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.     

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.     

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.     

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.

     

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

Susceptibility of different life stages of the European cherry fruit fly,Rhagoletis cerasi,to entomopathogenic fungi

The effects of six fungus isolates on the mortality of different life stages of the European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), were assessed in a series of laboratory experiments to find an isolate suitable for biological control. In a first step, the effects of fungus treatments on mortality, mycosis and fecundity of adult flies at a concentration of 10⁷ conidia/ml were evaluated. All fungus isolates caused mycosis but virulence varied considerably among the isolates. Beauveria bassiana and Isaria fumosorosea caused 90–100% mortality and had the strongest influence on fecundity. Metarhizium anisopliae also induced high rates of mortality, while the pathogenicity of Isaria farinosa was low. The effects of lower conidia concentrations and the influence of the age of flies were assessed in a second step. Higher conidia concentrations generally resulted in a higher mortality. B. bassiana was most efficient at low concentrations. Young flies showed lower mortality rates than older flies but, sub‐lethal effects on eclosion rate of eggs were greater in younger flies. Finally, the effects on L₃ larvae were tested: none of the fungus isolates induced mortality in more than 25% of larvae. As L₃ larvae and pupae are not susceptible to fungus infection, field control of R. cerasi should be focused on adult flies.     

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.     

THE EFFECT OF SOME PHYSIOLOGICAL AND ENVIRONMENTAL FACTORS ON SCLEROTIAL ASPERGILLI

Rudolph , Emanuel D. (Ohio State U., Columbus.) The effect of some physiological and environmental factors on sclerotial Aspergilli. Amer. Jour. Bot. 49(1): 71–78. Illus. 1962.—The effect of varying conditions of carbon-nitrogen balance, temperature, pH, and light upon the formation of sclerotia by 6 species of Aspergillus (A. alliaceus, A. avenaceus, A. flavus, A. quercinus, A. sclerotiorum and A. wentii) was studied. On Czapek's agar, optimal growth as well as maximum production of sclerotia and conidia took place at high sucrose and nitrate concentrations. In general, fewer sclerotia were formed with glucose than with sucrose, and very poor growth took place with lactose. Sclerotia were formed best at temperatures that were optimal or below optimal for mycelial growth. The ranges of pH through which sclerotia were formed were narrower than those through which conidia and mycelia were formed. Light had no effect upon sclerotium formation. The formation of sclerotia in A. alliaceus was found to represent the strand-type development. A number of UV-induced strains and a spontaneous mutant strain of A. alliaceus showing varying amounts of sclerotium and conidium production are characterized. It is suggested that the sclerotia in Aspergillus are sterile stromata.     

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH₄)₂SO₄ (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH₂PO₄. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.     

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.     

Phoma sp. FOM-8108, a producer of gentisylquinones, isolated from sea sand

A fungal strain, FOM-8108, that was isolated from sea sand was found to produce gentisylquinone and chlorogentisylquinone, inhibitors of neutral sphingomyelinase. The fungus grew well under normal conditions, in the darkness, or on various agar media, but typical morphological characteristics were not observed to determine its taxonomy. Therefore, culture conditions were studied extensively, resulting in formation of a number of pycnidia by the fungus on the media containing seawater or on natural substrates such as hydrangea leaves, gardenia leaves, and rice straw under natural light or near-ultraviolet radiation exposure. Eventually, from the morphological characteristics of pycnidia, conidiogenous cells, and conidia, strain FOM-8108 was considered to belong to the genus Phoma. Culture studies showed seawater in the fermentation medium was necessary for the strain to produce gentisylquinones. Particularly, a full production of chlorogentisylquinone, which has a chloride ion in the structure, was performed in the medium with higher concentration (75%–100%) of seawater. Received: June 27, 2001 / Accepted: December 14, 2001     

一株维氏气单胞菌发酵培养基优化及其制备疫苗免疫效力比较

采用单因素试验、响应面试验法对维氏气单胞菌(Aeromonas veronii)发酵培养基的氮源、碳源、无机盐和磷酸盐成分及用量进行优化组合,确定优化培养基组成：胰蛋白胨10.8 g/L,葡萄糖5.0 g/L,牛肉膏3.0 g/L,磷酸二氢钾2.0 g/L,硫酸镁0.4 g/L,NaCl 5.0 g/L。并与基础培养基的发酵活菌数、制备的灭活疫苗免疫效力进行比较,经过验证试验绘制维氏气单胞菌在优化培养基条件下的7 L发酵罐生长曲线。在优化发酵培养基条件下,维氏气单胞菌活菌数为5.94×10⁹ cfu/mL,比基础培养基增幅43.13%;制备的灭活疫苗相对保护率为77.78%,比基础培养基提高了14.81%。7 L发酵罐发酵培养10 h,活菌数达到最大8.85×10⁹ cfu/mL。通过对发酵培养基的优化,可以获得低成本、优质高效的维氏气单胞菌发酵菌液,为今后维氏气单胞菌灭活疫苗规模化发酵培养提供参考。     

The Infection Process of Pestalotiopsis Longisetula Leaf Spot on Strawberry Leaves

Pestalotia leaf spot, caused by the fungus Pestalotiopsis longisetula Guba, has become the major disease affecting strawberry production in Brazil. Strawberry seedlings with 4–5 leaves were inoculated with a conidial suspension of P. longisetula (2 × 10⁵ conidia/ml), and leaf samples were collected at 48, 72, 96 and 144 h after inoculation (hai) for observation in the scanning electron microscope. Conidia germinated within 48 hai. At 72 hai, conidia had formed very long germ tubes over the epidermal cells without any evidence of appressorial formation nor direct penetration. At 96 hai, fungal hyphae grew inter‐ and intracellularly in the lacunous parenchyma and also through tracheary elements. Pycnidia were first observed on the leaf surface at 96 hai. At 144 hai, conidia of P. longisetula were first liberated from the pycnidia. This study adds new information to better understand of the infection process of P. longisetula that may help in developing more effective disease control strategies.     

Effect of glucose on Listeria monocytogenes biofilm formation,and assessment of the biofilm’s sanitation tolerance

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v^–1) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.     

Promotion of conidial head formation in Aspergillus oryzae by a salt

Addition of KCl to medium at 0.1 M or higher promoted the formation of conidial heads in Aspergillus oryzae. When higher concentrations of KCl were added, a larger number of conidial heads were formed. NaCl and MgCl₂ were slightly less effective than KCl. The effect of salt on the formation of conidial heads on a minimal medium was as high as on a potato/dextrose medium but slightly higher than that on a complex medium when 1 M KCl was added.     

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.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.     

Impact of nutritional conditions on yields,germination rate and shelf-life of Plectosporium alismatis conidia and chlamydospores as potential candidates for the development of a mycoherbicide of weeds in rice crops

The effect of nutritional conditions on spore qualities was investigated in order to select which propagules, conidia or chlamydospores, would be most suitable for mycoherbicide development. Plectosporium alismatis was grown in a liquid basal medium supplemented with glucose and a mineral nitrogen source (sodium nitrate) or an organic nitrogen source (casamino acids). Conidial and chlamydospore yields, germination rate and shelf-life were compared. Two growth models were developed: on one hand, sodium nitrate added as the sole nitrogen source was partially utilised (8%), resulting in poor growth (1.77±0.02 mg mL^?1; 6±1.7×10⁵ conidia mL^?1). Under these conditions, P. alismatis produced dense, melanised-like aggregates that contained chlamydospores (12.4±0.7×10⁴ chlamydospores mL^?1). Germination rates of chlamydospores and conidia produced under these conditions was high (80%). Twenty percent of chlamydospores were able to germinate after 4 months storage at 25°C, while survival of conidia declined rapidly (<2%). When casamino acids were added to the liquid medium as the sole nitrogen source, P. alismatis produced sparser pellets resulting in high dry weights (5.37±0.09 mg mL^?1 and high conidia numbers (9.6±1.5×10⁶ conidia mL^?1), while no chlamydospore were observed. The germination rate of conidia produced in casamino acids was low (33±13%) after 8 h incubation and microcycle conidiation occurred. Five percent of these conidia germinated after 4 months storage. These data indicate that chlamydospores may be suitable for mycoherbicide development, provided further optimisation of yields is achieved.     

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.

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A procedure for isolating Pyrenochaeta terrestris from onion roots

Surface sterilised stelar tissues from onion roots exhibiting typical pink root symptoms were plated on water agar and incubated under cool white fluorescent light (CWFL) for 12 h day^-1 at 20°C. After 4–5 days, the plates were examined with the 10? objective of a compound microscope and tissues producing conidia of Fusarium spp. were discarded. Sub-cultures of isolates suspected to be Pyrenochaeta terrestris, grown from Fusarium-negative tissues, were transferred to chloroamphenicol-amended (500 ppm) corn-meal (CCMA) agar plates and incubated at 24°C under CWFL for a further 10–14 days. Cultures with setose pycnidia were identified as P. terrestris. Plates were again scanned for conidia of Fusarium spp., and cultures negative for Fusarium spp., but not producing pycnidia, were suspected to be P. terrestris. These were compared with known isolates on CCMA. On this medium growth of P. terrestris was slow, producing appressed, pinkish colonies which were circular with a smooth margin. All isolates of P. terrestris isolated by the procedure caused pink root of onion when tested, whereas none of the Fusarium spp. isolated produced the disease. However, a mixed inoculums of P. terrestris and Fusariurn solani produced typical pink root symptoms.