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similar to those which obtain in the onion fields when the plants are wet with dew or rain for two or three days and cloudy weather
INFECTION OF GROWING PLANTS BY MEANS OF SPORE
On July 8, 1915, twenty-three onion plants, started from white onion sets and growing in sterilized soil in three-inch pots, were placed in a large inoculation chamber. These plants were then thoroly atomized with a suspension of the onion Botrytis spores in sterile water. After inoculating the plants in the chamber, three additional plants which had not been atomized with spores were added to the lot to serve as checks. On July 12 every inoculated plant showed infection from the Botrytis on some of its parts. A number of the leaf tips were dead and showed abundant infection when examined a few days later. Every inoculated plant showed the fungus fruiting on the neck, in most cases at the edge of the leaf sheath at a point where it clasps the neck. The tissue at that point appeared soft and water soaked and of a faint yellowish color. Possibly the outer leaves may have been slightly on the decline, that is, beginning to yellow at the time of inoculation, but this certainly was not apparent. On these plants the fungus clearly showed varying degrees of parasitism, being able to attack the green tissue and also to live on the softened tissue. Certainly, the host tissue was killed in advance of the hyphae. From the behavior of the plants it was plainly evident that most of the infections occurred thru the neck of the bulb and not thru the upper portions of the leaves. The uninoculated checks remained healthy. After these plants were killed by the onion Botrytis and had fallen over to the ground the loose-growing, common Botrytis appeared on some of them as a saprophyte on the dead plants. This experiment was repeated a number of times with similar and convincing results, which proved conclusively that it is comparatively easy to bring about infection of growing onion plants. Apparently the only limiting factor is that the drop of water or suspension does not dry off too quickly.
On April 29, 1915, a deep, greenhouse flat containing 15 onion plants growing vigorously in sterilized soil was placed in the inoculation chamber with a fairly high relative humidity, and atomized with a spore suspension of the onion Botrytis in prune juice. The spore suspension was made by pouring a tube of sterile prune juice upon an agar slant culture which was fruiting profusely and after rubbing off the spores and possibly some pieces of the aerial growth with a sterile platinum needle the liquid was poured into the receptacle of a De Vilbis atomizer. When atomizing the suspension upon the plants some small pieces of the mycelium came up thru the atomizer. Later all of these produced infection wherever they struck. Three days after atomizing, a considerable number of the leaves showed small white spots about 1 to 2 mm. in length (for they had a tendency to run lengthwise of the leaf) scattered over the leaf. By carefully peeling off the outer epidermis of a leaf a typical Botrytis mycelium, 6.6 microns wide, was found penetrating the tissue and growing along the leaf in the direction of its growth. Other typical infections revealed a similar condition. The chlorophyl quickly disappeared from these spots. After remaining in the moist atmosphere of the greenhouse for six days the plants went down rapidly, the fungus fruiting abundantly on the dead and dying, water-soaked-appearing plant parts. After a lapse of one month all of the plants placed in the box were dead, the tops remaining as dried material covered with abundant conidia. It was also evident from this experiment in which sterile prune juice was used instead of sterile water as a medium in which to convey the spores to the plants, that infection takes place readily from the drops of spore suspension.
Ward (1888) in his infection experiments with the conidia of the Botrytis causing a lily disease noted that the young germ tubes, altho unable to infect green leaves and bulbs, made a stronger attack if some saprophytic nourishment was at hand. In order to determine whether there was any difference in the rate of infection from conidia when placed in either sterile water or prune juice and then sprayed on the plants some experiments were made. One of these was as follows: Thirty healthy onion plants about 6 to 8 inches high and growing in a greenhouse bench at the Geneva Station were covered with large glass lantern globes. Ten of these plants were then atomized with the onion Botrytis spores suspended in water, ten with spores in prune juice and ten were retained as checks. All of the glass globes were then closed at the top by wrapping cotton loosely about the projecting leaves and covering the remaining space with a glass plate. Five days later there were unmistakable signs of infection on both the prune-juice-sprayed and the water-sprayed plants, but it could not be seen that the rate of infection was any greater in the case of the prune-juice-sprayed plants except possibly in one or two instances. Typically some of the infections took place on the leaves about the necks and thru those which were very slightly yellowing. At this point it is well to call attention to the work of Brooks (1907, 1908) who found that the conidia of Botrytis cinerea were able to infect lettuce leaves which were slightly yellowing, that is, in the incipient stages of decay, while they were unable to attack normal green leaves. This author suggests three explanations for this phenomenon.
In another series of tests comparing prune juice and water as a medium in which to convey the spores to the plants, prune juice gave a slightly greater percentage of infections. Even tho infection of growing plants took place readily when drops of water were present it was evident that the results of these tests were slightly in favor of the prune juice suspension. Apparently, a slight amount of nutrients in the spore suspension has an influence upon the rate of infection.
EFFECT OF SIZE OF DROP OF SUSPENSION UPON INFECTION.
In an attempt to determine the effect of size of drop of spore suspension upon infection of the onion eight healthy plants were selected. Three of these were atomized with spores of the onion Botrytis in water, the atomizer covering the plants with a very fine mist. Three of the plants were sprayed with an onion-Botrytisspore suspension using a tin atomizer of the hinge type which placed the suspension on the glaucous leaves of the plants in small drops ry similar to drops of rain or dew. Two plants were retained as checks. All of the plants were then covered with bell jars. In the course of six days the inoculated plants showed abundant infection on the leaves and stems. The checks remained healthy. While there was not a marked difference shown in amount of infection thru either the small or the large drops, it was clearly demonstrated that the drops of liquid must be of sufficient size to prevent their drying off quickly, a condition such as obtains in the onion fields during a wet season. Observation has shown that following such a season there is considerable neck rot of the bulbs in storage.
INFECTION BY PLACING SPORES BETWEEN LEAF SHEATH AND
The possibility of infection taking place by dry spores dropping down in the leaf sheath was, in one instance, tested as follows: Eighteen apparently normal onion plants growing in a greenhouse bench were selected; then, with a sterile platinum needle, dry spores from a pure culture of the onion Botrytis were carefully placed down in the leaf sheath, where it clasps the neck, the leaf being gently drawn aside during the operation. Some of the leaves fitted tightly while others did not. Untreated plants were retained as checks. Fifteen days from the date of inoculation seven of the fifteen plants showed infection, that is, the leaf was wilted, drooping, and appeared as if it had been scalded. On eight of the plants no noticeable infection had taken place. Thirty days from the date of inoculation nine of the fifteen plants showed severe injury from the Botrytis infection. The leaves were dead and nearly dried up and the fungus mycelium was found to have passed down into the necks of the bulbs. The checks remained healthy. It was evident from these and similar tests that the dry spores lodging in the axils of leaves or in the leaf sheath very readily found moisture conditions suitable for germination and later caused infection. It seems fair to assume that this may happen when spores of the neck-rot fungus from piles of decaying onion refuse near onion fields are air borne and find lodgment on the plants of the growing crop.
INFECTION THRU INCISIONS.
Some preliminary experiments designed to test the possibility of securing infection by placing the Botrytis spores in incisions in the side of the bulbs and necks showed conclusively that infection takes place very readily thru such wounds when they are covered with wax or some other similar material to prevent a drying out of the exposed tissue. Following the preliminary experiments eighteen onion plants which had formed bulbs about two inches in diameter were selected. After cleaning the neck with a cloth soaked in mercuric chlorid solution and then washing off with sterile water, an incision was made into the neck about one and one-half inches above the surface of the soil. Dry spores from a pure culture of the onion fungus were then placed in the incisions which were immediately covered with grafting wax. Spores were omitted from some of the incisions, these to serve as controls. Seven days after inoculation every inoculated plant, with a single exception, showed that the fungus had made an attack upon the tissue of the neck. All of the control plants remained healthy. The infections showed as a whitening of the tissue around the point of inoculation. In some cases the diseased area extended one-half to three-fourths inch above the wax covering, and on some leaves the whitened or scalded appearance extended up along the leaf, in one case to a distance of three inches above the point of inoculation. In the course of twenty-one days the disease had progressed to the point where nearly every inoculated plant was completely killed. An examination of the leaf tissue about two inches above the point of inoculation showed it to be completely filled with the fungus mycelium, making it impossible to determine whether it was interor intracellular. The tissue became soft and of a pale yellowish color above the areas in which the mycelium could be found. Evidently infection takes place readily thru wounds of this type.
INFECTION BY MEANS OF SCLEROTIA.
That infection of bulbs can take place by means of sclerotia was clearly shown by making incisions into the sterilized sides and necks of healthy bulbs and inserting into the wounds sclerotia picked from pure cultures. These were then covered with wax. The results with bulbs so inoculated were positive while the checks which received no sclerotia gave negative results: Evidently, sclerotia also must be looked upon as a source of infectious material.
INFECTION THRU THE FLOWERS.
While working with inoculation experiments upon the leaves, necks and bulbs of the onion in the Geneva greenhouse a number of seed bulbs or “mother bulbs" placed in a bench produced seed heads. It was noticed that as soon as the flower heads opened, some of them were attacked by the onion Botrytis which was fruiting upon onion stems in the same house. The young germ tubes readily attacked the stigmas, anthers and petioles of the flowers producing a blasting of the flower heads very similar to that which occurred in the onion seed fields.
In experiments designed to test the pathogenicity of the onion Botrytis for the flowers, dry spores placed in freshly opened flowers