A i 9.3;^ U. S. DEPARTMENT OF AGRICULTURE, BDREAD OF PLANT INDUSTRY BULLETIN NO. 225 B. T. GAL1 "V. IY, ( S eau. A SPOT DISEASE OF CATLTFL LUCIA Mel II I OCH, Scientific Assistant, I t of Plant I'nibology. [881 i:i> AtJGl ST 29, 1911. f ». t * DEPOSITORY WASHINGTON: GOVERNMENT PRINTING OFFICE. 1911. BUREAU OF PLANT INDUSTRY. < "hh j 'of Bureau, Beverly T. Galloway. Assistant Chief of Bureau , William A. Taylor. Editor. J. E. Rockwell. Chitf Clerk, James E. Jones. Laboratory or Plant Pathology. SCIENTIFIC STAFF. Erwin F. Smith. Pathologist in Charge. R. E. B. McKenney, Special Agent. Florence Hedges, Assistant Pathologist. A. W. Giampietro, Assistant Physiologist. Nellie A. Brown, Lucia McCulloch, and Mary ^Catherine Bryan, Scientific Assistants. 225 2 LETTER ()Y TRANSMIT) \L ['. S. I >EP \i:i mi.\ i in A.GRK mi Bi reai "i Plant I ndustry, Ofi [< : OF in i: Cini'.i Washington, P. ( .. May 81, nil. Sir: I have the honor to transmit herewith a manuscripl entitled "A Spol Disease of Cauliflower," l>\ Mi is. Lucia McCulloch, scientific assistant in tin' Laboratory of Plant Pathol d recommend its publication as Bulletin No. 225 of the series of this bureau. This paper deals with a disea e which is 3hown to Ik' of bacterial origin and which has no) been reported hitherto. The investigations of tins disease have been carried out according to the advice and suggestions of Dr. Erwin V. Smith. Respectfully, Ww A. T UXOR, Acting ' Tiiefof Bureau. lion. .) \mi> V\ ii 30 S( in tary oj . Ygriculiun . Digitized by the Internet Archive in 2013 http://archive.org/details/spotdiOOunit CONTENTS Page. i ii lion 7 hi", nl 7 ption of the cauliflower leaf-spol organism Morpholog) Reaction to stains 10 t 'ultural characters 10 10 r stabs L0 Agar Maul- LO Bei i b • i ill' in II A id bouillon II Ukaline bouillon II Bouillon \\ iili mm limn chlorid II Beei bouillon over chloroform H Brum II ( ohn'e solution II Fermi's solution II Nutrient gelatin 12 l.i i in us milk 1- Milk 12 l i bii ! olution 12 Other i haracti ri tics L2 Fermentation tubes 12 Ammonia production 12 Ml ral'~ 13 I dol r 13 Bydr in sulphid L3 Tempera! ure relations 13 Thermal death point I" Optimum temperature 13 Maximum temperature. .; 13 Mini in ii i 1 1 ure 13 Effect of desiccation L3 Biinlighl L3 I : freezing 13 \ iialiiv "ii culture media I l i|> number I l hi M Latin II Summary I" ILLUSTRATIONS. Page. Plate I. Upper surfaces of cauliflower leaves, showing natural infection with Bacterium macul icol urn 1 (i II. Under surfaces of cauliflower, showing midribs spotted by natural infection with Bacterium maculicolum ]P> III. Under surfaces of cauliflower leaves from hothouse, inoculated v\ ilh Bacterium maculicolum 16 225 6 B. P. 1 V SPOT DISEASE OF CAULIFLOWER, INTRODUCTION. In April and again in May, 1909, diseased cauliflower plants were senl to the Laboratory of Plant Pathology from a farm in southeast- ern Virginia. In both lots the leaves were closely covered with brownish to purplish gray spots I to 3 millimeters in diameter (PI. I). There were also larger diseased area- due to the coalescing of spots. All parts of the leaves were affected. "Where the midribs and veins were badly attacked the tissues had contracted, giving a puckered appearance to the leaves (PI. II). From the spot- a. bacterium was secured in pure cultures 1>\ means of petri-dish poured plates on agar, and subcultures from colonies thus obtained were used for inoculating healt by cauliflower plants. The cauliflower heads from the same plants were noi iii good con- dition, but no success attended the efforts to secure from them the same kind of bacterium that was found in the leal' spots. Whal appears to he the same disease was also received once on cauliflower from Florida. INOCULATIONS. All inoculations were made by spraying the plant- with pure cul- tures of the bacterium suspended in water (24 to 4S hour agar -hints washed oil' in sterile water'. Young, healthy cauliflower plants 6 to 10 inches high were used, some being kept in infection cages and some merely on a bench in the greenhouse. The infection shows first on the lower surface of the leaves as sunken water-soaked spots. 1 These are visible on the third day after inoculation. In I or 5 days the spots are dark purplish-gray and -how on both surface-. | n transmitted light the centers are thin, almosl coli.iT'—. and surrounded 1>\ a dark border. In size they vary from mere point- to spots l ."> millimeter- in diameter. In shape they are irregularly angled; the spread of the disease appears to be stopped or hindered by the veins of the leaf. The individual 1 Somo infections from old cultures thai bad probably lost much of their virulence were first Indicated ciftei • • by tiny water-soaked elevations. As the disease progressed the tissues collapsed and a. sunk ilted. 7 b A SPOT DISEASE OF CAULIFLOWER. spots do not become more than 2 to 3 millimeters in largest diameter, though where crowded the spots usually coalesce, forming diseased areas of considerable size. "When only the upper surface of leaves was subjected to the inocu- lating spray very few infections resulted, while inoculations on the lower surface gave numerous infections (PI. III). So far as observed in sections the infection takes place only through stomata, and mostly from the lower surface. The older and the very young leaves appear to be partially or even completely immune, while those of intermediate age (on the same plant) may be seriously affected. The diseased leaves become yellow and fall off in from three to five weeks. The younger leaves and new growth are healthy. Under our rather dry hothouse conditions in no case was there evidence of infection on any but the inoculated leaves. From spots developing on leaves inoculated with the original culture the organism was repeatedly isolated by means of petri- dish poured plates, and subcultures from colonics were again used for inoculations, always with the production of the characteristic infection. The checks in all cases remained healthy; also numerous other cauliflower plants in the same greenhouse. Cabbages inoculated with this organism became infected in the same manner as the cauliflower with one exception, viz, the spots were darker. From spots appearing on the inoculated cabbages the organism was isolated and tested on cauliflower, with the result of the production of the disease. During May and June of 1909 all inoculations resulted in successful and typical infections. On July 10, 1909, seven plants were inoculated in the usual manner and, contrary to expectations, no infections resulted. The conditions were the same as in previous inoculations, except that the tempera- ture was higher at this time, 26° to 34° C. (78° to 93° F.). Subse- quent experiments show that the bacterium causing the disease refuses to grow in artificial media at 29° C. (84° F.) or above. It is probable, therefore, that in this case the high temperature prevented infection. After July no more plants were available for inoculation until Janu- ary 24, 1910, when 10 plants were inoculated. No infections resulted. On February 2, 1910, 10 more plants were inoculated and no infec- tions resulted. All conditions seemed favorable, and loss of virulence in (he culture was suspected as the cause of failure to infect. The cultures used for inoculation were descended through numerous transfers made during the winter. An agar-stock culture which had not been transferred since September, 1 ()()(), was then tried. Fresh agar-slant cultures were made from the September stock and used 225 DESCRIPTION OF TIIK LEAF-8POT ORGANISM. 9 when 24 hours old (February 12, 1910) for inoculating cauliflower plants. 'I'll is time infections resulted. The infections weret ery slight, however, and the spots too few in number to cause any noticeable injury t" the plants, bu1 the bacteria plated from these spots pro- duced typical colonies on agar and characteristic growth in artificial media. Cauliflower plants inoculated with this new strain became very generally infected, which Tact seem-, t . indicate that the organ- ism had increased in virulence by passage through the host. On December 6, L910, cabbage, cauliflower, turnip, rutabaga, rad- ish, and mustard were inoculated in cages by spraying with sterile water to which had been added agar-streak cultures :» days old. All o!' these plants were young. There were two to lour of each sort. The materia] for inoculation was obtained from a cauliflower inocu- lated November _'-'. 1910. Infections were obtained on cabbage and cauliflower (six days), but not on the other plants. Sections of the spots made on the tenth day showed them to be full of bacteria. On March 1, 1911, three cauliflower plants were again inoculated h\ spraying with sterile water to which had been added 2-day-old agar-slanl cultures. These plants were about 8 inches high and \m. healthy. They were kept in an infection cage for two days. At the end of ; (i daj s t here were very small dark speck- on each of I he plants. These speck- were in the center of a small semil ransparent eleval ion. A microscopic examination showed bacteria present m these -pots. 'The flower stalks of plant 101 also showed elongated water-soaked spot s, darker in the center. These also contained bacteria and plates made from them yielded the organism in pure culture. The flower- ing part of plant 104 bore no spots, although it had been drenched with the spray. The check plant remained healthy. Several attempts to inoculate the head- of the caulillower gave no satisfactory results. Growing heads were copiously inoculated and kept under moist conditions, but no infections occurred. Infection spots similar to those on leafstalks and midribs occurred on some of tin" larger stalks of the flower head, while the flowering parts remained free from infection. Mature heads from the market were abo inoculated, hut as decay of the tender surface was general in the checks as well as in the inoculated beads, the results are not conclusn e. DESCRIPTION OF THE CAULIFLOWER LEAF-SPOT ORGANISM. MORPHOLOGY. II rganism is a short rod. forming long in some media. Ends rounded. Size from leaf I .."> to 2.4 " l>\ 0.8 to 0.9 ft. Size in 24-hour beef-agar culture, temperature 20 to 25 C, 1.5 to •'! " by 0.9 /£. No spores are produced. The organism is activelj motile by 225 10 A SPOT DISEASE OF CAULIFLOWEK. means of one to five polar flagella, which are two to three times the length of the rod. (Stained by Van Ermengem's method; also by Hugh Williams's method.) Motility occurs in most artificial media. In beef-bouillon cultures grown and kept at 0.5° to 1.5° C. for four months the organism is still motile. Involution forms were found in alkaline beef bouillon (-17 on Fuller's scale). Pseudozoogloeae occur in Uschinsky's solution and in acid beef bouillon. REACTION TO STAINS. The organism does not stain by Gram. Modified Gram, using amyl alcohol, gives a deep blue stain. It stains readily and strongly with carbol fuchsin, with an alcoholic solution of gentian violet, and with a stain obtained from Dr. Kinyoun which contains methylene blue, silver nitrate, azure I, and azure II. It is not acid fast. CULTURAL CHARACTERS. Agar plates ( + 15 peptonized beef bouillon with 1 per cent agar). — Tbe colonies are visible on the second day as tiny white specks (temperature 23° C). In three to four days the colonies are 1 to 3 millimeters in diameter, white (opalescent in transmitted light), round, smooth, flat, shining, and translucent, with edges entire. Structure, under hand lens, coarsely granular with internal reticulations. Buried colonies small, lens shaped. With age the colonies become dull to dirty white, slightly irregular in shape, the edges undulate, slightly crinkled, and with indistinct radiating marginal lines. The internal reticulations disappear and the coarsely granular appearance changes to finely granular. In thinly sown plates 7-day-old colonies are 6 to 8 milli- meters in diameter; 15-day-old colonies are 12 to 15 millimeters in diameter. Agar stabs. — The surface of the agar is covered in two days (22° to 24° C.) by a thin white growth. For several days the stab shows a moderate growth in the upper 8 to 10 millimeters, but this does not continue. Finally, the stab is almost, if not quite, invisible. Crystals appear in the stab and on the surface. Agar slants. — In smear cultures the surface is covered in two days (temperature 19° to 21° C.) with a thin white growth, glistening, coarsely and irregularly pitted. White sediment in the V. In streak cultures in two days (temperature 19° to 21° C.) the streak is 3 to 5 millimeters wide, white, margins slightly undulate. The internal reticulations seen in colonies on plates are present in the streak cultures. At right angles to the streak are fine lines extending from center to margin. Agar cultures become slightly greenish. 225 DESCRIPTION OF TJIK l.K AK-si'oT <>U<;.\NI8M. 11 Beef bouillon. — Peptonized beef bouillon + l"> held at 24 to 25 C, it' inoculated from young, vigorous bouillon cultures, clouds thinly in 6 hours and is moderately to heavily clouded in 24 hours. The growth is besl at the surface, where a white layer is formed. This is not a true pellicle, as if disintegrates when the cultures are handled. 1 No zoogloese arc present. There is no rim. Tn two days there are heavy clouds and a moderate amount of white flocculenl precipi- tate. After several weeks the precipitate is white and slimy, mod- erate in quantity, and with small crystals in it. The medium becomes slightly greenish. After several months the precipitate is viscid. Acid bouillon. -In neutral heel' bouillon plus vegetable acids, growth occurs until an acidity of - ; :;i for oxalic acid and +86 for malic and citric acids (Fuller's scale) is reached. There is no rim or pellicle. Occasionally pseudozoogloeffl are formed in the more acid media. Microscopic examination shows most of the organisms greatlj reduced m length, some so short as to be spheroidal. That these were not contaminations was proved by plating out and by tests on other culture media. ATkalim bouillon. — Tn alkaline beef bouillon (XaOTT used > the organism grew well in — 17. - 10. —22, less in —23, and not at all in 25, -26, and 28 (Fuller's scale), [nvolution forms and filaments were present in —17 beef bouillon when two weeks old. Bouillon with sodium chlorid. - In beef bouillon plus 2 percent XaCl the growth is as good as in plain beef bouillon. With (he addition of more \a( 1 the growth gradually lessens until it is scarcely noticeable in a 5 per cent solution. When grown in a 1 per cent solution, the '-an is not motile. In a 2 per cent solution the organism is motile, but less so than in beef bouillon without XaCl. Beef bouillon "<•,/■ chloroform. — For the. first 24 hours the growth is somewhat retarded. By the end of 48 hours no difference could be seen between culture- over chloroform (5 c. c. of chloroform with 10 c. c. of beef bouillon i h >t shaken) and those in plain beef bouillon. Loeffler's />/"<»/ serum.— Growth of stroke is moderate, smooth. shining; color creamy; margins finely crinkled. No liquefaction. After three months the whole medium was slightly browned. Cohn' 8 solution. -Moderate clouding and white precipitate: no rim, pellicle, or zoogloese; no fluorescence. After some weeks feather- like crystals of considerable size (5 to 10 by 2 to 6 mm.) are formed. /■'< rm'i's solution. — Moderate clouding at first. Precipitate mode- rate to abundant, white, flocculenl. Pellicle white, tender, sinking in strings and masses. Finally the medium is densely clouded and 1 Old cultures kept [or several mouths at 0.5° to 1.5° C. had a delicate pellicle. 12 A SPOT DISEASE OF CAULIFLOWER. pale green-fluorescent (between water green, and greenish glaucous, Kepertoire de Couleurs, Paris, 1905); more precipitate than in beef bouillon. Nutrient gelatin, (+10 on Fuller's scale). — The stab cultures lique- fied in 8 to 10 days (temperature 17° to 18° C). Growth from sur- face crateriform. Slight, white, granular precipitate. Slight green fluorescence. The plate cultures showed no signs of growth in 24 hours at 17° to 18° C. In three days well-isolated colonies vary from mere points to round growths 2 millimeters in diameter. The gelatin is liquefied in cuplike hollows. Margin of smaller colonies entire, of larger colonics fimbriate. Thickly sown plates entirely liquefied in two days at 15° to 16° C. Litmus milk. — The medium becomes dark blue at the surface in 12 to 24 hours. The darkening proceeds downward in definite layers until in 8 to 10 days the whole medium is dark blue with a slight white precipitate. During six months' observation the medium remained dark blue (reflected light) and liquid. Finally by evaporation the medium becomes thickened, but there is at no time any separation into curd and whey. A few cultures showed a trace of reduction of litmus at the bottom. Milk. — As in the litmus-milk cultures, growth and color-change in the milk begin at the surface, proceeding downward in definite layers. In 15 to 20 days the whole tube (10 c. c. of milk) is yellow (near fiidgway's Naples yellow, but somewhat duller and with a greenish tinge) and translucent. No separation into curd and whey. Fat not changed. In four months the medium is quite dark (reddish- brown) and somewhat thick (evaporated to about 5 c. c.). Small tyrosin crystals are formed. These are distinctly visible only with a lens. Uschinsky's solution. — Growth moderate to copious; pellicle white, tender, breaking and sinking easily. Pseudozoogloese are present. There is a greenish fluorescence. The old cultures are much like those in Fermi's solution. OTHER CHARACTERISTICS. Ft r mentation tubes. — The organism is aerobic and does not form gas. It was tested in fermentation tubes in the presence of dextrose, saccharose, lactose, maltose, glycerin, and mannit, each of these carbon compounds being added to a basal solution consisting of 1 per cent of Wltte's peptone dissolved in water. It did not grow in the closed end of the fermentation tubes in the presence of any of these substances. Ammonia 'production. Moderate. DESCRIPTION OF THE LEAP-SPOT ORGANISM. 13 Nitrates. — Nitrates are doI reduced. Indol. — Iiulol production is feeble. Hydrogen sulphid. — Hydrogep sulphid is not formed in culture- on beef-peptone agar, potato cylinders, turnip cylinders, or in beef bouillon or milk. TKMl'Ki: \ i I RE RE] vi CONS. 'Flu final death point. — The thermal death point is 46° C. The following tests were made: Newrj inoculated beef-bouillon (+15) cultures in tubes were suspended in a hot-water hath where they were kepi for 10 minutes ai a constant temperature, then removed to room temperature (20° to 24° C). First, temperatures ranging from 40° to 50° ('. were tried, and, the thermal death point seeming to lie about halfway he! ween, trials were again made of 45°, 46°, and 47° C. More than half of the cultures exposed to 45° C. for 10 minutes clouded in 3 to 5 days. Of cultures exposed to 46° C. 1 out of 12 clouded after 1 1 days. The others never clouded. Of 20 cul- tures exposed to -J7° ('., none clouded. Optimum temperature. — The optimum temperature for growth is 24 to25°C. Maximum temperature. — The maximum temperature for growth is very low, viz, 29° C. Minimum temperature 'The minimum temperature for growth is below 0° C. The organism was dead after exposure for 3^ days at 33 to 36° C. in heel' houillon. EFFECT OF DESICCATION. When young, well-clouded beef-bouillon cultures were dried on cover glasses and kept in a dark place at temperatures of 22° to 25° ('., 75 per cent were killed in 24 hours and 90 per cent in 48 hours. All were dead in five days. EFFECT OF SUNLIGHT. Four minutes' exposure to sunlight killed all organisms in thinly sown agar poured plate- exposed bottom up on ice, one-half of each plate being covered as a check. EFFE< I OF 1 REEZING. Freezing by mean- of sail and pounded ice for two and five hours in +-15 beef bouillon had no eil'ect in reducing the number or the vitality of the organisms, as shown by poured-plate cultures made before and after freezing. Beef bouillon (10 C C.) inoculated and at once frozen and kept at temperatures of —4° to — 1N J (.'. (average —12° C.) for nine days 14 A SPOT DISEASE OF CAULIFLOWER. showed no growth during this period, but clouded moderately three days after removal to temperature of 18° to 20° C, and plates from this tube gave pure cultures of the cauliflower organism. Plates poured before and after 10 days' freezing showed considerable reduc- tion in the numbers of organisms, but the growth of the living ones was not retarded. Some tubes of beef bouillon, inoculated with a 3-millimeter loop from a 48-hour bouillon culture and kept frozen for seven days, cloudea within 48 hours after removal to a temperature of 18° to 20° C. Another frozen for 22 days did not cloud after removal to temperatures of 18° to 20° C. The check clouded. The organism grows readily at low temperatures, e. g., beef-bouillon cultures clouded in seven days when kept at temperatures of 0° to 1 ° C. VITALITY ON CULTURE MEDIA. This organism remains alive for six to eight months at tempera- tures varying from 18° to 24° C. on beef agar, Loeffler's blood serum, and potato cylinders, and in peptonized beef bouillon (+15), beef gelatin ( + 10), milk, Uschinsky's, Fermi's, and Cohn's solutions. Evaporation was not prevented in these cultures and the media became concentrated, often dry, and yet the organism was frequently alive. Beef-agar and beef-gelatin cultures at temperatures of 12° to 15° C. and subject to less evaporation (in refrigerator) were dead after eight months. In media less favorable for the growth of this organism, as beef bouillon plus salt, alkali, or acid, the bacteria live but two to three months. GROUP NUMBER. The group number, according to the descriptive chart of the Society of American Bacteriologists, is 211.3332023. NAME OF ORGANISM. This organism appears to be an undescribed form, and because of the characteristic spotting of the affected leaves the name Bacterium maculicolum (n. sp.) is suggested. LATIN DIAGNOSIS. BACTERIUM MACULICOLUM (n. SP.). Baculis in hospite brevibus, cylindricis, apicibus rotundatis, soli- tariis, saepe binis (in agar-agar quandoque 10-30 baculis in filamenta conjunctis); baculis 1.5-3.0 /* x 0.8-0.9 [x; 1-5 flagellis polaribus mo- bilibus; aerobiis, asporis. Habitat in foliis vivis Brassicae oleraceae in maculis 1-3 mm. latis, purpureo-griseo colore. Coloniae gelatinam liquefacientes. Colo- niae in agar-agar rotundae, albae, nitentes. Si baculi in petri-vasibus rare seruntur, in septimo die colonae 6-8 mm. diam. sunt. Baculi 225 SUMMARY. 15 methodo Gram non colorantur. Nitrum uon redigitur. Lac sterile alcalinum fit; initio translucidum, flavum pallidum demum opacum, brunnen e< gelatum; casein non segregatur. Enter temperaturam 29° ( '. el temporal uram - 5° ( '. cult urae crescunt. Si cultural' iu>\ ae in in l'i Kin in 1 carnis | horam in temporal ura 16° C. tenentur, moriuntur. Enter temperaturas —5° C. et — 15° C. per decern dies non moriuntur. Si bacuL' siccantur vel soli exponuntur, celeriter moriuntur. In foliis vivis Brassicae oleraceae aspergendo inoculatis, maculae in 3 I diebus producuntur. Contagium in stomatibus fit. SUMMARY. The leaf— pot disease of cauliflower described in t be preceding page9 is due to a bacterial organism, which was secured in pure culture from the leal' spots and inoculated into healthy cauliflower plants, with production of the disease. Healthy cabbages inoculated with the organism also showed similar infection. Inoculations during July, 1909, were unsuccessful because of the higher temporal are. The heads of cauliflower gave DO satisfactory 'results when inocu- lated wit li t he organism. The name BacUnum dkicuHcoIihh has heen suggested for this organ- ism. It is a schizomycete, pathogenic to crucifers, causing numerous small --pots on cauliflower and cabbage. Entrance by way of the stomata. Organism white, but causing a greenish fluorescence in some media (beef bouillon +15, beef gelatin +10, beef agar +15, Uschinsky and Fermi). Motile by means of one to several polar Bagella. A short rod (1.5 to 3 n by 0.9 /«), single or in chains in some media (10 to 30 >>. long on agar; 10 to 24 fi long in heel' bouillon plus 1 per cent sodium chlorid). Does not stain by Gram; stains deeply with amyl Gram. No spores. Involution forms (found in alkaline beef bouillon) and pseudozoogloese. Aerobic. Liquefies gelatin slowly. Does not liquefy Loeffler's blood serum. Not gas forming. Feeble produc- tion of ammonia, indol, and hydrogen sulphid. Nitrates no! reduced. Tolerates acids, citric and malic to +36 and oxalic to +34 (fuller'- scale). Tolerates sodium hydroxid in beef bouillon to —25 (Fuller's scale). Optimum temperature 24° to 25° C. Thermal death point 46° C. Will not grow in heel' bouillon (+15) or on agar (+15) at 1".)° ('. Grows at 0° C. and below. Grows well in bouillon over chloroform. Grows in Cohn's solution. Blues litmus milk. The mos< si i iking fact - about t be organism are it- ahilit v to grow ,it temporal ures below freezing and its failure to grow at a common sum- mer tempera! ure I 85° F. I. The leaves of the attacked plant- fall off. o Bui. 22! Plate I. Upper Surfaces of Cauliflower Leaves from Virginia, Showing Natural Infection with Bacterium maculicolum. Photographed May 4, 1909. UNIVERSITY OF FLORIDA 3 1262 08928 3229