plant inoculations, to fulfil Koch's postulates, are

Transcription

1 THE VALUE OF SEROLOGICAL TESTS FOR THE IDEN- TIFICATION OF PSEUDOMONAS MALVACEARUM Wellcome Tropical Research Laboratories, Khartoum Received for publication April 15, 1931 The chief objects of the present investigation were to make a serological study of various strains of P. malvacearum and to determine the specificity of the agglutination test in the identification of this organism, for it was felt that sonie such test was desirable in view of certain important bacteriological studies in these laboratories concerned with the epidemiology of Black Arm disease under field conditions. To those acquainted with bacteriological investigations of P. malvacearum, many difficulties present themselves, more especially with regard to the definite identification of this organism. In the literature, stress has been laid on its cultural characteristics on various media, its optimum temperature, thermal death point, staining reactions etc. but however helpful these may be, it has hitherto been the accepted rule in these laboratories that successful plant inoculation experiments were essential before an organism possessing such characteristics could be positively stated to be P. malvacearum. Unfortunately, plant inoculations, to fulfil Koch's postulates, are time consuming, and may give unreliable results for several reasons; chief among these are unfavourable climatic conditions at certain seasons, when low soil temperature and high humidity do not prevail; moreover, the difficulty of obtaining clean seed for raising healthy plants must be borne in mind. To render this enquiry complete it was deemed advisable to obtain not only strains of P. malvacearum from different districts of the Sudan, but also from other countries. Thanks to the assistance of the Curator; National Collection of Type Cultures, Lister Institute, it was possible to obtain strains other than those of Sudan origin. 287 JOURNAL OF BACTERIOLOGY, VOL. XXII, NO. 4

2 288 The majority of the strains were isolated from different sources in the Sudan and conformed in all morphological and cultural characteristics to the standard description of Ps. malvacearum. ISOLATION AND PROVENANCE Several strains were supplied by Mr. Massey, Government Botanist; the remainder were isolated from fresh boll lesions. The strains isolated by, or received from, Mr. Massey are numbered XI to XII, while all strains marked Y were isolated by the writer from green bolls coming from the Gezira (December, 1930): (XI) Gubba boll lesion. (X2) From Shambat lint twelve months old; pathogenicity proved. (X3) From flood water; pathogenicity proved. (X4) From suspension of a Shambat lint culture which remained alive in distilled water for a month. (X5) Received as a "rough" strain of X2. (X8) Received as a "smooth" strain of X2. (X7) Gezira flood water; pathogenicity proved. (X8) A Shambat strain; pathogenicity proved. (X9) Egyptian strain; pathogenicity proved. (X10) Kassala strain. (Xll) An old Shambat strain which was isolated several years ago and has remained constant in cultural characteristics and pathogenicity. Yl to Y9. All these strains were isolated from boll lesions from Gezira Research Farm, Wad Medani. Method of isolation. After many experiments it was found that the most satisfactory method was as follows: Soak the green boll in absolute alcohol and flame, open under sterile condition, remove small pieces of the infected areas inside with dissecting needles, and place on potato slopes. (The same method is applied to the infected lint.) Growth was usually visible in twentyfour hours, and subcultures on to fresh potato slopes were immediately made. This was found necessary as in many cases a growth of Malvacearum which was visible in twenty-four hours had been completely overgrown in thirty-six to forty-eight hours by other concomitant organisms. The potato slopes were plated out on agar or McConkey

3 IDENTIFICATION OF P. MALVACEARUM 289 medium and single colonies picked. It was found extremely difcult in many cases to obtain a pure culture, as Malvacearum often seems to exist in closest association with a Gram-positive bacillus; even, in some cases, what appeared to be single colonies of Malvacearum on subculture showed the other organism. Other pigmented organisms. It is well known that other yellow pigmented and non-pigmented organisms of the Pseudomonas group are associated with Malvacearum in the plant lesions. Several of these (P1, P2, P3, P4, P5) were isolated with a view to studying the serological association, if any, with Malvacearum. In addition to the above, the following strains were received from the National Collection of Type Cultures: Campestre (EFS), Campestre (Paine), Malvacearum (EFS), Malvacearum (Trinidad), Hyacinthi, and Pear Blossom. Antisera were readily prepared by giving rabbits three or four weekly intravenous inoculations of a moderately thick suspension of the organism. Both living, and phenolized emulsions were used for inoculation; there appeared to be no significant difference in the final titer. Sera were prepared against X3, X5, Xll, Y2 (Malvacearum EFS). Agglutination was carried out by the macroscopic method the tubes being left six hours in a water bath at 52 C. and afterwards on the bench for twenty-four hours. Final readings were made after twenty-four hours, the final titer being taken as the last trace of clumping visible with a hand lens. This was quite easy as the control tubes of the suspension always remained perfectly homogenous. The results with Malvacearum are given in table 1. Type of agglutinins. As would be expected from flagellate organisms, both floccular and granular or somatic agglutinins were present in all antisera. The so-called Rough Strain X5 gave a perfect emulsion and like the other strains contained both agglutinins. There was no trace of the "R" agglutinin and the strain is undoubtedly a normal smooth one but with a tendency towards formation of granular looking colonies. Agglutination emulsions. Twenty-four-hour agar plate cul-

4 290 tures emulsified readily in saline. These were used fresh or, in some cases, made up with 0.5 per cent phenol for floccular agglutination and with alcohol (Bien's method) for granular agglutination. Agglutinin absorption tests. Forty-eight-hour agar plate cultures were used, as growth after twenty-four hours is too scanty for the purposes of making strong emulsions. For the absorption dose at least 15 plates were used for every cubic centimeter of SERA HOMOLOGOUS TITER TABLE 1 x1-x11 ALL STRAINS FROM y1-y9 X3 5,000 f., g.* 5,000 5,000 X5 2,500-f., g. 2,500 2,500 Xll 2,500 f., g. 2,500 2,500 Y2 2,500 f., g. 2,500 2,500 * f-floccular agglutination. g - granular or somatic agglutination. TABLE 2 RBSIDUAL AGGLUTINA- SERUM ABSORBED BY TION AGAINST AL STRAINS Xli Xli Nil (L50) Xli XI, X2, X3, X4, Y7, Y8 Nil (L50) X3 X3 Nil (L50) X3 Xl, X2, X6; X9, XIO, Y7, Y8, Y9 Nil (LS0) Y2 Y2 Nil (LS0) Y2 Y9, Y8, Y7, X2, X6, X9, X10 Nil (L50) serum-doses smaller than this were found insufficient to clear all agglutinins. The absorbing emulsions were left in contact with the serum for at least six hours, of which one hour was spent at and the remainder at room temperature (average 3200.). As absorptions were in all cases perfectly regular it will be sufficient to give a summary of the results. It will be seen from table 2 that absorption is completely reciprocal, any one strain removing all agglutinins from a given serum, both for itself and for all other strains. There would thus

5 IDENTIFICATION OF P. MALVACEARUM appear to be complete antigenic identity of all the Sudan and Egyptian strains of P. malvacearum. Other strains. P. hyacinthi, Pear Blossom and Campestre (Paine) emulsions were all negative against any Malvacearum serum. Malvacearum (Trinidad) agglutinated up to full titer of all sera and appeared to be antigenically identical with the Sudanese strains. SERUM TABLE 8 SERUM EMUSIION TIR EFS Y2 125 tr. EFS Y9 125 tr. EFS X5 125 tr. EFS Xli 125 tr. EFS EFS 5,000 Xli EFS 0 X3 EFS 0 Y2 EFS 0 TABLE 4 EMULIDONS P1 P2 P3 P4 Xli X Y Malvacearum (EFS). This strain gave anomalous results. Cultures on agar did not appear typical of Malvacearum, being white in appearance with an entire absence of stickiess when touched with a loop. The organism grew with great difficulty on potato agar slopes. Like other Malvacearum strains it had no action on carbohydrates. (See table 3.) It is difficult at present to explain this one-sided agglutination; possibly the American strains differ somewhat from those of the Sudan, or else this strain has altered antigenically with repeated subculture. 291

6 292 Yellow pigmented organism Pl, 2, 8, 4 isolated from green bols were tested against Malvacearum sera with results as shown in tables 4 and 5. It is obvious that no kind of antigenic relation exists between these organisms and Malvacearum. DISCUSSION There are a few references in the literature to the serological study of members of the Pseudomonas group, in particular the TABLE 5 TABLE 6 To exclude the possibility of non-specific (normal) agglutination with Malvacearum BBEIRUM ZMULSbIONS Y1.Y2.. Normal rabbit (6 specimens) Normal guinea pig (3 specimens) No agglutination with any of the sera (1:25 dilution) Normal human (6 specimens) group of yellow pigmented orga isms of which Malvacearum is a member. Brooks et al. (1925) showed a remarkable degree of cross relationship between some strains of Campestre, Malvacearum and Phaseoli. Link and Link (1928) state that the agglutination test can be used to distinguish Malvacearum from other yellow organisms such as Campestre, Phaseoli, Citre, etc., while Malvacearum is more closely related to Sojense than to Campestre. Like Brooks, they place Malvacearum, Campestre, and Phaseoli in one serological group. Y9

7 IDENTIFICATION OF P. MALVACEARUM 293 The group relationship of Malvacearum scarcely falls within the scope of the present paper but a few agglutinations carried out between Malvacearum and Campestre (Strain EFS) showed some degree of cross agglutination. With Campestre (Paine), Hyacinthi, Pruni, the results were completely negative. Strains of Phaseoli were unfortunately not available. SUMMARY AND CONCLUSIONS 1. Plant inoculation experiments for the identification of P. malvacearum may give misleading results. 2. The agglutination test is a simple and highly specific method for the identification of this organism. 3. All African strains examined fall into one serologicallyhomogenous group. 4. There is no serological relationship between P. malvacearum and the yellow pigmented organism commonly found in association with it in the lesions of Black Arm disease. I would like to thank Major R. G. Archibald, C.M.G., D.S.O., Director of the Wellcome Tropical Research Laboratories, for many useful suggestions and assistance during the course of the above work, and to Mr. R. E. Massey, Government Botanist, I am much indebted for kindly providing many of the strains used. REFERENCES BROOKS, NAIN AND RHODES Jour. Path. and Bact., 28, 203. LINK AND LINK Bot. Gazette, 85 (2), 178.