Reference strains of Neurospora intermedia

Reference strains of N. intermedia Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This note on stocks is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol20/iss1/48 &rkinr,D. D. and B.C. Turner. Reference Species-reference strains believed to be authentic derivatives of the typestrains of Neurospora intermedia. material or of material conspecific with the type are avoiloble for N. ritophila and N. crassa (Perkins 1972 Neurorpora Newsl. 19: 2Bond 305: m believe that we have now estoblirhed reliable rtandords also for the third described heterothallic species, E intermedia. The need for reference strains. Vegetative morphology is completely inadequate in our experience for distinguishing the ertablirhecl heterathollic species of Neurorporo from one another. The published descriptions hove implicitly reccgnized this by placing greater emphoris on the sexual cycle than on vegetative traits. Taxonomic diagnosis stresses the size and shape of arcorporer and perithecicl. Yet even there structures may not be reliable because they can vary considerably within the some species, and although diagnostically useful quantitative differences may well exist between species, numeriwl values overlap even in plblirhed descript ions. Moreover, two hoploid isolates of opposite mating type must be avoiloble to complete the diploid phase of the life cycle before perithecio and ascospores can be obtained for measurement and diognorir. Yet in collections from nature only o single mating +ype may be present. Shear and Dodge recognized the boric importonce of crossing behwior for taxonomy, and once having ret up representatives of ritophila and crassa, they assigned other rtrains to one or the other species, not on the basis of their morphology, but of their fer-with one or another of the established strains (Table 6. Shear and Dodge 1927 J.&r. Rer.34: 1019). In our experience also, the production of fertile psrithecio and abundant asci with viable black spores, when an isolate is crossed to authenticated speciesreference strains, is the most procticol and convenient method of determining that the isolate belcmgs to one or onother of the ertoblished heterothollic species. It is probably also the most meaningful biologically. Unfortunately, failure to cross is not in itself D sufficient basis for excluding that two isolates belong to the same species, becoure intraspecific factors sometimes impair fertility. However, if an unknown is infertile with one species-reference but maker viable black spores abundantly with onother, there is no problem of assignment. The search for an historically authentic strain of N. intermedia. When cultures lobelled os N. intemwdia from various culture collections were intercrossed, numerous combinotionr were infertile. Also, some strains Iobelle~N. intermedio proved to be indistinguishable from tester strains known to be authentic N. craw or N. sitophila, and were com&tely fertile with them. For example, “N. intermedio” strain 268.36, obtained by the Fungol Genzcr Stock Center (FGSC*2055) in 1971 from Centmalbureau voo~Schimmelculhner (CBS),Bawn, Netherlands, is indistinguishable from N. crarro and is completely fertile with rtondord crossa strains, producing nearly lW% black orcospcres. Strain 108 (FGSC1580T obtained by FGSC from Dodge’s laboratory oftemeoth, and designated N. intermedia by FGSC on the basis of its label, is in fact N. ritophilo by the criterion of full fertility and black-spore p&u&n when crossed with the historically outhentictlted ritophx reference strain described in 1972 (Neurorporo Newt. 19: 28). (Strain IOB was unfortunately selected by Dutta et al. to reprerentE intermedia in their study of DNA differences between species, reported in 1971 ( N eurorpom Newsl. lB:6) and 1972 (Molec.Gan. Genet. 114:232 ). From among the supposed intermedia stocks examined, we believe that two ore authentic vegetative derivatives of the original rtroin described by Tai (1935 Mycolcgia 27:328) and deposited in CBS by Dodge in April 1936 0s strain 1OB. The first is a strain designated N. intermedia Tai which was deposited in the Fungal Genetics Stock Center by J.Weijer, University of Alberta, in 1963, and assigned FGSC “962. The strain was from the collection of E.S. (Dowding )Keeping, University of Alberta, where it was labelled “N. intermedia, Washington, 1928, Wilcox. ” E. S. Keeping has kindly written us: “I recall that in the twenties 1 received a culi& from Dodge with a note that it was from ‘war torn China. ’ It may very well have been N. intermedia. ” (M. 5. Wilcox was associated with Dodge at the Bureau of Plant Industry, U.S. D.A., Woshington,D.C. in 19283 A second probably authentic culture of N. intermedia was cbtained from CBS in 1956 by A.M. Srb. This has since been maintained vegetatively at Cornell, and was d$sited by Srb in 1969 in FGSC (“1754). ( The CBS cotologue lists only one N. intermedio stock, 268.36, deposited by Dodge. Yet the stock obtained from CBS OS intermedia by Srb in 1956 is markedly diGrent from the stock obtained from CBS os intermedia by FGSC in 1971. We find that the Iotter is indistinguishable from N. crosso. ) The two putative intermedia stocks, from Alberta via FGSC, and from CBS via Srb, were obtained and examined by us in 1972. They ore strikingly similar to one another, and are uniquely different in appearance from any other Neurorporo strain we have handled. Both ore 0. Both are female-sterile. Surface growth is dense, with conidiation close to the surface and little aerial growth. Color is bcght saffron (a golden yellow). The behavior of both is identicol when they are crossed os fertilizing parents to wmerous other strains used for testing. It is difficult to explain such remwkoble similarity unless the two strains hod o common origin, representing subcultures that have been maintained reparotely since 1928. The material on which Toi bored his brief 1935 description was isolated from o corn cob in Nonking, China in 1927. He stated that oscorpores resembled ritophilo while perithecio resembled crass, and that intermedia hybridized rather readily with the other species. Conidio of Toi’s intermedia strain a were described 01 saffron-colored, and distinctly different in color from z or ritophilo. Mating type z is mentioned expl’;itly, but not A. Tai’s description is not sufficiently detailed to tell whether the vegetative morphology resembled that of the saffron-colors a stocks we now hove from Weijer and Srb. Tai published only one other paper on Neurorporo (1936 Mycologic 28:24), in which only the mating-type z strain of intermm was used, although both A and a mating types were represented for other species involved in the some study. This suggests that only the a strain was still extant & 1936. All there facts ore consistent with our conclusion that the 0 strains from Weijer and from Srb & authentic representatives of the original E intermedia material used by Tai. Choice of A and a strains for use os testen. For several rea~)ns it is not practical to use the original strain for carrying out routine mating tests to ascertain the species status of other strains. Only one mating type is represented, and it is female-sterile. Also, experience has shown that the original strain differs greatly in vegetative morphology from all of the many “narmol”appearing strains which we have collected from nature and with which it is highly fer.tile and apparently irorcquentiol. (Similarity of chromosome requence is inferred from the fact that arcosporer are nearly loOO~ black, whereas the presence of gross reorrongementr would result in at least 25% or 50% defective, white ascorpores.) It is our belief that the porticulor strain on which the species description happened to be bored was morphologically highly atypical of the species as it is usually found in the wild. We have therefore chosen o Fair of highly fertile, morphologically “normal”Aand 0 rtroinr, which we propose to use on a trial basis as standard species-references for N. intermedia. Eventually it may be desirable to replace them with material from the original type-locality, but such strains a< not now available. Description and crossing behavior of the reference strains. The two strains proposed as intermedia standards ore: P420 A FGSC”2316 Collected from burned wgar cane near Clewirton, Florida in March 1970. P405 a FGSCtl940 Collected from burned grass near Lo Belle, Florida in March 1970. These were selected from among other candidates because of high fertility, good spore production, and uniform morphology among prcgeny from crosses with each other. The new reference strains resemble the standard cvxso and ritophila wild types in vegetotie morphology and appearance. Conidia and mycelia ore usually orange, but con be dirtinctly ymnged under some conditions, although never the intense yellow seen in the Weijer or Srb strains. Conidiation is better at 25’C and below than at 34’ or dmve. Arcorporet from P405 x P420 measure 28.5 x 16.5 pm (mean based on 125 spores, cross on synthetic cross medium, 25’ C ). Those from P420 X the strain from Srb measure 27.5 x 17.4 pm (50 spores ). Ascorporer from both crosses are thus larger than the size range given by Tai (19-26 x 12-15 pm), but the length/breadth ratio is similar. Both strains are female-fertile. They are apparently irosequentiol with one another, and theAstandard is fertile and isosequentiol with the strains from Weijer and Srb, which ore believed to be historically authentic. When progeny are obtained from crosses between the Weijer and Sub strains and the proposed references, a vast array of phenotypes is seen, varying markedly in pigment, morphology, conidiation, and vigor; and the saffron/orange carotenoid difference segregates. Crosser between intermedio and standard crassa produce many perithecia. Ortioler are formed, and numerous orcorparer are shot from the perithecio

-believe that we have now estoblirhed reliable rtandords also for the third described heterothallic species, E intermedia.
The need for reference strains.
Vegetative morphology is completely inadequate in our experience for distinguishing the ertablirhecl heterathollic species of Neurorporo from one another.The published descriptions hove implicitly reccgnized this by placing greater emphoris on the sexual cycle than on vegetative traits.Taxonomic diagnosis stresses the size and shape of arcorporer and perithecicl.
Yet even there structures may not be reliable because they can vary considerably within the some species, and although diagnostically useful quantitative differences may well exist between species, numeriwl values overlap even in plblirhed descriptions.Moreover, two hoploid isolates of opposite mating type must be avoiloble to complete the diploid phase of the life cycle before perithecio and ascospores can be obtained for measurement and diognorir.
Yet in collections from nature only o single mating +ype may be present.
Shear and Dodge recognized the boric importonce of crossing behwior for taxonomy, and once having ret up representatives of ritophila and crassa, they assigned other rtrains to one or the other species, not on the basis of their morphology, but of their fer--with one or another of the established strains (Table 6.Shear and Dodge 1927 J.&r.Rer.34: 1019).In our experience also, the production of fertile psrithecio and abundant asci with viable black spores, when an isolate is crossed to authenticated speciesreference strains, is the most procticol and convenient method of determining that the isolate belcmgs to one or onother of the ertoblished heterothollic species.It is probably also the most meaningful biologically.
Unfortunately, failure to cross is not in itself D sufficient basis for excluding that two isolates belong to the same species, becoure intraspecific factors sometimes impair fertility.However, if an unknown is infertile with one species-reference but maker viable black spores abundantly with onother, there is no problem of assignment.
The search for an historically authentic strain of N. intermedia.When cultures lobelled os N. intemwdia from various culture collections were intercrossed, numerous combinotionr were infertile.Also, some strains Iobelle~N.intermedio proved to be indistinguishable from tester strains known to be authentic N. craw or N. sitophila, and were com&tely fertile with them.For several rea~)ns it is not practical to use the original strain for carrying out routine mating tests to ascertain the species status of other strains.Only one mating type is represented, and it is female-sterile.Also, experience has shown that the original strain differs greatly in vegetative morphology from all of the many "narmol"appearing strains which we have collected from nature and with which it is highly fer.tile and apparently irorcquentiol.(Similarity of chromosome requence is inferred from the fact that arcosporer are nearly loOO~ black, whereas the presence of gross reorrongementr would result in at least 25% or 50% defective, white ascorpores.)It is our belief that the porticulor strain on which the species description happened to be bored was morphologically highly atypical of the species as it is usually found in the wild.We have therefore chosen o Fair of highly fertile, morphologically "normal"Aand 0 rtroinr, which we propose to use on a trial basis as standard species-references for N. intermedia.Eventually it may be desirable to replace them with material from the original type-locality, but such strains a< not now available.But the ascorpora ore nearly all white and inviable, with block spores ranging in frequency from <I% to perhaps 10%.This description applier both to crosses using the Weijer or Srb strains (as fertilizing parents), and the proposed new reference strains.Thus crc~ssa X intermedia crosses ore often highly fertile if the criterion of fertility be numbers of productive perithecia and numbers of escorporer; but they are quite infertile with respect to the production of viable block orcorporer.
Crosser have been made also behveen the intermedia references and strains which are believed to be authentic N. ritophilo (PsD85A and P8086a).
There resemble the intermedia X crasso crosses in producing abundant perithecia, but theyTiffer being relatively barren and producing few orcorporcr.

General distribution of the
),B awn, Netherlands, is indistinguishable from N. crarro and is completely fertile with rtondord crossa strains, producing nearly lW% black orcospcres.Strain 108 (FGSC1580T obtained by FGSC from Dodge's laboratory oftemeoth, and designated N. intermedia by FGSC on the basis of its label, is in fact N. ritophilo by the criterion of full fertility and black-spore p&u&n when crossed with the historically outhentictlted ritophx reference strain described in 1972 (Neurorporo Newt.19: 28).(Strain IOB was unfortunately selected by Dutta et al. to reprerentE intermedia in their study of DNA differences between species, reported in 1971 (N -eurorpom Newsl.lB:6) and 1972 (Molec.Gan.Genet.114:232 ).From among the supposed intermedia stocks examined, we believe that two ore authentic vegetative derivatives of the original rtroin described by Tai (1935 Mycolcgia 27:328) and deposited in CBS by Dodge in April 1936 0s strain 1OB.The first is a strain designated N. intermedia Tai which was deposited in the Fungal Genetics Stock Center by J.Weijer, University of Alberta, in 1963, and assigned FGSC "962.The strain was from the collection of E.S. (Dowding )Keeping, University of Alberta, where it was labelled "N.intermedia, Washington, 1928, Wilcox." E. S. Keeping has kindly written us: "I recall that in the twenties 1 received a culi& from Dodge with a note that it was from 'war torn China.' It may very well have been N. intermedia." (M. 5. Wilcox was associated with Dodge at the Bureau of Plant Industry, U.S. D.A., Woshington,D.C. in 19283 A second probably authentic culture of N. intermedia was cbtained from CBS in 1956 by A.M. Srb.This has since been maintained vegetatively at Cornell, and was d$sited by Srb in 1969 in FGSC ("1754).( The CBS cotologue lists only one N. intermedio stock, 268.36, deposited by Dodge.Yet the stock obtained from CBS OS intermedia by Srb in 1956 is markedly diGrent from the stock obtained from CBS os intermedia by FGSC in 1971.We find that the Iotter is indistinguishable from N. crosso.) The two putative intermedia stocks, from Alberta via FGSC, and from CBS via Srb, were obtained and examined by us in 1972.--They ore strikingly similar to one another, and are uniquely different in appearance from any other Neurorporo strain we have handled.Both ore 0. Both are female-sterile.Surface growth is dense, with conidiation close to the surface and little aerial growth.Color is bcght saffron (a golden yellow).The behavior of both is identicol when they are crossed os fertilizing parents to wmerous other strains used for testing.It is difficult to explain such remwkoble similarity unless the two strains hod o common origin, representing subcultures that have been maintained reparotely since 1928.The material on which Toi bored his brief 1935 description was isolated from o corn cob in Nonking, China in 1927.He stated that oscorpores resembled ritophilo while perithecio resembled crass, and that intermedia hybridized rather readily with the other species.Conidio of Toi's intermedia strain a were described 01 saffron-colored, and distinctly different in color from z or ritophilo.Mating type z is mentioned expl';itly, but not A. Tai's description is not sufficiently detailed to tell whether the vegetative morphology resembled that of the saffron-colors a stocks we now hove from Weijer and Srb.Tai published only one other paper on Neurorporo (1936 Mycologic 28:24), in which only the mating-type z strain of intermm was used, although both A and a mating types were represented for other species involved in the some study.This suggests that only the a strain was still extant & 1936.All there facts ore consistent with our conclusion that the 0 strains from Weijer and from Srb & authentic representatives of the original E intermedia material used by Tai.Choice of A and a strains for use os testen.
Description and crossing behavior of the reference strains.The two strains proposed as intermedia standards ore: P420 A FGSC"2316 Collected from burned wgar cane near Clewirton, Florida in March 1970.P405 a FGSCtl940 Collected from burned grass near Lo Belle, Florida in March 1970.These were selected from among other candidates because of high fertility, good spore production, and uniform morphology among prcgeny from crosses with each other.The new reference strains resemble the standard cvxso and ritophila wild types in vegetotie morphology and appearance.Conidia and mycelia ore usually orange, but con be dirtinctly ymnged under some conditions, although never the intense yellow seen in the Weijer or Srb strains.Conidiation is better at 25'C and below than at 34' or dmve.Arcorporet from P405 x P420 measure 28.5 x 16.5 pm (mean based on 125 spores, cross on synthetic cross medium, 25' C ).Those from P420 X the strain from Srb measure 27.5 x 17.4 pm (50 spores ).Ascorporer from both crosses are thus larger than the size range given by Tai (19-26 x 12-15 pm), but the length/breadth ratio is similar.Both strains are female-fertile.They are apparently irosequentiol with one another, and theAstandard is fertile and isosequentiol with the strains from Weijer and Srb, which ore believed to be historically authentic.When progeny are obtained from crosses between the Weijer and Sub strains and the proposed references, a vast array of phenotypes is seen, varying markedly in pigment, morphology, conidiation, and vigor; and the saffron/orange carotenoid difference segregates.Crosser between intermedio and standard crassa produce many perithecia.Ortioler are formed, and numerous orcorparer are shot from the perithecio.
species.A survey has been started crossing the reference standards to wild-collected stroinr from various rourcer.Isolates that are apparently N. intermedia hove been found omong Neurospom collected both in the New World and in locolitier ranging from Japan to AustraE, and from Hawaii to Indonesia and India.Sampler of Neurorporo collected by ~DDP from ontiom in and near Bogor, Indonesia, all appear to be N. intermedia, and intermedia appears to be the prevalent species in those ports of the Eastern Hemisphere where collections have && mode.Ho C.t. informs us that ontjam cultures recently collected by him in Java also behave in crosses as if they are conspecific with the proposed intermedio references.The cultures listed below appear to be N. intermedia on the basis of their crossing behavior with the proposed references.The survey is incomplete, and other FGSC rtroxr of uncertain species-rtotur still remain to be tested.FGSC stocks classified OI Nof Biological Sciences, Stanford University, Stanford, California 94305.