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OFFICERS AND STAFF
BOARD OF CONTROL.
... Orange George A. Hopson, Secretary...
Mount Carmel Wm. L. Slate, Jr., Director and Treasurer.
New Haven Joseph W. Alsop.
...Avon Elijah Rogers..
Southington Edward C. Schneider.
Middletown Francis F. Lincoln.
G. P. CLINTON, Sc.D., Botanist in Charge.
WALTER O. FILLEY, Forester in Charge.
DONALD F. JONES, S.D., Geneticist in Charge.
M. F. MORGAN, M.S., Investigator
P. J. ANDERSON, PH.D., Pathologist in Charge.
THE WILSON H. LEE Co.
Culture and the Stimulation of their Production by Extracts from other Fungi.
FLORENCE A. McCORMICK.
During a disease survey of tobacco in the summer of 1920, Thielavia basicola Zopf was found to be unusually abundant, the heavy rains in the early part of the season favoring its development. Perithecia were plentiful and, in some cases, they were found to be deeply embedded in the tissues of the host. It, therefore, seemed worth while to attempt to establish more definitely the connection of the ascospore stage with the chlamydospores and endoconidia, all three spore forms being known by the name Thielavia basicola Zopf, since Peglion, in 1900, (19) has given the only published record of perithecia in artificial culture. Aside from its scientific interest, it is of economic value to know whether there are two fungi or only one, possessing all spore forms, parasitic on its agricultural hosts. Thielavia basicola Zopf is a well known parasite, chiefly serious on tobacco, the violet and many of the Leguminosae. Johnson mentions thirty-nine hosts compiled by previous investigators and gives sixty-six additional ones.
The literature cited below concerns, in the main, the perithecia commonly assumed to be the perfect stage of the fungus producing chlamydospores and endoconidia known as Thielavia basicola Zopf and is only a small part of the mass of publications bearing chiefly on the economic problems connected with that fungus.
In 1850 Berkeley and Broome (2) found the chlamydospores at the base of stems of peas and Nemophila auriculata and gave the fungus the name of Torula basicola n. s. In 1876 Zopf (33) published an article in which he described a fungus growing on Senecio elegans L. in the Botanical Garden of Berlin and he recognized the chlamydospores as identical with those described and figured by Berkeley and Broome. In addition to the chlamydospores, he described endoconidia, ascospores and spermatia. The last spore form has not been observed by other workers and Zopf himself omitted it from a later publication. Due to the association of perithecia with the chlamydospores and endoconidia, Zopf, in his publication of 1876, changed the name, Torula basicola, given by Berkeley and Broome, to Thielavia basicola and placed the fungus in the Perisporiaceae. Since the date of that publication the fungus bearing chlamydospores and endoconidia, noted above, has also been known as Thielavia basicola Zopf and the perithecia occasionally found with them have been considered the perfect stage of that fungus. In August, 1876, Sorokin (30) announced the finding of a new fungus on the roots of Cochlearia armoracia. He described the chlamydospores, but, not recognizing them as belonging to the fungus found by Berkeley and Broome twenty-six years before, nor knowing of Zopf's discovery, named the fungus Helminthosporium fragile sp. n. Saccardo lists the fungus in three different places and under as many different names. He retains the name for the chlamydospore stage given by Berkeley and Broome (23), but he changed the name Helminthosporium fragile Sorok., given by Sorokin, to Clasteros porium fragile (Sorok.) Sacc. (24). He also gives a description of Thielavia basicola Zopf (22), including in it perithecia along with the other spore forms.
In his paper of 1891, Zopf (34) gives a detailed account of a new disease of lupines which he found to be due to Thielaria basicola. He did not make cultures but, since perithecia were again found associated with chlamydospores and endoconidia, he still considered the fungus undoubtedly an ascomycete and retained the name Thielavia basicola. This opinion has been generally accepted although the relationship of perithecia to endoconidia and chlamydospores has not been considered completely established, since perithecia are not obtained in artificial cultures and only infrequently found in nature. In classification of fungi, however, Thielavia basicola Zopf is invariably placed with the ascomycetes, on the supposition that chlamydospores, endoconidia and perithecia belong to the same fungus, and taxonomists agree that it is closely related to the Aspergillaceae.
Previous to the report from this Station in 1921(14), Peglion (19) is the only person who had obtained perithecia in a culture. He found perithecia only once, these occurring after he moistened a three year old culture on potato plugs with a 6% tartaric acid solution. A week later he discovered that perithecia had developed. Prof. Thaxter, 1891, (31), who found this fungus on violet roots and who was the first to report its presence in America, did not find perithecia and he has never been fully convinced of their relationship to the other two spore forms. Sorauer (29), 1895, does not question the relationship. Oudemans (18) considers Thielaria basicola Zopf as belonging to the Perisporiaceae and he illustrates only a mature perithecium, a mature ascus and a young ascogonium, omitting any reference to the asexual spores. CappellutiAltomare (4) found perithecia associated with chlamydospores on tobacco roots. Aderhold (1) failed to obtain perithecia in his cultures and did not find them in plants infected with that fungus. In discussing perithecia Clinton, 1906, (5) says, "So far as our own observations go we could not positively assert their relationship to the other spore forms of the root rot fungus, but from their presence and the observations of others there seems to be no reason for doubting this relationship." In his report of the following year
Dr. Clinton (6) further says “During the past year and a half the fungus has been under observation in cultures with various media in an attempt to develop the ascospore stage. This has not been obtained, though the fungus was grown on tobacco roots on which this stage occurs in nature in Connecticut. Fresh tobacco roots containing the fungus were sent to Professor Thaxter, who tried to isolate the ascospores by the Barber method and obtain cultures directly from them-our cultures having come originally from the endospores, or possibly from the chlamydospores—but he was not successful in obtaining such cultures. Professor Thaxter has, on the other hand, a culture of another species of Thielavia which forms ascospores, but never the endospores and chlamydospores. These facts possibly may indicate that the ascospore stage has no relationship to our fungus, and that it occurs on the tobacco roots as a parasite of the fungus rather than as a stage of it." Duggar(8) says, “The association of the ascosporous stage with the others and the apparent continuity of mycelium are believed to show genetic connection.” Gilbert (12) also accepts this hypothesis and states that he was fortunate to obtain a perithecium attached to the same hypha which bore an endoconidiophore, but that he never saw an ascus. Foex (10) and Reddick (21) found violets infected with Thielavia basicola Zopf and accepted the relationship of perithecia with the other spore forms. Johnson (15) considers association of the perithecia with chlamydospores in nature as an evidence of their relationship. Peters (20), who did not obtain perithecia in his cultures and only once found them in his investigations in the field, questions their connection with the chlamydospores and endoconidia.
Ferraris (9) distinguishes the asexual stage from the ascospore stage, applying the name Thielaviopsis basicola (Berk.) Ferraris to the fungus which bears the chlamydospores and endoconidia and the name Thielavia basicola Zopf to the ascospore stage, not, however, implying two distinct fungi. To simplify the nomenclature in this paper the writer is following hereafter the terminology of Ferraris for these different stages.
EXPERIMENTS WITH A CONIDUM-CHLAMYDOSPORE CULTURE.
In August, 1920, a culture of Thielaviopsis basicola (Berk.) Ferraris, culture No. 396, was isolated from tobacco roots in which there were large numbers of perithecia associated with chlamydospores. Pieces of the infected roots were thoroughly washed in running water, followed by sterilized water, and put on oat agar containing some tobacco leaf extract. An endoconidium-chlamydospore culture was obtained, but no perithecia were produced.
Peglion's result indicated that nutrition might be a factor in the development of perithecia. Repeated efforts were made, therefore, to induce the formation of perithecia by the use of various kinds of media, by the provision of an excess of food, by slow starvation and, finally, by partial starvation followed by an abundance of food.
It was thought that tobacco extract might be a favorable food, and agar was made with it, alone and in combination with other substances. Sterilized tobacco seedlings were used alone and made up
agar and soil. Stock agars were made with ground peas, beans, oats, corn, peanuts, carrots, potatoes and beef extract. These media were used alone and also in combination with other nutrients, such as glucose and yeast, and a series of cultures, grading from slightly acid to slightly alkaline, was also tried. Plugs of carrot, potato, pineapple and pea pods gave an excellent growth of the fungus, but orange and lemon plugs gave little or no growth. Transfers from luxuriant cultures to water agar were made, with the hope that a change from an abundance of food to a negligible quantity might be effective, and transfers were also made from one highly nutritive medium to another equally so, but of a different kind. With some cultures gelatine was substituted for agar. The results of these tests were strictly negative, not a single
culture showed a development of perithecia. Eighteen cultures on various media in protected test tubes, were planted outdoors in the spring and were left throughout the summer to see if the natural temperature of the soil might be a factor. Here again the results were entirely negative.
PERITHECIA IN CULTURE. In view of the negative results of the nutrition experiments it seemed worth while to determine whether or not perithecia of Thielava basicola Zopf were the product of sexual strains. It was the intention of the writer to secure during the summer of 1921 as large a number of strains as possible for the purpose of making mixed cultures, but work on tobacco wildfire prevented making such collections. No other culture in addition to culture 396 was procured until later in the summer when some violet plants, heavily infected with chlamydospores of Thielaviopsis basicola (Berk.) Ferraris and perithecia of Thielavia basicola Zopf, were sent to Dr. Clinton from a greenhouse near Hartford. The writer visited the greenhouse and secured fresh material for culturing
On August 9th pieces of these roots were cultured like the tobacco roots from which culture 396 was obtained, put into four Petri dishes and given the culture number 1351. On August 15th a slight growth of Thielaviopsis basicola was perceptible and transfers were made to three tubes of pea meal agar and three tubes of carrot agar. Two days later additional transfers were made to seven tubes of carrot agar. Nothing further was done with these cultures until October 6th. On that date one of the transfers made on pea meal agar, August 15th, showed chlamydo