PSEUDOMONAS AERUGINOSA; ITS ROLE AS A PLANT PATHOGEN'

R. P. ELROD mNm ARMIN C. BRAUNDepartment of Animal and Plant Pathology, Rockefeller Institute for Medical Research,

Princeton, N. J.

Received for publication May 14, 1942

The idea that some organisms may have the ability to establish themselvesand thrive within both plant and warm-blooded-animal tissues has received theattention of comparatively few workers. The vast gulf between the two formsof life, in structure, composition, and many environmental factors, has seemedto preclude the thought that both could be favorable hosts to the same organism.Nevertheless, attempts have been made to show that such a dual pathogenicitycan occur.The most striking results were obtained by Benham and Kester (1932) using

the fungus Sporotrichum. Employing strains isolated from both animals andplants, they found a few which would attack members of both kingdoms. Theorganism, S. schenckii, causing the human disease was transmitted to carnationand rose buds producing a rot similar to that caused by S. poae. After livingsaprophytically or parasitically in plants, S. schenckii retained its virulence foranimals. In order to insure infection, however, it was necessary to provokesome slight injury before or at the time of the fungus injection. Ciferri andBaldacci (1934) inoculated 22 human pathogenic fungi and one insectivorousfungus into tomato fruits and found 18 that gave positive infection. Theyconsidered that their work not only confirmed that of Benham and Kester, butfurnished additional evidence as regards the adaptability of human pathogensto plant hosts.With bacterial pathogens less positive results have been obtained. Baldacci

and Ciferri (1934) found two of 23 organisms of human source able to producesome evidence of infection in tomato fruits. These organisms were Proteusvulgaris and Bacillus pyocyaneus. In neither case was the infection severe, nordid it approach the type usually associated with the apical rot of this fruit.Pseudomonas aeruginosa (B. pyocyaneus) has been a recognized human patho-

gen for a good many years. Its most distinguishing feature is the formation ofthe diffusible, chloroform-soluble, blue pigment, pyocyanin. This organismis widely distributed in nature, usually as a harmless saprophyte. On occasion,however, it can become a dangerous pathogen. Generally, it appears as asecondary invader and is often associated with suppurative lesions of variousparts of the body. There are, nevertheless, numerous instances wherein it hasbeen shown to be the primary cause of a fatal infection in man and other animals.Experimentally many domestic animals, rabbits, goats, mice, and guinea pigsare susceptible to infection. The animal pathogenicity of the organism is, there-fore, a well established fact.

1 This paper was presented in part on December 29, 1941, before the Society of AmericanBacteriologists in Baltimore.

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R. P. ELROD AND ARMIN C. BRAUN

Scattered throughout the literature are reports concerning the pathogenicityof P. aeruginosa for plants. For the most part, these accounts have not consistedin a systematic attempt to prove the actual phytopathogenicity of the organism.Rather, they are instances concerning the isolation of the bacterium from plantpathological conditions.Verona and Passinetti (1936) obtained slight infection with B. pyocyaneus and

Bacillus fluorescens-liquefaciens in lettuce. In each case, however, infectionoccurred only in plants injured by frost. Paine and Branfoot (1924) describeda disease of lettuce caused by a bacterium which they concluded was identicalwith Bacterium marginale. A short time later, in the same laboratory, Mehtaand Berridge (1924) pointed out that on the basis of morphological and culturalcharacteristics B. marginale was identical with B. pyocyaneus. The latter wasshown to be capable of attacking young lettuce leaves and producing diseasesimilar to that due to B. marginale.

Brooks, Nain, and Rhodes (1925) utilized the B. marginale culture obtainedfrom Mehta in a serological comparison with B. pyocyaneus isolates of animalorigin. In 5 anti-pyocyaneus sera B. marginale failed to agglutinate, nor didany of the 11 B. pyocyaneus organisms agglutinate in the B. marginale antiserum.

Desai (1935) described an organism producing a soft-rot of sugar cane whichcaused extensive damage in India. To this organism he gave the name "B.pyocyaneus saccharum" (Phytomonas dexaioma) and pointed out its apparentclose relationship to B. pyocyaneus. He found that his organism was associatedwith a saprophyte and that the two organisms together produced greater infec-tion than the pathogen itself.

In this country Clara (1934) included a single isolate of B. pyocyaneus in hisstudy of the green-fluorescent plant pathogens. This culture did not prove to bepathogenic for the plants tested.

It is evident that the phytopathogenic nature of Pseudomonas aeruginosa hasnot previously been well established. However, our concern in the problemwas initiated indirectly. In the course of a serological study of the green-fluorescent group of bacterial plant pathogens, it was found that one organism,Phytomonas polycolor, was extremely virulent for small laboratory animals(Elrod and Braun, 1941). The suspicion arose at that time that the bacteriumwe were dealing with was in reality P. aeruginosa. Subsequent experimentsreported here have shown this to be true. With this fact established, we thenbelieved it expedient to reinvestigate the phytopathogenic potentialities of P.aeruginosa. This report concerns such experiments, as well as those whichdemonstrated P. polycolor to be identical with P. aeruginosa.

CULTURES EMPLOYED2

The P. aeruginosa cultures used in this work were selected as representing awide range of original habitats. Emphasis has been placed on cultures derivedfrom animal sources, either normal or pathological.

2 The writers are indebted to Dr. W. H. Burkholder, Miss Helen Knott, and Mr. Ed.Adams for supplying some of the cultures used.

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PSEUDOMONAS AERUGINOSA

Gil, received as P. polycolor. The animal pathogenicity of this isolate wasthoroughly investigated (Elrod and Braun, 1941). Produces an abundance ofpyocyanin on glycerol-peptone agar after several days' incubation.PP2, received as P. polycolor. Produces only small amounts of pigment at

first; this property greatly enhanced by growth on glycerol-peptone agar.Those known to be P. aeruginosa were:Rab., isolated from a lung abscess in rabbit. Produces an abundance of

pyocyanin on virtually all media.Ky., stock culture from University of Kentucky. Atypical in that pyocyanin

formation is overshadowed by the production of a brownish-black pigment.Chick., isolated from the ovary of a chicken with a primary Salmonella pullorum

infection. A strong pigment producer.W., from intestinal tract of a normal man. A strong pyocyanin former.Chi., originally from stock collected of University of Chicago via Ohio State

University. Produces a light emerald green pigment.OSU, stock culture from Ohio State University. A mediocre pyocyanin pro-

ducer.Birk., isolated from a middle-ear infection. A brilliant pigmenter.RIH, isolated and suspected in gastro-enteric disturbance. Produces a large

amount of pyocyanin.Al, A2, AS, all isolated from water. Form deep blue pigment on virtually

all media.97, 256, 257, 260, originally from the A.T.C.C. all derived from pathological

lesions. These cultures produce no pyocyanin and but little fluorescin.Our 17 isolates were all fatal to mice by intraperitoneal injection. Usually

.05 ml. of an 18-bour broth culture was sufficient to kill the mouse in 12 hours.Those tested against rabbits and guinea pigs also proved to be fatal. In eachcase a bacteremia resulted and the organism was always cultivable in pure cul-ture from the heart's blood. Previous experiments (Elrod and Braun, 1941) haveshown that there is a definite multiplication ofP. polycolorin the infected animals.

IDENTITY OF PHYTOMONAS POLYCOLOR WITH PSEUDOMONAS AERUGINOSA

P. polycolor was isolated and described by Clara (1930) as being the etiologicalagent of an economically important disease of tobacco prevalent in the Philip-pines. In the field and experimentally the organism produced a severe leafspotting and necrosis, while in the seed beds it very often manifested itself as asoft-rot in the stems of young plants.

This species differs in many respects from the ordinary members of the green-fluorescent group of phytopathogenic bacteria. The two cultures in our posses-sion (G11 and PP2) produce large amounts of pyocyanin. The pyocyanin iseasily extracted after several days' incubation from either broth or agar slopecultures with chloroform. Pyocyanin formation is greatly enhanced by growingthe culture on glycerol-peptone agar. Clara (1930), in his original descriptionof the organism, noted this blue coloration on glycerol agar, but in a later comAparative study (Clara, 1934) of certain members of the plant group failed to

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636 R. P. ELROD AND ARMIN C. BRAUN

isolate pyocyanin. According to Gessard, who is supported in this by Meader,Robinson, and Leonard (1925), the presence of pyocyanin is sufficient to identifyan organism as B. pyocyaneus. Clara noted, as we have, that the organism growsmore luxuriantly at 370C. than at lower temperatures.

In an attempt to understand more fully the relationship between P. polycolorand P. aeruginosa, biochemical and serological studies were made. The carbo-hydrates used in the fermentation studies were incorporated in a syntheticmedium to prevent any obliteration of acid formation by these strongly pro-teolytic organisms. This medium was made up of 0.2 g. magnesium sulfate,0.1 g. calcium chloride, 0.2 g. sodium chloride and 0.2 g. dipotassium phosphateper liter. Incubation was conducted at both 37CC. and at room temperature.

TABLE 1Biochemical reactions of Phytomonas polycolor and Pseudomonas aeruginosa

SUCROSE MANNI- GLYC- S RANRA XYOSE ARABI- MAT INDOLSE GELATINTOL EROL NOSE NOSE TOSE

Gil. .~~~A- - - - - A A - - +FF2....A....... A A A - +Rab.. A - - - -A A A - +Birk........... A A- +Chick.......- - - - - - A - - - +

97..A.......A- A A - - +

260.. A --.-.- A A - - +

2,57...Af-no v ec- A A - - +

256....A.-...... A -A - +

W....A........ A A A - +

Al . . ~~~A- - - - - A - - - +

A2 . . ~~~A- - - - - A A - - +

A3..- - - - - - ~~ ~~~~~AA - - +

Chi....A....... A A A - +

OSU . ~~~A- - - - - A A - - +

Ky . . ~~~A- - - - - A A - - +

RIH. .~~~A- - - - - A A - - +

A, acid formed; -, no visible change; +,gelatin liquefied.

As one can see in table 1, glucose, xylose, and arabinose were fermented withthe formation of acid only by almost all of the organisms. Sucrose, mannitol,maltose, glycerol, salicin, and raffinose were not acted upon. Indole was notformed, but gelatin was rapidly liquefied. The latter two reactions are consid-ered characteristic of P. aeruginosa.There is considerable difference of opinion among various authorities as to

the actual fermentative capacity of P. aeruginosa. Moltke (1927), using 4isolates, concluded that none of the more common carbohydrates were fermented;Bergey et al. (1939) evidently adhere to this belief. Other writers, Zinsser andJ3ayne-Jones (1939), Topley and Wilson (1931), and Sandiford (1937) agree thatglucose is attacked. Whatever the fermentative abilities of the bacterium, itmust be admitted that our 15 isolates of P. aeruginosa and 2 of P. polycolor are

PSEUDOMONAS AERUGINOSA

in close agreement. Clara (1934) found his isolate of P. polycolor far moresaccharolytic than is shown by our studies. According to him, glucose, galac-tose, levulose, mannose, arabinose, xylose, mannitol, and glycerol were all fer-mented. At the same time, however, the P. aeruginosa culture he utilizedfermented all of these carbohydrates, as well as salicin. In the original descrip-tion of the organism (Clara, 1930) xylose, arabinose, glucose, and mannose weresaid to be the only sugars fermented. This is similar to the results reported here.The antigenic characteristics of P. aeruginosa are likewise in a state of con-

fusion. It is generally agreed by most authorities that the species is serologi-cally heterogeneous. Aoki (1926) using 50 isolates concluded, on the basis ofagglutination tests, that the organisms were antigenically dissimilar. He em-

TABLE 2Agglutination tests

SERA PREPARED AGAINST:ORGANISM

AGGLUTINATEDBirk. 257 Gil Rab.

Birk....... . ...3200* <100 <100 <100A2............... 3200 <100 <100 <100Chick ....... 3200 <100 <100 <100A3........... 3200 < 100 < 100 <100W ......... 3200 <100 <100 <100Al............... 1600 <100 <100 <100OSU ........ 1600 <100 <100 <100RIH ........ <100 <100 <100 <100257........... <100 3200 25600 25600260 . . ..........<100 3200 25600 12800PP2. .. <100 1600 25600 12800Gil. .. <100 1600 25600 2560097 ......... <100 3200 25600 25600Clii. ..........<100 3200 25600 25600Rab........... <100 3200 25600 25600256........... <100 3200 25600 25600Ky........... <100 <100 12800 6400

* Titers indicated as reciprocals.

ployed 37 antisera and found that 22 separate groups were formed; some ofthese groups had but a single member, the largest 10.Meader, Robinson, and Leonard (1925), by means of agglutinin-adsorption,

stated that the B. pyocyaneus group was serologically uniform. They foundwide variations in agglutinability in different sera, but adsorption with any ofthe isolates brought about complete reduction in both homologous and heterol-ogous reactions.

In our study antisera were prepared against one isolate of P. polycolor (G11)and 3 of P. aeruginosa (Birk., Rab. and 257). Agglutination tests were per-formed with these 4 immune sera and the 17 isolates available. It is to beobserved in table 2 that there are two large groups demonstrable. Included in

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638 R. P. ELROD AND ARMIN C. BRAUN

the first group are those organisms which agglutinate in only anti-Birk. serum,but in none of the other three sera. This group includes: A2, OSU, Chick., A3,W, Al, and Birk. In the second group are those organisms which failed toagglutinate in Birk. antiserum but agglutinated in each of the other three sera.Rab., 260, 97, Chi., 256, 257, G11, and PP2 comprise this group. Only twoorganisms fall outside of these two groups. The isolate RIH seems more closelyallied to the first inasmuch as it failed to agglutinate in any of the sera, whereasKy. appears closer to the second as it agglutinated strongly in both Gll andRab. antisera but not in 257 antiserum. In each case the heterologous reactionswere as strong, or nearly so, as the homologous. Complement fixation testshave confirmed every detail of the agglutination experiments.

TABLE 3Agglutinin-adsorption experiments

Gil ANTISERUM ADSORBED 257 ANTISERUM AD- RAB. ANTISERUM ADSORBED BIRK. ANTISERUMORGANISM AG- ITH SORBED WIH WITH ADSORBED WITH

GLUTINATED

Rab. 257 PP2 Birk. Gll PP2 Birk. Gll 257 PP2 Birk. Gll 257 Chick.

257..... <100 <100<10025600 400 800 3200< 100 < 100 < 100 25600 *260. <100<100 <10025600 400 800 3200<100 <100<100 12800PP2 ....... < 100 1600< 100 25600 < 100 < 100 1600 < 100 3200 < 100 12800Gil ....... < 100 3200 < 100 25600 < 100 < 100 1600 < 100 3200 < 100 2560097 ......... < 100 < 100 < 100 25600 800 800 1600 < 100 < 100 < 100 25600Chi. < 100 3200 < 100 25600< 100 < 100 1600 < 100 1600 < 100 25600Rab. < 100 3200 < 100 25600 < 100 < 100 3200 < 100 3200 < 100 25600256...... <100<100<10025600 800 800 3200<100 <100 <10025600Ky.<... <100 1600 <100 1280 <100 1600<100 6400Birk 3200 3200 <100A2.....3200 3200 <100Chick no3200 1600a<100A3.....1600 3200 <100W.....3200 3200 <100Al.....1600 1600 <100OSU... 1600 1600 <100RIH....

*Blank =no agglutination.

Agglutinin-adsorption tests have revealed that certain isolates are antigenicallyidentical and others apparently so, but have failed to confirm the opinion ofMeader et al. (1925) that by this method the group is serologically homogeneous.The results of the adsorption experiments are found in table 3.With the 4 prepared sera certain facts are evident. By reciprocal adsorption

the P. aeruginosa culture, Rab., and the P. polycolor isolate, G11, are antigeni-cally identical. Likewise, the other strain of P. polycolor, PP2, is serologicallyvery close to these two organisms. Adsorption with this bacterium removes allthe agglutinins from both Gll and Rab. antisera. That the Gil, Rab., 257agglutination group is very likely complex is demonstrated by the adsorptionof Gll serum with 257 and the latter antiserum with Gil. In each case there

PSEUDOMONAS AERUGINOSA

is a severe reduction in the homologous titer, but some residual agglutinins areleft. G11 and Rab. apparently have factors in excess of those common to 257,and, at the same time, 257 has factors in excess of the other two.

Adsorption with the non-agglutinating (in Gil and Rab. antisera) Birk. failedto remove any agglutinins, either homologous or heterologous. Likewise, ad-sorption of anti-Birk. serum by G11 and 257 did not reduce the titer for Birk.nor remove any of the heterologous agglutinins.The above adsorption experiments add evidence to the facts obtained from

the agglutination and complement fixation studies. By them it is possible todemonstrate common antigenic factors, and at the same time the two serologicalgroups are further emphasized. It is apparent that one of the groups (G11Rab., 257, etc.) can be split still further by means of adsorption. The same maybe true of the Birk. group.

PHYTOPATHOGEMICITY OF PSEUDOMONAS AERUGINOSA

Inasmuch as P. polycolor had been isolated from tobacco, this plant becamethe choice in testing the phytopathogenicity of our P. aeruginosa cultures. Wehave produced on tobacco all of the symptoms described by Clara with certainof the P. aeruginosa isolates, as well as the two alleged P. polycolor organisms.Koch's postulates can always be fulfilled in regard to these experiments. Clara(1934) with his one B. pyocyaneus strain failed to get infection on any of theplants he tested, including tobacco.

Inoculation of the plants by needle puncture (both stem and leaves), leafsmears, and spraying were all effective to varying degrees. The most favorablemethod was that of the smear. The application of a loopful of a fresh agar slopeculture to the surface of the leaf produced severe necrosis (fig. 3), followed bythe destruction of the whole leaf. This technique was efficacious 100 per centof the time and with all 17 isolates. A culture of P. fluorescers produced feeblelesions (easily distinguished from those caused by P. aeruginosa) by the smearmethod, but none by any other means. This "blunderbuss" method probablyis most effective due to the amount of the inoculum used and to the transfer ofcertain toxic metabolites. Needle puncture inoculations into the leaves of youngplants produced lesions in about 40 per cent of the cases. Usually only thehighly pigmented forms were active by this method. Stem punctures broughtabout a destructive soft-rot and wilt (fig. 2), typical of that seen in the seed bedsby Clara (1930). Here again the pigmented forms were the most potent.Both smear and needle puncture inoculations, although accepted phyto-

pathological techniques, are subject to criticism. In neither case can one becertain that the organisms are actually invasive. The inefficiency of the needlepuncture technique compared with that of the smear method seems due both tothe introduction of a smaller amount of inoculum and to a decrease of toxicproducts, both of which apparently aid in infection. The introduction of theinoculum by spraying obliterates these objections and at the same time servesas a better index of the invasiveness of the organism. In our hands (and inClara's), however, spray inoculations have not proved especially effective. Nev-

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R. P. ELROD AND ARiIN C. BRAUN

ertheless, we have been able to produce the disease by this method (fig. 1). Theefficiency of the method can be greatly increased by first water-soaking theleaves of the plant. In such water-soaked tissues infection took place readilyand there was an active multiplication and spread of the bacteria. This re-sulted after 3 or 4 days in large, brown, necrotic areas.

Clara clearly recognized the necessity of certain predisposing environmentalfactors before P. polycolor became excessively destructive. He was not able,however, to define these. We have found that a temperature of 220 to 250C.(and probably higher), a highly humid atmosphere, as well as a water-soakedcondition, are beneficial for the establishment and spread of the organisms in thetobacco plant. The phenomenon of water-soaking undoubtedly plays a majorrole in promoting a spread of the organisms under natural conditions. Braunand Johnson (1939) have observed extensive natural water-soaking due tointernal pressures in tobacco plants grown in seed beds as well as in many otherplant species during periods of high humidity and high soil moisture. Clayton(1936) has reported that water-soaking results under field conditions from theimpact of hard-driven rain on the tobacco leaf surface. When these meteoro-logical conditions prevail, an epiphytotic can easily result. All these conditionsdo occur in the Philippines where damage resulting from this disease has beenmost severe.We have noted, as did Clara, that the lower leaves on the plant are the most

easily infected; this is due in part to the greater ease with which these leavesbecome water-soaked. The disease in the field is probably spread by means ofrain-splashed contaminated soil and plants. The susceptibility of the lowerleaves aids, therefore, in the spread of the organism.The linking of B. marginale to B. pyocyaneus by Mehta and Berridge (1924)

prompted us to attempt infections of the plants attacked by B. marginale withour P. aeruginosa isolates. This organism was originally isolated by Brown(1918) from lettuce. It usually manifests itself by a marginal wilt of the leavesand a progressive soft-rot. Red spots and streaks, not usually necrotic, areoften scattered over the leaves. According to Dowson (1941), this organismis also effective in causing storage rots, and he lists the organism with theErwinia carotovora group in this respect. He found potato, onion, and cucumberto be effectively attacked.Spray inoculations of lettuce by P. aeruinosa (certain isolates) produced the

same type of rot and wilt that was held due to B. marginale (fig. 4). The reddishspots were readily produced. It is our opinion that the red coloration is due tothe formation of acid pyocyanin. Here again the pigmented forms were mosteffective. A large number of the cultures produced a rot of potato, cucumber,and onion seemingly identical with that ascribed to B. marginale. In theseexperiments sterile slices of the above-mentioned vegetables were inoculated by

8 Water-soaking refers to the injection or flooding of the intercellular spaces of planttissues with water. This condition was induced artificially by us by either driving waterinto the stomata of plants with an atomizer or by the application of water under pressureto the root system or cut stem ends of tobacco plants.

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PSEUDOMONAS AERUGINOSA 641

smearing the culture. Incubation was carried out at room temperature for aweek or more. The rot produced is very similar to that caused by E. carotovorabut progresses more slowly. This action is very likely due to the liberation ofprotopectinase.Not all of the 17 isolates affect the plants, nor are the effects always the same.

For the most part the highly pigmented organisms are the most infective. How-ever, pyocyanin isolated as the pure base has not evoked any of the symptomsdescribed. A summary of the plant inoculations is found in table 4. It wasalso noted that, whereas rough forms of the various strains had lost their patho-genicity for animals, they had retained the ability of attacking plant tissues.Passage through plants did not alter the effectiveness of the phytopathogenic

TABLE 4Plant inoculations

TOBACCO

ORGANISM TESTED ONION CUCUMBER POTATO LETTUCENeedle Smear Spraypuncture

Gil . + + + + + + +PP2................. + + + + + +Rab. + + + + 4 + +Birk.+ + + + + + +Chick.+ + + 4 + + +97........... - + - - + - -

260.- + - + -

257.......... - + -

256.................. - + - + +

A3..+ + +Chi.............. + + + + + +_osU.......... + + +-Ky.+ + -_ +_RIH..............+ + + + + + +

+, good reaction; [, weak reaction;-, no visible change; blank = not done.

powers of the organisms. Likewise, plant passage did not detract from theability of the various isolates to infect animals.

DISCUSSION

From the results of the biochemical and serological tests it is evident that thetwo isolates of P. polycolor are indistinguishable from certain strains of P.aeruginosa. The heterogeneity of the antigenic character of the group and theminor inconsistencies in the fermentative abilities do not detract from thisrelationship. Also, the production of pyocyanin by the two plant cultures con-firms still further their true identity. Likewise, the pathogenicity for smallanimals of P. aeruginosa is also shared by P. polycolor. From the above facts

R. P. ELROD AND ARMIN C. BRAUN

it would be impossible to separate P. polycolor from P. aeruginosa, and we con-clude that the two are identical.The similarity between the plant and animal isolates is further emphasized by

plant inoculations. Both produce similar pathological conditions on tobacco.The lesions produced by us experimentally are identical with those described byClara as occurring naturally and produced in the laboratory by him with P.polycolor. The production of soft-rot of onions, cucumbers, and potatoes byour cultures links them with B. marginale. Mehta and Berridge (1924), how-ever, have already pointed out the similarity of B. pyocyaneus and B. marginale.Their experiments, and ours, would indicate that the 3 organisms are the same.In view of the serological heterogeneity of the group, the agglutination resultsof Brooks, Nain, and Rhodes (1925) would not invalidate this relationship.

Infection of plants, as of animals, by P. aeruginosa occurs best in a weakenedhost. Thus, a storage rot must be considered as a passive pathogenic process,implying little more than the ability to grow and excrete certain products (inthis case the enzyme protopectinase) for inducing the pathological condition.In actively growing plants, also, a weakened state is conducive to good infection.Water-soaking must be looked upon as a debilitory state. Likewise, excessivelyhigh temperature and high humidity contribute to a weakened condition. Itmust be remembered, too, that the less actively growing lower leaves are themost susceptible. Such debilitating conditions have their analogies in virtuallyall P. aeruginosa infections in animals, wherein the organism serves either as asecondary invader or attacks a previously weakened host, e.g., undernourishedchildren.

Neither in its plant pathogenicity, nor in its animal infections, can P. aeru-ginosa be considered an aggressive pathogen. As we have pointed out, theamount of the inoculum must usually be excessive or the host weakened. Butin plants, as well as in animals, when conditions are favorable, the disease canbe acute and widespread. The presence of P. aeruginosa in the soil very likelyserves as a continual reservoir of infection. The susceptibility of the lowerleaves likewise would aid in spreading and maintaining the disease. Brown(1918) recognized B. marginale as a soil organism and was confident that infec-tion does occur by contaminated soil. The marginal aspect of the disease inlettuce would seem to bear this out. Soil contamination could well account forthe presence of the organism in storage rots.

It appears likely that the phytotoxic factors of the organism are not the sameas the toxic substances that induce animal disease. This was emphasized by theaction of rough variants which, though not fatal to animals, retained theirpathogenicity for plants. To date we have not been able to isolate the phyto-toxic factor. Patty (1921) made an interesting observation concerning theproduction of HCN by B. pyocyaneus in culture and in the animal body. Ifthis material is produced in sufficient quantity, it might well account for thenecrotic lesions in plants.

It has been our experience, and that of others, to lose rabbits occasionallyduring a course of immunization with phytobacteria. Such deaths are ap-

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PSEUDOMONAS AERUGINOSA

parently due to an accumulative toxemia, the animals rapidly losing weightand becoming less active. On autopsy the organisms are not recoverable. Suchis not the. case with P. aeruginosa where a fulminating bacteremia is produced;showing the ability to adapt conditions within the animal body. In theplant, also, the organism thrives and multiplies. Without doubt the resultinglesions and other evidences of destruction are due to the production of toxicfactors. Nevertheless, this in no way detracts from the ability of the organismsto adapt themselves to the conditions as set up in the plant. We have, there-fore, in P. aeruginosa a bacterium which serves as a dual pathogen, and which,we believe, is unique in the field of bacteriology.

SUMMARY

Two isolates of Phytomonas polycolor have been found to be indistinguishablefrom cultures of Pseudomonas aeruginosa.On the basis of pyocyanin formation, growth at 37TC., and animal patho-

genicity, they react as does P. aeruginosa. Agglutination, complement fixation,and agglutinin-adsorption experiments have shown the two plant organisms tobe serologically identical with at least one animal isolate and closely allied toothers. A brief biochemical comparison has also indicated singleness of thegroup.We have failed to confirm the view of Meader et al. that by agglutinin-adsorp-

tion the P. aeruginosa group is serologically uniform.Fifteen isolates of P. aeruginosa derived from many sources have shown the

ability to attack tobacco. The type of lesions produced is identical with thatproduced by P. polycolor. Many of these organisms produce a soft-rot ofvegetables like that ascribed to Bacterium marginale. We concur with Mehtaand Berridge in the identity of P. aeruginosa and B. marginate.The ability of P. aeruginosa to thrive in plant tissues as well as in warm-

blooded animals makes it unique in the field of bacteriology.

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Aon, K. 1926 Agglutinatorische Einteilung von Pyocyaneus-Bazillen, welche beiverschiedenen Menschenerkrankungen nachgewiesen wurden. Zentr. Bakt. Para-sitenk. I, 98, 186-495.

BALDACCI, E., AND CIFERRI, R. 1934 Intorno alla pathogenicity di alcuni batteriidell'uomo per il frutto del Pomodoro. Boll. soc. ital. biol. sper., 9, 197-200.

BENHAM, R. W., AND KESTER, B. 1932 Sporotrichosis, its transmission to plants andanimals. J. Infectious Diseases, 50, 437-458.

BERGEY, D. H., BREED, R. S., AND MURRAY, E. G. D. 1939 Manual of DeterminativeBacteriology, 5th Ed., The Williams & Wilkins Co., Baltimore.

BRAuN, A. C., AND JOHNSON, J. 1939 Natural water-soaking and bacterial infection.Phytopathology, 29, 2-3.

BROOKS, R. ST. J., NAIN, K., AND RHODES, MABEL 1925 The investigation of phytopatho-genic bacteria by serological and biochemical methods. J. Path. Bact., 28, 203-209.

BROWN, N. A. 1918 Some bacterial diseases of lettuce. J. Agr. Research, 13, 367-388.CIFERRI, R., AND BALDACCI, E. 1934 Intorno alla pathogenicity di alcuni miceti dell'uomo

per il frutto del Pomodoro. Boll. soc. ital. biol. sper., 9, 200-202.

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CLARA, F. M. 1930 A new bacterial leaf disease of tobacco in the Philippines. Phyto-pathology, 20, 691-706.

CLARA, F. M. 1934 A comparative study of the green-fluorescent bacterial plant patho-gens. Cornell Univ. Agr. Expt. Sta. Mem., 159.

CLAYTON, E. E. 1936 Water-soaking of leaves in relation to development of the wildfiredisease of tobacco. J. Agr. Research, 52, 239-269.

DEeS, S. V. 1935 Stinking rot of sugarcane. Indian J. Agr. Sci., 5, 387-392.DOWSON, W. J. 1941 The identification of the bacteria commonly causing soft-rot in

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ton-Century Co., New York.

PLATE I

(Photographs by J. A. Carlile)FIG. 1. A leaf of tobacco from a plant inoculated by spraying. Lesions are typical

necrotic type. Culture Rab.FIG. 2. a. Control tobacco plant inoculated into the stem with P8eudomonasafuorescens.b. Tobacco plant inoculated by stem puncture. Culture Birk.FIG. 3. Tobacco plant inoculated by smear method. Culture 257.FIG. 4. Leaf from lettuce plant inoculated by spray technique. Culture Chick.

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