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germinated, and readily attacked and destroyed the non-cuticularized floral parts. The fungus then worked down thru the receptacle and invaded the seed stalk. In the greenhouse, at least, the initial infection of the flowers took place by means of wind-borne spores from diseased plants in adjoining benches. It seems fair to assume that infection of flowers in the onion seed fields could occur in a similar manner. Field observations and all indications confirm this idea.
MYCELIUM IN NECKS OF INFECTED BULBS.
At the conclusion of the greenhouse experiments in 1915 there was a considerable quantity of onion bulbs at hand some of which had matured. Some of these bulbs had shown infection from the neck-rot fungus early in the season, but after the neck and some of the dry, outer skins had been removed the specimens appeared sound even tho somewhat undersized. A careful examination was made of a considerable number of these bulbs in search of the mycelium in the necks. In nearly every instance the mycelium was found between the outer three or four fleshy scales at the necks of the bulbs. In the case of one apparently healthy bulb a mat of mycelium was found between the second and third fleshy scales where it had caused no discoloration of the tissue. Isolations from this mycelium gave a prompt and vigorous growth. When such bulbs were placed in a moist chamber the neck rot developed rapidly.
INOCULATION EXPERIMENTS WITH OTHER PLANTS.
Species of Lilium, including L. tigrinum, L. candidum, and L. grandiflorum were dusted with dry spores and sprayed with various spore suspensions of the onion Botrytis on leaves, buds, and open flowers. These gave negative results in every instance, indicating that under the varying conditions under which the tests were conducted the onion Botrytis is not pathogenic for these species of Lilium.
Greenhouse flats of lettuce plants were placed in an inoculation chamber, at a number of different times, and sprayed with spores of the onion Botrytis suspended in sterile distilled water. In none of the trials did infection from the onion Botrytis follow the inoculations.
Garden peas were started in greenhouse flats filled with sterile sand from a cutting bench. These peas were started for the purpose of testing germination and were planted very close together. When the plants were about five inches high the flats were placed in a moist chamber and atomized with a spore suspension of the onion Botrytis. No indications of infection were apparent until six days later when it was observed that infection had taken place only on the yellowing leaves and stems. After infection had taken place the infected parts became flaccid and water soaked. The fungus then progressed to other parts of the plants killing the tissue in advance of its growth. Attention has already been called to the observations of Brooks (1908) who noted a similar instance in the case of Botrytis cinerea on lettuce plants.
SUMMARY AND CONCLUSIONS.
The results of inoculations of cry onion bulbs, and of growing plants, by dusting with dry spores, were negative. However, positive results were obtained from inoculations made in the following ways: (1) By spraying spores on the growing plants, and on the bulbs; (2) by placing dry spores between leaf sheaths and the neck; (3) by placing spores in incisions in the necks and sides of the bulbs; (4) by placing sclerotia in incisions in the necks and sides of bulbs; and (5) by dusting dry spores on the freshly opened flowers. Evidence was secured that drops of liquid in which the spores were suspended must be of sufficient size to prevent their drying off quickly. Some evidence was also brought forth which indicated that a small amount of nutrients in the drop of spore suspension slightly favors infection. An examination of bulbs from plants used in the infection experiments showed the mycelium of the fungus to be present in the necks.
A summary of the inoculation experiments brings forth but one conclusion, viz.: That spores in large drops of water (or sufficient inoisture) on leaves, in wounds, and chiefly on the necks of onions (and only onions) produce the typical neck-rot disease. Sclerotia must be looked upon as a source of infectious material. The leaves attached to and clasping the lower part of the neck are very susceptible to attack, especially when they are on the decline, i. e., slightly yellowing.
With the results of the inoculation experiments in mind it was possible to go into the onion fields and storage houses and explain most of the conditions found there. It is believed that in the field the greater part of the infections above ground take place thru the lower leaves and the neck and then work down into the bulbs, the mycelium going into the storage houses in the necks of the bulbs, where it develops rapidly under favorable conditions.
Mycelium.— The mycelium is large and coarse, septate, and varies considerably in diameter in the same hypha. In diseased onion bulbs the mycelium frequently occurs as only 4.5 microns wide, while in diseased seed stalks from the field it is often 9 microns wide, the average width being approximately 6.5 microns. It has no regular method of branching, and is not only intercellular but intracellular, it being impossible in many cases to determine this point because of the disintegrated condition of the host tissue (Plate XX, fig. 11). In the bulbs and necks of the host plant the young mycelium is practically colorless. With age the walls become of a sepia tint bordering on a light brown color. Various types of cross connections may be found in the host tissue (Plate XX, fig. 12). The mycelium when crowded down between the bulb scales forms a dirtywhite mat. Here, in one instance, it gave an average width of 6.3 microns for a large number of measurements. The mycelium is filled with a dense, fine-grained or minutely vacuolate protoplasm, frequently appearing granular. When grown in solid media weak in nutrients, and in synthetic solutions, the mycelium shows considerable variation in form and method of growth. Fig. 14 on Plate XXI, shows a type of mycelium common in solid media. A type of heavy mycelium is frequently found in pure cultures (Plate XXI, fig. 26). Sporulating filaments not unlike those shown by Beauverie and Guilliermond (1903) and giving the same staining reactions to hacmatɔxylin and methylene blue were found (Plate XXII, figs. 33, 34). The mycelium showed"corpuscular bodies" similar to those shown by these investigators (Plate XXII).
The short, erect conidiophores arise directly from the mycelium eitlier singly or in clusters (Plate XX, fig. 6). They turn from hyalin to light brown and later to brownish black. Upon the host plant the conidiophores are usually unbranched (Plate XX, figs. 6, 9), while in cultures there appears to be a tendency toward branching (Plate XX, fig. 8). The compact clusters of conidia are borne upon vesicles which have a tendency toward a spiral arrangement (Plate XX, fig. 7), and usually upon the upper portion of the conidiophore (Plate XX, fig. 10).
Appressoria.- While studying the behavior of the fungus as it worked its way down the neck of inoculated onion plants it was noted that appressoria or organs of attachment were formed rather profusely. These appeared to have an important relation to the discoloration and solution of the cell walls of the host. The writer's attention was first attracted to these bodies while examining an onion leaf which had been sprayed with a spore suspension of the onion Botrytis. The leaf appeared green and perfectly normal with the exception of small white spots where infection had taken place, and one particular area where the fungus was fruiting abundantly. Around this latter spot the leaf tissue for a distance of about 5 mim. appeared as if it had been scalded. By making thin sections thru the leaf, at a point where the diseased and healthy areas adjoined, and then unrolling the sections, the inner side of the leaf tissue could be examined. This examination revealed a profusely branched mycelium growing upon the inner epidermis of the leaf and extending along in the same direction as the long diameter of the cells, i. e., in the direction of growth. A considerable number of the tips of the hyphae showed knobs or enlargements of various shapes and sizes, many of them being simply enlargements of the tips of the hyphae (Plate XXI, figs. 15-22). In most cases these bodies were full of dense, bright protoplasm, while the mycelium just back of them was vacuolate, and markedly so. The tips of these bodies were surrounded by a film of material which was very refractive to light and which could be rather easily demonstrated by staining in weak gentian violet for a few seconds. This condition as described was found to be very common in the inoculated plants and those showing natural infection from the field. Quite frequently the mycelium thickens slightly just back of the appressorium. The young mycelial elements may also become markedly flattened where they come in contact with the culture dish (Plate XXI, fig. 32). The bodies appear simply to apply themselves to the host tissue following which there is a softening of the tissue and it takes on a water-soaked appearance.
The chlorophyl disappears and the mycelium branches and pene
Aderhold (1896) mentions the fact that the appressoria of Ven-
Blackman and Welsford (1916) have demonstrated that the germ tubes of Botrytis cinerea, when they have reached sufficient length (about one spore-diameter), “possess a mucilaginous investment” by means of which the germ tube is aided in exerting sufficient pressure to penetrate the leaf tissue. These authors further state that this method of penetration is impossible in the absence of the mucilaginous investment of the germ tube, but that “penetration can occur without the development of an appressorium.'
Sclerotia.- The typical, firm, black sclerotia may be found upon any or all parts of the onion bulbs in storage. These vary in size from 1 mm. in width to large crusts covering nearly the entire side of a bulb (Plate VII). The sclerotia vary in shape from small roundish bodies 1 to 2 mm. wide and flattened on one side to large,