Ethylene glycol treatment of conidia

Ethylene glycol treatment of conidia Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This technical note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol19/iss1/14 Mylyk,O.M. and S.F. H.Threlkeld. The use of Certain morphological and biochemical mufonts ore female sterile, i.e., heterokoryonr in facilitating crosses between fail to produce perithecio and spores, or cross poorly when used or fe&ie Neurospro strains having reduced female fertility. porentr. Reciprocal crosses between two such mutants moy either foil completely or moy be extremely slow. Our work with female sterile mutants suggests that heterokoryonr moy be effective in overcoming these difficulties in many inrtonces. Horowitz et 01. (1960 J. Mol. Biol.2: 96) were suecersful in making o cross between the female’rterile tyrorinose mutants fy-l ond ‘y-2 when ty-I was in o heterokoryon’aed os o femole parent ond t& was the mole parent. In our laboratory heterokoryonr having all pairwire combinations, except one, of seven or eight different female sterile mu+ontr were tested for their ability to produce perithecio and spores when the heterokoryonr were used os female porentr. bin&ion foiled repeatedly, and it has not been established that the mutonh ore non-ollelic. Only one cornAll other combinations produced on abundance of perithecio and oscosporer, with over 95% of the crosses shooting oscosporer within two weeks after fertilization. Only rarely did o cross involving on effective combination of mutants foil, probably due to a distorted nuclear ratio in the heterokoryon; further ottempts in each case produced rubstonticl numbers of perithecio and oscospores. The female sterile mutants used are all morphologicolly different from the wild type. One is the mu+on+ leu-I (33757). in which female sterility and abnormal morphology seem to be inseporoble from the biochemical requirement. One mutant has o somewhat colonial morphology ot 25’C and does not grow ot all ot 34OC. The others, including leu-I, ore subtly different from the wild type. They grow more slowly, ore less pigmented, and form o more continuous culture than the type ( the wild types 74-OR23-IA and 74-ORB-lo used for comparison form o dense bond of conidio above o gop of relatively sparse aerial growth in a IO x 75 mm tube.) The mutants pan-2 (83) ond nit-3 (Y31881 ) to affect growth chorocteristics or fertility. were used os h&e&or/on forcing markers, since they ore non-leaky and do not seem 2% sucrose ond 1.5% ogar. The heterokoryons were grown on a Westergoord ond Mitchell crossing medium having After seven days they were fertilized with o conidiol suspension of one female sterile mutant or, in seporote tests, with al-2 (15300). A typical cross was: (nit-3 fs-m A +& fs-n A)$ x in which h, fs-n and ti represent different female s+erilemut.nts. fgo (oro&o)* Most cro= produced on abundance of orcosporer, suggerting that most feae sterile mutants co,, be tronrmitted through crosses when used os female parents in heterokoryonr. This is supported by the fact that most of our female sterile mutants were originally detected os a result of segregation in crosses where stmins used os female parents hod accumulated the mutonh. We tested further to see if one of the female sterile mutants could be recover ed from o cross where it was present in both parent nuclei. The following was attempted: (pan-ZA + nit-3 fsA) $ x pen-2 fr o 07 The nit-3 mufont was recovered among the progeny, demonstrating that the h x 2 component of the cross hod token place. The success with which the use of heterokaryonr in the above crosses resulted in the production of perithecio and orcosporer wggertr that progeny moy be recovered from crosses between various other morphological or biochemical mutontr which have reduced female fertility if heterokoryonr ore similarly used. In making such crosses, for instance between the mutants mut-I and mut-2 the following format using & os on oscorpae color marker is convenient: ( “;I’! + nit-3mut-l A)F x ~mut-2oo~ Arcosparer having on-2 do not develop pigment in crosses mode on o medium ackang pontothen~c acid (Threl e d 965”. J. Genet.Cytol.7: I71 ‘iHence oil orcorporer from the pan x pan component and half of those from the nit-3 mot-l x pow2 mut-2 component will be pole if the crosser are mode on minGi mium. All of the dork spores will represent the mut-I x mot-2 component, which is desired. The dark and pole spores con be eerily distinguished on o block of ogor under o disxg microrcope when illuminated from above. To enhonce the germination of nit-3 orcospores, nicotinamide con be odded too cross tube at the time of fertilization. This is done by preparing the conidiol svspenrion of the mole parent in o solution of 0.04 mg/ m nicotinamide, adding opproximotely I .5 I ml to o 15 x I50 mm cross tube containing 5 ml of medium, and spreading the suspension by rhoking the tube. If pow2 must be recovered from such o cross, o similar amount of pantothenic acid con be added. will be indirtinguirhoble from &, but th’ This will cause pon oscospores to darken so they $6 ts necerrory since pole oscorporer usually show redux germination. We ore deporiting pan-2 and nit-3 mutants crossed into o St. Lawrence (Oak Ridge) genetic bockground with the Fungol Genetics Stock Center. Deportment of Biology, McMorter University, Homilton, Ontorio, Conodo. (Present address of OMM: Deportment of Biologicol Sciences, Stanford University, Stanford, Colifornio 94305 ). WiIron,J. F.ond W.K.Boter. Ethylene glycol Recently we described some effects of treatment of Neurosporo conidio treatment of Neurorpom conidio. with theylene glycol (Bates and Wilson 1972 Genetics 68:s4). This treatment results in conidio which enlarge, with concomitant weight gain, and which become osmoticolly sensitive after hvo or more days. Cmtotic disruption of these cells yields large numbers of intact nuclei and mitochondrio, while groduol removal of the ethylene glycol rewits in approximately 75% germination within one hour. We now present some details of the methodology involved. The conidio routinely used ore from seven-to-fwrtecn-day-old cultures, grown ot 3O’C on Vogel’s minimal ogar medium, with supplements os required for mutonh. The strain used for most of our studies is o m-isolate of the Oak Ridge wild type. Additional studies with me-3 (36104) FGSCb502, inos (37401 ) FGSCY406, and [mi-I] ( poky, mi-l-1.8) FGSC”l578, with oppropriote sup7 plements, ho=dicoted that the effect II not limited to one strain, although voriotionr do occur in the degree of the response. Conidia ore harvested in sterile wohr, filtered through four loyers of sterile gone to remove hyphol fragments, and the concentmtion is determined with o hemacytometer. The conidial suspension is allowed to stand for (I+ leosf one hour a+ 25’C before the conidia ore transferred +o ethylene glycol medium. This pre+reo+men+ with wo+er results in foster and more uniform enlargement of the conidia in response to ethylene glycol. Pre-treo+ment periods longer than one hour produce no odditionol effect, The formulation for 100 ml of the ethylene glycol medium is: 2 ml of 50X Vogel’s minim.1 medium; 80 ml of distilled water; 18 ml ethylene glycol, reagent grade (20 g rams); 1.5 g sucrose. We routinely double these amounts to obtain 200 ml ethylene glycol medium, and use this volume in 500 ml Erlenmeyer +ype flasks with stainless steel closures (Delong culture flasks). All components are outocloved together in the flask. We inoculate at 1-3x Id conidio per ml medium (2-6x I09 per flask) by centrifuging the volume of aqueous suspension of conidia necessary for each flask in o sterile screw-copped tube and decanting the water from the conidiol pellet. The conidio ore then re-suspended in o part of the contents of o flask of ethylene glycol medium and transferred bock to the flak. Thus, inoculation is achieved without dilution of the medium. Flasks ore then placed on o rotor/ shaker ot 2YC with carriers mounted at o 15’ angle and ore shaken continuously a+ 150 rpm. Osmotic sensitivity is demonstrable ot 48 hrs, and both size and osmotic sensitivity continue +o increase for ot least IO days. We hove observed more than 80% viability after 8 days of this treatment. Osmotic disruption is occomplirhed by centrifuging a suitable portion of the suspension ond re-suspending the pellet in o hypotonic solution to opproximotely 10% of the original volume. Disruption occurs within o few seconds. For mitochondrio, +he pellet is resuspended in 2% sucrose-ImM EDTA at 5% of the original volume, followed by on equal volume of 28% sucrose-1mM EDTA ot 30 seconds. It should be noted, however, that such mitochondrio ore not identical +o those prepared by s&d grinding. For studies involving germination (including sorbore plating) it is necessary to dilute the ethylene glycol gmdually, allowing the conidio to equilibrate o+ the lower concentrations. We hove accomplished this with minim.1 disruption by non-linear rates of oddition of water or minimal Tedium, according to the following schedule: 10 ml conidiol suspension in 20% ethylene glycol in 125 ml Erlenmeyer flask on magnetic stirrer at room tempemture. Add diluent ot I ml/min for IO min to yield 10% solution, Add diluent ot 2 ml/min for IO min +o yield 5% solution, Add diluent-at 4 ml/min for 15 min to yield 2% solution. Diluent is added by-o peristaltic pump, osepticolly if necessary. If faster dilution is required, rates of addition con be doubled with only a slight increase in disruption. Ethylene glycol treated conidio ore much more susceptible to disruption by sand grinding than ore untreated conidia, os judged by compamtive extraction yields. This allows preparation of extroctr when osmotic shock is not desirable, or in the prepomtion of mitochondrio. The procedure is: dilute os described above; centrifuge to concentrate the conidio; re-suspend in the ex

The conidio routinely used ore from seven-to-fwrtecn-day-old cultures, grown ot 3O'C on Vogel's minimal ogar medium, with supplements os required for mutonh. The strain used for most of our studies is o m-isolate of the Oak Ridge wild type. Additional studies with me-3 (36104) FGSCb502, inos (37401 ) FGSCY406, and [mi-I] ( poky, mi-l-1.8) FGSC"l578, with oppropriote sup-7 plements, ho=dicoted that the effect II not limited to one strain, although voriotionr do occur in the degree of the response.
Conidia ore harvested in sterile wohr, filtered through four loyers of sterile gone to remove hyphol fragments, and the concentmtion is determined with o hemacytometer. The conidial suspension is allowed to stand for (I+ leosf one hour a+ 25'C before the conidia ore transferred +o ethylene glycol medium. This pre+reo+men+ with wo+er results in foster and more uniform enlargement of the conidia in response to ethylene glycol. Pre-treo+ment periods longer than one hour produce no odditionol effect, The formulation for 100 ml of the ethylene glycol medium is: 2 ml of 50X Vogel's minim.1 medium; 80 ml of distilled water; 18 ml ethylene glycol, reagent grade (20 grams); 1.5 g sucrose. We routinely double these amounts to obtain 200 ml ethylene glycol medium, and use this volume in 500 ml Erlenmeyer +ype flasks with stainless steel closures (Delong culture flasks). All components are outocloved together in the flask.
We inoculate at 1-3x Id conidio per ml medium (2-6x I09 per flask) by centrifuging the volume of aqueous suspension of conidia necessary for each flask in o sterile screw-copped tube and decanting the water from the conidiol pellet.
The Osmotic sensitivity is demonstrable ot 48 hrs, and both size and osmotic sensitivity continue +o increase for ot least IO days. We hove observed more than 80% viability after 8 days of this treatment.

Osmotic disruption is occomplirhed by centrifuging a suitable portion of the suspension ond re-suspending the pellet in o hypotonic solution to opproximotely 10% of the original volume. Disruption occurs within o few seconds. For mitochondrio, +he pellet
is resuspended in 2% sucrose-ImM EDTA at 5% of the original volume, followed by on equal volume of 28% sucrose-1mM EDTA ot 30 seconds. It should be noted, however, that such mitochondrio ore not identical +o those prepared by s&d grinding.
For studies involving germination (including sorbore plating) it is necessary to dilute the ethylene glycol gmdually, allowing the conidio to equilibrate o+ the lower concentrations. We hove accomplished this with minim.1 disruption by non-linear rates of oddition of water or minimal Tedium, according to the following schedule: 10 ml conidiol suspension in 20% ethylene glycol in 125 ml Erlenmeyer flask on magnetic stirrer at room tempemture. Add diluent ot I ml/min for IO min to yield 10% solution, Add diluent ot 2 ml/min for IO min +o yield 5% solution, Add diluent-at 4 ml/min for 15 min to yield 2% solution.

Diluent is added by-o peristaltic pump, osepticolly if necessary. If faster dilution is required, rates of addition con be doubled with only a slight increase in disruption.
Ethylene glycol treated conidio ore much more susceptible to disruption by sand grinding than ore untreated conidia, os judged by compamtive extraction yields. This allows preparation of extroctr when osmotic shock is not desirable, or in the prepomtion of mitochondrio. The procedure is: dilute os described above; centrifuge to concentrate the conidio; re-suspend in the extraction medium; and grind with sand with o mortar and pestle.
Although various modifications will be necessary to suit specific experiment.1 conditions, the methods outlined above should prove odequote for preliminary studies. A more complete chomcterizotion of these conidio and the extracts obtained from them will be presented elsewhere.