Some Genetic Techniques for Gibberella zeae

Leslie, J. F. 1983. Some genetic techniques for Gibberella zeae. Phytopathology 73:1005-1008. Gibberella zeae, an important plant pathogen, is exploited commercially dependent, glutamate dehydrogenase-deficient mutant. Protoplasts were in the production of zearalenone, a fungal sex hormone. Genetically, G. liberated by hyphal digestion with commercially available f-glucuronidase zeae is relatively intractable. A variety of mutants induced by ultraviolet and chitinase. Protoplast fusion, mediated by polyethylene glycol 4000, irradiation were recovered by using a high-sorbose, filtration-enrichment produced stable heterokaryotic colonies under proper selective conditions. technique. These mutants included: adenine-, arginine-, and histidine Development of these mutants and techniques lays the groundwork for auxotrophs; a non-nutritional, heat-sensitive mutant; and an NADPHstudying the genetics of G. zeae. Additional key words: auxotrophic mutants, Fusarium roseum 'Graminearum,' protoplast fusion. Gibberella zeae (Schw.) Petch, the perfect state of Fusarium Protoplast regeneration medium (PRM) was described by Acha et roseum (Link emed.) Snyder and Hansen 'Graminearum,' causes al (I). Auxotrophic mutants were grown on a minimal medium stem rot of carnation, stalk and ear rot of maize, and head blight of supplemented with 200 mg of the required nutrient(s) per liter. small grains. Some strains of this fungus are used commercially to Solid media contained 2% agar (w/v) except for Coon's synthetic make the fungal sex hormone zearalenone (13,32), which is medium (33), which contained 5% agar (w/v). All media were subsequently processed to zearalanol. Zearalanol is marketed as sterilized by autoclaving at 121 C and 1.02 k Pa (15 psi) for 20 min. RALGRO® (International Minerals and Chemical Corporation, Enzyme solutions were filter sterilized. Terre Haute, IN 47808), an anabolic agent for cattle and sheep Mutagenesis. Ten milliliters of a macroconidial suspension (13,23). (107-10' macroconidia per milliliter) in a 2.5% (v/v) Tween-60 Little is known of the basic genetics of G. zeae. Its homothallic solution were placed in a sterile glass petri dish. Mutations were reproductive system and fastidious requirements for laboratory induced by exposure to ultraviolet irradiation from a new UVSLproduction of perithecia have steered geneticists to more amenable 25 Mineralight lamp (Ultraviolet Products, Inc., San Gabriel, CA fungi. In this report, protoplast fusion is proposed as an asexual 91778). At a 3-cm distance (44 ergs/sec/mm ), a 60to 90-sec alternative to the conventional sexual cycle. Techniques are exposure gave 80-90% kill. described for auxotroph induction and recovery, for protoplast Auxotroph recovery. A filtration-enrichment procedure similar formation and fusion, and for regeneration of colonies from to those for Neurospora crassa Shear and Dodge (3,34,35) and protoplasts. Cochliobolus heterostrophus Drechsler (15,35) was used to increase the proportion of auxotrophs in the population examined. MATERIALS AND METHODS The irradiated macroconidial suspension was added to 250 ml NSC Strains. Four primary strains of G. zeae were used: 'Dewar' and sorbose medium in a 500-ml Erlenmeyer flask. This flask was 65-338B (two wild types from the IMC/CSC culture collection); completely wrapped with aluminum foil during the first 24 hr of 65-38B twowil tyes r the C/SC ultre ollctin); incubation. After 4-6 hr incubation at 28 C on a rotary shaker ( 120 251-15 (a strain freshly isolated from a corn kernel by Dr. J. Tuite, icbto te 4-6 hr intion t2Con a rotary sha er1 Purdue University); and ATCC 20273 (a patented strain used for rpm), the suspension was filtered through a 105-4m-mesh zearalenone production [14]). Dewar, 65-338B, and 251-15 are polypropylene filter (Spectrum Medical industries #146436; Los morphologal lyTye production [14]). while AT 6C52238, is m o aly Angeles, CA 90054), returned to the flask, and placed back on the morphologically Type A (7), while ATCC 20273 is morphologically shaker. Thereafter the filtration was repeated at 12-hr intervals for Type B (7). 72-96 hr with 30-tim-mesh nylon (Spectrum Medical Industries Media. Bennetts NmediumI, Ng gdextrose, 2 gyNZ Aminex[Type A, #146506). The final filtrate was mixed with 250 ml of a double Sheffield Products, Norwich, NY 13815], 1 g yeast extract [Difco strength, 4% sorbose, complete medium or appropriately Laboratories, Detroit, MI], 1 g beef extract [Difco Laboratories, supplemented minimal medium with agar that had been held at 48 Detroit, MI], 2.5 g NaCl and distilled water to make 1 L) was used C. The mixture was poured into sterile petri dishes and incubated at whenever a complete medium was required. A modified 21 C for 5-8 days. Surviving colonies were tested for temperature formulation of Coon's synthetic medium (33) and Neurospora sensitivity by growth at 28 C and for nutritional auxotrophy by synthetic crossing medium (NSC) (31) were used as the minimal media. NSC sorbose medium (NSC sorbose) contained 0.2% (w/ v) growth on NSC agar. mediae.2% (wv) frb mediumoe andC 6%(wv) corboseain plaed ofthe Preliminary identification of auxotrophs was by auxanography dextrose,0.2%(w/v) fructose,and 6%(w/v)sorbose inplace ofthe (18). After colonies were identified as auxotrophic, the specific normal sucrose. Protoplasting medium (PPM) was modified from nutritional requirement was identified by embedding at least 10' that of Miller ( 17) and consisted of 16 mg cysteine, 2 g sucrose, 14 macroconidia in 20 ml of NSC agar in a sterile petri dish and mg NaH2PO 4,300 ugstreptomycin sulfate, 500 ugchioramphenicol, spotting test substances on the surface. Growth responses could be 10 mg chitinase (Sigma Chemical Co., St. Louis, M0 63178), 0.75 scored after 72-96 hr. ml 83-glucuronidase (Sigma), and distilled water to make 10 ml. Formation of perithecia was examined using the techniques of The publication costs of this article were defrayed in part by page charge payment. This Tschanz et al (27-29), and Wolf and Mirocha (33). article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § Protoplast formation. Strains were grown at 28 C in a 125-ml 1734 solely to indicate this fact. Erlenmeyer flask containing 25 ml of appropriately supplemented @1983 The American Phytopathological Society NSC broth and inoculated with > l0 macroconidia. After 18-36 hr Vol. 73, No. 7,1983 1005 the contents were filtered through a coarse sintered glass filter and Samples were collected after each filtration and the number of the retained hyphae were washed twice with sterile distilled water. colony-forming units (cfu) of each type remaining was determined The washed hyphae were placed in a 50-ml Erlenmeyer flask (Fig. 1). In all cases, no detectable ATCC 20273 remained after 72 containing 20 ml of PPM and incubated with gentle shaking for hr, while at least 100 ade-1 cfu/ml were retained. 18-24 hr. Protoplasts were separated from hyphal debris by In developing this filtration enrichment a number of important filtration through two 60-pm-mesh nylon filters (Spectrum variables were identified. These included: 1) Frequency of Medical Industries #146494) over a coarse sintered glass filter and filtration-filtering approximately every 12 hr gave the best results. then washed three times by centrifugation (3,000 g, 10 min) in 0.6 M At 24-hr intervals, the accumulated hyphal mass was large enough KCI. This procedure yielded 105-106 protoplasts; the yield varied to plug the filter and retain nongrowing or slowly growing isolates. with the strain. Protoplasts regenerated in 2-6 wk on solid PRM as Filtering more frequently than every 12 hr did not increase the described by Acha et al (1). percentage of auxotrophs recovered and increased the chance of Protoplast fusion. Protoplasts to be fused were prepared as contamination. 2) Filter type-both nylon and polypropylene described above. These protoplasts were then mixed in filters were satisfactory. The high surface tension of Teflon@ filters approximately equal numbers, centrifuged (3,000 g, 10 min), and greatly retarded the rate of filtering. 3) Filter size-a "large" mesh resuspended in 1 ml of sterile 90 mM CaC12 with 25% (w/v) filter, 100 pm or larger, was required for the first filtration because polyethylene glycol (PEG) 4000 (Sigma). After 30 min at 4 C, the of the large hyphal mass to be removed. Afterwards, a smaller mesh mixture was plated on unsupplemented PRM and incubated at 28 filter was required for efficient removal of the prototrophic C up to 6 wk. colonies. 4) Molten agar temperature-this temperature should be Strain preservations. Cultures of both mutant and wild strains as low as possible. When the agar temperature exceeded 60 C the were preserved on carnation leaf disks as described by Fisher et al number of colonies decreased. (10). Nutritional supplementation of the auxotrophic mutants was Mutants recovered. Mutants were induced with ultraviolet light not required. and identified by their inability to grow on NSC at 28 C. From nearly 1,000 survivors of 12 independent mutagenesis experiments RESULTS using ATCC 20273, 24 morphological mutants, 25 auxotrophic mutants, and one non-nutritional temperature-sensitive mutant Filtration enrichment technique. The high-sorbose filtration were identified (5% mutants among the survivors). The technique of Applegate et al (3) and Yoder (35) was modified for G. morphological mutants were preserved on carnation leaf disks (10) zeae. The efficiency of this modified technique was assessed with and not studied further. Auxotrophic deficiencies were 10:1 and 100:1 mixtures of ATCC 20273 and ade-1 (IMC125). characterized by auxanography (18). Among the auxotrophs, eight mutants required adenine, one mutant required arginine, one mutant required histidine, one mutant required glutamate, and 14 mutants had undiagnosed requirements. On complete medium, 106 I I I I I I auxotrophs were slower than wild types to produce the characteristic reddish purple pigment. Representative mutant 105 phenotypes, genotypes, and ATCC strain designations are given in Table 1. Gdh will be described in more detail elsewhere (Leslie and Kinzel, unpublished). Reversion rates of the mutants examined