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Serologic studies of the maize stalk rot pathogen Erwinia carotovora f. sp. zeae
Zbl. Bakt. II. Abt., Bd. 133, S. 80-85 (1978)
[Department of Botany, Ranchi University, and Department of Plant Pathology, Ranchi Agricultural College, Kanke, Ranchi, India]
Serologic Studies of the Maize Stalk Rot Pathogen Erwinia carotovora f. sp. zeae M. Prasad and S. K. Sinha
Summary With the help of serological techniques, namely microprecipitin, agglutination, and gel diffusion, sixteen isolates of maize stalk rot pathogen were proved to be identical. Serological techniques have thus been utilized as an additional tool for identifying the pathogen Erwinia carotovora f. sp. zeae. It was also established that the pathogen does not perpetuate in the seed, either externally or internally. It could, however, be found through serological tests that infected ears carried the pathogen and transmitted it as a contaminant to the healthy seeds.
Zusammenfassung Mit Hilfe serologischer Techniken, wie Mikrofallung, Agglutination und Geldiffusion, wurden sechzehn Isolate des Maisstengelfaule-Erregers als identisch ermittelt. Serologische Techniken wurden so als ein zusatzliches Instrument zur Identifizierung des Erregers Erwinia carotovora f. sp. zeae benutzt. Es wurde auch festgestellt, daJ3 del' Erreger sich nicht immerwahrend in dem Saatgut aufhalt, wedel' auJ3erlich noch innerlich. Es konnte jedoch durch serologische Priifungen festgestellt werden, daJ3 infizierte Ahren den Krankheitserreger als Verunreinigung auf gesunde Saat iibertrugen.
Serology has found wide application in the identification and classification of phytopathogenic bacteria (FRIEDMAN 1953; MUSHIN, NAYLOR, and LAHOVARY 1958; LOVREKOVICH and KLEMENT 1961; LOVREKOVICH, KLEMENT, and DOWSON 1963; GUTHRIE, HUBER, and FENWICK 1965; SAVULESCU and LAZAR 1967; GUTHRIE 1968; MORTON, DUKES, and JENKINS 1965; LUCAS and GROGEN 1969; SKRYPAL 1969; OTTAL and ENGLISH 1969, 1971; HILDEBRAND 1971; COLENO, NORMAND, and BABZIC 1972; NOMEKATA and OLIVERIA 1972; GRAHAM 1971; LAZAR 1968,1972; SAALTINK, SLOGETEREN, and KAMERMANN 1972). It is proposed here to present the techniques of serological studies, made on the maize stalk rot pathogen Erwinia carotovora f. sp. zeae Sabet (redesignated as Er'l})inia carotovora var. chrysanthemi [(BURKHOLDER et al.; Dye) by DYE 1969)]. Findings with regard to the establishment of taxonomic identity and the presence of this phytopathogenic bacterial form in the seeds have been included.
Serologic Studies of the Maize Stalk Rot Pathogen
81
Materials and Methods Agglutination, microprecipitin, and OUCHTERLONY (1949) gel diffusion techniques have been adopted.
a) Production of antigen The four bacterial isolates of Erwinia caroto'Vora f. sp. zeae, namely 84(1-1), 88(1-5), 89(1-6), and 93(1-10), obtained from Prof. Dr. A. KELMAN of the University of Wisconsin, U.S.A., who had originally received them from Dr. PAYAK oflARI, New Delhi, were used for preparation of the antigens. Each of these isolates was pre-tested for their virulence and pathogenicity on maize plants. Each antigen-source was first streaked on agar for singlecolony isolation and afterwards increased in broth shake culture. After 36 hours the culture was centrifuged at 2500 g and washed twice in sterile saline water. It was re-centrifuged and finally suspended in 50 ml of formaline saline water. The bacterial antigen was then diluted to be adjusted to a 3 or 4 and 6 McFarland. This was estimated against a McFarland scale.
b) Preparation of antisera i) Immunization of rabbits without adjuvant. Healthy white adult rabbits, 2 to 3 kg in weight, without damaged ears were selected and maintained in a sanitary environment with adequate nutrition. After few days of acclimatization to the new surroundings they were used for immunization. A pre-immunization normal serum was collected from the heart of the rabbit prior to antigen injection. The serum was stored by freezing after proper labelling. Immunization schedule, which was followed in the present study, was as follows Day
Volume of antigen 4 McFarland
1 4 8 11 15
0.1 0.3 0.5 1.0 2.0
ml ml ml ml ml
These amounts were inoculated into the ear of the rabbit, beginning at the extremity of the ear with subsequent injections nearer to the base of the ear. A 5 ml syringe with 25 gauge needle was used for this purpose. All the time sterile equipment and aseptic technique was followed. Five days after the last antigen injection a test bleeding was made and the antibody content was determined in comparison to the initial bleeding. Dilutions of antiserum of 1: 640 was found active in agglutination test. ii) Immunization schedule with adj uvant. Equal parts of Freund's complete adjuvant and antigen (6 McFarland density) were emulsified by repeated passing through 18 gauge needle in a 20 ml syringe. When the emulsion became milky and a drop of it could be held in water forming bubbles, then it was used for injection. 0.5 ml of emulsified bacterial suspension was injected subcutaneously in the shoulder region of the rabbits at 5 different sites. For this, a 20-gauge needle was used for penetrating the previously shaved, disinfected skin. After 4 to 6 weeks, the antisera were collected and titers were determined by agglutination test and one of 0.5 ml of 3 McFarland density was given if the titer was of inadequate strength after being observed in test bleeding. Again, test bleed was made after 3 - 6 days and finally the antisera was harvested from the heart of the rabbits. The blood was allowed to clot at room temperature. The clotted blood was then centrifuged and the antiserum was withdrawn, leaving small pellets of erythrocytes at the bottom of the tube. The antisera were stored, after proper labelling, either by freezing or were mixed with 1 : I part of glycerol and kept in a refrigerator. The antisera were prepared from the bacterialisolates 84 (I-I) of Udaipur, Rajasthan; 88 (I-5) from Pantna. gar, U.P., 89(1-6) of Pantnagar, U.P., and 93 (1-10) from Dhaulakuan, H.P. 6
ZbI. Bakt. II. AM., Bd. 133
82
M. PRASAD and S. K. SINHA
I. Taxonomic identification of the pathogen For ascertaining similarity among isolates, sixteen of the isolates of Erwinia carotovora f. sp. zeae Sabet, obtained from maize plants (sources mentioned in Table 1) which included also the four mentioned above from which the antisera were prepared, were tested serologically by applying the microprecipitin, agglutination, and gel diffusion tests. Two more isolates of Erwinia carotovora obtained from potato were used as a check. Antigens of the sixteen isolates were tested with the antisera of the four isolates. For microprecipitin test equal quantities of 1 : 10 antisera and 1 : 10 antigen, placed in the well of serological plates, were mixed for 30 minutes at 25 °0. Each test was examined under 10 X dissecting microscope. The tube agglutination tests were made in 5 X 50 mm tubes, containing equal parts of I : 100 antisera and 1: 10 dilution of concentrated antigen at 25 °0. The reactions were examined at the end of 1 and 4 hours. For gel diffusion test, 0.2 ml of antiserum was placed into the centre of the well of the agar plate (15 g of agar was dissolved in 1000 ml of saline 8.5 g NaOl. To this melted agar, 10 ml of 1 % merthiolate and 10 ml of Orange G 1 % aqueous solution were added. The pH of this agar should be 7.7 - 8.0_ 15 ml of this agar was then poured into a 10 em diameter petri dish. 15 mm diameter wells were cut with a cork borer). 0.2 ml of the prepared antigens were placed into the peripheral wells around the antiserum. The plates were placed in a humidified incubator at a temperature of 280 to 32 00 for the development of precipitation bands. However, several bands may diffuse into each other and appear as one band, reducing thereby the precision of the test. By comparing the number and site of the precipitation bands, related bacteria may be identified. All tests were repeated thrice.
II. Detection of pathogen in and on maize seeds and in ears Bacterial stalk rot and ear rot are both caused by the pathogen Erwinia cal'otovora f. sp. zeae Sabet. The test here, in order to determine the presence of the pathogen, consists of soaking a small sample of maize seed that has been surface-sterilized in a solution of chlorox and alcohol (5 %) in order to detect if the pathogen was internally seed-borne. A certain percentage of seeds were not surfacesterilized in case they were to be externally seed-borne. Such seeds were incubated in sterile water for about 36 to 48 hours. Similarly treatment was given for known ear-rot infected seeds which served as a check. A drop of the liquid surrounding the maize seeds, both in surface-sterilized as well as in non-surface-sterilized seeds, were mixed with a drop of antiserum. If the bacterium is present within or on the maize seed, rapid reproduction occurs so that within 48 hours the concentration is large enough to give positive agglutination and micropreciptin tests. In order to obtain results with greater accuracy, tests involving bacterial cultures grown on selective agar medium have also been used. This method consists of taking a sample of broth, inoculated with both surface-sterilized and non-sterilized seeds, along with known ear-rot-infected seeds as check and streaking it on solid agar plates and selecting isolates that appear to be Erwinia. The colonies were re-streaked on solid agar plates and incubated for 36 hours. A large loopful of cells from the agar surface was mixed with 1.0 ml of formalized saline solution to produce a cloudy suspension of cells. This suspension was then tested serologically, employing the agglutination and microprecipitin tests for a final determination of identity. A known isolate of Erwinia carotovora f. sp. zeae was also tested in order to confirm the above results.
Results and Discussion Table 1 shows the sources of maize varieties from which the isolates were obtained. Table 2 gives the results of serological tests, and Table 3 shows the detection of the pathogen in maize seeds. As is evident from the results presented in Table 2, the sixteen isolates of the maize stalk rot pathogen, obtained from the diseased maize plants, showed positive reaction with four antisera of Erwinia carotovora f. sp. zeae in both microprecipitin and agglutination tests and gave a distinct precipitation band with gel diffusion test. The two
83
Serologic Studies of the Maize Stalk Rot Pathogen Table 1. Isolates of Erwinia used, their source and the location from where obtained Source
Location
Symbol
1. 2.
Kishan Jowahar
3. 1. 5.
Sona Ganga-5
Experimental Station, Pantnagar University farm, Beni, Pantnager University farm, Westzone, Matkota Agronomy block, Pantnagar
K-4 J-2 S-I
CM 500 CM 601
Breeding Block, Pantnagar Breeding Block, Pantnagar
Vijay Ganga-2 CM-200
University farm, Haldi
S.N.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
G·5 1-5 1-6 V-I G-2 1-2
Model Agronomy, Pantnagar University farm, Eastzone, Matkota Farmer's plot, Rudrapur
Ganga-3 Amber
G-3 A-I J l -4 84(1-1)
University farm, Nagala, Pantnagar
J1-White Maize Maize
I.C.A.P. (Block), Pantnagar Udaipur, Rajasthan Pantnagar, Uttar Pradesh
Maize Maize
Pantnagar, Uttar Pradesh Dhaulakuan, Himachal Pradesh
Potato
Bhuvali, Nainital, Uttar Pradesh
93(1-10) P-I
Potato
Simla, Himachal Pradesh
P-2
88(1-5) 89(1-6)
Table 2. .,."licroprecipitin and tube agglutination reaction of 16 isolates of Erwinia carotovora f. sp. zeae against antisera of 84(1-1),88(1-5),89(1-6) and 93(1-10) Symbol
K-4 J·2 S-I S-I G-5 1-5 1-6 V-I G-2 1-2 G-3 A-I J l -4 84(1-1) 88(1-5) 89(1-6) 93(1-10) P-I (Potato) P-2 (Potato) M = Microprecipitin; A reaction. 6*
84(1-1)
88(1-5)
89(1-6)
93(1-10)
M
A
M
A
M
A
M
A
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + +
=
Agglutination. -
±
+ + + + + + + + =
±
+ + + + + -I-
+ + No reaction,
± =
Weak reaction,
+=
-I-
+ + + Positive
84
M. PRASAD and S. K. SINHA
Table 3. Serological detection of maize pathogen in and on maize seed by using microprecipitin and agglutination tests Source of bacterial isolates used Bacteria isolated from surface-sterilized seeds Bacteria isolated from non-surface-sterilized seeds Bacteria isolated from known affected ear rot seeds (Control) 88(I-5), a known bacterial isolate of Erwinia carotovora f. sp. zeae (also control)
1. 2. 3. 4.
+
=
positive reaction, -
=
Microprecipitin
Agglutination
+
+
+
+
no reaction.
isolates of Erwinia carotovora, obtained from potato, gave negative results in all the three tests. All the stalk rot of maize pathogen isolates gave similar serological responses by all the three methods employed; it may be concluded that, within the limitations of these tests, all isolates are serologically identical and Erwinia carotovora f. sp. zeae. The potato isolate gave negative serological reaction. Therefore Erwinia carotovora, the potato isolate, is definitely distinct from the maize pathogen E. carotovora f. sp. zeae. NOMEKATA and OLIVERIA (1972) found serological differences between Xanthomonas citri and X. citri f. sp. aurantifolia. On the other hand, SAVULESCU and LAZAR (1967) considered Erwinia cytolytica and E. chrysanthemi to be similar organisms because of serological relationships and certain other biochemical tests. Recently, LAZAR (1972) studied the serological relationship between "amylovora", "carotovora" and "herbicola." groups of the genus Erwinia. All these studies and our findings here show the significance of additional serological tests in establishing the identity of the pathogen. In another set of experiments, the results of which are presented in Table 3, the importance of serological studies becomes evident in detecting the presence of the pathogen. It was found that only ear-rot-infected seeds that were used as control gave positive serological results, both in microprecipitin and agglutination tests. We got negative results in case of surface-sterilized and non-sterilized maize seeds, thus excluding the possibility of the perpetuation of the bacterial pathogen, either attached externally to the surface of the seed or being present inside the seed. This indicates that maize seed does not carry the bacterium and, therefore, it is not the source of inoculum, either internally or externally. But the infected ear which carries the bacterium may serve as a source of contamination to healthy seeds, either during the time of threshing or as plant refuse after harvest. Acknowledgement The authors are grateful to Professor Dr. Y. L. NENE, the Head of the Department of Plant Pathology, G.B. Pant University of Agriculture and Technology, Pantnagar, U.P., for extending research facilities to the second author in his laboratory. We also extend our thanks to Professor Dr. A. KELMAN of the University of Wisconsin, U.S.A., for sending us the bacterial isolates.
References -COLENO, A. L., NORMAND, M., and BARZIC, M. R., A qualitative study on the antigens involved in the complementfixation reactions among some phytopathogenic Pseudomonas. Proc. of 3rd Internat. Conference on Plant Pathogenic Bacteria, Wageningen 1972, 143-150. DYE, D. W.: A taxonomic study of the genus "Erwinia. II. The Cartovora" group. New Zealand J. of Sci. 12 (1969), 81-97.
Serologic Studies of the Maize Stalk Rot Pathogen
85
FRIEDMAN, B. A.: Serological tests with some phytopathogenic species of Pseudomonas. Phytopatho. logy 43 (1953), 412-414. GRAHAM, P. H.: Serological studies with Agrobacterium radiobacter, A. tumefaciens, and Rhizobium strains. Arch. Mikrobiol. 78 (1971), 70-75. GUTHRIE, J. W.: The serological relationship of races of Pseudomonas phaseolicola. Phytopathology 58 (1968), 716-717. - HUBER, D. M., and FENWICK, H. S.: Serological detection of halo blight. Plant Dis. Reptr. 49 (1969),297-299. HILDEBRAND, D. C.: Pectate and pectin gels for differentiation of Pseudomonas sp. and other bacte· rial plant pathogens. Phytopathology 61 (1971), 1430. LAZAR, 1.: Serological relationships of Corynebacterium. J. Gen. Microbiol. 52 (1968),77-83. Serological relationships between "amylovora", "carotovora" and "herbicola" group of the genus Erwinia. Proc. of 3rd Internat. Conference on Plant Pathogenic Bacteria, Wagenigen 1972, 131 to 141. LOVREKOVITCH, L., and KLEMENT, Z.: Species specific antigens of Pseudomonas tabaci. Acta Microbiol. Acad. Sci. Hung. 8 (1961),303-310. - - and DOWSON, W. J.: Serological investigations of Pseudomonas syringae and P. morsprunorum strains. Phytopathol. Z. 47 (1963), 19-24. LUCAS, L. T., and GROGAN, R. G.: Serological variation and identification of Pseudomonaslachrymans and other phytopathogenic Pseudomonas nomenspecies. Phytopathology 59 (1969), 1908-1912. MORTON, D. J., DUKES, P. H., and JENKINS, S. F., Jr.: Serological identification of Pseudomonas solanacearum in four solanaceous hosts. Phytopathology 55 (1965), 1191-1192. MUSHIN, R., NAYLOR, J., and LAHOVARY, N.: Studies on plant pathogenic bacteria. II. Serology. Austral. J. BioI. Sci. 12 (1958), 233-246. NAMEKATO, T., and OLIVERIA, A. R. De: Comparative serological studies between xanthomonas citri and a bacterium causing canker on Mexican lime, X. citri f. sp. aurantifolia. Proc. of 3rd Internat. Conference on Plant Pathogenic Bacteria, Wageningen 1971, 365p. OTTAL, T. D., and ENGLISH, H.: Pathological and serological determination of Pseudomonas syringae. Phytopathology 59 (1969), 1043. - - Serology and pathology of Pseudomonas syringae. Phytopathology 61 (1971), 443-452. OUCHTERLONY, 0.: Antigen-antibody reaction in gels. Acta Pathol. Microbiol. Scand. 26 (1949), 507. SAALTINE, G.l., VAN SLOGTEREN, D. H. M., and KAMERMANN, W.: Serological identification ofbacteria in bulbs. Proc. of 3rd Internal. Conference on Plant Pathogenic Bacteria, Wageningen 1971, 365pp. SAVULESCU, A., and LAZAR, I.: Investigations on bacterium Erwinia cytolytica Chester. Revue roum. Bioi. Ser. Bot. 12 (1967), 379-383. SKRYPAL, I. G.: Serological properties of Pseudomonas sp. causal agents of bacterial disease of fruit trees. l\fykrobiol. Zh. 31 (1969), 588 - 595. Authors' addresses: Dr. MAHENDRA PRASAD, Department of Botany, Ranchi University, Ranchi - 834008 (Bihar), India; Dr. SATISH KUMAR SINHA, Department of Plant Pathology, Ranchi Agricultural College, Kanke, Ranchi (Bihar), India.
[Department of Botany, Ranchi University, and Department of Plant Pathology, Ranchi Agricultural College, Kanke, Ranchi, India]
Serologic Studies of the Maize Stalk Rot Pathogen Erwinia carotovora f. sp. zeae M. Prasad and S. K. Sinha
Summary With the help of serological techniques, namely microprecipitin, agglutination, and gel diffusion, sixteen isolates of maize stalk rot pathogen were proved to be identical. Serological techniques have thus been utilized as an additional tool for identifying the pathogen Erwinia carotovora f. sp. zeae. It was also established that the pathogen does not perpetuate in the seed, either externally or internally. It could, however, be found through serological tests that infected ears carried the pathogen and transmitted it as a contaminant to the healthy seeds.
Zusammenfassung Mit Hilfe serologischer Techniken, wie Mikrofallung, Agglutination und Geldiffusion, wurden sechzehn Isolate des Maisstengelfaule-Erregers als identisch ermittelt. Serologische Techniken wurden so als ein zusatzliches Instrument zur Identifizierung des Erregers Erwinia carotovora f. sp. zeae benutzt. Es wurde auch festgestellt, daJ3 del' Erreger sich nicht immerwahrend in dem Saatgut aufhalt, wedel' auJ3erlich noch innerlich. Es konnte jedoch durch serologische Priifungen festgestellt werden, daJ3 infizierte Ahren den Krankheitserreger als Verunreinigung auf gesunde Saat iibertrugen.
Serology has found wide application in the identification and classification of phytopathogenic bacteria (FRIEDMAN 1953; MUSHIN, NAYLOR, and LAHOVARY 1958; LOVREKOVICH and KLEMENT 1961; LOVREKOVICH, KLEMENT, and DOWSON 1963; GUTHRIE, HUBER, and FENWICK 1965; SAVULESCU and LAZAR 1967; GUTHRIE 1968; MORTON, DUKES, and JENKINS 1965; LUCAS and GROGEN 1969; SKRYPAL 1969; OTTAL and ENGLISH 1969, 1971; HILDEBRAND 1971; COLENO, NORMAND, and BABZIC 1972; NOMEKATA and OLIVERIA 1972; GRAHAM 1971; LAZAR 1968,1972; SAALTINK, SLOGETEREN, and KAMERMANN 1972). It is proposed here to present the techniques of serological studies, made on the maize stalk rot pathogen Erwinia carotovora f. sp. zeae Sabet (redesignated as Er'l})inia carotovora var. chrysanthemi [(BURKHOLDER et al.; Dye) by DYE 1969)]. Findings with regard to the establishment of taxonomic identity and the presence of this phytopathogenic bacterial form in the seeds have been included.
Serologic Studies of the Maize Stalk Rot Pathogen
81
Materials and Methods Agglutination, microprecipitin, and OUCHTERLONY (1949) gel diffusion techniques have been adopted.
a) Production of antigen The four bacterial isolates of Erwinia caroto'Vora f. sp. zeae, namely 84(1-1), 88(1-5), 89(1-6), and 93(1-10), obtained from Prof. Dr. A. KELMAN of the University of Wisconsin, U.S.A., who had originally received them from Dr. PAYAK oflARI, New Delhi, were used for preparation of the antigens. Each of these isolates was pre-tested for their virulence and pathogenicity on maize plants. Each antigen-source was first streaked on agar for singlecolony isolation and afterwards increased in broth shake culture. After 36 hours the culture was centrifuged at 2500 g and washed twice in sterile saline water. It was re-centrifuged and finally suspended in 50 ml of formaline saline water. The bacterial antigen was then diluted to be adjusted to a 3 or 4 and 6 McFarland. This was estimated against a McFarland scale.
b) Preparation of antisera i) Immunization of rabbits without adjuvant. Healthy white adult rabbits, 2 to 3 kg in weight, without damaged ears were selected and maintained in a sanitary environment with adequate nutrition. After few days of acclimatization to the new surroundings they were used for immunization. A pre-immunization normal serum was collected from the heart of the rabbit prior to antigen injection. The serum was stored by freezing after proper labelling. Immunization schedule, which was followed in the present study, was as follows Day
Volume of antigen 4 McFarland
1 4 8 11 15
0.1 0.3 0.5 1.0 2.0
ml ml ml ml ml
These amounts were inoculated into the ear of the rabbit, beginning at the extremity of the ear with subsequent injections nearer to the base of the ear. A 5 ml syringe with 25 gauge needle was used for this purpose. All the time sterile equipment and aseptic technique was followed. Five days after the last antigen injection a test bleeding was made and the antibody content was determined in comparison to the initial bleeding. Dilutions of antiserum of 1: 640 was found active in agglutination test. ii) Immunization schedule with adj uvant. Equal parts of Freund's complete adjuvant and antigen (6 McFarland density) were emulsified by repeated passing through 18 gauge needle in a 20 ml syringe. When the emulsion became milky and a drop of it could be held in water forming bubbles, then it was used for injection. 0.5 ml of emulsified bacterial suspension was injected subcutaneously in the shoulder region of the rabbits at 5 different sites. For this, a 20-gauge needle was used for penetrating the previously shaved, disinfected skin. After 4 to 6 weeks, the antisera were collected and titers were determined by agglutination test and one of 0.5 ml of 3 McFarland density was given if the titer was of inadequate strength after being observed in test bleeding. Again, test bleed was made after 3 - 6 days and finally the antisera was harvested from the heart of the rabbits. The blood was allowed to clot at room temperature. The clotted blood was then centrifuged and the antiserum was withdrawn, leaving small pellets of erythrocytes at the bottom of the tube. The antisera were stored, after proper labelling, either by freezing or were mixed with 1 : I part of glycerol and kept in a refrigerator. The antisera were prepared from the bacterialisolates 84 (I-I) of Udaipur, Rajasthan; 88 (I-5) from Pantna. gar, U.P., 89(1-6) of Pantnagar, U.P., and 93 (1-10) from Dhaulakuan, H.P. 6
ZbI. Bakt. II. AM., Bd. 133
82
M. PRASAD and S. K. SINHA
I. Taxonomic identification of the pathogen For ascertaining similarity among isolates, sixteen of the isolates of Erwinia carotovora f. sp. zeae Sabet, obtained from maize plants (sources mentioned in Table 1) which included also the four mentioned above from which the antisera were prepared, were tested serologically by applying the microprecipitin, agglutination, and gel diffusion tests. Two more isolates of Erwinia carotovora obtained from potato were used as a check. Antigens of the sixteen isolates were tested with the antisera of the four isolates. For microprecipitin test equal quantities of 1 : 10 antisera and 1 : 10 antigen, placed in the well of serological plates, were mixed for 30 minutes at 25 °0. Each test was examined under 10 X dissecting microscope. The tube agglutination tests were made in 5 X 50 mm tubes, containing equal parts of I : 100 antisera and 1: 10 dilution of concentrated antigen at 25 °0. The reactions were examined at the end of 1 and 4 hours. For gel diffusion test, 0.2 ml of antiserum was placed into the centre of the well of the agar plate (15 g of agar was dissolved in 1000 ml of saline 8.5 g NaOl. To this melted agar, 10 ml of 1 % merthiolate and 10 ml of Orange G 1 % aqueous solution were added. The pH of this agar should be 7.7 - 8.0_ 15 ml of this agar was then poured into a 10 em diameter petri dish. 15 mm diameter wells were cut with a cork borer). 0.2 ml of the prepared antigens were placed into the peripheral wells around the antiserum. The plates were placed in a humidified incubator at a temperature of 280 to 32 00 for the development of precipitation bands. However, several bands may diffuse into each other and appear as one band, reducing thereby the precision of the test. By comparing the number and site of the precipitation bands, related bacteria may be identified. All tests were repeated thrice.
II. Detection of pathogen in and on maize seeds and in ears Bacterial stalk rot and ear rot are both caused by the pathogen Erwinia cal'otovora f. sp. zeae Sabet. The test here, in order to determine the presence of the pathogen, consists of soaking a small sample of maize seed that has been surface-sterilized in a solution of chlorox and alcohol (5 %) in order to detect if the pathogen was internally seed-borne. A certain percentage of seeds were not surfacesterilized in case they were to be externally seed-borne. Such seeds were incubated in sterile water for about 36 to 48 hours. Similarly treatment was given for known ear-rot infected seeds which served as a check. A drop of the liquid surrounding the maize seeds, both in surface-sterilized as well as in non-surface-sterilized seeds, were mixed with a drop of antiserum. If the bacterium is present within or on the maize seed, rapid reproduction occurs so that within 48 hours the concentration is large enough to give positive agglutination and micropreciptin tests. In order to obtain results with greater accuracy, tests involving bacterial cultures grown on selective agar medium have also been used. This method consists of taking a sample of broth, inoculated with both surface-sterilized and non-sterilized seeds, along with known ear-rot-infected seeds as check and streaking it on solid agar plates and selecting isolates that appear to be Erwinia. The colonies were re-streaked on solid agar plates and incubated for 36 hours. A large loopful of cells from the agar surface was mixed with 1.0 ml of formalized saline solution to produce a cloudy suspension of cells. This suspension was then tested serologically, employing the agglutination and microprecipitin tests for a final determination of identity. A known isolate of Erwinia carotovora f. sp. zeae was also tested in order to confirm the above results.
Results and Discussion Table 1 shows the sources of maize varieties from which the isolates were obtained. Table 2 gives the results of serological tests, and Table 3 shows the detection of the pathogen in maize seeds. As is evident from the results presented in Table 2, the sixteen isolates of the maize stalk rot pathogen, obtained from the diseased maize plants, showed positive reaction with four antisera of Erwinia carotovora f. sp. zeae in both microprecipitin and agglutination tests and gave a distinct precipitation band with gel diffusion test. The two
83
Serologic Studies of the Maize Stalk Rot Pathogen Table 1. Isolates of Erwinia used, their source and the location from where obtained Source
Location
Symbol
1. 2.
Kishan Jowahar
3. 1. 5.
Sona Ganga-5
Experimental Station, Pantnagar University farm, Beni, Pantnager University farm, Westzone, Matkota Agronomy block, Pantnagar
K-4 J-2 S-I
CM 500 CM 601
Breeding Block, Pantnagar Breeding Block, Pantnagar
Vijay Ganga-2 CM-200
University farm, Haldi
S.N.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
G·5 1-5 1-6 V-I G-2 1-2
Model Agronomy, Pantnagar University farm, Eastzone, Matkota Farmer's plot, Rudrapur
Ganga-3 Amber
G-3 A-I J l -4 84(1-1)
University farm, Nagala, Pantnagar
J1-White Maize Maize
I.C.A.P. (Block), Pantnagar Udaipur, Rajasthan Pantnagar, Uttar Pradesh
Maize Maize
Pantnagar, Uttar Pradesh Dhaulakuan, Himachal Pradesh
Potato
Bhuvali, Nainital, Uttar Pradesh
93(1-10) P-I
Potato
Simla, Himachal Pradesh
P-2
88(1-5) 89(1-6)
Table 2. .,."licroprecipitin and tube agglutination reaction of 16 isolates of Erwinia carotovora f. sp. zeae against antisera of 84(1-1),88(1-5),89(1-6) and 93(1-10) Symbol
K-4 J·2 S-I S-I G-5 1-5 1-6 V-I G-2 1-2 G-3 A-I J l -4 84(1-1) 88(1-5) 89(1-6) 93(1-10) P-I (Potato) P-2 (Potato) M = Microprecipitin; A reaction. 6*
84(1-1)
88(1-5)
89(1-6)
93(1-10)
M
A
M
A
M
A
M
A
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + +
=
Agglutination. -
±
+ + + + + + + + =
±
+ + + + + -I-
+ + No reaction,
± =
Weak reaction,
+=
-I-
+ + + Positive
84
M. PRASAD and S. K. SINHA
Table 3. Serological detection of maize pathogen in and on maize seed by using microprecipitin and agglutination tests Source of bacterial isolates used Bacteria isolated from surface-sterilized seeds Bacteria isolated from non-surface-sterilized seeds Bacteria isolated from known affected ear rot seeds (Control) 88(I-5), a known bacterial isolate of Erwinia carotovora f. sp. zeae (also control)
1. 2. 3. 4.
+
=
positive reaction, -
=
Microprecipitin
Agglutination
+
+
+
+
no reaction.
isolates of Erwinia carotovora, obtained from potato, gave negative results in all the three tests. All the stalk rot of maize pathogen isolates gave similar serological responses by all the three methods employed; it may be concluded that, within the limitations of these tests, all isolates are serologically identical and Erwinia carotovora f. sp. zeae. The potato isolate gave negative serological reaction. Therefore Erwinia carotovora, the potato isolate, is definitely distinct from the maize pathogen E. carotovora f. sp. zeae. NOMEKATA and OLIVERIA (1972) found serological differences between Xanthomonas citri and X. citri f. sp. aurantifolia. On the other hand, SAVULESCU and LAZAR (1967) considered Erwinia cytolytica and E. chrysanthemi to be similar organisms because of serological relationships and certain other biochemical tests. Recently, LAZAR (1972) studied the serological relationship between "amylovora", "carotovora" and "herbicola." groups of the genus Erwinia. All these studies and our findings here show the significance of additional serological tests in establishing the identity of the pathogen. In another set of experiments, the results of which are presented in Table 3, the importance of serological studies becomes evident in detecting the presence of the pathogen. It was found that only ear-rot-infected seeds that were used as control gave positive serological results, both in microprecipitin and agglutination tests. We got negative results in case of surface-sterilized and non-sterilized maize seeds, thus excluding the possibility of the perpetuation of the bacterial pathogen, either attached externally to the surface of the seed or being present inside the seed. This indicates that maize seed does not carry the bacterium and, therefore, it is not the source of inoculum, either internally or externally. But the infected ear which carries the bacterium may serve as a source of contamination to healthy seeds, either during the time of threshing or as plant refuse after harvest. Acknowledgement The authors are grateful to Professor Dr. Y. L. NENE, the Head of the Department of Plant Pathology, G.B. Pant University of Agriculture and Technology, Pantnagar, U.P., for extending research facilities to the second author in his laboratory. We also extend our thanks to Professor Dr. A. KELMAN of the University of Wisconsin, U.S.A., for sending us the bacterial isolates.
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Serologic Studies of the Maize Stalk Rot Pathogen
85
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