated Fresh or After Cold Storage with Single Airborne Conidia of Botrytis cinerea.

Infection of grapes by different densities of airborne conidia of Botrytis cinerea was investigated on table grapes (cultivar Dauphine) harvested ripe (16°Brix) and inoculated fresh, or after SO2 treatment and 8-week storage at -0.5°C. Berries were detached at each inoculation and dusted with dry conidia in a settling tower. Following inoculation, the fresh berries were incubated for 24 h at high relative humidity (≥93%), or were overlaid with wet sterile paper towels. Cold-stored berries were incubated at high relative humidity. The effect of conidial density on surface colonization, penetration, and lesion formation was determined by surface sterilization, isolation, and freezing studies on fresh berries. Only symptom expression was determined on cold-stored berries. Fluorescence microscopy of skin segments showed that conidia were consistently deposited as single cells, and not in pairs or groups, on berry surfaces. Individual conidia, at all densities tested, readily infected the cold-stored berries and formed separate lesions after 2 days. Although the cold-stored berries were highly susceptible, lesion numbers were not related to conidial density at low inoculum dosages (0.67 to 2.60 conidia per mm2 berry surface). Lesion numbers tended to increase exponentially at higher dosages (3.24 to 3.88 conidia per mm2 berry surface). Individual conidia, however, did not induce any disease symptoms on fresh berries. Removal of the pathogen after 24-h incubation from the surface of fresh berries by ethanol, and subsequent incubation of excised skin segments revealed that, irrespective of the conidial density or the wetness regime, less than 2% of skin segments were penetrated. Furthermore, increasing densities of conidia did not lead to higher rates of surface colonization and skin penetration. The low incidence of disease caused on fresh berries and high disease incidence induced after prolonged cold storage indicated that infection was not governed by conidial density on berry surfaces, but by the level of host resistance.

Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by self-inhibitors and requires surface attachment.

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of Magnaporthe grisea, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in Colletotrichum gloeosporioides and Colletotrichum trifolii differentiation. After calmodulin cDNA and genomic DNA from M. grisea were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of M. grisea appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.

The survival and saprophytic competitive ability of the Botrytis spp. antagonist Ulocladium atrum in lily canopies

In 1995, conidia of Ulocladium atrum were applied to a canopy of green lily (Lillium spp.) leaves in order to investigate its survival, colonisation of artificially induced necrotic leaf tissues and competitive ability against Botrytis spp. and naturally occurring saprophytes. U. atrum conidia density cm-2 at the top and middle canopy levels was not significantly different following application of the antagonist with a propane powered backpack sprayer. In repeat experiments, conidia density on leaves at the lower canopy level was 18% to 20% of that deposited onto leaves at the top of the lily canopy. There was a significant (P < 0.001) linear decline of U. atrum conidia over time and after 21 days conidia density had declined by up to 73%. Germination of U. atrum on green leaves in the field reached a maximum of 81%, seven days after antagonist application. Conidial viability, measured as germination potential, declined slightly (100% to 88%) after seven days exposure to field conditions but there were no further changes in the germination potential even after 21 days of field exposure. The germination potential was not affected by canopy level. The ability of surviving U. atrum conidia to colonise necrotic tissues, artificially induced with paraquat, was measured. U. atrum colonisation was consistently highest on necrotic leaves at the top level of the canopy and consistently lower on leaves from the bottom canopy level. Necrotic leaf colonisation by U. atrum decreased over time from 51% (necrosis induced immediately after antagonist application) to 21% when necrosis was induced 21 days after antagonist application. A significant (P < 0.001) linear relationship (R2 = 0.713) between colonisation of necrotic tissues and conidia density prior to induction of necrosis was detected. When necrosis was induced immediately after antagonist application, U. atrum outcompeted commonly occurring saprophytic Alternaria spp. and Cladosporium spp. The ability of U. atrum to significantly reduce colonisation by Alternaria spp. was maintained for up to 21 days. Botrytis spp. did not occur in these field experiments. It was concluded that U. atrum had the ability to survive and persist in the phyllosphere for up to 21 days in the field and provided further evidence that U. atrum has the necessary survival characteristics to be a successful biological control agent of Botrytis spp.

Cuticular waxes relieve self-inhibition of germination and appressorium formation by the conidia ofMagnaporthe grisea

We tested whether germination and appressorium formation by the conidia ofMagnaporthe grisearequires a hydrophobic surface. On specially cleaned cover glass surfaces conidia ofM. griseagerminated but did not form appressoria. Addition of rice leaf wax, other plant surface waxes or syntheticn-C22fatty acid, fatty alcohol or alkane induced appressorium formation. The requirement for the presence of hydrophobic material to induce appressorium formation was dependent on the conidial population density. As the conidial concentration decreased from 105ml−1to 104ml−1, the need for the lipid decreased to the point of not requiring any lipid to obtain nearly complete appressorium formation, suggesting possible presence of self-inhibitors on the conidia. This suggestion was supported by the observation that the surface lipids washed out of the conidia inhibited germination and appressorium formation in a dose-dependent manner. This inhibition could be reversed by plant surface wax. These results suggest that conidia ofM. griseacarry lipophilic self-inhibitors which prevent germination at the site of sporulation, and that the plant cuticle can relieve this self-inhibition when conidia land on the plant surface.

Biological Control of Rose Powdery Mildew with the Antagonist Yeast Tilletiopsis pallescens

The efficacy of Tilletiopsis pallescens Gokhale, a naturally occurring ballistosporeforming yeast isolated from mildew-infected leaves, was evaluated as a biological control agent against rose powdery mildew [Sphaerotheca pannosa (Wallr.:Fr.) Lév. var. rosae Woronichin]. Two trials were conducted on potted rose (Rosa sp.) plants (1-year-old cv. Cardinal Pink) under commercial greenhouse-growing conditions during the summer (June to September) when mildew was most severe. Mildew-infected plants were subjected to one of four treatments: a T. pallescens spore suspension applied three times (3–4 d apart), distilled water (applied three times), one application of T. pallescens spore suspension, or one application of culture filtrate without spores. Two weeks after treatment began, mildew development was evaluated by enumerating conidial density on sampled leaflets. Sporulation was significantly reduced (by 97%–98%) on plants treated with three applications of T. pallescens spore suspension, compared to a 47%–57% reduction on plants treated with three applications of distilled water. There was no significant difference in conidial density between plants treated with one application of T. pallescens spore suspension and plants treated with one application of its culture filtrate, with a 78%–94% reduction in conidia, which was significantly higher than for the water treatment. The mode(s) of action of T. pallescens appears to be eradicant and associated with enzymes or metabolites produced in the culture filtrate. The results from this study demonstrate the potential for biological control of rose powdery mildew under commercial growing conditions in British Columbia.

PERSISTENCE OF BEAUVERIA BASSIANA IN SOIL FOLLOWING APPLICATION OF CONIDIA THROUGH CROP CANOPIES

AbstractThe influence of three formulations, water, oil, and a 5% oil emulsion, and two crops, alfalfa and crested wheatgrass, on the deposition and subsequent persistence of Beauveria bassiana (Balsamo) Vuillemin conidia in soil was investigated. The alfalfa canopy was considerably denser than that of wheatgrass. Leaf area indices for alfalfa ranged from 1.8 to greater than 2, those for wheatgrass ranged from 0.24 to 0.55. Initial populations of conidia averaged 1.2 × 103 to 2.6 × 104 colony-forming units (cfu) per gram of dry weight of soil under alfalfa, and 5.5 × 103 to 3.4 × 104 cfu per gram of soil under wheatgrass. There was no consistent influence of formulation or application method (high or ultra low volume) on penetration of conidia through the canopy of either crop. However, conidial populations under wheatgrass were larger than those under alfalfa in two of three trials. After 225–272 days (over winter), substantial populations (87 to 4.3 × 104 cfu/g) were recovered from soil. Although conidial densities decreased over time, reductions in population size over this period were generally less than one order of magnitude; neither crop nor formulation consistently influenced conidial persistence. In most instances, a rapid decrease in conidial populations was observed within approximately 20 days but thereafter, the rates of population decline abated. The initial decrease in conidial numbers did not appear to be related to precipitation. This study demonstrates that substantial numbers of B. bassiana conidia infiltrate crop canopies, are deposited on the soil surface, and subsequently persist in a clay–loam soil. The aerial application of B. bassiana conidia to vegetated roadsides may prove useful for the management of ovipositing grasshoppers and emerging nymphs.

Inoculum Density and Infection Efficiency of Conidia and Conidiophores of Isolates ofPyrenophora tritici-repentis

Glass slides and wheat leaves were inoculated with conidia and conidiophores of Pyrenophora tritici-repentis isolates to compare the density (no./cm2) of propagules on nonhost and host surfaces. Regression functions of the density of each propagule form on glass slides, as a function of the inoculum concentration, overestimated the density of each propagule form on wheat leaves by three to four times. Subsequently, conidia and conidiophores of three isolates were inoculated at equal rates of propagule density on wheat cultivars TAM 105 (susceptible) and Red Chief (resistant) to compare lesion incidence resulting from the different forms of propagule. Conidia caused 26 times more lesions than did conidiophores, and differences among the isolates for lesion incidence were significant (P less than or equal to 0.05). Finally, the infection efficiency of the three isolates was determined utilizing their conidiophores and conidia in separate inoculum suspensions. Infection efficiency was determined from the slope of the regression of lesion incidence as a function of the density of propagules per unit area of inoculated leaf. Infection efficiency for conidia of the isolates ranged from 0.91 to 0.55 whereas infection efficiency for their conidiophores was not significantly different from zero or was extremely variable. Results indicate that studies of epidemiological parameters of P. tritici-repentis are more precise when based on estimates of conidial density on host leaf surfaces, and when conidiophores are excluded from inoculum suspensions. The estimates of infection efficiency should prove useful in the identification of virulent isolates of P. tritici-repentis and should lead to improved identification of resistance to tan spot.

The Role of Organic Matter in the Population Dynamics of the Endoparasitic Nematophagous Fungus Drechmeria Coniospora in Microcosms

Addition of organic matter to soil may indirectly enhance nematophagous fungi. The effect of organic amendments on the population dynamics of the endoparasitic nematophagous fungus Drechmeria coniospora was studied in soil microcosm experiments. In one experiment, soil was amended with lucerne meal, and in a second experiment the root system of barley served as the source of organic matter. Indigenous soil bacteria, the bacterivorous nematodes Rhabditis sp. or Acrobeloides butschlii, and D. coniospora were added as representatives of the first, second, and third trophic level. In lucerne meal amended soil, the conidial density of D. coniospora increased in response to increase in number of nematodes. Rhabditis sp. appeared to be a more susceptible host than A. butschlii. Nematode population density was suppressed by D. coniospora. In the rhizosphere of barley, a small but significant increase in the conidial density was measured as compared to the control without nematodes, but nematode numbers were not suppressed. In rhizosphere soil from two fields, the densities of endoparasitic and trapping fungi were not related to numbers of nematodes. Drechmeria coniospora was not detected in the field. We conclude that introduction of conidia of D. coniospora in combination with high input of organic matter may lead to suppressive levels of the fungus. The possible application for control of plant-parasitic nematodes is doubtful because of the narrow host range of the fungus.

Dimensions, Density, and Settling Velocity of Entomophthoralean Conidia: Implications for Aerial Dissemination of Spores

The epizootic behavior of entomopathogens is determined, in part, by their capacity for spatial spread in a host population. Among the entomophthoralean fungi, this may be accomplished through various mechanisms, including passive aerial dissemination of conidia. In this study we measured the size, shape, density, and settling velocity in still air of conidia of three species of entomophthoralean fungi: Zoophtora radicans, Conidiobolus obscurus, and Conidiobolus thromboides. In addition, the aerodynamic behavior of Z. radicans conidia was observed. Size and shape, measured by light microscopy, were consistent with previously reported values. Conidial densities ( 1.10 g cm -3 for Z. radicans and 1.06 g cm -3 for Conidiobolus spp.) were determined from the refractive index of spores measured by interference microscopy; these are the first reports in the literature of the density of entomophthoralean conidia. Settling velocity was measured by allowing fungal cultures to sporulate into a glass chamber, while falling conidia were observed by video microscopy. Images of falling spores were analyzed by a computerized motion analysis system, and settling velocities were determined. The system was calibrated by measuring the settling velocity of polystyrene microspheres of sizes similar to those of the spores and comparing observed velocities to those predicted by Stokes' Law. These are the first reports of settling velocity for entomophthoralean conidia. The nearly spherical spores of C. thromboides fell at velocities expected from Stokes' Law (median, 1.4 cm sec -1), while the spherical, but larger, spores of C. obscurus fell more slowly than expected (median, 4.6 cm sec -1), for reasons that are unclear. Conidia of Z. radicans fell at a velocity equal to that of spheres of the same volume and density (median, 0.4 cm sec -1), with little or no correction needed for their ellipsoidal shape. The settling velocity of ellipsoidal particles depends on their orientation. The falling orientation and aerodynamic behavior of conidia of Z. radicans were observed by high-resolution video microscopy; the spores consistently fell in a stable orientation with their major axis vertical. Our measurements of conidial size, shape, density, and settling velocity in still air provide information needed for models of particulate transport, which can be used to predict spore dispersal under field conditions involving wind and turbulence above and within plant canopies.

Adhesive knob formation by conidia of the nematophagous fungus Drechmeria coniospora.

We studied conidiogenesis and adhesive knob formation (maturation) by newly developed conidia of the nematophagous fungus Drechmeria coniospora. Upon conidiogenesis on infected nematodes or during saprophytic growth of the fungus in axenic cultures compact clusters of conidia developed. Less than 10% of such clustered conidia matured; mature conidia were invariably located on the periphery of the clusters. The kinetics and rate of maturation of conidia were studied in in vitro systems and in soil. In both cases adhesive knobs were formed; the rate at which knobs were formed appeared to be determined by the age of the conidia, the temperature and the soil moisture. In addition, knob formation was suppressed at increasing conidial densities. Under favorable conditions, however, over 90% of the conidia matured within a period of 3 days. The rate of knob formation was neither influenced by the presence of nematodes nor by that of exogenous nutrients, which suggests that maturation is an autonomous process. Electron-microscopical analysis indicated that budding of the conidia at the initial stage of maturation occurred simultaneously with the deposition of the sticky, adhesive layer around the wall of the developing knob. The ecological significance of the time- and spatially separated maturation of conidia after conidiogenesis is discussed with respect to survival of the conidia.

Conidial biomass and desiccation tolerance of Trichoderma harzianum produced at different medium water potentials

Abstract Fungal biomass to be used for biological control of plant pathogens must be economical to produce, able to withstand desiccation, and stable under storage conditions. Polyethylene glycol (PEG) 200 was added to modified Richard's medium (RMS) to create different medium water potentials for producing Trichoderma harzianum. Water potentials of RM8 and RM8 with 6% (v/v) PEG 200 were −0.82 and −1.88 MPa, respectively. As medium water potentials were lowered with increasing concentrations of PEG 200 in RM8, the biomass dry weights decreased. However, conidial density and colony forming units (cfu) in the dry biomass were significantly improved to a maximum at −1.88 MPa. The total cfu number (biomass dry weight × cfu/g) in the dry biomass produced in RM8 at −1.88 MPa was increased on the average about 14-fold over that produced in RM8 alone. Although the cultivation temperatures did not affect the yield of the biomass, raising the cultivation temperature to 30°C and lowering the medium water potential to −1.88 MPa at 24 h of fungal growth enhanced conidial formation and cfu numbers after drying. Under these growth conditions, conidiophores were formed within 12 h after PEG 200 was added; intensive conidiation occurred within 48 h and reached the maximum level in 84 h of incubation. Trehalose levels have been correlated with desiccation tolerance of T. harzianum. The concentration of trehalose in the dried conidial biomass produced in this manner was higher than that produced in RM8 alone, and the germination percentage of the dried conidia was 65%.

Water potential, temperature, and clay-coating of Beauveria bassiana conidia: Effect on Spodoptera exigua pupal mortality in two soil types

When Spodoptera exigua pupae were buried for 10 days at 24°C in nonsterile soil inoculated with Beauveria bassiana conidia, 1 × 10 7 conidia/cm 3 of soil was the lowest conidial density to produce disease in pupae and adults. At water potentials of −37 and −200 bars (24°C), adult emergence was reduced when 3.2 × 10 7 or 1 × 10 8 conidia/cm 3 was used in Yolo fine sandy loam (YFSL) and Staten peaty muck (peat). In these dry soils, adult emergence was as low as 6% at 1 × 10 8 conidia/cm 3. At water potentials of −0.3, −2, and −15 bars, a concentration of 1 × 10 8 conidia/cm 3 was necessary to reduce adult emergence. In these treatments, 18 to 62% of the pupae eclosed to the adult stage. Thus, adult emergence was greater at water potentials from −0.3 to −15 bars that at −37 and −200 bars. Also at −0.3 and −2 bars, adult emergence was greater in peat than in YFSL. Uninoculated control soils had 85–94% adult emergence. Adult emergence increased as the temperature decreased from 25° to 8°C. At 8°C, adult emergence was the same in inoculated and uninoculated soil. Clay-coating of conidia, to increase conidial longevity, did not influence S. exigua mortality.

Taxonomy and biology of the lettuce anthracnose fungus

The morphology and development of the causal organism of lettuce anthracnose, Marssonina panattoniana , was compared with Microdochium nivale, M. oryzae and M. stoveri . The species are congeneric and the new combination Microdochium panattonianum is proposed. Conidia from fresh lettuce or cultures differed significantly in size from those in herbarium material. Germination occurred at temperatures between 3–26°C and pH 4–5.2. Germination decreased following exposures > 9 h at 32°, 4 h at 34° and 1 h at 36°. Germination did not occur in the absence of free water, at −4 MPa water potential, or at conidial densities of 5100 mm −2 . Germ-tube growth was optimum at 20° and pH 5.2; at this temperature germination commenced after 4 h, the second germ-tube appeared after 20 h and sporulation at 44 h. Germ-tube growth was inhibited following 1 h at 32°, 34° and 36°. Colony growth was linear with time and was optimum at 15–20° with growth on PDA > MEA > TWA. Conidial production increased logarithmically 2–7 d after incubation at 20°.

Effect of peanut mottle virus on reaction of peanut cv. Tamnut 74 to Cercospora arachidicola

Abstract Two-week old seedlings of Tamnut 74 peanut were inoculated with peanut mottle virus (PMV) or left nontreated, and four weeks later were sprayed with a conidial suspension of Cercospora arachidicola (2 × 104 conidia/mL) prior to placement in a polyethylene enclosure in a growth chamber (22–30 C, 100% RH). After three weeks, number of lesions/leaflet, leaflet area (mm2), necrotic area (mm2)/leaflet, percent necrotic area, conidial density (conidia/mm2) of necrotic area), and conidia/leaflet, was determined on four leaflets for each of 12 PMV-infected and 12 virus-free plants. Conidia/leaflet (one important parameter in evaluating reaction of peanut genotypes to C. arachidicola), lesions/leaflet, area of lesion, and leaflet area was reduced on PMV-infected plants compared to virus-free plants in each of 4 experiments. Similarly, necrotic area/leaflet were reduced in 3 of 4 experiments. Conidia/mm2 of necrotic area was reduced in 2 of 4 experiments. PMV was not detected in conidia of C. arachidicola from PMV infected plants by inoculation to a PMV indicator host, serology, electron microscopy or electrophoresis.

Effect of Auxin and Gibberellin on Conidial Germination in Neurospora crassa II. “Conidial Density Effect” and Auxin

Effect of Auxin and Gibberellin on Conidial Germination in <italic>Neurospora crassa</italic> II. “Conidial Density Effect” and Auxin

Roles of low pH, carbon and inorganic nitrogen source use in chlamydospore formation by Fusarium solani.

Citrate and malate were poorer sources of exogenous carbon than several hexose, pentose, or disaccharide sugars for supporting macroconidial germination by Fusarium solani at high conidial density (1 X 10(5) condia/ml). Only citrate, however, failed to block chlamydospore morphogenesis to a degree comparable to glucose or other readily used sugars. Mostly immature chlamydospores were formed in the presence of citrate. At low conidial density (5 X 10(3) conidia/ml), exogenous carbon-independent macroconidial germination and subsequent rapid chalmydospore formation on germ tubes was not inhibited by ammonium or nitrate nitrogen. The citrate-phosphate buffered, low pH (4.0) medium of Cochrane induced more immature chlamydospore formation by F. solani than a pH 6.0 medium, but few mature chlamydospores were formed in either medium. Condensation of hyphal cytoplasm into developing chlamydospores, a character typical of chlamydospore formation, did not occur extensively and macroconidia, hyphae, and immature chlamydospores stained deeply with Sudan III, suggesting lipid biosynthesis. This inhibition of chlamydospore maturation may be due partly to nitrogen deficiency imposed by the high C:N ratio of the medium and to the presence of citrate. Only vesiculate hyphal cells were formed by F. solani f. sp. phaseoli in both media. Field soils to which the clone of F. solani used is indigenous had mean pH values ranging from 5.2 to 6.0.

Entomophthora species infecting pea aphis

SUMMARY Three species of Entomophthora infected the pea aphis, Acyrthosiphon pisum, on lucerne at Rothamsted Experimental Station, Harpenden, Hertfordshire, between 1967 and 1972 and caused a maximum mortality of 83% in July 1968. E.thaxteriana, the commonest species, occurred each year and frequently infected aphids during cool weather in spring and autumn. E.aphidis and E.planchoniana were absent in some years and were most common during warm weather in midsummer. The incidence of infection by the three species was correlated with the density of conidia in the air above the crop, host density and rainfall, but not with any other weather factor considered. Infection at any time was much more closely related to environmental factors during the preceding 12 than 3 days. The regressions, however, were weak and the relative importance of different environmental factors for the spread of infection can probably only be determined under controlled conditions in the laboratory.

Tyrocidin inhibition: effect of Tween 80 and conidial density

Tyrocidin inhibition: effect of Tween 80 and conidial density

Exogenous carbon and nitrogen requirements for conidial germination by Aspergillus flavus.

In a phosphate-buffered (pH 5.7) inorganic salts solution (B), nearly full dependence on exogenous carbon was found for germination of washed, Tween 20-treated conidia of Aspergillus flavus over a range of conidial densities. No exogenous carbon-independent germination was observed for settling tower-deposited dry conidia. At equivalent carbon, an amino acid mixture supported highest germination of washed conidia; proline, alanine, or glucose plus NH4Cl supported higher germination than several other single amino acids or single sugars plus NH4Cl tested. When exogenous carbon was supplied as glucose, total and percentage germination of washed conidia were influenced more by exogenous nitrogen, supplied as NH4Cl, as the conidial density increased and as the glucose and NH4Cl concentrations increased. At equivalent nitrogen, proline alone, or certain amino acid mixtures supported highest germination of several nitrogen sources in B solution plus glucose. Germination decreased as the conidial density decreased when exogenous carbon and nitrogen were supplied at each density at constant amounts per conidium. Removal of CO2 from the germination medium inhibited germination. Tween 20 and Tergitol NPX stimulated germination of settling tower-deposited conidia. "Amino acid carry-over" for conidial germination was not demonstrated. The possible relation of these findings to the ecology of conidial germination in soil is discussed.

Exogenous carbon and nitrogen requirements for chlamydospore germination by Fusarium solani: dependence on spore density

Chlamydospores were formed on germ tubes of macroconidia of F. solani after germination at 3 × 105 conidia/ml in an inorganic salt solution (pH 5.7) containing glucose (40 μg C/ml) plus NH4Cl (2.6 μg N/ml), and at 3 × 103 conidia/ml in salt solution containing no glucose or NH4Cl. After 5 days, chlamydospores formed in the low conidial density system required an exogenous source of carbon for high germination, whereas chlamydospores formed in the high conidial density system required both exogenous carbon and nitrogen for high germination. Chlamydospores formed in the high conidial density system did not require exogenous nitrogen for high germination when the spore density (chlamydospores plus some ungerminated conidia) was reduced from 3 × 105 to 3 × 104 spores/ml; high germination was observed at 3 × 103 spores/ml in the absence of both exogenous carbon and nitrogen.