The Genetics of Ceratostomella radicicola and the Phylogenetic Relationship between Chalaropsis and Chalara

The Genetics of Ceratostomella radicicola and the Phylogenetic Relationship betweenChalaropsis and ChalaraAuthor(s): Arif S. El-AniSource: American Journal of Botany, Vol. 45, No. 3 (Mar., 1958), pp. 228-232Published by: Botanical Society of AmericaStable URL: http://www.jstor.org/stable/2446454 .

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THE GENETICS OF CERATOSTOMELLA RADICICOLA AND THE PHYLOGENETIC RELATIONSHIP BETWEEN CHALAROPSIS AND CHALARA1

Arif S. El-Ani

CERATOSTOMELLA RADICICOLA, the perfect stage of Chalaropsis, is a heterothallic pyrenomycete with long-necked perithecia and hyaline single-celled ascospores. Asexual reproduction is accomplished by means of two types of conidia, the microconidia (fig. 6) produced endogenously, and the macroconidia (fig. 2, 3) borne on sympodially branched macroconidiophores. Previous studies (Bliss, 1941; El-Ani et al., 1957) have revealed the occurrence of two distinct strains on roots of the date palm, the black strain (fig. 1) producing dark macroconidia (fig. 3) and black perithecia (fig. 5b), and the brown strain (fig. 1) in which both macroconidia (fig. 2) and perithecia (fig. 5a) are brown. The ascospores are uninucleate (fig. 7), and single-ascospore cultures are hermaphroditic, selfsterile, and of two compatibility types (A and a), which must be brought together before perithecia can be produced. When black and brown strains are mated, the progeny has been found to consist of black and brown thalli in a 1:1 ratio. Thus the two strains differ genotypically from each other by black to brown has been observed in vitro. Recently two mutants (fig. 1), gray and tan, which arose spontaneously in vitro were used with the other strains for further genetic investigation, the results of which are described in the present paper.

MATERIALS AND METHODS. Cultures of C. radicicola derived from single ascospores were grown on two media, potato-dextrose-yeast agar (2g. yeast extract/I.) on which rapid vegetative growvth at temperatures of 27-30?C. ensures morphological distinction of each strain; and Bacto prune agar (48 g./l.) used mainly for perithecial production. In crosses between two thalli, conidia suspended in distilled sterile water were transferred from one thallus to another of different compatibility. Peri- thecia with mature ascospores were formed within four days. Single-ascospore cultures grown on the potato-dextrose-yeast agar medium were assorted according to their morphological characters within a week. In order to determine the mating type and perithecial color of each single-ascospore culture, a small conidial inoculum was transferred from the unknown thallus to each of two tester thalli of the same perithecial color but differing in compatibility factors. Tester thalli with perithecial color presumably different from that of the tested culture were chosen. For example, if the single-ascospore a single gene. As yet, however, no mutation from

1 Received for publication September 17, 1957. The author is grateful to Prof. Spencer W. Brown, and

Prof. Everett R. Dempster for criticism of the manuscript and to Dr. Henry Schneider for the use of the facilities in his laboratory.

culture to be tested was black, thalli of A and a compatibilities bearing the gene for brown perithecia were used as testers. The appearance of black perithecia among the brQwn in one of the two testers as a result of reciprocal fertilization, as frequently occurred, would of course reveal the compatibility type and perithecial color of the tested thallus. On the other hand, if fertilization proceeded in one direction and perithecia were produced only by the tester thallus, the tested culture whose compatibility had thus already been determined would be grown on a prune agar slant and fertilized by the compati- ble tester. Perithecia would be produced within a few days and their color would be recorded.

CULTURAL TYPES.-In the present studies four different strains (fig. 1), which can be distinguished morphologically by macroscopic examination, were used. The genotype of each strain, as determined by the genetic studies, and a brief description of its morphology are given below.

Black (b+ m+).-This is the common strain of C. radicicola isolated from roots of date palm as a heterokaryon from which thalli of A and a comnpati- bilities were separated (El-Ani et al., 1957). It produces dark macroconidia and black perithecia, which are either glabrous and embedded in the medium, or superficial and, frequently, with black appendages (Bliss, 1941).

Brown (b m+). As already pointed out, brown differs from black by the color of perithecia and macroconidia. The latter are predominantly brown, but hyaline macroconidia are also produced in a considerable proportion. Perithecia and perithecial appendages, if produced, are also brown.

Gray (b+ m) .-Gray (fig. 1) was obtained as a mutant single-ascospore isolate from the cross black X brown, and has never been encountered in nature. The main character that distinguishes gray from the two natural strains is the complete absence of macroconidia. It produces black perithecia like those of the black strain. Microconidia en masse and abundant protoperithecia (fig. 4) give the thallus its grayish color.

Tan (b m). Tan (fig. 1) was also obtained as a mutant single-ascospore isolate from the cross black X brown. Like gray it produces no macroconidia, but it differs from the latter by its tan color and by producing brown perithecia similar to those of the brown strain..

RESULTS OF CROSSES.-The color locits (b+).- (A) brown a X black A, black A X brown a.-As already pointed out, black and brown differ by a single gene. The question immediately arises as to whether all black and brown cultures produced in a ratio of 1:1 from these crosses are respectively

228



March, 1958] EL-ANI-CERATOSTOMELLA GENETICS 229

identical with the parents in the color of perithecia and macroconidia or whether recombinant types in which the two structures have different colors can be found among the progeny. The inheritance of color and compatibility has not previously been investigated. The results from these crosses have revealed that the color of the perithecia in each single-ascospore culture is always identical with the color of the macroconidia, both being either brown or black. It is apparent, therefore, that the color of these two structures is transmitted as a

single hereditary character. This in turn may im- ply that the pigments embedded in the wall of the macroconidium are the same as those in the wall of the perithecium.

Although the four groups (table 1) which consti- tute the progeny from brown a X black A fit approximately a 1:1:1:1 ratio, one can hardly escape noticing the excess of the non-parental types (55 per cent) over the parental types. In the reciprocal cross, however, the recombination percentage drops to 50 per cent. Therefore, it is likely that differences

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Fig. 1-7. Ceratostomella radicicola.-Fig. 1. Brown and gray strains crossed (left), followed by 2 brown, 2 gray, 2 black and 2 tan cultures that appear in the progeny.-Fig. 2. Macroconidia from a brown strain. Note the presence of hyaline and colored ones. X400.-Fig. 3. Macroconidia from a black strain. X400.-Fig. 4. Perithecial initials (protoperithecia) from a tan strain. X200.-Fig. 5. Brown (left) and black (right) perithecia. X50---Fig- 6. Microconidia, dark and hyaline from a graa culture. X40O.Fig. 7. Uninucleate ascospores X1800.



230 AMERICAN JOURNAL OF BOTANY [Vol. 45

TABLE 1. The inheritance of color, compatibility, and macroconidial formation in Ceratostomella radicicola.

Crosses Cross Cultural types Progeny no. crossed Black (b+ m+) Brown (b m+) Gray (b m+) Tan (b m) Total

1. Brown a X Black A 215 223 438 (b m+ a) (b+ niz+ A) 94A 121a 123A lOOa

2. Black A X Brown a 80 76 156 (b+ m+ A) (b m+ a) 43A 37a 42A 34a

3. Gray A X Tan a 104 104 208 (b+ m A) (b m a) 47A 57a 56A 48a

4. Tan a X Brown A 36 35 71 (b m a) (b m+ A) 17A 19a 17A 18a

5. Gray a X Black A 69 73 142 (b+ m a) (b+ mn+ A) 29A 40A 34A 39a

6. Black A X Tan a 233 240 254 211 938 (b+ m+ A) (b m a) 113A 120a 128A 112a 128A 126a 113A 98a

7. Tan a X Black A 140 147 133 146 566 (b m a) (b+ m+ A) 66A 74a 84A 63a 70A 63a 73A 73a

8. Brown A X Gray a 54 59 41 53 207 (b m+ A) (b + m a)

in ascospore viability, rather than linkage and posi- tive interference in chiasma formation, are the cause of the high percentage of recombinants in the first cross. The percentage of ascospores that germi- nate on potato-dextrose-agar after 15 hours at 26?C. (the same conditions for making single ascospore cultures) was found to vary from 81-92 per cent. The possibility that the recombinant ascospores, b+ m+ a and b m+ A, are more viable than the parental types is indicated in other crosses too (as will be shown later).

(B) Gray A X tan a.-The fact that gray and tan are inherited in a 1:1 ratio (table 1) obviously indicates that the two mutants differ by a single allelomorphic gene which controls color. Because all tan thalli produce brown perithecia, and all gray thalli produce black perithecia, it can be concluded that the mechanism of pigment formation in the perithecial and macroconidial walls has re- mained unimpaired despite the loss of macroconidia. The difference in color between the two mutants is due to the color of their respective microconidia (fig. 6), protoperithecia, and endoconidiophores. The color of these structures is governed by the same gene as that in the previously mentioned crosses of black and brown, hence, the formation of macroconidia and the synthesis of the pigments rembedded in their walls are two different processes that go on independently. In sum, gray has arisen from black and tan from brown, each through a single gene mutation that suppresses the formation of macroconidia and thus brings about the re- markable change in thallus color. Yet the two mutants have retained the gene for color, whose function is still made manifest not only by the color of the perithecia but also by the thallus color.

Therefore, the mode of inheritance of color and compatibility is essentially the same as that observed in the cross black X brown.

The macroconidium locus m+.-The two crosses gray a X black A, and tan a X brown A were made with a twofold purpose: (1) to find out how each mutant differs from its corresponding natural strain, and (2) to determine whether linkage exists between the compatibility gene and the gene that controls the formation of macroconidia. The results recorded in table 1 show that in both crosses the mutant differs from the strain with which it is crossed at one locus, which segregates independently of that for compatibility. All cultures from the cross gray a X black A produce black perithecia, and those from the cross tan a X brown A produce brown perithecia. The data from these two crosses can be treated as a single sample of progeny since the two mutants used are of the same compatibility type and each differs from the wild type with which it is crossed by the factor that governs macroconidial formation.

Color (b+), macroconidia (m+), and compatibility.-Black A X tan a, tan a X black A, brown A X gray a.-From these crosses segregation of three pairs of allelomorphic genes controlling color, compatibility and macroconidial formation can be studied. The results (table 1) provide further sup- port for the conclusion already reached that production of macroconidia and synthesis of the pigments in their walls are two different processes controlled by different genes. Since the four types, black, brown, gray, and tan are produced in all crosses in approximate equality, it can be concluded that color (b+) and macroconidial formation (m+) are inherited independently.



March, 1958] EL-ANI-CERATOSTOMELLA GENETICS 231

In considering compatibility and macroconidia alone, the four genotypes mn+ A, n+ a, m A, m a are produced in the ratio of 241: 232: 241: 224 from the cross black A X tan a, and in the ratio of 150: 137: 143: 136 from the reciprocal cross tan a X black A. Accordingly, the ratios of non-recombinants to recom-binants in the two crosses are respectively 465: 473 and 286: 280. Therefore, the two loci A/a and m+/m segregate independently. Of particular interest is the segregation of color and compatibility which accounts for the appearance of the four genotypes b+ A, b+ a, b A and b a, in each of the two crosses black A X tan a and tan a X black A. The ratio among the four genotypes is 241: 246: 241: 210 in the first cross and 136: 137: 157: 136 in the second, and the recombination percentage in either cross is approximately 52. It must be noted that 55 per cent of brown and black and 49.8 per cent of gray and tan thalli from the cross tan a X black A are recombinants. Hence the recombination percentage in the progeny as a whole is 52.4. Such fluctuation in the recombination percentage from one group to another in the same progeny provides further evidence for differences in ascospore viability as suggested previously in the cross brown a X black A. It is interesting that the percentage of recombinants in the latter cross is also 55, and in both crosses the mated thalli can be designated, with respect to color and compatibilty as b a and b+ A. Therefore, segregation of A/a and b+/b loci can be described as independent.

DisCUSSION.-Chalaropsis was erected as a new genus in-1916 by Peyronel on a type species called Chalaropsis thielavioides. The new genus, as the name indicates, is a Chalara-like fungus which was described by Peyronel as a Thielaviopsis in whicb the macroconidia are not formed in chains. The three genera are undoubtedly closely related. The microconidia produced by each of the three genera present convincing evidence for their relationship, whereas the macroconidia, lacking in Chalara but present in Chalaropsis and Thielaviopsis, provide the bases for generic identification. The distinction between Chalaropsis and Thielaviop,sis is based on the fact that the macroconidia are single in the former genus and catenulate in the latter. Obvi- ously, the loss of macroconidia in either genus would change it to Chalara. While certain species of Chalara and Thielaviopsis have been known for a long time as the imperfect stages of the asco- mycete Ceratostomella, the perfect stage of Chala- ropsis had not been discovered until 1941 when Bliss described C. radicicola as a new species. The present investigation reveals a likely phylogenetic relationship between the imperfect genera Chalara and Chalaropisis in demonstrating the origin of the first genus from the latter through a single gene mutation. Accordingly, the two genera should be classified as a single genus, the name of which on

a priority basis would be Chalara, and Chalaropsis would he a synonym.

The appearance of four different types of thalli from the cross black X tan presents a striking re- semblance to certain aspects of inheritance in Venturia inaequalis (Keitt et al., 1943) particularly the cross normal X tan non-conidial in the latter genus. It must be noted that brown and tan of C. radicicolla correspond respectively to tan and tan non-conidial mutants of V. inaequalis. The loss of conidia in both ascomycetes is not associated with any sex mutation as it is in Hyp,omyces solani f. cucuzrbitae (Hansen and Snyder, 1943). The conclusion of Hansen and Snyder (1943) that sex (culture type), compatibility and perithecial color in Hypomyces solani f. cucurbitae are inherited independently was based on the fact that from crosses involving the three characters eight types were produced in the progeny. Since no ratio was given among the eight types, crossing over as a possible explanation can not be entirely ex- cluded. Recently these two authors (Snyder and Hansen, 1954) claimed that the male and female factors "inherit independently of one another." It is beyond my comprehension how these two sex factors being situated on the same chromosome and showing 22 per cent recombination (El-Ani, 1954) can be inherited independently.

The perithecial wall in C. radicicola is identical in color with that of the receptive thallus and is produced, perhaps entirely, by the latter. In their detailed and well-illustrated studies of the perithecial development in Glomerella cingulata, McGahen and Wheeler (1951) come to the conclusion that the perithecial wall is built up by the proliferation of a single initial. It is interesting that in a uniform heterokaryon arising from brown and black thalli of two different compatibilities in C. radicicola, all perithecia produced are black. Such a thallus can hardly be distinguished from a homokaryotic black thallus, as the macroconidia produced are predominantly dark. Therefore this heterokaryotic condi- tion, which can be shown to prevail in the macroconidia (El-Ani, unpublished), would undoubtedly also account for the formation of the exclusively black perithecia.

Although it has been shown that Chalara may arise from Chalaropsis as a result of a single gene mutation, the evolutionary change may have proceeded in the reverse direction through the acquisi- tion of macroconidia by Chalara-like ancestors of Chalaropsis. In the closely related species Cerato- stomella paradoxa, the perfect stage of Thielaviop'- sis paradoxa, macroconidia and microconidia are also produced. Moreover, hyaline and dark microconidia are produced by the same endoconidiophore (Dade, 1928). According to Klotz and Fawcett (1932) , distinctions between macroconidia and microconidia are artificial as gradations in size, color and shape can be found, and "all the conidia



232 AMERICAN JOURNAL OF BOTANY [Vol. 45

are probably produced endogenously. . . ." Thus it seems likely that the macroconidia are here in a transitional form, evolving from microconidia. In contrast to this situation, striking distinctions exist between the two types of conidia in C. radicicola. Many attempts to cross C. radicicola with C. paradoxa have been unsuccessful.

In view of the foregoing discussion, it would be interesting to find out what biochemical mechanism is involved in the formation of macroconidia and how mutation at m+ locus brings about their loss. Such information would contribute much to our knowledge of morphogenesis and taxonomy of imperfect fungi. It is noteworthy that thiamine which is essential for perithecial formation in Cerato- stomella fimbriata (Barnett and Lilly, 1947) is re- quired for the growth of C. (Endoconidiophora) paradoxa (Robbins and Ma, 1942) and C. radicicola (El-Ani, unpublished).

SUMMARY

Four strains of Ceratostomella radicicola were used in the present genetic investigation; the macroconidia-producing strains black and brown and the macroconidia-lacking mutants, gray and tan. It was

found that gray arose from black and tan from brown, each as a result of a single gene mutation that suppressed the formation of macroconidia. The loss of macroconidia transmuted Chalaropsis, the imperfect stage of C. radicicola, to Chalara. Segregation of compatibility factors, macroconidial formation, and color of both macroconidia and perithecia was studied. The results show that the color of macroconidia in any culture is always identical with the color of perithecia, both being either dark or brown. The difference in color between gray and tan is due to the color of their respective protoperithecia, microconidia and endoconidiophores. The color of these structures is governed by the gene for the color of macroconidia and perithecia. Analyses of progenies from the crosses black A X brown a, brown a X black A, and gray A X tan a show that color and compatibility are inherited independently. Since eight types of cultures are obtained in approximate equality from the crosses black A X tan a and tan a X black A, it is evident that color, compatibility, and macroconidial formation segregate independently.

DEPARTMENT OF PLANT PATHOLOGY

UJNIVERSITY OF CALIFORNIA

RIVERSIDE, CALIFORNIA

LITERATURE CITED

BARNETT, H. L., AND V. G. LILLY. 1947. The relation of thiamin to the production of perithecia by Ceratosto- mella fimbriata. Mycologia 39: 699-708.

BLISS, D. E. 1941. A new species of Ceratostomella on the date palm. Mycologia 33: 468-482.

DADE, H. A. 1928. Ceratostomella paradoxa, the perfect stage of Thielaviopsis paradoxa (de Seynes) von H6hnel. Trans. Brit. Mycol. Soc. 13: 184-194.

EL-ANI, ARIF S. 1954. The genetics of sex in Hypomyces solani f. cucurbitae. Amer. Jour. Bot. 41: 110-113.

- -, L. J. KLOTZ, AND W. D. WILBUR. 1957. Hetero- thallism, heterokaryosis, and inheritance of brown perithecia in Ceratostomella radicicola. Mycologia 49: 181- 187.

HANSEN, H. N., AND W. C. SNYDER. 1943. The dual phe- nomenon and sex in Hypomyces solani f. cucurbitae. Amer. Jour. Bot. 30: 419-422.

KEITT, G. W., M. H. LANGFORD, AND J. R. SHAY. 1943.

Venturia inaequalis (Cke.) Wint. II. Genetic studies on pathogenicity and certain mutant characters. Amer. Jour. Bot. 30: 491-500.

KLOTZ, L. J., AND H. S. FAWCETT. 1932. Black scorch of the date palm caused by Thielaviopsis paradoxa. Jour. Agric. Res. 44: 155-166.

McGAHEN, J. W., AND H. E. WHEELER. 1951. Genetics of Glomerella IX. Perithecial development and plasmo- gamy. Amer. Jour. Bot. 38: 610-617.

PEYRONEL, B. 1916. Una nuova mallattia del lupino pro- dotta da Chalaropsis thielavioides Peyr. nov. gen. et nova sp. Staz, Sper. Agrarie Ital. 49: 583-596.

ROBBINS, W. J., AND ROBERTA MA. 1942. Vitamin deficien- cies of Ceratostonella and related fungi. Amer. Jour. Bot. 29: 835-843.

SNYDER, W. C., AND H. N. HANSEN. 1954. Species concept, genetics, and pathogenicity in Hypomyces solanii. Plyt:opath. 44: 338-342.



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The Genetics of Ceratostomella radicicola and the Phylogenetic Relationship between Chalaropsis and Chalara