MUTATION IN THE GENUS ALTERNARIA BY ORDA ALLEN PLUNKETT A. B. Wabash College, 1920 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS IN BOTANY IN THE GRADUATE SCHOOL OF THE UNIVERSITY OF ILLINOIS, 1922 URBANA, ILLINOIS Digitized by the Internet Archive in 2016 https://archive.org/details/mutationingenusaOOplun I z) (^ fa PH UNIVERSITY OF ILLINOIS THE GRADUATE SCHOOL Jlay -igi 2 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Orda Allen Plunke tt ENTITLED. Mutation in the Genua Alt exnaria^ BE ACCEPTED AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE DEGREE OF Recommendation concurred in* Committee on Final Examination* '‘Required for doctor’s degree but not for master’s . 'v y i O VOVjo ■ 4 . ' Table of Contents Page I. Introduction 1 II. The present status of the mutation question in fungi and bacteria 3 III. Source of materials 20 IV. Methods employed for isolation and growth 21 Appearance of mutations 22 Modifications 24 V. Methods employed for contrast studies of mutants and originals 35 Color of colonies on agar 36 Zonation of colonies 27 Rate of growth 20 Color of mycelium 20 Color of medium 29 Aerial mycelium 29 Length of conidia 30 Width of conidia 32 Cross septa 33 Longitudinal septa 35 Color of conidia 36 Conidial production 37 Permanency of mutants 38 Frequency of mutation 38 VI. Conclusions 39 VII. Literature cited 41 VIII. Explanation of plates 45 - 1 - Mutation in the Genus Alternaria I. Introduction The present study arose from an interest which the writer had in the appearance of a sector of decidedly different color in a supposedly pure culture of Alternaria growing in a Petri dish on corn-meal agar. Single spores of this culture were grown and the colonies examined for further evidence of this phenomenon. It was found that quite often in these single- spore cultures variant sectors were formed, which retained the characteristic variation and remained different from the parent colony throughout many transfers. This led to a tentative assump- tion that these variant sectors represented mutants or saltants of more or lees permanent nature, and a more detailed study of this phenomenon was therefore undertaken. The writer has cultured on corn-meal agar, some 15 strains of Alternaria isolated from different hosts, to see if the phenomenon of variant sectors is equally common in all these species or races. These strains have all been grown from single spores to avoid the possibility of error arising from mixed cul- tures. The variants found in the study of several strains have been carefully examined and compared in relation to each other - . - 2 - and to the original form. II. The Present Status of the Mutation Question in Fungi and Bacteria Bud variations, vegetative variations, sports, mutations and saltations have been known and studied among the higher plants for eome time. In these cases such variations may or may not be connected with an intervening sexual act. That similar cases appear among bacteria and fungi is evident from a number of arti- cles bearing on this subject published in the last few years. The mutations occurring in bacteria have been divided by Dobell (16) into two classes, ’’those in which the change is functional (changes in the power of producing ferments or pigment e) and those in which the change is structural”. The greater number of mutations recorded for bacteria belong to the first class. Massini (26) obtained from a case of enteritis an organ- ism which at first produced whitish colonies on Endo's medium. After a time, however, red, daughter colonies appeared within the parent colony. Massini convinced himself, by carefully plating out the original culture, and by other means, that he had not been working with a mixed culture. The organism in the red daugh- ter colonies had permanently acquired the power to ferment lactose and always produced red, and never white colonies. Even after transplanting to other media, free from lactose, they never lost this oower. The trohosus-l ike original therefore, had given rise - . . -3- to a number of new individuals which closely resembled the original, but which had acquired the power to ferment lactose. The white parts of the colonies containing the red, daughter colonies be- haved exactly like the original when transplanted. The white race was therefore constantly undergoing a partial, mutation into a pure red race. Twort (42) recorded independently that certain coli- typhosus organisms were able to acquire the power to ferment cer- tain sugars if grown in them for a sufficiently long time. Ey this method he was able to get a strain of B. typhosus which could ferment lactose and dulcite. The organism which had acquired this power retained it permanently, even after passage through a guinea- pig and cultivation in a dulcite-free medium. Burke (10), Sauerbeck (33), Benecke (4) and Kowalenko (23) have isolated organisms similar to the one studied by Maesini (26). Their results check his and in addition make an important addition to them, since they succeeded in testing individual organ- isms. Kowalenko (23) found that a single individual , which at the outset was unable to split lactose, produced in the presence of this substance offspring, which were in part like itself, and in part able to eplit lactose. The only objection to Massini’s results was therefore removed. Single individuals from white colonies gave rise to mutating races; single individuals from "red" colonies retained their characters in following generations. if ■ -4- Their properties were unaltered by passage through animals, by changes of temperature, or by phenol or other drugs. Muller (27) demonstrated that all typical races of B. typho sus are unable to ferment rhamnose. When grown in a med- ium containing this sugar, however, the colonies develop daughter colonies (nodules) consisting of individuals which have permanent- ly acquired the power of splitting rhamncse. A single non-rhamnose splitting individual gives rise in the presence of this substance to offspring which are partly like itself and partly able to fer- ment rhamnose. Mutations of this sort invariably occur from single individual cultures and under no conditions do the rhamnose split- ting individuals lose their power. SchrOter and Gtltjahr (35) record the following observa- tions. A race Y of coli typhosus-group organisms cannot ferment maltose. After cultivation in a medium containing this sugar, 4 . however, it partially acquires the power, thus coming to resemble Flexner’s bacilli. They found similar changes in related organ- isms; for example, Shiga-Kruse bacilli may acquire the power of splitting both maltose and saccharose when grown in media contain- ing these sugars. The change appears to be permanent, for once acquired by the race the property is never lost. Sobernheim and Seligmann (36) have isolated an oa^nism which behaves exactly like Mass ini’s B. co li m utabil e. In addi- tion they record that they have obtained four different pure races from one pure original race grown from a single individual. From r . 1 -5- a coli-typhosus-group organism they have obtained in pure culture (l) a true Gartner strain, (a) a similar strain, but differing in agglutinating power, (3) a typical typhosus, and (4) a strain al- most identical with B. coli mutab ile (Maseini). The papers of Burri and Duggeli (13) and Burri and And- re;) ew (12) may be considered as parts of Burri' s admirable paper of 1910 (ll). Burri (ll) has isolated a race of organisms of the coli-typhosus group which are unable to ferment saccharose and lactose. He calls this race Bacte rium imper fectum . It never ac- quired the power of splitting lactose, but on the other hand, when grown in media containing saccharose, some colonies acquired the power of splitting this sugar. Burri’s (ll) observations were originally made upon organisms grown in "shake cultures" and not on Endo's medium. The cultures were made in the ordinary way and also from single indivi- duals isolated by his Indian ink method. Each non-saccharose splitting mutating organism was found to give rise to only a very low percentage of saccharose-splitting colonies. The latter, how- ever, had acquired the power permanently, and always retained this character in following generations, even after any number of sub- cultures in many different media. By a number of ingenious experiments Burri (11 ) attempt- ed to determine what percentage of the offspring of B. imper f ectum underwent mutation into B. per fectum. He concludes that all of the individuals of the imperf ectum race are able, under suitable ■ . - - 6 - cultural and environmental conditions, to mutate into perfectum forms. In another set of experiments he has shown that the change from the imperfectum to the perfectum type is not a sudden change but a rather gradual one. Between the non-saccharose-spl it- ting imperfectum and the saccharose-splitting perfectum intervene many generations of individuals showing every transitional stage. This power once acquired by a race is never lost. Burri (ll) supposes that the power to ferment saccharose is latent in every B. imper fectum individual, probably in the form of a zymogen or pro-ferment of some sort. The enzym is produced gradually by the constant action of the sugar on successive genera- tions. He thinks that the newly acquired power of attacking sac- charose does not represent a regeneration of a power originally present in the race but temporarily lost. Bernhardt and Markoff (5) obtained a col i-typhosus-like organism which was capable of fermenting lactose, but in which, after culturing for some time, and passage of the organism through mice and rabbits, the power of fermenting lactose was lost. Thus in this case it would seem that a lactose-fermenting organism which usually remains constant has reverted, or lost this power. t Barethlein (3) found a very similar case to that above cited in several races of the coli-typhosus group: viz that many of the lactose-splitting organisms, when grown for some time on ordinary media, lost the power of fermenting lactose. His results * 'W$l ■ . ■■ -7- seem to disagree with those of most other writers. The reason for this may be that he was dealing with different races of appar- ently the same organism which do not all behave alike. Revis (30) in studying certain organisms of the coli- typhosus group which produce both acid and gas when grown on pep- tone broth containing lactose, was gradually able to acclimatize these organisms to grow in a medium containing 0.1 per cent mala- chite green. These organisms completely lost their power to form gas in the original medium although they still produced acid. The dye appears therefore to have made a lasting change in their method of attacking certain food substances. Wolf (45) carried out a. number of extensive experiments to induce mutation in pigment production. He found that the same chemical substance often produced several kinds of mutations. Thus mercuric chloride produced a dark red race and a pure white race in B. p r odidgiosue . Here the mutation seems to have been in oppo- site directions; in one case the intensifying of color, and in the other the loss of color. Similarly cadmium-nitrate produced a con- stant dark red mutant and a white reverting strain which did not remain constant in following generations. Eisenberg (17) by cultivating Bacillus a nthr acis on glycerin-agar obtained a race of organisms which did not produce spores and which remained constant in this character. Barber (2) in studying Bacillu s coli found that there ■ . . . - 8 - were certain long individuals, longer than the ordinary B. col i. constantly present in his cultures. By isolating and growing these individuals he found that the greater number of them pro- duced colonies of individuals typically like B. coli ; however, in a single instance he obtained one which retained the character and gave only long individuals. It seems from his results that the long individuals were of two kinds, though outwardly more or less alike. The majority represented merely temporary modifications which did not produce like individuals in the following generations while only a few were permanently mutated organisms. Revis (31) maintains that by growing B ♦ coli on peptone broth to which malachite green had been added, he produced a strain which was structurally different, as well as morphologically differ ent, from the original culture. His experiments were not carried on by isolation of single individuals, and although the purity of the culture is guaranteed his work is open to some criticism and should be repeated using single individuals. Brierley (8) has divided the previous records of muta- tions in fungi into two groups. "In the first are those changes in the genetic constitution which appear to bear a direct and purposive relation to certain conditions which have operated dur- ing the development of the organism or that of the preceding gen- eration. The second category of mutations include those cases in which the genetic change is apparently quite fortuitous, and this group ie divisible into two sub-groups. In the first of these . ' . ■ ' B -9- the change is associated with certain conditions which interfere with the normal metabolic reactions of the organism. For example, by treating the organism with toxic substances, or by subjecting it to extremes of temperature or dessication, various intermediate morphological or physiological changes are induced, such as alter- ations in spore dimensions or coloration, or growth on standard media. In the second sub-group the changes occur spontaneously under, so far as may be judged, perfectly normal conditions of development.” The latter sub-group would include the various mutant forms of Alternaria discussed in the present paper. The best known case of purposive mutation, perhaps, is that discussed by Massee (25). In this case Massee sowed spores of Trich otheci um candidum on leaves injected with a sugar solution and from the growth thus secured inoculated other injected leaves. This was done for several generations of the fungus, after which spores from the last growth were sown on normal leaves and infec- tion obtained. "This means that after 12 generations of the fungus educated to grow in Begonia by means of a chemotrophic substance (a solution of cane sugar) the faculty of parasitism had been ac- quired for this particular host plant. By similar means a para- sitic fungus can be induced to become parasitic on a new host." Similar to the findings of Massee (25) are those describ- ed by Salmon (32) on the adaptive parasitism of Ery s iphe gram inis by means of growth upon bridging hosts, or the parallel results of Marshall Ward (43), Freeman (20), Freeman and Johnson (21), . . ' . ' ■ - 10 - Johnson (22) , Pole Evans (19) and others, obtained with various rust fungi. None of the above work, however, was carried on with single-spore strains of the organism in question and is therefore open to criticism. The work of the latter writers has been contra- vened by the work of Reed (29), Stakman (37), Stakman and Piemeisel (38), Stakman, Parker and Piemeisel (39), and Stakman, Piemeisel and Levine (40). As a result of their investigations the latter state; "The results of the experiments with P. gram ini s tri tici- compac t i show that barley which both theoretically and from the results obtained by previous investigators might be expected to increase the infection range does not do so. Even susceptible varieties of wheat do not change the parasitic capabilities of the rust so as to enable it to attack a normally resistant variety. Furthermore the rust does not acquire additional virulence when associated for a long time with a given host. Although it is possible that rusts may change and new biologic forms may develop, it seems more probable that the change is either a very gradual one, extending over long periods of time, or that they change by mutation. No evidence of mutation, however, was obtained in the present investigation. The difference may be one of evolution as compared with experimentally induced change. For practical pur- poses, however, it seems perfectly safe to say that no certain and marked changes in biologic forms need be expected as a result of growing on bridging hosts ; nor does it seem probable that biologic forms are able to gradually adapt themselves to semi- - • * . . ' - 11 - congenial hosts by constant association with those hosts. The writers unsuccessfully tried to get evidence of such adaptation.” The records of instances of mutation in fungi induced by exposure to unfavorable conditions are few in number. Arci- chovskij (l) obtained a form of Asperg i llu s niger with yellow- brown spores, which rose in a pure culture of the black form grow- ing in Haulin' s fluid to which 0.0001 per cent of zinc sulphate had been added. This form produced spores more quickly and was of faster growth than the original culture. He mentions that he has carried the form through a number of generations, but does not make clear, whether it was grown on a medium containing zinc, or on a normal medium. Schiemann (34) obtained various mutants of Aspergillus niger from a single-spore culture by growth on media containing potassium bichromate, and by exposure to extreme temperatures. The potassium bichromate was added in a concentration of 1 to 2,000 and 1 to 20,000 parts to a medium prepared from cane-sugar, pep- tone, potassium nitrate, magnesium sulphate and potassium di-hydro- gen phosphate. From the first generation of a culture on a medium containing potassium bichromate, 1 to 2,000, a brown form was iso- lated and cultured on malt-agar. This mutant remained constant through 32 generations. From the 11th generation of a culture growing on a medium containing potassium bichromate, 1 to 20,000, a form was isolated which varied from a dirty white to a bright- brown in color. This mutant remained constant through 23 genera- . . . ' - 12 - tione on malt-agar. From a culture growing on malt-agar slants free from any poisonous substance, but exposed to a maximum temper- ature of 44° - 45° C. , a deviating growth was produced. The colony of this form was of faster growth than the original. It had a loose mycelium with many aerial hyphae and very long conidiophores; (3-4 mm. as compared to 1-2 mm. of the original). This mutant re- mained constant through 17 generations when cultured on malt-agar at normal temperature. The brown mutant appeared three times; twice in the med- ium containing potassium bichromate and once in a culture which had neither been exposed to high temperature nor contained potass- ium bichromate. Thus it seems that mutation occurs both with and without these unfavorable conditions for growth. Waterman (44) by cultivating Penicil lium glaucum , of a supposedly pure race, in the presence of p-oxybenzoic acid, pro- tocatechetic acid, salicylic acid and trichloracrylic acid was able to induce mutation. The mutant thus produced was distinctly lighter in color, when grown on malt-gelatin, in contrast to the original culture. It differed also from the primitive form in having fewer spores, a decidedly different odor, as well as a slow r er rate of growth. In continued cultivation on malt-agar the mutant, which in all cases seemed the same, remained constant. He also found that a pure culture of Aspergillus niger when grown for some timie in a 2 per cent solution of galactose gave three distinct forms; namely, the original black form, a ■ v. raofe* - I T . ’ I e-.T . . ■ * -13- brown form and a white one. The brown and white forme when isola- ted on malt-agar gave fewer spores than the original, and the white form fewer than the brown one. There was also a decrease in the color intensity of the spores in the mutants, the spores being brown instead of black. There was also a difference in the metab- olism of the original and the mutants, the latter showing a more vigorous combustion of p-oxybenzoic acid to carbonic acid. In this respect the white form had a more vigorous combustion than the brown one. Brierley (8) reports that he has tried carefully to repeat the above experiments of Arcichovskij (l), Schiemann (34) and Waterman (34), using single-spore cultures of Asper g illus niger and Penicillium itali cum . He was unable to do so and states, "Although modifications in the color of the spores, general morph- ological facies, and physiological reactions were observed, these affected the whole growth acted upon to an equal degree; but the reproductive bodies of the modified fungi, gave in every case the original result. These changes were phenotypic and not genotypic and throughout the whole series of experiments not a single muta- tion occurred. " Stevens (41) tested the effect of the amount of the medium, humidity, temperature extremes, and content of the medium upon cultures of Helminthosporium. He found that, although there were variations produced by these environmental changes, they were mere modifications and did not remain constant when the fungus was ' . I . . . -14- returned to favorable environmental conditions. Crabill (14) attempted in four ways to induce mutation artificially in Conio thyrium pirinum but without success. The literature dealing with the general question of muta- tion in the Eumycetes has been fully discussed by Stevens (41), and it is not deemed necessary to go into a detailed review of these papers here. There are, however, a few which he has not mentioned and some which have a more or less direct bearing upon the present problem which deserve some consideration. Crabill (14) working with single-spore cultures of Coniothyrium pirinum described two strains which he designated as plus and minus. These strains differed markedly in several charac- ters, particularly in size and abundance of pycnidia, and in color of colony. He found in his different strains variations ranging from many, large, fully developed pycnidia to few small pycnidia, verging on complete sterility. He says; "The cultural studies show that minus strains may rise from plus strains by sudden sport- ing or mutation. An objection might be raised that these cultures were impure, i.e. mixtures of two strains. In anticipation of such an idea it seems desirable to state that frequent pourings of dilution cultures were used to preclude such a possibility. Pro- geny were then selected only from well-isolated plants, microscopic examination of which showed that each had been derived from a single spore. — - Both strains have repeatedly arisen from the pro- geny of a single plus spore. When once purified the minus strain -15- remains constant from generation to generation. The variation is apparently occurring in only one direction. The only explana- tion which remains is that the minus strain is a sport or mutant arising from the plus strain at irregular and unprognosticable intervals. " The plus strains by sudden variation give rise to the minus strains but the minus strains were not seen to give rise to the plus strains. Contrary to the findings of Stevens (41) and myself he states that the variation apparently occurs in the spores and not in the mycelium. He pictures sectoring and colony differ- ences quite similar to those shown by Stevens (41) for Helmintho- sporium and those pictured in this paper for Alternaria. Burger (9) in studying Colleto trichum gloeosporioides found variant sectors occurring in certain of his cultures. Upon transferring mycelium from these sectors he obtained a strain dis- tinctly different from the original in morphological cultural characters. Since the sectoring was of common occurrence in his cultures he thought of the possibility of mixed cultures and grew colonies from single spores. He found that the sectoring contin- ued and states; "One might be led to conclude from the foregoing data that Coll etotriohum gl oeospo rioides is constantly giving off new types under natural conditions, as well as in artificial cul- tures. Brierley (8) has reported an albino mutant of Botry t ia cinerea. This strain developed by forming a colorless eclerotium . ; -16- in a culture which normally formed black sclerotia. The parent colony originated from a single spore and had been under cultiva- tion for some time. Brierley states in summary: "The colorless form arose spontaneously without any evident relation to external conditions or stimuli, in a single sclerotium of a culture which on accepted criteria was a pure line. The new form is apparently differentiated from its parent in respect to a single character only, that of color, and this albinism would appear to be associa- ted with the absence of cromogen. The change has occurred once only, and has given rise to a form unknown in nature and perfectly constant under all conditions. It would seem possible to place only one interpretation on these facts, that of mutation, and accordingly when I was privileged to exhibit this form to the Linnean Society of London on April 3, 1919, I described it as 'an albino mutant of Botrytis cinerea.'" Blakeslee (6) reports numerous variations in single- spore cultures of Mucor . Many of these mutants tend to revert to the original form when cultured; however, he has found two mutants which are more stable and maintain the varying characteristics. Stevens (41 ) while studying a species of Helminthospor- ium causing a foot-rot of wheat noticed the prevalence of sectors of different color and growth occurring in his cultures. Single- spore cultures of the original culture were made and carefully studied. He found that saltation occurred quite frequently in this species. The saltants differed from the original strain in ' ■ . ■ -17- a number of ways, particularly however, in color of colony, rate of growth, number of spore septatione and length of spores. The case he describes for Helminthosporium and the pictures and plates presented are quite similar to those presented for this paper. There exists among the various writers on the mutation question differences as to the usage of the term mutation. The bacteriologists denote (Dobell (16)) " a permanent change - how- ever small it may be - which takes place in a bacterium and is then transmitted to subsequent generations. All other changes which are impermanent depending generally upon changes of the en- vironment - and hereditarily fixed, are called modifications." Brierley (7) defines mutation as "a genotypic change in a pure line". In the present paper the term mutation is used to denote a sudden morphological change which is transmittable to subsequent generations. In this respect it does not necessarily imply a nuclear change, and is similar to the terms saltation as used by Stevens (41), or mutation as used by Blakeslee (6), Grabill (14) and Burger (9). From the above summaries of the mutation question in bacteria and fungi one may draw certain conclusions. If we consid- er that the work of the above writers has been carefully done, then we must conclude that mutations occur under cultural conditions and it is reasonable to suppose that such phenomena occur in nature. Many of the above papers are undoubtedly open to criticism, espec- ially those dealing with bacteria, because of the loose methods . . , ' . - - 13 - employed or conclusions drawn from too scanty evidence. However, in such work as that of Burri (11) and Barber (2), both of whom worked with single-spore cultures and carried on their work under the best of conditions it can hardly be denied that mutation has taken place. Brierley (7) is not inclined to accept the recorded mu- tations for bacteria on the grounds that little or nothing is known of the genetic constitution of bacteria, and also since the strains in question were not derived from pure lines. He looks upon the variations recorded as being mere segregations of hetero- gynous strains and not mutations in his sense of the term. A possible method by which these strains might be of mixed genetic constitution has been brought out by the work of LOhnis and Smith (24) on the existence of life cycles in bacteria. / They state: ”The life cycle of each species studied is composed of several sub-cycles showing wide morphological and physiological differences. They are connected to each other by the symplastic stage. Direct changes from one sub-cycle into another occur, but they are rather rare exceptions.” In this respect it is not in- conceivable that in the bacterial life cycle there may exist some phase more or less comparable with the sexual fusions of other organisms. LChnis and Smith (24), for example note a 83 'Tnplastic stage in which, ”the living matter previously inclosed in the separate cells undergoes a thorough mixing either by a complete disihtegrat ion of the cell walls, as well as cell content, or by ■ \ -19- the melting together of the contents of many cells which leave their empty cell walls behind them". Furthermore they state, " another mode of interaction between the plaemic substance in bacterial cells was obeerved consisting in the direct union of two or more individual cells. This conjunction seems to be of no lees general occurrence than the process first mentioned". Such conditions as these, if existing in the bacteria may offer an explanation of the apparent mutations in bacteria. The suggestion has been offered that the mutations and saltations of Crabill (14), Burger (9), Stevens (41) were due to segregation in a heterozygous strain. It is true that in all these cases cultural work was begun from a single asexual spore which to all external appearances was homozygous. Yet it is true for some, and possibly true for all, that an ascigerous or sexual stage exists. It is also conceivable that, if the spores from which these cultures originated had been derived in the not far distant past from an ascigerous stage, the possibility of their being he tercz 3 rgous exists. Stevens (4l) states, "this possibility is purely hypothetical but it appears to offer a possible explana- tion for the saltations in Helminthosporium". Dastur (15) found variation in Gloeosporium pip eratum a common occurrence only in strains recently derived from perithecia. This fact would tend to support such an hypothesis. It may be that seme of my cultures of Alternaria which are mutating have been recently through a sexual stage, while • * * ' j - 20 - others which show no signs of mutation have not. All of my cul- tures have been secured from asexual spores and none of them have shown signs of producing perithecia. One point against such an argument is the fact that my culture No. 1 which has been under constant cultivation for over 14 months and during that time has been grown three times from single spores still continues to mu- tate. Brierley (8) offers entirely different suggestions as to the possible explanation of his mutation in Botrytis cinerea , in which sexuality is unknown. He suggests the possibility of a nu- clear transference due to anastomosis of the mycelia, or the cyto- plasmic contamination by such anastomosis. He presents no evidence for his suggestions, and although anastomosis is common among fungi it would take detailed cytological study to prove a nuclear transference or fusion. III. Source of Materials The strains or species of Alternaria used in these studies have been secured from widely different and varied sources. Strain 1 was obtained from germinating millet purchased from a Champaign seed-house. Strain 7 was isolated from a ripe wheat- head from Granite City, Illinois. Strain 10 was secured from dead onion seed— stalks from Crawf ordsville, Indiana. Strain 15 was iso- lated from decaying applee obtained from a grocery in Urbana. Other etraine which have failed to show signs of mutation have been isolated and studied from, leaf-spots on Datura, Lilium and . ■ ■ ' . H . - 21 - Yucca, from fruits of okra, morning glory, snow berry, mango and pepper, and from dead blades of grass and corn. IV. Methods Employed for Isolation and Growth Cultures of Alternaria from the different sources were usually obtained by planting infected tissue of the host on agar, or by scraping spores from the host into a few drops of sterile water which was then added to a tube of agar and poured into a sterile Petri-dish. The growth thus secured was examined and trans- ferred until, as far as could be ascertained, a pure culture had been obtained. Single-spore cultures were then made in order to be sure that the cultures were pure. This was accomplished in the following manner. Spores from the cultures were transferred to a tube containing about 10 cubic centimeters of sterile water. It was quite essential that only a few spores be placed in the water. In order to ascertain the approximate number of spores per oese in the suspension, two oeses were transferred to a slide and examined under the microscope. If the number of spores thus obtained was under 12 the suspension was considered satisfactory. Two oeses of the suspension were then transferred to a tube of melted agar at 42° C. , thoroughly mixed by rolling between the hands, and poured into a sterile Petri-dish. As soon as the agar had hardened the poured plates were examined by inverting them under the low power of the microscope. In this way single spores, well isolated from other spores, were located and their position marked by draw- ing a circle around them with Indian ink, on the bottom of the . . . . - 22 - Petri-dish. Great care was used in making sure that only one spore was inside the circle. The agar within the circle was carefully lifted out, transferred to another sterile agar plate and again examined to make sure that only one spore had been transferred. The single spore was then allowed to germinate and produce a colony. Transfers from the colonies originating from single spores have been made to plates of corn-meal agar and grown at room temper- ature to observe whether mutation would take place or not. In all the cultural work corn-meal agar, made by the following formulae, has been used. Fifty grams of corn-meal were added to 1,000 cc. of dis- tilled water and cooked for one hour over a water bath at 60° C. , then filtered through cloth and filter paper and 13 grams of agar shreds added. The above medium was then cooked for one and one- half hours over a water bath, filtered through cotton, tubed and sterilized in the autoclav for 20 minutes at fifteen pounds pressure. Care has been taken to have the media as nearly constant as possible to avoid variations due to the quality of the medium. Appearance of Mutations The mutations herein reported always arose as sectors differing in color from that part of the parent colony adjacent to them. Transfers were made from these variant sectors to poured sterile agar-plates, and a transfer of the normal colony placed in the same plate some distance from the variant transfer. The variant transfer was marked as M and the normal transfer designated as 0. * . ' . * . 4 p -23- The colonies produced from such transfers were allowed to grow until they filled the plate when other transfers were made, and so on for a large number of generations. Ey this method it was possible to study the mutant and the original under approximately the same conditions. The two colonies grown in this way differed most markedly in color, presence or absence of aerial mycelium, and in conidial production. In practically all cases such mutants arising from distinct sectors have remained constant, though occa- sionally one is obtained which in turn gives off sectors which are apparently reversions to the original form. In the contrast stud- ies of the mutants and the originals care has been taken that the thickness of the medium be the same all over the place, since it was found that the thickness of the medium plays an important part in the appearance of the colony. Throughout the paper M denotes a mutant while 0 denotes the original culture. I have given the different strains of Alternaria tested for mutation a serial number. Thus 0 10, M2 refers to a mutant, No. 2, arising from original culture No. 10. A generation is taken to mean the growth produced on a sterile plate by a transfer of mycelium from another culture. In original culture No. 10 mutant 1 appeared in the 2nd generation. This culture has been giving rise to mutants for more than 14 months. Some 18 of these mutants have been isolated and studied but they appear to be the same, or ver}*- closely alike, and are similar to mutant 1. Mutant 15 appeared in the 22nd gener- ation of the original culture and in the 4th generation after re- . . ' . • -24- isolation from a single spore. In culture No. 7 mutant 1 appeared in the 12th genera- tion. This mutant has appeared but once and has repeatedly given off sectors which appear to be reversions to the original, since no significant differences are brought out by contrast studies. In culture No. 10 two distinct mutants have been iso- lated from among the many variant sectors given off by the original culture. Mutant 1 appeared in the 4th generation of the original culture; mutant 2 in the 9th generation. Other mutants have arisen from this culture but in contrast studies have not appeared to be distinct from mutant 1. Thus it might be said that mutant 1 has appeared several times. Mutant 2 has arisen but once and has re- mained constant. Modifications Throughout these cultural studies certain modifications have arisen. The most common modifications are in the form of small sectors of different color at the very edge of any old colony which is drying out. In most cases transfers from these small sectors give normal colonies like the original. A form of modi- fication less common than the above is the appearance of light or dark radiating streaks in original cultures Nos. 7 and 10. Trans- fers made from these streaks produce normal colonies. In a normal colony which has grown to the edge of the plate white aerial mycel- ium is often produced on the sides of the plate. This upon trans- fer does not maintain its characteristic white appearance but gives ' -25- a normal colony. In cases where the medium is not the same thick- ness throughout the plate certain modifications are produced which do not maintain themselves when transferred. Modifications have been induced by the addition of certain chemicals and toxic sub- stances to the medium. Transfers from such modifications, grown on a normal medium, produce normal colonies. V. Methods Employed for Contrast Studies of Mutants and Originals In the following studies for the contrast in color of colonies, zonation of colonies, rate of growth, presence or ab- sence of aerial mycelium and abundance of conidial production, the mutants have been grown side by side with each other and with their parent, under practically the same conditions. For the contrast of length and breadth, conidia were measured under the microscope by means of an ocular micrometer having a value of 3.4 mic. per space. A mechanical stage was used to move the slide on the micro- scope, All conidia were measured which came between certain lines on the ocular micrometer. In this way unconscious selection of conidia was avoided. The conidia were divided into classes and plotted on cross section paper according to the number of ocular spaces occupied. In cases where conidia fell midway between spaces note was made of them and they were equally divided between the two classes. The results of these measurements and the results of counting the number of cross and longitudinal septations have been plotted in the form of polygons and curves and compared biometrie- -26- ally. Ifr contrasting the color of the conidia, mycelia and media, two Leitz microscopes of approximately the same magnification were used. The fields of these two microscopes containing the desired material were brought together by using and E. Leitz, Wetzlar comparison ocular. In this way the different shades and tints could be quite accurately compared. In view of the fact that much variation is caused by the quality of the medium, (Planchon (28)) the media used in all cul- tures has been made as nearly constant as possible. To avoid differences due to the age of the cultures, colonies of the same age have been used in all contrast studies. By this method it has been possible to add some significance to the differences in color of spores and mycelium, which would not be justifiable if cultures of unequal age had been used. Color of Colonies on Agar Culture No. 1. The original culture gives a very light grey qjOLony with white aerial mycelium. Mutant 1 has a reddish-brown colony. Mu- tant 15 gives a gray colony, but much darker in hue than the orig- inal, and it has dark aerial mycelium. Culture No. 7. The original colony of this culture is lead colored. Mutant 1 is light-brown with a bluish tint. The mutant is much darker than the original but has not nearly as much brown color as mutant 1 of culture No. 10, or mutant 1 of culture No. 15. -27- Culture No. 10. The original culture has a colony that is dark gray approaching black. Mutant 1 has a reddish-brown colony much light- er in color than the original. Mutant 2 gives a colony similar in general color to the original but of brown tint. It is of much darker hue than mutant 1 but does not have the reddish-brown cast of mutant 1. Culture No. 15. The original culture gives a bluish-gray colony; mutant 1 a reddish-brown colony similar in appearance to mutant 1 of cul- ture No. 1, but lighter in hue than mutant 1 of culture No. 7 and mutant 1 of culture No. 10. Zonation of Colonies Culture No. 1. There is no zonation in the colonies of the original ; a slight zonation in the colonies of mutant 1 and a very distinct zonation in the colonies of mutant 15. Culture No. 7. Colonies of the original show a slight zonation and light streaks radiating from the center. Colonies of mutant 1 show a distinct zonation which is more prominent than in the original. The radiating streaks present in the original are absent in the mutant. Culture No. 10. Colonies of the original culture show no marked zonation. . . -38- Colonies of mutant 1 show a distinct and marked zonation, while those of mutant 2 show a slight zonation which ie not so pronounoed as that of mutant 1. Culture No. 15. Colonies of the original culture show very slight zona- tion while those of mutant 1 show distinct zonation. Rate of Growth Contrast studies of the different mutants and their originals have shown little or no difference in their rate of growth. There appears to be a slight difference between original 15 and its mutant 1 in that the mutant grows slightly faster than the original. The difference here is very slight and not readily apparent. Color of Mycelium Culture No. 1. The original culture has a hyaline mycelium. The mycel- ium of mutant 1 ie yellowish-brown. The mycelium of mutant 15 is gray being much darker than that of the original. Culture No. 7. The mycelium of mutant 1 is thicker and slightly darker than that of the original culture. Culture No. 10. The mycelium of the original is darker than that of either of the mutants. The original culture has a dark-gray mycel- ium; mutant 1 a nearly hyaline mycelium with a yellowish cast; mu- -29- tant 2 a light-gray mycelium. Culture Mo. 15. The mycelium of the original culture is lead-colored while that of mutant 1 is much lighter with a yellowish cast. Color of Medium. In cultures Nos. 1, 7, and 15 neither the originals nor i A the mutants color the medium which they are growing. In culture Mo. 10 the medium ie not colored by the original culture or mutant 2, but is colored slightly yellow by mutant 1. Aerial Mycelium Culture Me. 1. There is an abundance of aerial mycel ium produced by the colonies of the original culture. The colonies of mutant 1 do not produce aerial mycelium; those of mutant 15 produce aerial mycelium, but not so abundantly as colonies of the original. The mycelium of mutant 15 is quite flocculose, a character entirely absent in the original culture and in mutant 1. Culture No. 7. There is an abundance of aerial mycelium produced by the original culture, together with numerous white tufts. Mutant 1 produces considerably less aerial mycelium, and the white tufts are entirely lacking. Culture Mo. 10. The original culture produces an abundance of aerial mycelium; this is lacking in mutant 1 and is produced in less abundance in mutant 2 than in the original. The mycelium of mu- . . -30- tant 2 is quite flocculose, a character lacking in the original and in mutant 1. Culture No. 15. The original culture produces abundant, long, aerial mycelium. Mutant 1 produces some aerial mycelium which is short and with age comes to lie flat on the suface, producing a felt- like weft. The following mathematical calculations represent the mean (M) , standard deviation (