Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in Ceratocystis Ulmi

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Microbiology Papers in the Biological Sciences 3-1-2004 Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in Ceratocystis ulmi Jacob M. Hornby University of Nebraska-Lincoln, [email protected] Sarah M. Jacobitz-Kizzier University of Nebraska-Lincoln Donna J. McNeel University of Nebraska-Lincoln Ellen C. Jensen University of Nebraska-Lincoln, [email protected] David S. Treves University of Nebraska-Lincoln See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro Part of the Microbiology Commons Hornby, Jacob M.; Jacobitz-Kizzier, Sarah M.; McNeel, Donna J.; Jensen, Ellen C.; Treves, David S.; and Nickerson, Kenneth W., "Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in Ceratocystis ulmi" (2004). Papers in Microbiology. 37. https://digitalcommons.unl.edu/bioscimicro/37 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Jacob M. Hornby, Sarah M. Jacobitz-Kizzier, Donna J. McNeel, Ellen C. Jensen, David S. Treves, and Kenneth W. Nickerson This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ bioscimicro/37 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2004, p. 1356–1359 Vol. 70, No. 3 0099-2240/04/$08.00ϩ0 DOI: 10.1128/AEM.70.3.1356–1359.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved. Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in Ceratocystis ulmi Jacob M. Hornby,† Sarah M. Jacobitz-Kizzier,‡ Donna J. McNeel,§ Ellen C. Jensen,¶ David S. Treves,ʈ and Kenneth W. Nickerson* School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0666 Received 25 June 2003/Accepted 10 December 2003 We studied the inoculum size effect in Ceratocystis ulmi, the dimorphic fungus that causes Dutch elm disease. In a defined glucose-proline-salts medium, cells develop as budding yeasts when inoculated at >106 spores per ml and as mycelia when inoculated at <106 spores per ml. The inoculum size effect was not influenced by inoculum spore type, age of the spores, temperature, pH, oxygen availability, trace metals, sulfur source, phosphorous source, or the concentration of glucose or proline. Similarly, it was not influenced by added adenosine, reducing agents, methyl donors, amino sugars, fatty acids, or carbon dioxide. Instead, growing cells excreted an unknown quorum-sensing factor that caused a morphological shift from mycelia to budding yeasts. This yeast-promoting effect is abolished if it is extracted with an organic solvent such as ethyl acetate. The quorum-sensing activity acquired by the organic solvent could be added back to fresh medium in a dose- dependent fashion. The quorum-sensing activity in C. ulmi spent medium was specific for C. ulmi and had no effect on the dimorphic fungus Candida albicans or the photomorphogenic fungus Penicillium isariaeforme.In addition, farnesol, the quorum-sensing molecule produced by C. albicans, did not inhibit mycelial development of C. ulmi when present at concentrations of up to 100 ␮M. We conclude that the inoculum size effect is a manifestation of a quorum-sensing system that is mediated by an excreted extracellular molecule, and we suggest that quorum sensing is a general phenomenon in dimorphic fungi. Fungal dimorphism is defined (20) as an environmentally 250 ␮M prevents the yeast-to-mycelium conversion, resulting controlled reversible interconversion of the yeast and mycelial in actively budding yeasts without influencing cellular growth morphologies. Interest in this phenomenon derives from the (9). Farnesol exhibits general cross-reactivity within C. albicans prevalence of dimorphism among those fungi exhibiting patho- in that supernatants from strain A72 are active on five other genicity towards plants and animals. Numerous chemical and strains of C. albicans and vice versa. environmental parameters have been reported to shift the In Ceratocystis ulmi (12), the causative agent of Dutch elm yeast-mycelium dimorphism. Among these have been temper- disease, the nitrogen source controls dimorphism. At cell den- ature (18), pH (18), glucose levels (2, 3, 5, 18), nitrogen source sities of Ն106/ml in a defined liquid medium containing phos- (12, 22), carbon dioxide levels (2), and transition metals and phate salts and either glucose or sucrose, proline (10 mM) chelating agents (3, 8, 17, 18), as well as the inoculum size or induced the yeast morphology, while a 10 mM concentration of cell density employed (3, 12, 19, 24). either ammonium, asparagine, or arginine induced the myce- We have been studying quorum sensing in the regulation of lial morphology (12). For both the ammonium- and arginine- yeast-mycelium dimorphism in fungi. In Candida albicans,we containing media, inoculum size (103 to 108 blastospores per recently showed (9) that the inoculum size effect results from ml, final concentration) had no effect on morphology; mycelia production of farnesol. Farnesol is continuously excreted by C. were produced in all cases. With proline, budding yeasts albicans during growth in amounts roughly proportional to the formed only when the cell concentration was Ն106 blasto- number of CFU per milliliter. At a sufficiently high level (1 to spores per ml. Smaller inoculum sizes produced a transient 5 ␮M), farnesol prevents mycelial development during growth. mycelial stage with the mycelium length inversely proportional It also blocks germ tube formation caused by three chemically to inoculum size (12). We termed this phenomenon the “inoc- distinct triggers: L-proline, N-acetylglucosamine, and serum. In ulum size effect” (12). Throughout we will refer to the extra- all cases, the presence of farnesol at concentrations of up to cellular cell density-dependent signals produced by C. ulmi and C. albicans as “quorum-sensing molecules” (QSMs), in part because there is little information on the mode of action of * Corresponding author. Mailing address: School of Biological Sci- these factors. Our objective in this study was to determine how ences, University of Nebraska, Lincoln, NE 68588-0666. Phone: (402) 472-2253. Fax: (402) 472-8722. E-mail: [email protected]. similar the inoculum size effect in fungi is to quorum sensing in † Present address: Division of Natural Sciences, Lewis-Clark State bacteria, whether dimorphic fungi other than C. albicans use College, Lewiston, ID 83501. similar signaling systems, and whether farnesol could initiate ‡ Present address: Department of Bacteriology, University of Wis- cross talk between C. albicans and those other fungi. consin, Madison, WI 53706. § Deceased 27 November 2000. ¶ Permanent address: Biological Sciences, College of St. Benedict’s MATERIALS AND METHODS and St. John’s University, Collegeville, MN 56321. ʈ Present address: Department of Biology, Indiana University South- Organism. C. ulmi (Buism), C. Moreau, was obtained from the National east, New Albany, IN 47150. Center for Agricultural Utilization Research (NRRL 6404 and 6405) Peoria, Ill., 1356 VOL. 70, 2004 EXTRACELLULAR CONTROL OF YEAST-MYCELIUM DIMORPHISM 1357 as was the photomorphogenic fungus Penicillium isariaeforme (NRRL 2639). C. TABLE 1. Effect of inoculum size on cell morphology in C. ulmi at ulmi also is known as Ophiostoma ulmi. Cultures were maintained at 5°Con 20 h of growth potato dextrose agar (PDA) (1) slants; culture transfers to fresh PDA slants were a made every 6 months. Inoculum size % of mycelia with growth on : Growth medium. The defined liquid medium contained the following per liter cells/ml GPP GPR of distilled water: glucose (or sucrose), 20 g; KH2PO4,4g;Na2HPO4, 3.2 g; ⅐ ␮ ⅐ ␮ ⅐ ϫ 7 MgSO4 7H2O, 0.5 g; biotin, 20 g; thiamine HCl, 200 g; pyridoxine HCl, 200 2 10 16 95 ␮ ⅐ ⅐ ⅐ ϫ 7 g; ZnSO4 7H2O, 1 mg; MnCl2 4H2O, 1 mg; CuSO4 5H2O, 1 mg; FeCl3,1 1 10 15 95 ϫ 6 mg; 10 mM L-proline or 10 mM L-arginine ⅐ HCl (pH 6.0 Ϯ 0.2). The vitamins 8 10 20 95 6 were prepared as a 1,000ϫ stock mixture in 20% aqueous ethanol, and the trace 6 ϫ 10 20 95 6 elements were prepared as a 5,000ϫ stock solution (5 mg/ml in 0.1 N hydro- 4 ϫ 10 20 95 6 chloric acid). The medium ingredients, except carbon and nitrogen sources, were 2 ϫ 10 18 95 ϫ 6 dissolved in distilled water, dispensed into culture flasks as required, and auto- 1 10 27 95 ϫ 5 claved at 121°C for 15 min. The nitrogen sources were prepared as 100 mM stock 4 10 95 95 ϫ 5 solutions, autoclaved separately, and added aseptically to the medium to give a 1 10 95 95 ϫ 4 final concentration of 10 mM. The chemical nature of the nitrogen source was 4 10 95 95 important, but the actual number of nitrogen atoms in the nitrogen source was a Filamentous growth is given. Only differentiated cells were counted; thus, the not (12). For proline, asparagine, and arginine (one, two, and four nitrogens, percentages of mycelial-filamentous growth and percentage of yeasts sum as respectively), cell morphology did not vary over the range from 1 to 100 mM (12). 100%. GPP medium contains proline as the nitrogen source, whereas GPR Thus, 10 mM was chosen for routine use. Similarly, glucose (or sucrose) was medium contains arginine. Values shown are averages of data from triplicate prepared as a 20% stock solution, autoclaved separately, and added aseptically to experiments performed earlier (14). Similar results agreeing with Ϯ10% were the growth medium to give a final concetration of 2%. (Hereafter, the abbrevi- obtained by four researchers using two strains (6404 and 6405) over a span of 20 ations SPP, GPP, GPR, and GPA represent sucrose-phosphate-proline, glucose- years.

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