« PreviousContinue »
contoured and folded crusts covering the diseased bulbs. Frequently there are indications of striations on the sclerotia. These run in the same direction as the veins of the bulb scales. It is not uncommon to find the sclerotia massed together in the form of a sclerotial crust encircling the neck of the bulb. Sclerotia were found which presented the appearance of an inseparable layer of dull black enamel over the onion scales. Typically, the sclerotia have a tendency to round off on the upper surface while the lower side is flattened or even slightly hollowed out and inseparable from the onion bulb scales or tissue. When found between the bulb scales they often take the shape of the cavity which they fill. The sclerotia are of the cartilaginous type, having a hard outer cortical covering or layer filled with a white mass of mycelium closely pressed together forming a pseudoparenchyma. Cultural experiments, to be discussed later, show that the presence of a certain amount of moisture is favorable to the development of sclerotia. Sclerotia are not formed readily in cultures except where certain conditions prevail. They are formed where the mycelium is pressed together at the point of contact of fungus and glass, very often appearing as centrifugally formed crusts. It was observed that in many cases the appressoria are incipient stages of the sclerotia (Plate XXI, figs. 23, 24). At first the sclerotia are dark flesh color rapidly becoming black.
The sclerotia germinate by the ends of hyphae pushing out from the pseudoparenchyma and forming tufts of conidiophores bearing conidia similar in shape, size, color, and germination to those found on the mycelium (Plate XX, fig. 13). All attempts to induce the sclerotia to germinate in any other manner than by pushing out typical conidiophores which bear conidia or by the formation of a mycelium were failures with the exception of one remarkable instance which will be described in a later paragraph. No apothecia were formed on any of the sclerotia. Sclerotia taken from onions in various storage houses and from pure cultures were sterilized in several different ways, then placed in test tubes of sterile soil, in deep and shallow dishes of sterile sand and loamy soil, and kept in the laboratory, out-of-doors, and in the ice box for varying lengths of time; but in all cases the tufts of conidiophores were produced when germination took place. Sterilized sclerotia which were thrust into sterilized cornmeal moistened with water, produced, in germi
nation, an abundant mycelium which gave a fairly profuse crop of sclerotia thruout the medium. In other experiments sterilized sclerotia were floated upon sterile water and prune juice. Some of the sclerotia sank to the bottom of the liquid; but many of those which remained at the surface produced conidiophores and conidia. When the sterilized sclerotia were thrust into prune, potato, and onion agar in petri dishes, pure cultures of the fungus were invariably obtained.
A number of sclerotia were separated into two lots before sterilizing - one lot comprising the small sclerotia, the other the larger ones. When placed on moist sterile sand to germinate. both sizes responded with the usual crop of conidiophores and conidia. However, it was noticed that the small sclerotia were slightly more viable than the larger specimens.
Sclerotia subjected to the stimulating action of drying, and to fumes of chemicals such as ether, chloroform and alcohol gave results no different from those previously noted.
From the germination tests made upon hundreds of sclerotia the following points were secured: The most rapid and the highest percentage of germination occurred when the sclerotia were kept out-of-doors or in an ice box. A fairly low temperature (about 10° C.) greatly stimulates germination. Drying and exposure to sunlight reduced the viability of the sclerotia. If the sclerotia placed on sterile, moist sand or similar media did not germinate, or even after they had produced a crop of conidiophores, they readily broke down leaving the outer cortical layer in the form of a crust.
The length of time during which the sclerotia remain alive is very important. A fairly large percentage of the sclerotia were found to be dead at the end of one year. One lot of sclerotia kept for two years in the laboratory showed a very weak germination in a very few instances at the end of that time. Results of the tests with sclerotia indicate that under average conditions the life of the sclerotium in moist soil is but a few months, possibly only a few weeks.
It was noted above that in one particular instance germination took place other than by the production of the typical tufts of conidiophores or the formation of mycelium. This was the case with a lot of sclerotia picked from onion bulbs in storage early in 1913. These were sterilized by being soaked in sterile distilled
water, then immersed in a 0.1 per ct. mercuric chlorid solution for two minutes, and again washed in sterile distilled water. The sclerotia were then picked from the water with sterile forceps and placed upon moist sterile sand in petri dishes. Some of the dishes were kept in the laboratory in a dark closet, the others were placed in an ice box. On June 6, 1913, it was noted that two of the plates in the ice box showed that germination had taken place by the production of tufts of conidiophores bearing conidia, while upon a third plate there were no typical conidiophores but instead there were formed stalk-like bodies with masses of conidia at their tips. (Fig. 1, plate XXVIII). The stalks of these peculiar structures were black while the tips were white. These bodies were 20 mm. high and 3 mm. wide. Cultures made from the knobs of these structures upon onion agar gave pure cultures of the Botrytis. The conidia found on these knobs gave an average measurement of 11.3 microns long and 5.7 microns wide. A further analysis of these structures showed the stalk of one of them to be made up of 16 conidiophores. No further development was noticed in these structures.
Conidia.--- The pale colored or hyalin conidia are borne upon stout, short, erect conidiophores, these being very rarely over 1 mm. high, usually much shorter. The conidia are oblong to elliptical in form, often slightly tapering at both ends (Plate XIX, fig. 1). The short tip or sterigma may often be seen at the narrower end of the conidium. The conidia have been found to vary in size from 7.1-16.2 x 3.8-6.3 microns, the average from hundreds being 10.3 microns long and 5.1 microns wide.
The conidia fail to germinate in a saturated atmosphere. In water they are wetted with difficulty, but soon absorb water and germinate rapidly, usually by one, occasionally by two, germ tubes, forming considerable mycelium in 24-36 hours (Plate XIX, fig. 3). The mycelial branches unite very frequently, forming unions (Plate XIX, fig. 4), also the tips of hyphae form cross connections (Plate XIX, fig. 5).
While examining some old cultures of the fungus there were found at the bottom of the slant in the test tube, round, dark brown conidia about 3 microns in diameter and having very thick walls, while at the upper part of the slant typical elliptical conidia were formed. It appeared that conditions of nutrition caused the formation of these peculiar conidia. Price (1911) described some peculiar spore forms of Botrytis. Perhaps these round conidia observed by the writer are similar to the microconidial form of fructification mentioned by Istvanffi (1905), and Brooks (1908) who worked with Botrytis cinerea.
Comparative morphology.— The cultural characters of the onion Botrytis were critically compared with those of other forms or species of Botrytis, since during the course of the investigation of the onion Botrytis fungus the writer had opportunity to isolate a considerable number of forms or possibly species of Botrytis from a number of hosts. Some of these were living saprophytically on dead plant parts, while others were active parasites. At the outset, the possibility of the onion Botrytis being the common gray mold found upon greenhouse plants presented itself. As soon as the onion Botrytis was placed in pure culture and compared with the so-called Botrytis cinerea Pers., it was plainly evident that the organisms compared were entirely distinct species. The onion Botrytis possessed a low, dense, felt-like aerial mycelium bearing elliptic, smoke-gray conidia on very short conidiophores, while B. cinerea showed the loose, cottony, cinereous mycelium with long erect conidiophores, bearing the pale, globose conidia (Plate XIX, figs. 1,2). The writer has isolated the loosc-growing common Botrytis from dead onion leaves and from the tips of growing onion leaves where dying back had taken place due to the disease called " white tip." It was also isolated once from onion bulbs stored in a storage house along with a quantity of carrots which were severely attacked by Sclerotinia libertiana and overgrown with Botrytis. In another instance both the common Botrytis and the onion Botrytis were isolated from the perennial or tree onion growing in a private garden. Here, the onion Botrytis was acting as a destructive parasite killing the lower leaves and necks of the clusters of green bunching onions, while the common Botrytis was living as a saprophyte on the dead onion leaves. In Plate XXVII the onion Botrytis is shown as culture 12091 with its low form of growth, and the common Botrytis as culture 12091–1 with its loose, cottony mycelium completely filling the culture tube. Both cultures are of the same age and on the same kind of medium. Plate XXVII also shows four cultures of Botrytis picked at random from stock cultures. The cultures serve to show the dense, felt-like growth of the onion Botrytis as compared with the loose, cottony growth in the three other cultures, and also of that of a large number of isolations of Botrytis from a number of greenhouse plants. The writer will not venture a statement as to the classification of species of Botrytis showing the loose, cottony growth. While greatly resembling each other, it may be said that they present some distinctive cultural characteristics. The onion Botrytis has been carefully compared with forms of Botrytis from every available source among which were B. parasitica said to be the cause of the “Dutch bulb disease," and B. peoniae the cause of the Botrytis disease of peonies. None of these seems at all similar to the onion Botrytis. To summarize, the writer is confident that the onion Botrytis is responsible for the neck-rot disease of onions and is a separate species. Smith (1900, p. 397) appears to be the first in America to point out that the Botrytis causing the onion disease "was not a typical cinerea” because of its “low, dense manner of growth.” He gives some excellent illustrations of the conidia and conidiophores of the onion Botrytis comparing them with Botrytis cinerea.
The name of the fungus.- Considerable confusion has existed as to a name for this fungus since it has apparently been known under a number of different names. Sorauer (1876) appears to be the first to record a disease of onions which may be the neck-rot disease. In 1886 the same author described a disease of onions due to a Botrytis which evidently included a sclerotial stage. He referred the Botrytis stage to Botrytis cana (Pers.) Fr., and the sclerotial stage to Sclerotium cepae. Again, in 1908, Sorauer discussed a Botrytis disease of onions and referred the Botrytis stage to Botrytis cana Kze. et Schm., and the sclerotial stage to Sclerotium cepivorum Berk.
Frank (1880, 1896) reported a Botrytis disease of onions. He reiterated Sorauer's statements as to the possible identity of the Botrytis stage of the fungus with Botrytis cana Pers., and the sclerctial stage to Sclerotium cepae Berk. et Br. This author (1896) stated that he noted a Botrytis on Allium ursinum near Leipzig which was similar to that observed by Schröter.
Halsted (1890), in what appears to be the first report in America of an onion bulb disease due to a Botrytis, states that Botrytis parasitica Cav. may be the cause.
In 1894, Massee, writing in the Gardeners' Chronicle, described an onion disease due to a Botrytis with an ascogenous stage. This