A longisection thru a diseased bulb is shown in Figure 1, Plate VI, between the scales near the neck and extending down nearly to the center of the bulb one can see the mycelium woven together in the form of a dense weft or crust. All of the scale tissue below, altho no mycelium was found to be present, was discolored and the cells were broken down. The sprout in the center has been attacked at the upper end where it is in contact with the diseased scales and shows some discolored and softened tissue.

Stevens (1916) in a recent paper describes an almost identical condition as found from a study of the pathological histology of strawberries affected by Botrytis sp. This author concludes that the fungus is evidently capable of not only dissolving the middle lamella but of penetrating the cells themselves.

CELL WALLS OF THE HOST.

Since the fungus showed a very marked action upon the hostplant cell walls a brief study was made of the tissue of the necks and bulbs.

Tests for cellulose were positive in all cases, the best reactions being secured with cuprammonia or Schweizer's reagent in which the cellulose is soluble. A concentrated chloriodid of zinc solution, acting directly upon the sections and giving them a violet color, was also successfully used.

The middle lamellae of the host cell membranes were shown to consist of pectic acid, or its insoluble salts, by treating thin sections for twenty-four hours with a mixture composed of one part hydrochloric acid and four parts 95 per ct. alcohol. At the end of this time they were washed with water, then treated with a 10 per ct. solution of ammonia for a short time when the sections could be easily separated into their constituent cells by gentle pressure. When these sections were stained with methylene blue for a few minutes after treatment with the acid alcohol the middle lamellae stained very deeply. The results of these and other tests applied agree with the statements of Mangin (1892, 1893) that pectose is very pronounced in the cell walls of young tissue, but in older tissue calcium pectate comprises the greater part of the middle lamella.

PRODUCTION OF OXALIC ACID BY THE FUNGUS.

Preliminary to a study of the enzyms produced by the fungus it seemed necessary to make a study of the possibility of oxalic acid being the toxic substance produced.

De Bary (1886), in his study of the fungus Sclerotinia libertiana, reported that the hyphae were often coated with crystals of oxalates. He found as high as 0.39 per ct. oxalic acid in one sample of his fungus. It appears that this author discounted the possibility of oxalic acid being the toxic substance since he was unable to secure the same effect upon plant tissue with solutions of oxalic acid as was obtained with the fungus or an extract of the fungus cultures. De Bary was unable to demonstrate any free oxalic acid.

Ward (1888, p. 336) reports the presence of minute crystals upon some of the conidiophores in his Botrytis cultures upon raisin extract.

Wehmer (1891), in a valuable series of articles reporting his work upon the production of oxalic acid by a number of fungi, points out that the fungi studied vary as to their ability to produce this acid, depending upon their metabolism as influenced by different factors which he gives. He holds that oxalic acid is a type of excretion.

Nordhausen (1899), working with a Botrytis, permitted the hyphae of this fungus to grow thru pieces of the epidermis of Allium which had been previously stained with congo red. The absence of a blue acid dye around the penetrating hyphae was taken as evidence of the failure of the fungus to excrete an acid, at least one as strong as oxalic acid.

Smith (1902), working with Botrytis cinerea, stated that he found over 20 per ct. oxalic acid in the extracts of mycelium from old cultures. This author was inclined to believe that oxalic acid is responsible for part of the action upon plant tissues. He was unable to detect any difference between the action of boiled and unboiled mycelial extracts upon petioles of lettuce leaves. Working with solutions of oxalic acid he reported a bleaching effect from the acid, which was noticeable to the naked eye.

Peltier (1912), in his work with a Botrytis, was unable to detect any difference between the boiled and unboiled extracts. Applying several delicate tests which, however, he does not outline, he was. unable to detect the presence of oxalic acid. This worker also

tested the action of different concentrations of oxalic acid upon petioles of lettuce and found it to be somewhat like that of the mycelial extract, except that a bleaching of the tissues took place. He found this to be true of the organic acids and therefore concludes from his experiments that it is not oxalic acid, but may be some undetermined organic acid.

Cooley (1914), who gives his method of analysis, found oxalic acid to be produced in appreciable amounts by Sclerotinia libertiana when cultured on certain fruits.

Valleau (1915) studied the middle-lamella solvents concerned with the resistance of plums to brown-rot and reports some very interesting results with oxalic acid. He immersed blocks of tissue from various species of plants, among them onion and potato, in 0.015, 0.062, and 0.125 per ct. solutions of oxalic acid for stated periods of time. The various tissues showed varying degrees of resistance to the action of the acid, but all those affected showed that the softening was due to the solution of the middle lamella. The onion tissue softened in the 0.125 per ct. solution, while the potato did not soften even in a 0.25 per ct. solution. Valleau noted a marked bleaching effect produced by the acid and concludes from his experiments that oxalic acid "is not the sole toxic substance produced."

Brown (1916) found no evidence, at the end of twelve hours, of the action of a N/40 solution of oxalic acid or of N/20 potassium oxalate when placed on leaves of bean, or the petals of rose. He found, however, that the fungus (Botrytis cinerea) excreted an oxalate into the "infection drop," but that M/800 represented the upper limit of concentration. This author states that infection by the fungus could take place even tho the presence of a soluble oxalate was excluded.

Oxalate crystals in media. While examining the medium directly beneath some cultures of the onion Botrytis growing on lettuce-leaf agar plus peptone (1 per ct.), the writer found a large number of crystals distributed thruout the substratum. These tetragonal pyramids had every appearance of calcium oxalate crystals. When treated with concentrated hydrochloric acid they dissolved slowly. At first it was believed that these were present only in the agar cultures containing peptone since Emmerling (1903), from his study of oxalic acid formation by certain fungi, stated that abundant

production of oxalic acid takes place on substances containing peptone. A further examination of the substratum from cultures of the onion Botrytis and ten other strains or species of Botrytis from as many hosts, and growing upon onion agar, showed the presence of these crystals. Examinations of lettuce-leaf agar cultures to which no peptone was added showed these crystals to be present. The cultures examined varied in age from 30 to 60 days. When carefully examined microscopically and tested microchemically these crystals appeared to possess all the properties and characteristics ascribed to crystals of calcium oxalate by Zimmermann (1893), Tunmann (1913) and Molisch (1913). Examinations made by Mr. O. B. Winter, Assistant Chemist of the Michigan Experiment Station, using the polarizing microscope, showed them to be of the monoclinic or tetragonal system. Hence, his observations confirmed those of the writer, since calcium oxalate possesses just such properties.

Oxalic acid in the diseased tissues.— In order to determine whether any free oxalic acid could be detected in the diseased, decaying onion bulbs, microchemical tests were made using calcium nitrate which showed no crystals of calcium oxalate to be formed. Other microchemical tests for oxalic acid as given by Zimmermann, Tunmann, and Molisch gave negative results.

Oxalates in the diseased tissues. To ascertain whether any oxalates were produced by the fungus upon its host, chemical analyses were made of diseased and healthy onion bulbs. The analytical method used by Wehmer (1891) was employed. The soft, decayed tissue of onion bulbs overrun by the onion Botrytis was carefully cut away from healthy tissue. This material was then digested in cold hydrochloric acid, using in each lot 100 grams of the diseased onion tissue to 150 cc. of the acid. Controls were prepared by using 100 grams of healthy onion bulb scales sliced into 150 cc. of the acid. These lots were allowed to digest for 24 hours when the analyses were made.1 The oxalic acid or its soluble oxalate was precipitated out as a calcium salt, which was filtered off, dried to constant weight and weighed. Results as follows: Healthy onion tissue showed..

· Diseased onion tissue showed...

..0.0050 per ct. oxalic acid .0.0098 per ct.

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1The determinations for oxalic acid were made by Mr. O. B. Winter, Assistant Chemist of the Michigan Experiment Station, to whom acknowledgments are due.

The analyses of liquid culture media yielded some very interesting results. Two hundred grams of clean onion bulbs were added to one liter of distilled water. This was then autoclaved for twenty minutes at fifteen pounds pressure when the juice was pressed out and 500 cc. placed in each liter Erlenmeyer flask and again autoclaved for 15 minutes at 15 pounds pressure. Some of the flasks prepared in this manner were inoculated with the onion Botrytis spores, the remainder was retained as checks. The cultures were 56 days old when the analyses were made. The liquid from beneath the fungous crusts was filtered off and the determinations made exactly as indicated above. The results are as follows: Autoclaved onion juice, uninoculated......0.0040 per ct. oxalic acid Autoclaved onion juice, from beneath

.0.0115 per ct.

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growth. The results of the determinations show that the onion Botrytis, when grown upon liquid media or upon onion bulbs, produces appreciable amounts of oxalic acid.

Titration of the liquid media.— At the time the determinations for oxalic acid were made, 5 cc. portions of the liquid from beneath the cultures were removed and added to 45 cc. of distilled water. This was then titrated with N/20 NaOH using phenolpthalein as an indicator. It required less than 0.1 cc. of the N/20 NaOH to neutralize the free acid in the juice; in fact, one drop of the N/20 alkali brought a distinct pink color. The juice was recorded as being +1° acid on Fuller's Scale. This would seem to indicate that there was but little free acid present.

Acidity of sound and diseased onions. In the hope that the determination of the acidity of the diseased and sound onion bulbs would prove of value the following tests were made: Thirty-gram samples of the sound and diseased onion tissue were titrated with N/1 NaOH. The sound tissue was taken from several large healthy onions. The diseased tissue was taken from a number of onion bulbs attacked by the onion Botrytis. These lots of tissue were ground to a pulp in a mortar and then washed out with a large volume of distilled water into a large evaporating dish. The pulp and the water were thoroly stirred after which the titrations were made with N/1 NaOH using phenolpthalein as an indicator. The results of the titrations. given in terms of cubic centimeters of normal alkali required to neutralize the acid in 30 grams of pulp are as follows: