LIBRARY Michigan State University MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from «© your record. FINES will be charged if book is returned after the date stamped below. BACTERTAL SOrT ROT OF LETTUCE. Thesis presented for the Degree of Master of Science. Michigan Agricultural College. by Lionel k. Tisdale. 1921 ACKNOWLEDGMENTS . The writer is indebted to Doctors EH. A. Bessey and G. H. Coons for many valuable suggestions throughout the course of experiments, ana for thorough criticism and correction of the manuscript. braze Aaa ed fon Lr ee a re [f ‘way @ Table of Contents. Introduction mconomic Importance of Soft Rots oz Lettuce. Field Losses lizrket Losses Previously Described Soft Kots of Lettuce Symptons of the Disease Under Consideration pource of the Disease and luethods of Isolation pathozenicity Relations of the Disease +O Tip Bum vLifferences in Pathovenicity from rreviously described diseases temperature relations of the visease vistribution of the Soft Rot Urganism in the soil Description of the Pathozenes Urgenism Wo. lL Urge nism No. 2 Or; anism Wo. od Urecnism No. 4 on Urgenism No. m Uiganism No. Recomzendaticns for Control of the Disease Summa ry Litarature VCited Description of the Plates wo WwW ©O ~J © LO £4 BACTERIAL SOFT ROT OF LETTUCE. | INTRODUCTION. The growing of head lettuce (Lactuca sativa L.) on a commercial basis has proved very profitable, due to the ex- tremely high demands on-the market for this type of vegetable. Large areas in Michigan are well suited for growing ‘this crop, especially for the early and late summer market. Since the trade demands a fresh crisp product, and two of the large market centers, Chicago and Detroit, are so aceessible, there is great incentive for the development of the industry, with prospects of the state becoming one of the heading producers of this crop. One of the limiting factors in this development is the matter of plant diseases. Lettuce in this regard is no exception in the list of intensive crops, and is subject in to several very serious parasitic diseases. Various root rots are common and loss from them has been very discouraging to growers. But the most serious obstacle is found in the so- called soft or black rot which, although common in the field under certain conditions reaches its height under transportation conditions. In recent years some attention has been directed to this type of disease, and several organisims have been described as a result of this work. It is the purpose of this paper to show the results of experiments and investigations carried out during 1919, 1920 and 1921, which it is believed point to some of the underlying facts dealing with the cause of this disease and thus permit the making of some recommendations as to possible methods of prevention of loss. The investigations also lead to the dis-~ covery of six new pathogenes, and these are herein described. ECONOMIC IwPORTANCE OF Sort ROTS OF LETTUCE. Field Losses. Since the market demands a fresh, sound product free from blemish and disease, it is very evident that soft-rot is a major disease of this crop. There have been several reports of epidemics of this disease in the fields in recent years. It wags reported as completely destroying a 200 acre field in Louisiana during the winter of 1914-1915 (1) By actual count a field 33 acres in South Carolina (2) was found to be 98 per cent diseased, and another field of 17 acres suffered at least 60 per cent loss. Other cases have been reported from Virginia and along the Rio Grande Valley in Texas. It is a generally known fact that when a field is once infected it is useless to try to put the product on the market, unless special precautions are taken to select those heads which are absolutely free from disease, and this would mean a loss of considerable time and mons ye Market Losses. So common is the disease that at this Station during the course of these investigations it was difficult to procure head lettuce suitable for experimental work. Crate after crate secured on the market were rejected because of rot. Inspectious ~Za of stock at commission houses at various times commonly showed the stock on hand was suffering from rot, the outer leaves of the head being badly rotted and the inner leaves frequently partially decayed. Before this produce goes to the consumer, these outer rotted leaves are removed, along with such of the inner leaves as are rotted, and often times this shrinkage from blemished leaves amounts to pratically one-fifth to one- fourth of the entire head. Hence from this point of view it is the consumer who suffers the damage by receiving a smaller and inferior product. Taking this into consideration it is difficult to make an estimate of the amount of damage that is really done, but the loss may be constantly placed at from 25 to 50 per cent of the crop. Figures from the Plant Disease Survey Bulletin, Volume [II, 1919, show that car load shipments from different states, reveal from 1 to 100 per cent infection, often as high as 20 to 40 per cent of the shipment being worthless. PREVIOUSLY DESCRIBED BACTERIAL SOFT ROT OF LETTUCE. Bacterial diseases of lettuce of the type considered in this paper have been known in this country for a number of years. Such troubles have been reported from Vermont, Massachusetts, Louisiana, Florida, South Carolina, Virginia, Kansas and along the Rio Grande river in Texas. & bacterial stem-rot of lettuce was reported by L. R. Jones in 1893 (3). He found a large Bacillus in the diseased -4- stems but did not make isolations. He reproduced the disease: by setting plants in soil which had been inoculated with fragment of diseased plants; and also by pouring over the roots of healthy plants water in which a diseased lettuce head had been crushed. Pietro Voglino (4) reported a bacterial disease of lettuce in Italy in 1904, and named the causal organism Bacillus lactucae. This disease was reported as causing serious trouble in the gardens around Turin, and had occurred annually for ten years before definite study was made. Maximum damage was done where the plants were heavily fertilized. Voglino des- cribes the organism as a long rod form, producing on the lettuce gelatine, ivory white colonies which rounded up and after 15 days assume a rose-colored tint. In 1907, G. E. Stone (5) gave a brief account of a bacterial disease, which he found Accurring in the greenhouse on leaves of lettuce that had been repidly forced. stone believed this disease was the same as that investigated in his laboratory by Percival ©. brooks six years earlier. Brooks isolated an organism, and reproduced the disease by inoculations; but due to the belief that the disease was caused by the succulent growth of the plants, and was oi very minor economia:: importance, he did not make any studies of the orzanisnm. ¥F. Le. Stevens (6) in 1908 reported a bacterial disease of lettuce. He described the disease as causing a pale, green- yellow coloring of the leaf which finally becomes brown. ihe tissue of the diseased leaves when dry disintegrates, leaving a striking net work of veinsi Mjscropic examinations of dis- eased tissue revealed numerous long rod forms of bacteria. He isolated the bacterium, but was not successful in reproducing the disease. He did not describe the organism. In 1908 H. S. Fawcett (7) reported a bacterial disease of lettuce as producing dark brown irregular areas on the margin and other parts of the leaf. Occassioally these spots occurred only on one side of the leaf, while the opposite side remained green. Uther characteristics of the disease were browning of the midrib, often times starting on the leaves that had already headed up and progressing from leaf to leaf until finally the entire head was blackened. He grew the organism in pure culture and reproduced the disease by inoculations. The organ- ism stained well with carbol fuschin, Aqueous genetian violet, but with difficulty with Methylene blue. On standard agar the colonies had pearl white foci with irregular margins. O. F. Burger, in 1912-1913 (8) described a bacterial disease of lettuce which caused browning of the margin of the leaves or spotting along the midrib; the entire leaf often being spotted. Un the head the disease was observed to begin at the center, causing a blackening and softening of the head. fhe organism was large, motile, occurring in chains with endospore. Burger believed that the orgenism was a species of Pseudomonas. JInoculations of the organism upon healthy growing lettuce plants gave positive results. C. W. Carpenter (10) in 1916, reported a bacterial dis- ease of lettuce, from Texas along the Rio Grande Valley. He described the grass symptoms of the disease as (1) reddening of the older leaves and blanching of the younger central ones; (2) restricted development of newly forming leaves, accompanied by small, dark-colored blister spots along the border; (3) development of numerous, lateral adventitious shoots, and J4) dry and dead small rotts. He proved the absence of parasitic insects and fungi. in connection with the disease, but did not describe the organisn. In summing up the earlier investigations of the bacterial disease of lettuce, it appears that the descriptions of the pathogenes are very incomplete, and it is often impossible to make a comparison with those under consideration in this paper. Miss Nellie A. Brown (1) in 1915 was the first to des- cribe fully a bacterium causing a disease of lettuce. The diseased specimens were sent in to the U. S. Department of Agriculture from Louisiana. The disease was described as making its appearance first on the outer leaves which beoome spotted and darkened throughout. Positive results were produced by inoculating the organism which she isolated onto healthy plants. ‘The bacterium grew well on steamed potato, producing a dark blue-green color, and due to this pecularity the name Bacterium Viridilividum was suggested for the organism. In 1918 Miss Brown descrived two other new species of bacteria which were the cause of disease outbreaks in South Carolina, Virginia and Kansas. The diseased plants from South Carolina were described as being of a lighter green color than heal thy ones. in later Stages the head may show rot through the center, on the top, Or a general wilting of the’ head may occur without visible —__ . To ao a > = — Signs of spots; and again only the outer leaves may be affecte ed. The stems of the diseased plants were brittle, and in early stages a cross section through these stems showed a blue- green color md later it was brown. the name Bacterium Vitians was suggested for the organism. The same organism was isolated from diseased lettuce sent from Virginia. The symptoms of the Kansas lettuce disease were described as the wilting of the tips of the leaves, first appearing as very small areas, which very often coalesce and become quite large. The vascular system often showed browning, causing the appearance to be marred but no decay of the heads. Miss Brown Buggests the name Bacterium marginale for this organism. she also isolated the same organism from diseased lettuce sent in from Virginia. SYMPTOMS OF THE DISEASE UNDER CONSIDERATION. The disease under consideration in this paper usually follows tip-burm or some form of injury of the host plant. Vinen plants are affected the decay is rapid, often causing a rotting of the entire hsad. The older leaves are ordinatily attacked first, but in some instances the disease has been known to start on the younger leaves. The most pronounced signs of the disease are the spots appearing on leaves that have tip-bum. These spots are very small when first noticed, but in moist warm weather they increase rapidly in size and number and coalesce, until the entire leaf may become rotted. -8- The spots are first light green in color, are translucent with & water-soaked appearance. Soon they turn dark brown and slimy. When this rotted tissue becomes dry there remains the network of veins which are partially decomposed into stringy masses. On reaching the vascular system, a rapid browning ossurs through- out it. Cross sections of diseased main stems show a dark greenish-brown color of the vascular system, which on exposure becomes almost black. In the Hortigultural vardens at the Wichigan Agricultural College an out-break of disease occurred in the fall of 1920 in @ small patch of lettuce after a frost, which was followed by several warm, moist, cloudy days. The disease was first noticed on the eenter of the heads, which rapidly caused the decay of the entire head. In transportation the disease most commonly occurs on the outer leaves. these rotted leaves are soft, brown to almost black and slimy. When the rot once gets started it goes rapidly from the outer to the inne leaves; finally the entire head often becoming a slimy, rotted mass. SOURCE OF DISEASE AND METHODS OF ISOLATION, In September 1919 specimens of diseased head lettuce were brought to the Michigan Agricultural itxperiment Station from the greenhouse at Kalamazoo, Michigan. Un making miscropic examinations of sections from diseased tissue ,numerous bacterial - rods were found. ihe organism was isolated and numerous inoculations made by needle puncture onto healthy growing lettuce plants and also heads in moist chambers, with positive results. The organism was reisolated and proved to be the same. ‘wo weeks leter on visiting one of the commission houses at Lansing, it was observed that they had just received a shipment of Cafifornia Iceberg lettuce, which was very badly diseased. Two organisms were isolated from different heads of this lettuce and inoculations gave the same positive results. In October 1920 a bacterial disease of lettuce started in the Horticultural gardens at the Michigan Agricultural College. Isolations were made and from three different spots on the same head. Three different organisms were found, which produced the diseas from inoculation. In making isolations, two methods were ordinarily em- ployed vis. (1) method as described by Miss Brown. Small pieces of the diseased leaf were treated with Mercuric Chloride 1-1000 for one minute, washed in sterile distilled water, then crushed in a tube of broth, and dilutions were plated out on standard Nutrient Agar. (2) Im case where the heads had started rotting, the outer leaves were care- fully removed, and with a sterile platinum needle, bits of diseased tissue that had not bc.en exposed to the air were transferred to broth tubes and plated out as above. By béth methods it is possible to get an almost pure culture. The latter is preferred on account of being more convenient, mt in some cases where the disease only shows up on the outer lower leaves it is necessary to use the former method. -10- For convenience during these investigations, each organism was numbered, and is referred to, through this paper as follows: Kalamazoo organisms #1. Organisms isolated from diseased lettuce secured from commission house #2, and #3 and Organisms isolated from diseased lettuce from the Horticultural Gardens at Michigan Agricultural College #4, #5, and #6. PATHOGENICITY, Soon after the organisms were isolated numerous tests of their pathogenicity were made by inoculating head lettuce in moist chambers. in testing the different organims, in case of all experiments, reinoculations of the organisms were made, also microscopic examinations of the diseased tissue were made to prove the absence of any fungi or other invading parasites. There were two kinds of moist chambers ordinarily oem- ployed for carrying out the experiments; tin boxes 12 x 10 x 6 inches and glass dishes 10 inches in diameter. these chambers were always sterilized before use with 1 - 1000 mercuric chloride, and towel paper was placed inside md moistened with sterile water. The tin boxes proved to be much more convenient, where whole heads of lettuce were to be used dus the size, where as the glass dishes were preferred in case of single leaf in- Ocula tions. Sound heads of lettuce were selected, washed and then rinsed in sterile water. Single leaves and whole heads were placed in these moist chambers, and inoculated. The inoculate ions were made by placing a loopful of a 24 hour bouillon culture -ll- diluted in sterile distilled water on the leaf and then ruptur- ing the epidermis with a sterile platinum needle through the Grope In case of the whole heads, inoculations were made near the center. These inoculations were made in triplicate and kept at room temperature. The results of this experiment are Shown in Table I. The results were identical for the six organisims used. TABLE I, Pathogenicity: Test of Head Lettuce in Moist Chambers. obtulation _ —_ 2 days & days & days | 10-12 days Me ins of Results. Drop of 24 |. Light green | Rotted area | All outer) Head a hour Bouil- yellow brown, trans] leaves of] slimy lan culture | water soak- | lucent, head rot-| motted place on ed area voscular ted spots/ mass leaf and 1 mm in dia-/] system on inner | through epidermis meter at p browning leaves. out dark rupture with point of brown needle puncture. bad odor. through drop control Sound Sound Sound Sound As shown in the table in this experiment, in the inoculat- ions began to show light green yellow areas for 1 mm immediate- ly around the point of inoculation in two days. These areas had a water soaked appearance, turning brown on the third day, The vaseular system became brown in three days, and from this time on a rapid decay of the leaf followed, burning complete .- in five days. At this time the inner leaves were showing rotted areas. in 10-12 days the rot had progressed through the entire head, leaving a dark brown, slimy rotted mass. The checks remained sound. & series of healthy lettuce plants that were just beginning to form heads was selected. these plants were growing in 6 inch pots. inoculations with the organisms were made by placing a drop of bouillon culture diluted in sterile distilled water on one of the leaves in the center of the head, and rupturing epidermis with a sterile platinum needle. Large bell jars containing pieces of moistened filter paper were placed over the pots. Six plants were inoculated with each organism and the same number untreated were kept under similar conditions for control. ‘The results of this experiment are shown in fable II ~13- TABLE II Pathogenicity: Test of Growing Plants Under Bell Jars. | Method of | ion 2 days 35 days 5 days 2-14 day 1 aro 3 . : Light Vascular | Plants aouil.on | green system of/all dark giluted in yellow entire brown. sterile areas. inoculat-/ fallen 1 Gistilied water ed leaf | over, upidermis soe lice ares | punctured. ance 1 mmj1 mm in in dia- diameter irregujar 2 out of | All Vascular 6 plants ylants system. show very | show rot-|Al1l brown smali ted are a8|.spots on rotted at point |,inner 9 Doe areas. of inocul#leaves DO. Light tion. brown. Igreen. Vascular system browned if 2 of plants 3 Do As in #1 |4s in #1 Do 4 Do Do Do Do 5 Do As in #2 |As in #2 Do Out O 6 plants show very 6 Do small lighit As in #2] As in 72 Do green ellow Are ABe Control | Untreated Plants Plants Plants Plants healthy [healthy |healthy | healthy ~14- This experiment shows that infection took place from two to three days after inoculations were made. It indicates that organisms #2 and #6 are more virulent than No's 1,3,4 and 5,as infections were noted to take place one day earlier. The vascular system became brown in three days in these two cases, and in five days the wscular System was browned in all cases, the decayed areas of the leaf being typical as described in the preceding: experiment. In five days all the outer leaves were browned and om examination the inner leaves were found to be spotted. ‘the decaying progressed very rapidly, extending from leaf to leaf until at the end of twelve to fourteen days the plants were all dark brown, almost black, slimy masses and part of them had fallen down. the controls all remained healthy. After noting in the preceding experiments thet, when the heads of lettuce were once infected, the decay progressed from leaf to leaf until the entire head was decayed, a more careful experiment was planned. Heads were inoculated in moist chambers, care being taken to rupture the epidermis only. 4t was noted that spots began to appear under the inoculated areas in one to two days. this rot camtinued from leaf to leaf very rapidly through the entire head. Carrots and potatoes were selected, washed, dried and heated with mecuric chloride 1-1000 for one minute and then washed again in sterile water. with a cles knife they were cut up in disks about 1 om in thickmess. The outer layers of Onion bulbs were pealed away and disks cut in the same manner. Single leaves of cabbage wre also used, and lettuce was used as a check for the experiment. This material was placed in moist chambers and inoculeted. The inoculations were made on the carrot, potato and onion by placing a loopful of diluted bouillon culture on the cub surfaces of the disks. the cabbage and lettuce leaves wre inoculéeted by placing a drop of the diluted culture onthe leaf and rupturing the epidermis through the drop. Moist chambers were kept at room temperature. fhe results of this experiment are shown in table IIil Toa4u0d o- - - - - = = - - ~- - L844 B627UN Ss GU pe1,02L ferwga UpNods ensst4 moTttTes op é op + op +] op op + op 9 op & Op + - - OD & OD op + op G op + oD + - - oD + op op +r Ve V op + oD + - - Op & OD OP + OP ¢ OP é OP + = = OP & OP OP + OP g ‘axe cCUTPEEDV UMOL -eid wu B LOLOEWBTP LOpo 64902 UOT -Trems] eSeqavo pue *pe4zor Uy WuUg SATS pus sepnxe PONAZET ASTP tls +] go Sut -~Hetjo uoy, A + JO eoBy T ueyoBTg + - - IOA + |epnxete 16M -INS UO UOT 5 LENOg INO te gO dosg POT P2 POT P02 POT PE POT PERG POT .. Ve PT U0 F *e0n14eEL 65 B4140B0 o1810d uoTUg 1OLIBH 5 PBTNOOUT JUS TUGeIO gO pouyoNn ‘106EJ FO OU OYVOTDUT - ‘esudaaqno pus seoi1siod *suotuo « a o: Q10L IO SUTUUTSE GQ SOLSOTPUT WTS + Til ‘sqoziso JO 48e] alevib SAA TOTUSZOULB The experiment shows that the organisms are all pathogenic to carrots, onions and cabbage, while organism No. 6 is the only one pathogenic to potatoes. In case of the carrots swell- ing occurred on the surface of the disk in one day. In two days a clear watery exudation appeared which turned dark brown in three days, together with the tissue beneath the exudation. in ten days the rot had extended through to the under side of the disk, the whole disk being almost completely rotted. In three days 3st the point of inoculation on the onions appeared small water soaked areas, which turned black two to three days later. The disks were a slimy, rotted mass in twelve-fourteen days with a very offensive odor. Only organism No. 6 produced infections on potato. in three days there appeared an orange-yellow slimy growth on the surface of the disk. In five days the tissue had become brown Over the entire surface of the disk. In ten-twelve days there Was a sunken area in the disks at the point of inoculation, the rotted tissue being a grey, watery mass. In case of the cabbage there appeared blackened areas 1mm in diameter at the point of inoculation in two days. These spots never became very large. At the end of the experiment the Spots were about four to six mm in diamater, and had a dry appearance. The controls in this experiment all remained sound. During the course of these experiments it seemed advisable to test various methods of inoculating plants. In the follow- ing experiment, young plants were inoculated ana bell jars ~18— With pieces of moistened blotting paper in the tops, were placed over the pots. Inocluations were made as follows: (1) @ loopful of a diluted bouillon culture was placed on the leaf and then the epidermis was ruptured through the drop; (2) a transfer was made with the diluted culture with a needle direct into the tissue of the leaf; and (3) in each of the above cases moistened absorbent cotton was placed over the points of in- oculation. The rotting is indicated by a plus sign (+) them indicating the degree of the rot. dicates no rotting. TaBLi LV The The results of this experiment is shown in Table 1V number of A minus sign (-) in- hffect of Different Methods of Inoculations. Test of Growing Lettuce Plants. Organism Methods of Inoculation and Results. L) Drop of dilut- ed culture placed (2) Direct Trans- rtst sane as l. fer of culture by with moistene on leaf and epid= {puncture of leaf absorbent ermis ruptured thrujtissue with needle [cotton placed drope . over point of : ) ation 2d 52 5 d 2aqaiod 5d 2ajsd 1 t+ + it +++ - - + - - - 2 + tetelireeet — ¢ + + - - ~ 3 + le pele ete ef eg _ + - | —- - 4 t+ jt tFl+e+te tt — - + ~ {| -] 7 5 t+ it le + + +| - — + - - ~ 6 + | +t Fig weet] - + + + - —- |-+? This experiment shows that-the method of placing a dro:,-of culture of the leaf and then rupturning the epidermis is very successful; while the adding of absorbent cotten after inoculat- ions were made seems to retard or check the growth of the organism. Disease appeared in the former method in two days in every case; while only one inoculation wis positive in the latter fhe case of slow growth where the direct transfer was made with @ needle may be sxplained in the fewer number of organisms. There seems to be some difference due to the amount of inoculat- ion used. Similar experiments were performed with carrot disks. Carrots were prepared and disks were cut as already described. These disks were placed in moist chambers and inoculated as follows. (1) & loopful of diluted culture of the organism was Placed on the cut surface of the disk; (2) same as one, with @ small piece of moistened absorbent cotton placed over the point of inoculation. (3) same as one with a small filter paper cone placed over the disk and kept moistened with sterile water, Moist chambers were incubated at room temperature. The results of this experiment are shown in Table V 320- TABLE V Effect of Differznt Methods of Inoculation. Test on Carrot. + siens indicate degree of rottinz. - sisns indicate no rotting Organism Methods of Inoculation. (1) (2) (3 ) Loopful of Same as l, with Same as ] with lorzanism placed} moistened absorb- & small moistened fon cut surface ent cotten over filter paper cone fof disk | point of inoculat ion. over disk | Results Results Results l1dajf/2dafad ldfedadars5d P1ldyjed | 5 da ) 1 + [| + [e+e] - j- | - | + [+e Fret t+ - | | ) 2 1+ fF [e+ + - - | + | - f a sn al + + + P+ + + + 3 — + ++ + ~ ~ - + ++ [+ + + + } 4 = + + + + - - I+ + + + ++ + + -t in 6 5 - + + + + ~ ~ |. + + + +++ + + ~- 6 - + + + + - - [+ 1 in 6 + + + ++ + + ~ ® . ‘ ¢ ° ' 4 \ 1 : \ j . t “> m- ~21- This experiment checks with the preceding. It seesm that the absorbent cotton almost completely checks the growth of the Orgenisms. There were six inoculations made with each organism and only one of these inoculations with three of the organisms was positive, and the disease was very slow in mking its appearance. By placing a moistened filter paper cone directly Over the disk after inoculation, hastens infection. watery exudation appeared in case of each organism in one day. fhis experiment shows that the organism produced the greatest amount of damage under moist conditions. Theat there is considerable difference in the progress of the aisease, where the host is infected at different points is shown in the following experiment. Healthy growing lettuce plants and head lettuce in moist chamber were inoculated at the different points on the leaves as follows: (1) in the parenchyma tissue of the leaves between the veins; (2) in the small veins; and (3) in the midrib and main stems. Bell jars with moistened pieces of blotting paper in the top were placed over the growing plants. The results of this experiment were practically the same with each organism as shown in Table VI -- eee me eat --— ~~ oe ae ae =» eae h eee wo eee ~Low TABLE VI Lffect of Inoculating at Different Points: Test of Lettuce Plants. point in- Organism] oculated Results. _ 2 days 5-4 days | 5-8 days [10-le d. Paren- diseased disease | disease plants 1-6 chyma area 1 mm] reached | spread dark inc. tissue in diamet+ veins, through | brown between er browning] Vascular } and veins 1 cm system to] rotted. other leaves. browning | Vascular] kntire of veins system j|plant lL cm all dark Rotted 1-6 Small from brown brown mass in. veins point of in leaf inoculat- ion browning LO. 1 cm from also 1-6 midrib point of | browning; Do Do inc. inoculat-| of veins 10n in other leaves ontrol no Healthy Healthy Healthy | Heahthy treat- ment As shown in this experiment there is a marked difference in the progress ot the disease, when inoculatinns are mde at different points on the leaf, however, actual decay of the tissue at the point of inoculation begins about the same time, in two days. Im case where the parenchyma tissue was inoculated, the progress of the disease was rathe: slow until -25= , it reached the véins, then a rapid browming of the vascular system occurred, as in case of the direct inoculations of the veins and midribs. In two days in case of the direct inoculat- ions into the vascular system, browing occurred 1 cm from the point of inoculation. A decay of the accompanying paren- chyma tissue was observed to take place very rapidly. In two or three cases where the main stem was inoculated, the plants wilted in four to five dayS. Examinations showed that the Vascular system was plugged. Cross sections of diseased main stems in the early stage, show the vascular system as being dark greenish brown in color, and turning black on exposure to the air. 4 series of dilution flasks was prepared by adding 10 cc of bouillon culture of the organism to 90 cc of sterile distilled water, making 1-10 dilutions. From these flasks dilutions of 1-100, 1-1000, 1-10,000 and 1-1,000,000 were made in the ordinary way. MInoculations were made on healthy lettuce heads in moist chambers by placing a drop of each of these dilutions on the leaves and then rupturing the epidermis through the drop with a meedle. Heads were also inoculated with an undiluted bouillon culture of the organism. fhe moist Ghambers were incubated optimum temperature 24 to 26 C; maximum 34° C; minimum below 0° C. Thermal death point between 50° o C funder con- and 51 ditions stated); stains readily with basic anilin and aqueous dyes; is Gram-negative; not acid-fast; tolerates sodium hydroxide -20 Fuller's scale; does not tolerate acids to any marked degree; is not killed very readily by drying nor by sunlight; retains virulence for more than 8 months. Orgenism No. 5 The org:mism is a small rod, non-motile, occuring sinsly and in long chains. The single rods vary fro.i1.0 to 1.44 microns long by 0.40 to 0.65 microns wide, the average size being 1.12 x 0.53 microns. Capsulss were stained from old bouillon culture with welch's Capsule Stain. The absence of endospores .as tesied by staining and heating. Pseudozoogloeae occur in ola bouillon and potato cultures, and ere composed of masses of lon. chains attached tozether by gelatinous threads. No involution forms noted. Behavior Toward otesins. fhe org2nism stains well in all basic anilim and aqueous and alcoholic dyes. It is not acid-fast «na is Gram-negative. Cultural Characters. Beef-Agar Plates:- Colonies appear on peptonized beef- agar plates (ph. 6.6 ) in 24 hours at room temperature, 23° m5 b= to 25° C. The colonies are first*cream color and round. Later they are cream-yellow ana heve irregular margins, almost rhizoid, glistening, flat, smooth, 2-5 mm in diameter with a violet fluorescence. Beef agar Stroke:= Growth occurs in 18 to 24 hours at room temperature 25° to 25° C. The growth is Spreading, flat Slightly contoured, glistening, opaque and of a cream yellow color. Agab Stab:= Yellow growth occurs on the surfece of the stab in 24 hours. Growth along line of puncture is feeble and filiform. Beef Bouillon:- Peptonized beef bouillon (Bh. 6.8) is densely clouded in 24 hours. Wo other change in medium noted, until in 6 weeks the medium is clear with little sdeiment. Nutrient Gelatin:= Small round cream colored colonies appear in 2 days on peptonized gelatin (Ph. 6.57 kept at 11° to 12° GC. They are flat, entire ana glistening, Liquefaction begins in 4 days and is cup-Shaped. Liquefaction in gelatin stabs kept at 15° to 17° ¢ ve ins rapidly in 2 days. virst it is crateriform, ana then strateform The entire tube is liquefied in 10 to 12 days, Uskinsky's Solution:=- fhe orgunism does not grow in Uskinsky's solutione Cohbn'a Solutiag:- No growth. Kermi's Solution:= No growthe we CO om re Sterile Wilk:- Clearing begins without coagulation in 7 dayse Curdlike sediment in bottom. ‘In 6 weeks there is curd- like, flaky mass hanging through the meaium. the chear medium is yellow ocher color. Litmus Milk:- Im 5 days an acid reactiun is Shown at top of mediume The medium shows acid reaction througn out in LO days. In 6 wecks the medium is Liver-brovm in color, .ith a white sediment at bottome Sveamed Potato Cylincer:- Thee orcurs a scanty to moderate growth on poteto. The growth is spreading, flat, glistening, and of a mustard yellow color. The j;otato is Slowly changed to a light brown color. No diastatic action on the Sstarche further tests for diastatic action were made by making Streak cultures, of the organism on 0,2 percent starch agar, tests with Lugal's iodine solution in 5, 7 and 14 cays did not show lishnt areas near growthe Other Cultural weatures. ammonia:- Test for ammonia with Nessler's test solution were made in old bouillon cultures. these tests showed abundant production of ammonia. Hydrogen Sulphid:= Is not wroduced. Indol:- Is not produced. Nitrate Reduction:- Nitrates are not reduced, Toleratiaa of Acids:=- The organism does not tolerate acids very well. No growth occurred in bouillon to which o.l per cent of citric, malic or vartaric acids were added. -o—= Toleration of Sodium Hydroxide. Tests were made in neutral bouillon to which was added sodium hydroxide titrating -20, -25, -30 and -35 on s'uller's scale. Growth occurred in -20 and -25, but no growth in -d0 and =35 colution. Gas bormat ion The organism is a facultative anaerobe and does not produce gas. Tests were mde in peptone water und bouillon to wnich was added 1 per cent dextrose, lactose, saccharose, maltose, mannit and glycerine respectively. Growth occurred in both open ana closed ends of tubes, but no gas was formed. There waS no change in the Ph. of the meaia. further tests for anaerobism were made by depriving plates and tubes of oxygen with pyrogallic acid snd sodium hydroxide. Growth accurred in each case in 36 to 48 hours. Temperature nelatione Thermal Death Point:- The t:i.ermal death point of the Organism is between 52° and 53 C. for 10 minutes USing the Capillary tube metnode Miinimum Temperature:- The minimun temperature for growth be Low 0° Ce > cn Optimum lempe.ature:=- The optimum temperature for growth , 0 0 1s 20 to 24 0, Maximum Temperature:=- The maximum tempereture for growth . 0 is 39 C. Relations to Light. Tne org-nism is not very sensitive to sunlight. MThinly seeded plates weve ,oured fron bouillon cultures, ena half of each plate was covered With black paper. Tie plates were exposed to direct sunlight on crushed .ce, at noon-time in april and way. Plates exposed for 50 minutes, showed no grovth on uncovered side, wWiile there were 70-80 colonies on uncovered side. Plates exposed for 40 minutes had 4-6 colonies On uncovered side while there were 80-90 colonies on covered side. Relations to Moisture. The organism is not very readily killed by drying. wrops of 24 hour ola bouillon culture were placed on sterile cover Slips in petri aishes, and allowed to dry in the aark at room temperature. No growth occurred in bouillon tuoes receiving pieces o1 wtnese slips efter 3 Guys. Growth occured in those kpet for 2 dsaySe bLozs of Virulence. There is no loss of virulence of the organism 8 months after isolation. Pathogenicity. Lhe org:mism rots, letcwuce, carrots, onions and cabbage, in case of injury or following other disease. Group Number. according to the descri;tive bhart of the Society of American Bacteriolozists the group numoer if 221.6535605. Brief Technical Decription of the urg-nism. The orgenism is a@ ghort, non-motile rod with rounded ends; capsules, pseudozoogloeae; no spores, involution forms not noted; fecultative anserobe; agar colonieg crexum yellow — =00= with violet fluorescense, irregular margins. Growth on potato cylinders is scanty, mustard yellow; produces acid reaction in litmus milk; liquefies gelatin rapidly; .,roauces ammonia; does not produce indol nor hydrogen sulphid; does not reduce nitrates; no diastatic action on ,poteto stxrch; does not crow in Uskinsky's, Cohn's nor vermi's Solutions; thermal death point 52° to 53. CG, siaximum temperature for growth is 39° © minimum below O° U, Optimum 23° to 24° G. Is not acid-fast; is Gram-netative; and svzins Well with 211 basic anilin ayes. Not very sensitive to sunlight; is not killed very readily by drying; slight toleration of alkali and tartaric acid; retsins its virulence more than 8 months. Urg-mism No. 6 The orz.nism is a rod, with roundea ends, occasionally Slightly curved, motile by means of 1 to 2 flagella at one pole, more commonly one. «saverase size 1,57 x 0.65 microns vzeryins from 1.03 to 1.76 microns long and 0.42 to o.%?9 microns wide. Flagella weve stained with Van srmengen's flasellar stain. Capsules Wve Stained With velch's Capsule Svainana alm with Van urmengen's flagellar stain. The absence of endos; ores Was tested by staining and by heating old bauillon cultures. Pseudozoogloeae occur insome media, 2na are composed or masses of short chains attached to:ethc: by gelatinous threads. Vxrious Shaped organisms were noted in alkaline media, and also svollen and very small. Behavior Toward Stains. Tne organism steins Well with all basic anilin ayes; such as fuchsin and gentain violet. It is Giamenegautive and not acid-tast. Cultural Characters. Beef-agar Plates; Colonies avyecr on peptone beef agar pletes (Ph. 6.6) in 24 hours at 25° to 26. C. ney are first round bluish wnite. The colonies spresd rapidly having irregular auriculate margins, oecoming 3 mm to 2 @m in diameter, are flat, translucent glistening, finely granular and bluish-white. Beef-izgar otroke:=- Growth occurs in 24 hours at room temperaturee The growth is filifora, flat, glistening, simooth and translucent, with a decided odor. Beef Bouillon:- Peptonized beef bouillon (Ph. 6.8) is densely clouded in 24 hours at room tem erature with a ti:in White pellicle. ln 5 aays tne medium is veronese green, which disappears in 7 to 10 days. ‘he pellicle breaks up in 5-7 days, With wi.ite sediment. In 6 weeks the medium is clear crcam White, with a viscid Whive sediment. Nutrient Gelatin:- Colanies apvesr on ;eptonized gelatin (Phe 6.5) plates in 2 days at lL to 12 G. «Are very snell One-half to 2 mm in diameter, flat, entire, slistening, write and translucent. No liquefaction. Gelatin stabs sre not liquefied when «et at 15° to 17° c in w_ter bath.. Growth is best at Surface, ap.ecaring ia 2 days. The growth along line of puncture is feeble, and peaded Uskinsky'’s Solutica:- Uskinsky's solution is aensely Clouded in 24 hours, with white pellicle in 2 asys. Im © days there iS a veronese ring at top of meaium, which exvends throughout the medium in 7 to 8 usys. The-e is noved a change in the color of the medium in 4 weeks to a dull-green yellow. Pellicle has disappeared and a heevy viscid, white vedinent at bottom. No further chanse in 6 weeks. Cohn's Soluti-n:=- There is faint cloudiness in Cohn's solution in 3 GdaySe In 5 cays flake lixe particles are floate ing in medium. Im 2 weeks there is a flaky sediment, the medium being water green. Im 5 weeks the medium is still clouded and pale-green yellow. Hermits Solution:=- Slight cloudiness occurs in 24 hours, medium has a sky blue tinge. There is a white pellicle in 3 days, Whick becomes a heavy scum in 4 weexs and martius yellow. In 2 weeks ther: is a flooeulent cream colored sediment. In 6 weeks the medium is clear, citron yellow, dense cream color Sedimente sterile Milk:- No change detected in 6 weeks except a litle finely curdlike sediment. Litmus wilk:- alkaline ring ap,ears at the surface of medium in 24 hours, which extends through-out in 8 to 10 aays. In 10 days there is a white sediment. 6 weeks the medium is Windsor blue. Steamed ~otato Cylinders:- There is an abundant growth on Steamed poteto cylinders, spreading, first thin, then Slightly raised and contoured and glistening. ine growth is Urchraceous tavmy in 10 days, changing to tawny-olive in 5 or weexs and slimy. Zhe medium ig sluvly vrowned. Wo ciastatic action on ;otvxto starcn. further te.ts for diastatic action on poteto starch were made by m&.cins streak cultures on U.2 ; cr Cent &é541 plates. Tests were mede With Lugol's iodine solution after 5, 7 and 14 days énd no light colo: Was visibie near the growth. Uther Cultural #eatures. Indol:- ‘Yests for indol were made with cultures of vouilion and Dunhan's Solution with brlich's indol tect solution 5c c¢ of solution, I and 5 cc of coluvion II were adaed to 10 ce of Liquic culture, and no red color a; pcered sS:i.owiag th. t the Oorgsnism aoes not proauce indol. Ammonic rroduction:- abundant. Hyarogen Sulphia:=- Is not proauced. Nitrate neauctiwm:- Nitsates are not reduced. Toleration of acids. the orgunism does not tolerate citric, malic anu vurtaric agids, when 0.1 per cent “re adaded to pouillon. toleratim of Sodium Hyaroxide. The orgenism tolerates sodium hyaroxidae to -20 in bouillon Huller's scale. ‘esis weie made in -18, -20 ana -25 ana -80 bouillon. Growth occurred in -15 an -20 in five days, the medium is chen-ed to viridine ~reen. Wo sro.u: occurred in <2 and -50. Gas Formation. The organism is ea facultetive anaerobe anu uoes not produce gas. ‘Tests were mede in fermentation tubes in peptone water and bouillon to which was added 1 ¢er cant of lactose, ~64- dextrose, saccharose, maltoSe, mannit and glycerine, respectively. Growth ap,;cared in the closed arms of lactose “na sacchrose, and in the open arms of all tubes. No gas was formed. there wesa change in the Ph. of dextrose, from 6.5 to 5.6 indicating Slight acid reaction. further tests for anaerobism were made by depriving deep culture pletes and Giltner H. tubes of Oxygen with pyrogallic acid and sodium hyaroxide. Growth occurred in each case in 2 daySe Temperature Relations. Theimal Death Point: The thermal death point of the organism lies between 50% and 51° 6. Minimum Tempezature:= Tize minimum temperature for growth is below 0° C, Optimum Temperature:—- The optimum temperature for srowth is 24° to 25° 6 Maximum Yemperature:= The maximum temperature for growth is 34° C, Relation to Light. The organism is not very sensitive to sunlight. -Frlates that were half covered with black paper were exposed to direct sunlight on crushed ice at noon time in april and Imiay. after o5 minutes esposure, there were from 65-70 colonies on covered Side of plates and no growth on uncovered side. There were 2-5 colonies on uncovered side of plates exposed 50 minutes, and 40-50 colonies on covered side. sthere geeiied to be no differences in plates exposed for 40 minutes. relation to Lloisture e fie organism is not very readily killed wy drying. Tests were made by placing 2a drop of 24 hour ola pouillon culture on cover slips in petri dishes and alloviing them to ary in the dark at 22° to 25° CG. No growth occurred in bouillon when Slips were transferred that had been drying for 3 days. Growth occurred in those tubes reciving slips which had been drying for 2 days. Loss of Virulence. There is no loss of virulence 8 months after isolation. Tests we:e made by inoculating head lettuce in moist chambers. Pathovsenicity. The orzanism is pathogenic to lettuce, czurrots, potatoes, onions and cabbage.after injury or other diseases. Group Number. According to the descriptive chart of the Society @ American Bacteriologists the group numoer iS 222.25550535 Beief Technical Description of the Orgenism. The orgenism is a motile rod, slightly aurved with rounded -ends, flagella at one pole; capsules; no spores; pseudozoogloeae; involution forms few types; facultative anaerobe; agar colonies; bluish white, large irregular, thin and smooth. Growth abundant on poteto, tewny-olive, produces alkaline reaction in Litmus milk; does not Liquety gelatin; produces ammonia, does not produce indol nor hydrogen sulphid; does not reduce nitrates no diastatic action on potato starch; grows well in Uskinsxy's and Fermi's solutions; feeble growth in Cohn!g solution; | . - 0 thermal death point 50° to 51°C. wwinimum temperature below O U -66-= Optimum 24° to 25° C; maximum 34. C; does not produce gas, is Gram-negative and is not acid-fast. Stains well with all basic anilin dyes; is not killed very readily py dying; is not very senSitive to Sunlight; does not tolerate acid at O.l per cent, tolerates sodium hydroxide to -20 Fuller's scale; retains virulence tor mor than 8 months, RECOMMENDATIONS FUR CUNTRUL uF THu DISHASE, Through field experiments Levin (11) states that lettuce rot can be prevented and also that rot already in crogress will be checked by spraying plants with formaldehyde, 1 pint to 30 gallons of water. Vsrious strenths of formaldehyde have been tested at this stati.-n, and it was found thet solutions strong enough to prevent the discase, will cause burning of the Leaves. Under these conditions the use of formaldehyde as a control method is not advised until more thorough tests have been made. Since the disease under consideratiun ordinarily attacks plants troubled with tip-burm it seems safe to sey that Wien a control measure is discovered for tip-burno there will be a minimum amount of this type of soft rot under field conaitions. notation of crops, Sanitary cultivation methods and fertilizing With well decomposed manure are strongly advised. and where irrigation is used, the over head method is reagomnended. More care should ve exercised in harvesting and packing to prevent bruising Since the discase causes the maximum damage in transportatim «nd stora;e. The pathosenes are invariably present on the leaves and are ready to cause infection -67%- at the first injury of the host tissue. If plants in the field show any considrable amount of disease if is a generally known fact that it is useless to harvest, as very Slightly diseased neads will rot rapidly under transportation conditions. Uare should be taken to Select a perfectly sound product for market purposes, and in harvesting it is sdvisable to remove the lower older leaves. -~68= SULUIARY. le The bacterial soft rot disease of lettuce is very widely distributed and causes lar_-e losses in the lettuce crop each yesre ithe maximum daméze being aone in trans, orvation. Diseased specimens were examined from sast Lansing and Kalamazoo, Wwichigan and lettuce shipped from California. Six apparently new pathogenes were isolated and found to be in- fectious to the hozt only in case of injury or following some Other disease. These pathozenes are descrizsed in this paper. The progress of the disease reaches its height at fairly warm temperature from 22° to 28° Ge Pathogenes causing soft-rot were found ;resent in a lerge. number of soil samples collected in the near vicinity of the Michisan agriculturel sxperiment station fields. It is recommended that clean cultivation methods, &he 6leaning up and burning of old disessed leaves in the field, rotation of crops, fertilizing with well rotted manure and Overhead irrigatiq@ should pe employed for control of the Giscase. for market it is advised to select heads ,erfectly free from disease and to avoid bruising in pacxing the crates. (1) (2) (s) (4) (5) (6) (7) (8) (9) -0Y= LITeacAT vin CLTnD,. Brown, Nellie a. LOLS a Bacterial Viseuse of Lettuce. Journal agr. Research 4: 475-478 1918 Some Bacterial Viseases of Lettuce. Jourm,. Agr. Research 13: S67-588 (14 Pls.) Jones, L. &. 1893 A Bacterial "Stem-rot"™ of Lettuce. Vt. Agr. uxp. Sta. 6th ann. report 1892 87-88 Voglino, Pietro. 1904 Sulla Batteriosi Delle Lattughe ann. R. Accod, agr. Torhno 46: 25-33 4 fig. Stone, G. i. 1907 Bacterial Disease of Lettuce, Mass agr. uxp. Sta. 19th ann. report, 1906. 165-164 Stevehs, ©. L, : 1908 & Bacterial Disease of Lettuce. W. U. agr. Bxp. Sta. 30th ann. report 1906 107:29-50 Fawcett, H. 8. . 1908 Lettuce Visease. Fla. Agr. pxp. ote. Kept. 1907/08: LXXX-LXXXVII, pl. IV-V Burger, U. FE. L912 Lettuce Rot; Fla. agr. xp. Press. Bul 200 LDe : 1912 A Bacterial Lettuce Visease. Fla. agr. Lxpe Rpt. 1911/12 XCVIII-c (10) Crapenter, ©. W. 1916 Rio Grande Lettuce Visease. Phytopetnology (11) Levin, Ezra 1917 Control of Lettuce Kot. Phyotopathology. 13292-2935 -70- Plate I. Soft rot on New York head lettuce collected in Chicago, showing rotting on the tips of the leaves. (Photo by Ray Nelson.) Plate II. Crate of Western New York head lettuce showing soft rot on the heads. (Photo by Ray Nelson.) ~72- Plate III. Heads of California Iceberg lettuce showing soft rot on the outer leaves. Specimens collected at Lansing, Michigan. (Photo by Ray Nelson). Plate IV. Head of Iceberg lettuce showing soft- rot following tip-burn, at the tips of leaves forming the head. From inoculation experiments in greenhouse... (Photo by Ray Nelson). tii il Hi 4 =__o | LSS ——") + — 3 =~" -——- 5 = —"s ‘iil il ia , a) | 31 ——— ———— _—