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Interaction of tobacco callus tissue with Pseudomonas tabaci, P. pisi and P. fluorescens
Physkdogical Pkmt Patholqv (1978) 13,65-72
Interaction of tobacco callus tissue with Pseudomonas tabaci, P. pisi and P. fluorescens JENG-SHENG
HUANG
and C. GERALD VAN DYKE
Defiartment of Plant Padwlogy, North Carolina St& University Raleigh, KC. 27650, U.S.A.
(Accepedfor publicntionJanua~ 1978)
Pm&nom tobad, a tobacco pathogen, multiplied rapidly and colonized tobacco callus within 2 days after inoculation. P. piA, a pathogen of pea but not tobacco, and P.Juoresccnr, a saprophyte, multiplied slowly and remained at inoculation sites. Rates of multiplication of these three bacteria in tobacco callus were comparable to those in leaves kept in the dark and at high relative humidity following inoculation. Scanning electron microscopy showed P. tab& cells densely distributed over the tobacco callus. P. pi.ri cells formed aggregates and were trapped in a network of fibrillae on the callus surface. Some P.&mmn.s cells formed spherical bodies, apparently as a result of damage to bacterial cell walls. The spherical bodies eventually lysed. The entrapment of bacterial cells of P. pin’ by fibrillae and the damage to the cell wall of P.jhwrescen.smay be factors which limit the rate of multiplication of these species of bacteria in tobacco callus tissue.
INTRODUCTION Tissue cultures are suitable for study of host-parasite interactions in certain fungal and viral diseases. Helgeson et al. [4] demonstrated that calluses from tobacco plants susceptible to black shank disease were susceptible to the disease-inducing organism Phytophthora parasitica var. nicotianae, and that calluses from resistant plants were resistant to the pathogen. Russell & Halliwell [13] reported that rapid loss of cell integrity exhibited by cultured tobacco Samsun NN cells to infection by tobacco mosaic virus (TMV) paralleled the local lesion response of the intact plants. They also found that over a period of time TMV multiplied in Samsun cells without damaging the host, in accordance with the systemic symptom usually observed in Samsun plants. Systems involving tissue culture and bacteria have been investigated [7] but suitable models, with the exception of crown gall [I], have not been developed. Ultrastructural studies of the interaction between bacteria and plant cells have shown that in several incompatible combinations, bacteria are attached to plant cell walls and are engulfed by fibrillar and granular materials. This phenomenon has been used to explain the localization of incompatible bacteria and cessation of bacterial multiplication in tissues undergoing the hypersensitive reaction (HR) [3, 6 151. 0048-4059/78/0701-0065 5
$02.00/O
@ 1978 Academic Press Inc. (London)
Limited
J.-S. Huang and C. G. Van Dyke
66
Callus tissues would provide an excellent working model to study bacteriaplant cell interactions since most bacteria are in the intercellular space of the inoculated tissue, and do not penetrate host cells. The development of scanning electron microscopy (SEM) has made it possible to examine interactions between bacteria and the surface of callus cells, free of contaminating micro-organisms. The metabolites involved in the callus-parasite interaction can also be isolated with comparable ease. The objectives of this investigation were to follow the multiplication of Pseudomonas tabaci (Wolf and Foster) Stevens (compatible with tobacco), P. pisi Sackett (incompatible with tobacco) and P. jluorescens Migula (a saprophyte) in tobacco plants and in tobacco callus tissues, and to examine with SEM the topographical relationship of these three bacterial species to the callus cells. MATERIALS
AND
METHODS
Bacterial cultures Pseudomonastabaci, a pathogen of tobacco plants, P. p&i, a pathogen of pea but not tobacco, and P. jluorescens, a saprophyte, were obtained from R. N. Goodman, University of Missouri. All cultures were maintained in a lyophilized state. Bacteria grown on nutrient agar fortified with 0.5% yeast extract and 1% glucose at 24 “C for 16 h were washed 3 times with sterile distilled water, suspended in water and adjusted to lo9 cells/ml as determined with a Bausch & Lomb Spectronic 20. Tobacco callus tissues Plants of .Nkotiana tabacum Samsun NN, Hicks, Burley 21, and a hybrid of N. tabacum x N. glutinosa obtained from A. C. Hildebrandt, University of Wisconsin, were grown in vermiculite and irrigated with Hoagland’s solution in a greenhouse. Plants with five to six expanded leaves were used in this study. Sections O-5 to 1 cm long were cut from the stems of plants, immersed in 75% ethanol for 5 s, then in O.525o/o sodium hypochlorite for 25 mm, and finally rinsed thoroughly in three changes of sterile distilled water. The stem pieces were placed on the surface of 50 ml of Schenk & Hildebrandt (SH) medium [I41 in 125 ml wide-mouth Erlenmeyer flasks. Callus tissue that developed was transferred to fresh SH medium, incubated in the dark at 25 “C and subcultured once every 3 weeks. Bacterial inoculation and multiplication Two-week-old callus tissues, usually four uniform pieces per flask, were inoculated, without injuring the tissue, with 10 $ of bacterial suspensions containing 10’ cells/ml or sterile water. Three inoculated pieces of callus tissue were taken for bacterial counts immediately after inoculation and at 12 h intervals for 2 days. To determine the bacterial number, inoculated calluses were ground in 5 ml sterile water in the presence of a small amount of sand in a mortar. A lo-fold dilution series was made from the homogenate. One-tenth ml of the diluted suspension was pipetted onto the surface of a prepared nutrient agar plate and was spread over the surface with a glass rod [S]. The results were expressed as numbers of colonies
Tobacco callus tissue with Pseudomonas spp.
67
that developed on the nutrient agar plate per callus after 48 h incubation at 25 “C. These inoculations and bacterial counts were repeated 5 times. Tobacco leaves were infiltrated with bacterial suspensions containing 10s cells/ml by using a hypodermic syringe as described by Klement [9]. Inoculated leaves were detached from the plants, sealed in large Petri dishes (15 cm in diameter) and kept in darkness at 25 “C. Leaf discs were taken with a no. 7 cork borer (1 cm in diameter) immediately after inoculation and at 12 h intervals for 2 days for bacterial counts by using the standard dilution plate technique described earlier. The bacterial densities were expressed as number of colonies that developed on a nutrient agar plate per leaf disc. Scanning electron microscopy
Callus tissues of the hybrid tobacco were selected for SEM study since callus tissues from the different tobacco varieties showed similar results in population trends. Inoculated hybrid calluses were taken immediately after inoculation, and at 12 h intervals for 2 days as for cell counts, and prepared for SEM. Pieces of callus, c. 2 to 3 mn?, were taken from the site of inoculation and were fixed in a 0.1 M cacodylate buffer containing 3% glutaraldehyde and 4% paraformaldehyde (pH c. 7.2) for 12 h at 4 “C. The fixed pieces were washed in O-1 M cacodylate buffer and subjected to serial dehydration with ethanol and critical point drying with Freon 13. The samples were coated with gold, and were examined and photographed with an Etec autoscan microscope. RESULTS Multiplication
of bacteria in tobacco leaf tissues
P. tabaci, P. pisi and P. fluorescent multiplied at different rates in the infiltrated leaf tissues (Fig. 1). About 1 x lo6 cells/disc of P. tabaci, P. pisi and P. &rescens cells were recovered immediately after infiltration with 108 cells/ml. Forty-eight h later, 5 x log, 1 x 108 and 1 x 10’ cells/disc were recovered from leaves inoculated with P. tabaci, P. pisi and P. jluorescensrespectively. None of the inoculated leaf tissues showed symptoms at the end of 2 days’ incubation in the dark in the sealed Petri dishes. Multiplication
of bacteria in callus tissue
P. tabaci multiplied rapidly, and the bacteria moved from the inoculation point to other areas. Two days after inoculation, the callus was covered by bacteria and bacterial colonies were observed on the surface of the SH medium surrounding the inoculated callus tissue. Callus tissues inoculated with P. pisi and P. Juorescens were not covered by bacterial colonies (Plate 1). Callus tissues inoculated with P. pisi and P. jIuorescensturned light brown while the color of tissue inoculated with P. tabaci was unchanged after 48 h. Colony counts (Fig. 2) showed that P. tabaci, P. pisi and P. jluorescens multiplied at different rates in tobacco calluses. About 5 x lo5 cells/ callus were recovered for each bacterium immediately after inoculation. Approximately 1 x 10ls, 5 x 10s and 3 x 1O* cells/callus were recovered after 48 h incubation for P. tabaci, P. pisi and P. Juorescensrespectively. These differences in multiplication
J.-S. Huang and C. G. Van Dyke
66
“I
Hum after inoculation Fro. 1. Multiplication Racterial suspensions at tissues. Infiltrated leaves 25 “C. Results based on P. Jawm~, u-u.
of bacteria in leaves of a hybrid tobacco (N. nico&zm x N. glutinosa). concentrations of lo* cells/ml were infiltrated into tobacco leaf were detached, sealed in Petri dishes and incubated in the dark at average of five replicates. P. tubaci, O-O; P. phi, A-A;
I 12
I
I
I
24
36
48
Hours after inoculation
FIG. 2. Multiplication of bacteria in callus tissues derived from a hybrid tobacco (N. niGotimu x N. glutiaosa). Ten pl of bacterial suspensions containing 10’ ceils were placed on the surface of callus tissues. Pieces of inoculated callus tissue were incubated in the dark at 25 ‘C. Results based on average of five replicates. P. tab&, 0-e; P. /h-i, A-A; P. jhmmns, O-0.
PLATE 1. Multiplication of bacteria upper right, lower right and lower left containing 10’ cells of P. taban’, P. pisi received 10 pl of sterile distilled water.
in pieces of hybrid tobacco callus tissue. Tissues on were inoculated with 10 pl of bacterial suspensions and P. fluorescens, respectively. Tissue on upper left Photograph was taken 2 days after inoculation.
PLATE 2. Scanning electron micrograph elongated and had smooth surface. x 200.
of non-inoculated
callus cells. Most cells were
PLATE 3. P. tabaci cells on the surface of a callus cell immediately x 4000.
after inoculation.
PLATE 4. P. tab& cells multiplied and spread over the inoculated area. The sample was fixed 24 h after inoculation. x 1000. PLATE 5. Callus cells in the inoculated inoculation. X 1000.
area were covered heavily by P. tabaci 36 h after
PLATE 6. High magnification of P. tabaci cells showed that most of the bacteria retained their structural integrity 36 h after inoculation. Fibrillae were observed but they did not entrap the bacterial cells. x 4000. PLATE 7. P. pisi cells on callus surface immediately
after inoculation.
x 2000.
PLATE 8. P. pisi cells were not distributed evenly on the surface of callus 24 h after inoculation. Fibrillae appeared and were interwoven into bacterial aggregates. x 1000. PLATE 9. P. pisi cells entrapped by the fibrillae-forming tion. X 300. PLATE 10. High magnification x 4000.
aggregates at 36 h after inocula-
of a bacterial aggregate trapped in thick fibrillae network.
PLATE 11. P.jiuorescenscells on the surface of callus immediately
after inoculation.
x 4000.
PLATE 12. Some P. ~%orescenscells with spherical bodies (arrows) 12 h after inoculation. x 20 000. PLATE 13. The number of P. ~?aorescenscells with spherical bodies increased and the spherical bodies also increased their size at 36 h after inoculation. Bacterial cells with spherical bodies had protrusions (arrows) on the cell surface. x 4000. PLATE 14. Some spherical bodies lysed at 36 h after inoculation.
x 12 000.
PLATE 15. Light micrograph of P. Jaorescens cells prepared from callus tissue 2 days after inoculation. Many bacteria had spherical bodies (arrows). x 1000. PLATE 16. Light micrograph of P. fruorescenscells prepared from colonies grown on SH medium for 2 days. The bacteria did not produce spherical bodies. x 1000.
PLATES l-2
PLATES3-6
PLATES 7-l 0
PLATES 11-14
-PLATES
15-16
Tobacco callus tissue with Pseudomonasspp.
69
L I
I
I
I
I2
24
36
46
Hours after inoculation FIG. 3. Multiplication of bacteria on SH medium. One-tenth ml of bacterial suspensions at concentrations of 108 cells/ml were spread on the surface of SH medium. Plates were incubated in the dark at 25 ‘C. Results based on average of five replicates. P. tuba-i, ; P. juorescem, O---O. 0-O; P. j&-i, A -A
rate in calluses are assumed to be the result of interaction between callus and bacteria, since these three bacteria multiplied at approximately equal rates on SH medium (Fig. 3). There was no noticeable difference in response to bacterial inoculation between calluses grown in Petri dishes or in flasks; calluses grown on SH medium in a Petri dish are shown in Plate 1. Scanning electronmicroscopyof tobaccocallus cells Most tobacco callus cells were elongated, 20 to 70 pm in width and 100 to 500 e in length with smooth surfaces (Plate 2). Occasionally spherical cells, 50 w in diameter, were seen. Particulates of various shapes were found occasionally on the surface of some cells. Scanning electronmicroscopyof inoculated callus tissue Bacterial cells were not evenly distributed on the surface of inoculated callus cells immediately after inoculation with P. tabaci, P. pki or P. fruorescens(Plates 3, 7 and 11). Some areas had more bacterial cells than others. Cells of P. tabaci were typically rod-shaped, mostly single-celled immediately after inoculation (Plate 3). The number of bacterial cells increased after inoculation and by 24 h many callus cells were covered by a thin layer of bacteria (Plate 4). Thirty-six hours after inoculation, P. tabaci cells were densely and uniformly distributed over most inoculated areas (Plate 5). Cells of P. tabaci retained their normal rod shape, and bacterial aggregates were not observed. A few fine filaments were observed in some areas (Plate 6). Cells of P. pisi were also rod-shaped, mostly single-celled immediately after inoculation (Plate 7). The number of bacterial cells increased and they were unevenly
70
J.-S. Huang and C. G. Van Dyke
distributed on the callus surfaces by 12 h after inoculation (Plate 8). Cells of P. pi& aggregated into clumps with fibrillae interwoven into the bacterial aggregates, and by 36 h after inoculation, there was an increase in fibrillar thickness and bacterial aggregation (Plates 9 and 10). At 36 h, other areas of callus surface without bacteria were also covered by a fibrillar network. Cells of P.~uo~escens were typically rod-shaped, mostly single-celled, immediately after inoculation (Plate 11). Spherical bodies were observed associated with some P.juorescem cells 12 h after inoculation. The number of bacterial cells with spherical bodies and the size of the bodies increased by 24 h. After 36 h more than 50% of the bacteria in many areas had spherical bodies which measured l-0 to l-5 pm in diameter. Each bacterial cell usually produced only one spherical body. These bodies were attached either to the side or to the end of a bacterium (Plates 12 and 13). Most bacterial cells with spherical bodies had protrusions on the cell surface, c. O-1 pm in size (Plates 13 and 14). Some spherical bodies appeared to lyse at 36 h after inoculation and to release their cellular content (Plate 14). Light microscopic examination of suspensions prepared from callus tissues, 2 days after inoculation with P. fluorescens,revealed that most bacterial cells had spherical bodies (Plate 15), whereas P.JIuorescens cells grown on SH medium for 2 days did not have spherical bodies (Plate 16). DISCUSSION
Under ordinary laboratory and greenhouse conditions, tobacco leaves respond differently to the three Pseudomonads. Leaves infiltrated with P. pisi, at a concentration of lo6 cells/ml or higher, develop a typical HR within 12 h and the infiltrated areas usually dry within 1 to 2 days after infiltration. P. tabaci and P. jluorescenSdo not induce symptoms during this period, although the leaves infiltrated with P. tabaci develop wildfire disease 4 to 5 days later. P. tabaci multiplies in the infiltrated tobacco leaf tissue at a rapid rate, whereas P. pisi mutiplies only for a short period of time, followed by an abrupt inhibition of bacterial multiplication; this inhibition coincides with a rapid breakdown of host cellular organization. P. jluorescenris unable to multiply in the tobacco leaf [IO]. In the dark and at high relative humidity (r.h.), the tobacco leaves and the bacteria interacted differently from observations under ordinary conditions. P. pi.ri did not evoke typical hypersensitive symptoms even several days after inoculation. Both P. pisi and P. jluorescem multiplied for several days in infiltrated tobacco leaf tissues at rates lower than the one for P. tabaci. The observations, however, are in agreement with those reported previously by other workers [Z, II]. Goodman [,?I reported that P. @ i did not induce the HR on tobacco leaves and that the bacteria multiplied for 48 h if the inoculated leaves were kept in a humid chamber. Lovrekovich & Lovrekovich [II] also reported that P. jluorescens multiplied in tobacco leaves if the leaves were kept in the dark and at high r.h. Based on the population trends of P. tabaci, P. pisi and P. juorescens in tobacco callus and in leaves kept in the dark and at high r.h. (Figs 1 and 2), the reaction of tobacco callus with these three pseudomonads is similar to that of tobacco leaf tissue. Goodman [Z] reported that membrane damage and electrolyte leakage took place in tissue infiltrated with an incompatible bacterium whether it was kept
Tobacco callus tissue with Pseudomonas spp.
71
under ordinary, or dark and high r.h. conditions. Whether these ultrastructural and physiological changes occur in P. @ i-inoculated callus tissue has not been investigated. Cells of P. tubaci covered most of the surfaces of inoculated callus within 36 h after inoculation; bacterial cells retained their structural integrity throughout the entire experimental period. Fine filaments were observed in many areas, but they did not restrict bacterial growth. In observations of intact tissue, Goodman et al. [3] also reported that P. tabaci did not induce the formation of a bacteria-limiting wall cuticle in tobacco leaves, and the bacteria were not immobilized. The predominant characteristic of P. @ i callus cell interactions was the formation of thick fibrillae which entrapped the bacterial cells. It is apparent that the entrapment of P. pisi cells by the fibrillae may be one of the factors responsible for the localization of the bacteria. The origin and the chemical nature of the fibrillar material have not been determined. Many areas covered heavily by the fibrillae had no bacterial cells which suggests that fibrillae are probably of callus origin. Goodman et al. [3] examined tobacco leaves and observed that the cell wall cuticle of parenchyma cells separated from the cell wall in P. pi&inoculated tissue. The cuticle was initially filamentous but became progressively thicker and was impregnated by dense staining fibrils which eventually enveloped and immobilized P. pi& cells. Little is known about the interaction of plants with saprophytes. Misaghi & Grogan [12] reported that P. jluorescens is nutritionally more versatile and biochemically more active than many phytopathogenic pseudomonads. Klement et al. [IO] also noted that this saprophyte multiplies in juice expressed from tobacco leaves immediately following inoculation. However, it has been known for some time that P. jluorescens does not multiply in tobacco leaves under ordinary laboratory and greenhouse conditions. Although the saprophyte multiplies in tobacco leaves kept in the dark and at high r.h., it multiplies at a slower rate than for phytopathogenic pseudomonads. Wood [16] attributed the inability of saprophytes to multiply in plant tissue to the plant’s resistance, and assumed that the plant prevents the saprophyte growth. The morphological abnormality of P.Jluorescens observed in inoculated callus tissues, e.g. the formation of spherical bodies by the saprophyte (Plates 12, 13, 14 and 15), apparently is the result of an active resistance mechanism by the plant tissue to the bacteria and is not the effect of chemical constituents of SH medium, since the bacterial cells grown directly on the medium for 2 days did not produce spherical bodies (Plate 16). Induction of P. Juorescensto form spherical bodies may be one of the host resistance mechanisms which prevent bacterial multiplication. It has been known that under certain conditions, such as in the presence of penicillin and lysozyme, many bacterial cells transform to L-forms and spheroplasts. The transition from the normal to the L-form or spheroplast has been suggested to occur as the result of injury to the bacterial cell wall [a. The formation of spherical bodies is apparently a process similar to spheroplast formation. The principle responsible for the transformation, however, has not been determined. This work was supported in part by a grant to the senior author from the Faculty Research and Professional Development Fund, North Carolina State University.
12
J.-S. Huang and C. G. Van Dyke
Paper no. 5384 of the Journal Series of the North Carolina Agriculutral Station, Raleigh, N.C.
Experiment
REFERENCES 1, BRAUN, A. C. & WHITE, P. R. (1943). Bacteriological sterility of tissue derived from secondary crown-gall tumors. Phytopathology33,85-100. 2. GOODMAN, R. N. (1972). Electrolyte leakage and membrane damage in relation to bacterial population, pH and ammonia production in tobacco leaf tissue inoculated with Pscua’omonar jii Phytoj~a-&logy62, 1327-133‘1. 3. GOODMAN. R. N.. HUANG. Pt-Yu & WHITE. 1. A. 11976). Ultmstructural evidence for immobilization of ah incompatible bacterium, %udom&s &, in tobacco leaf tissue. Phytopathology 66,754-764. 4. HELGESON,J. P., HABERLACH, G. T. & UPPER, C. D. (1976). A dominant gene conferring disease resistance to tobacco plants is expressed in tissue cultures. Phytopathalogv66,91-96. 5. HENNING, U. & SCHWARZ, U. (1973). Determinants of cell shape. In Bacterial Membranes and WaZZs,Ed. by L. Leive, pp. 415-438. Marcel Dekker Inc., New York. 6. HUANG, Pt-Yu, HUANO, J. S. & GOODMAN, R. N. (1975). Resistance mechanisms of apple shoots to an avirulent strain of Erwinin amylovora. Physiological Plant Pathology 6,283-287. 7. KENNEDY, B. W., MEW, T. W. & OLSCN, L. (1972). Reaction of soybean tissue culture to pathogenie and saprophytic bacteria. In- P&eedi& of the Third Z&matiunal Confncnu & Plant Pathwnic &curia. Ed. bv H. P. Maas Geesteranus. DD. 201-202. Universitv of Toronto Press. KLEME&, Z., S&YMO.W, F. & VIRUS, J. (isib). Me hods in Plant Pkhologv. AkadCmiai 8. MLY,~., Kiado, Budapest. 9. KLEMENT, Z. (1963). Method for the rapid detection of the pathogenicity of phytopathogenic pseudomonads. .Nature 199,299300. 10. KLEMENT, Z., FARKAS, G. L. & LO~REKO~ICH, L. (1964). Hypersensitive reaction induced by phytopathogenic bacteria in the tobacco leaf. Phytopothology54, 474-477. 11. LOVREKOVICH,L. & Lovamco~o~, H. (1970). Tissue necrosis in tobacco caused by a saprophytic bacterium. Phytopathology60, 1279-1280. and biochemical comparisons of plant12. MISAGHI, I. St GROGAN, R. G. (1969). Nutritional pathogenic and saprophytic fluorescent pseudomonads. Phytojathalogy 59, 1436-1450. 13. RUSSELL, T. E. & HALLWELL, R. S. (1974). Response of cultured cells of systemic and local lesion tobacco hosts to micro-injection with TMV. Phytopathology 64, 1520-1526. 14. SCHENK,R.U.& I-hDEBRANDT, A. C. (1972). Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Canadian 3oumal of Botany 50, 199-204. 15. SEQUEIRA,L., GAARQ G. & DE ZOETEN, G. A. (1977). Interaction of bacteria and host cell walls: its relation to mechanisms of induced &stake. .Phy&logicaL Plant Pathology 10, 43-50. 16. WOOD, R. K. S. (1972). Introduction: disease resistance in plants. Proceedingsof the Royal So&p of London, B 181,213-232.
Interaction of tobacco callus tissue with Pseudomonas tabaci, P. pisi and P. fluorescens JENG-SHENG
HUANG
and C. GERALD VAN DYKE
Defiartment of Plant Padwlogy, North Carolina St& University Raleigh, KC. 27650, U.S.A.
(Accepedfor publicntionJanua~ 1978)
Pm&nom tobad, a tobacco pathogen, multiplied rapidly and colonized tobacco callus within 2 days after inoculation. P. piA, a pathogen of pea but not tobacco, and P.Juoresccnr, a saprophyte, multiplied slowly and remained at inoculation sites. Rates of multiplication of these three bacteria in tobacco callus were comparable to those in leaves kept in the dark and at high relative humidity following inoculation. Scanning electron microscopy showed P. tab& cells densely distributed over the tobacco callus. P. pi.ri cells formed aggregates and were trapped in a network of fibrillae on the callus surface. Some P.&mmn.s cells formed spherical bodies, apparently as a result of damage to bacterial cell walls. The spherical bodies eventually lysed. The entrapment of bacterial cells of P. pin’ by fibrillae and the damage to the cell wall of P.jhwrescen.smay be factors which limit the rate of multiplication of these species of bacteria in tobacco callus tissue.
INTRODUCTION Tissue cultures are suitable for study of host-parasite interactions in certain fungal and viral diseases. Helgeson et al. [4] demonstrated that calluses from tobacco plants susceptible to black shank disease were susceptible to the disease-inducing organism Phytophthora parasitica var. nicotianae, and that calluses from resistant plants were resistant to the pathogen. Russell & Halliwell [13] reported that rapid loss of cell integrity exhibited by cultured tobacco Samsun NN cells to infection by tobacco mosaic virus (TMV) paralleled the local lesion response of the intact plants. They also found that over a period of time TMV multiplied in Samsun cells without damaging the host, in accordance with the systemic symptom usually observed in Samsun plants. Systems involving tissue culture and bacteria have been investigated [7] but suitable models, with the exception of crown gall [I], have not been developed. Ultrastructural studies of the interaction between bacteria and plant cells have shown that in several incompatible combinations, bacteria are attached to plant cell walls and are engulfed by fibrillar and granular materials. This phenomenon has been used to explain the localization of incompatible bacteria and cessation of bacterial multiplication in tissues undergoing the hypersensitive reaction (HR) [3, 6 151. 0048-4059/78/0701-0065 5
$02.00/O
@ 1978 Academic Press Inc. (London)
Limited
J.-S. Huang and C. G. Van Dyke
66
Callus tissues would provide an excellent working model to study bacteriaplant cell interactions since most bacteria are in the intercellular space of the inoculated tissue, and do not penetrate host cells. The development of scanning electron microscopy (SEM) has made it possible to examine interactions between bacteria and the surface of callus cells, free of contaminating micro-organisms. The metabolites involved in the callus-parasite interaction can also be isolated with comparable ease. The objectives of this investigation were to follow the multiplication of Pseudomonas tabaci (Wolf and Foster) Stevens (compatible with tobacco), P. pisi Sackett (incompatible with tobacco) and P. jluorescens Migula (a saprophyte) in tobacco plants and in tobacco callus tissues, and to examine with SEM the topographical relationship of these three bacterial species to the callus cells. MATERIALS
AND
METHODS
Bacterial cultures Pseudomonastabaci, a pathogen of tobacco plants, P. p&i, a pathogen of pea but not tobacco, and P. jluorescens, a saprophyte, were obtained from R. N. Goodman, University of Missouri. All cultures were maintained in a lyophilized state. Bacteria grown on nutrient agar fortified with 0.5% yeast extract and 1% glucose at 24 “C for 16 h were washed 3 times with sterile distilled water, suspended in water and adjusted to lo9 cells/ml as determined with a Bausch & Lomb Spectronic 20. Tobacco callus tissues Plants of .Nkotiana tabacum Samsun NN, Hicks, Burley 21, and a hybrid of N. tabacum x N. glutinosa obtained from A. C. Hildebrandt, University of Wisconsin, were grown in vermiculite and irrigated with Hoagland’s solution in a greenhouse. Plants with five to six expanded leaves were used in this study. Sections O-5 to 1 cm long were cut from the stems of plants, immersed in 75% ethanol for 5 s, then in O.525o/o sodium hypochlorite for 25 mm, and finally rinsed thoroughly in three changes of sterile distilled water. The stem pieces were placed on the surface of 50 ml of Schenk & Hildebrandt (SH) medium [I41 in 125 ml wide-mouth Erlenmeyer flasks. Callus tissue that developed was transferred to fresh SH medium, incubated in the dark at 25 “C and subcultured once every 3 weeks. Bacterial inoculation and multiplication Two-week-old callus tissues, usually four uniform pieces per flask, were inoculated, without injuring the tissue, with 10 $ of bacterial suspensions containing 10’ cells/ml or sterile water. Three inoculated pieces of callus tissue were taken for bacterial counts immediately after inoculation and at 12 h intervals for 2 days. To determine the bacterial number, inoculated calluses were ground in 5 ml sterile water in the presence of a small amount of sand in a mortar. A lo-fold dilution series was made from the homogenate. One-tenth ml of the diluted suspension was pipetted onto the surface of a prepared nutrient agar plate and was spread over the surface with a glass rod [S]. The results were expressed as numbers of colonies
Tobacco callus tissue with Pseudomonas spp.
67
that developed on the nutrient agar plate per callus after 48 h incubation at 25 “C. These inoculations and bacterial counts were repeated 5 times. Tobacco leaves were infiltrated with bacterial suspensions containing 10s cells/ml by using a hypodermic syringe as described by Klement [9]. Inoculated leaves were detached from the plants, sealed in large Petri dishes (15 cm in diameter) and kept in darkness at 25 “C. Leaf discs were taken with a no. 7 cork borer (1 cm in diameter) immediately after inoculation and at 12 h intervals for 2 days for bacterial counts by using the standard dilution plate technique described earlier. The bacterial densities were expressed as number of colonies that developed on a nutrient agar plate per leaf disc. Scanning electron microscopy
Callus tissues of the hybrid tobacco were selected for SEM study since callus tissues from the different tobacco varieties showed similar results in population trends. Inoculated hybrid calluses were taken immediately after inoculation, and at 12 h intervals for 2 days as for cell counts, and prepared for SEM. Pieces of callus, c. 2 to 3 mn?, were taken from the site of inoculation and were fixed in a 0.1 M cacodylate buffer containing 3% glutaraldehyde and 4% paraformaldehyde (pH c. 7.2) for 12 h at 4 “C. The fixed pieces were washed in O-1 M cacodylate buffer and subjected to serial dehydration with ethanol and critical point drying with Freon 13. The samples were coated with gold, and were examined and photographed with an Etec autoscan microscope. RESULTS Multiplication
of bacteria in tobacco leaf tissues
P. tabaci, P. pisi and P. fluorescent multiplied at different rates in the infiltrated leaf tissues (Fig. 1). About 1 x lo6 cells/disc of P. tabaci, P. pisi and P. &rescens cells were recovered immediately after infiltration with 108 cells/ml. Forty-eight h later, 5 x log, 1 x 108 and 1 x 10’ cells/disc were recovered from leaves inoculated with P. tabaci, P. pisi and P. jluorescensrespectively. None of the inoculated leaf tissues showed symptoms at the end of 2 days’ incubation in the dark in the sealed Petri dishes. Multiplication
of bacteria in callus tissue
P. tabaci multiplied rapidly, and the bacteria moved from the inoculation point to other areas. Two days after inoculation, the callus was covered by bacteria and bacterial colonies were observed on the surface of the SH medium surrounding the inoculated callus tissue. Callus tissues inoculated with P. pisi and P. Juorescens were not covered by bacterial colonies (Plate 1). Callus tissues inoculated with P. pisi and P. jIuorescensturned light brown while the color of tissue inoculated with P. tabaci was unchanged after 48 h. Colony counts (Fig. 2) showed that P. tabaci, P. pisi and P. jluorescens multiplied at different rates in tobacco calluses. About 5 x lo5 cells/ callus were recovered for each bacterium immediately after inoculation. Approximately 1 x 10ls, 5 x 10s and 3 x 1O* cells/callus were recovered after 48 h incubation for P. tabaci, P. pisi and P. Juorescensrespectively. These differences in multiplication
J.-S. Huang and C. G. Van Dyke
66
“I
Hum after inoculation Fro. 1. Multiplication Racterial suspensions at tissues. Infiltrated leaves 25 “C. Results based on P. Jawm~, u-u.
of bacteria in leaves of a hybrid tobacco (N. nico&zm x N. glutinosa). concentrations of lo* cells/ml were infiltrated into tobacco leaf were detached, sealed in Petri dishes and incubated in the dark at average of five replicates. P. tubaci, O-O; P. phi, A-A;
I 12
I
I
I
24
36
48
Hours after inoculation
FIG. 2. Multiplication of bacteria in callus tissues derived from a hybrid tobacco (N. niGotimu x N. glutiaosa). Ten pl of bacterial suspensions containing 10’ ceils were placed on the surface of callus tissues. Pieces of inoculated callus tissue were incubated in the dark at 25 ‘C. Results based on average of five replicates. P. tab&, 0-e; P. /h-i, A-A; P. jhmmns, O-0.
PLATE 1. Multiplication of bacteria upper right, lower right and lower left containing 10’ cells of P. taban’, P. pisi received 10 pl of sterile distilled water.
in pieces of hybrid tobacco callus tissue. Tissues on were inoculated with 10 pl of bacterial suspensions and P. fluorescens, respectively. Tissue on upper left Photograph was taken 2 days after inoculation.
PLATE 2. Scanning electron micrograph elongated and had smooth surface. x 200.
of non-inoculated
callus cells. Most cells were
PLATE 3. P. tabaci cells on the surface of a callus cell immediately x 4000.
after inoculation.
PLATE 4. P. tab& cells multiplied and spread over the inoculated area. The sample was fixed 24 h after inoculation. x 1000. PLATE 5. Callus cells in the inoculated inoculation. X 1000.
area were covered heavily by P. tabaci 36 h after
PLATE 6. High magnification of P. tabaci cells showed that most of the bacteria retained their structural integrity 36 h after inoculation. Fibrillae were observed but they did not entrap the bacterial cells. x 4000. PLATE 7. P. pisi cells on callus surface immediately
after inoculation.
x 2000.
PLATE 8. P. pisi cells were not distributed evenly on the surface of callus 24 h after inoculation. Fibrillae appeared and were interwoven into bacterial aggregates. x 1000. PLATE 9. P. pisi cells entrapped by the fibrillae-forming tion. X 300. PLATE 10. High magnification x 4000.
aggregates at 36 h after inocula-
of a bacterial aggregate trapped in thick fibrillae network.
PLATE 11. P.jiuorescenscells on the surface of callus immediately
after inoculation.
x 4000.
PLATE 12. Some P. ~%orescenscells with spherical bodies (arrows) 12 h after inoculation. x 20 000. PLATE 13. The number of P. ~?aorescenscells with spherical bodies increased and the spherical bodies also increased their size at 36 h after inoculation. Bacterial cells with spherical bodies had protrusions (arrows) on the cell surface. x 4000. PLATE 14. Some spherical bodies lysed at 36 h after inoculation.
x 12 000.
PLATE 15. Light micrograph of P. Jaorescens cells prepared from callus tissue 2 days after inoculation. Many bacteria had spherical bodies (arrows). x 1000. PLATE 16. Light micrograph of P. fruorescenscells prepared from colonies grown on SH medium for 2 days. The bacteria did not produce spherical bodies. x 1000.
PLATES l-2
PLATES3-6
PLATES 7-l 0
PLATES 11-14
-PLATES
15-16
Tobacco callus tissue with Pseudomonasspp.
69
L I
I
I
I
I2
24
36
46
Hours after inoculation FIG. 3. Multiplication of bacteria on SH medium. One-tenth ml of bacterial suspensions at concentrations of 108 cells/ml were spread on the surface of SH medium. Plates were incubated in the dark at 25 ‘C. Results based on average of five replicates. P. tuba-i, ; P. juorescem, O---O. 0-O; P. j&-i, A -A
rate in calluses are assumed to be the result of interaction between callus and bacteria, since these three bacteria multiplied at approximately equal rates on SH medium (Fig. 3). There was no noticeable difference in response to bacterial inoculation between calluses grown in Petri dishes or in flasks; calluses grown on SH medium in a Petri dish are shown in Plate 1. Scanning electronmicroscopyof tobaccocallus cells Most tobacco callus cells were elongated, 20 to 70 pm in width and 100 to 500 e in length with smooth surfaces (Plate 2). Occasionally spherical cells, 50 w in diameter, were seen. Particulates of various shapes were found occasionally on the surface of some cells. Scanning electronmicroscopyof inoculated callus tissue Bacterial cells were not evenly distributed on the surface of inoculated callus cells immediately after inoculation with P. tabaci, P. pki or P. fruorescens(Plates 3, 7 and 11). Some areas had more bacterial cells than others. Cells of P. tabaci were typically rod-shaped, mostly single-celled immediately after inoculation (Plate 3). The number of bacterial cells increased after inoculation and by 24 h many callus cells were covered by a thin layer of bacteria (Plate 4). Thirty-six hours after inoculation, P. tabaci cells were densely and uniformly distributed over most inoculated areas (Plate 5). Cells of P. tabaci retained their normal rod shape, and bacterial aggregates were not observed. A few fine filaments were observed in some areas (Plate 6). Cells of P. pisi were also rod-shaped, mostly single-celled immediately after inoculation (Plate 7). The number of bacterial cells increased and they were unevenly
70
J.-S. Huang and C. G. Van Dyke
distributed on the callus surfaces by 12 h after inoculation (Plate 8). Cells of P. pi& aggregated into clumps with fibrillae interwoven into the bacterial aggregates, and by 36 h after inoculation, there was an increase in fibrillar thickness and bacterial aggregation (Plates 9 and 10). At 36 h, other areas of callus surface without bacteria were also covered by a fibrillar network. Cells of P.~uo~escens were typically rod-shaped, mostly single-celled, immediately after inoculation (Plate 11). Spherical bodies were observed associated with some P.juorescem cells 12 h after inoculation. The number of bacterial cells with spherical bodies and the size of the bodies increased by 24 h. After 36 h more than 50% of the bacteria in many areas had spherical bodies which measured l-0 to l-5 pm in diameter. Each bacterial cell usually produced only one spherical body. These bodies were attached either to the side or to the end of a bacterium (Plates 12 and 13). Most bacterial cells with spherical bodies had protrusions on the cell surface, c. O-1 pm in size (Plates 13 and 14). Some spherical bodies appeared to lyse at 36 h after inoculation and to release their cellular content (Plate 14). Light microscopic examination of suspensions prepared from callus tissues, 2 days after inoculation with P. fluorescens,revealed that most bacterial cells had spherical bodies (Plate 15), whereas P.JIuorescens cells grown on SH medium for 2 days did not have spherical bodies (Plate 16). DISCUSSION
Under ordinary laboratory and greenhouse conditions, tobacco leaves respond differently to the three Pseudomonads. Leaves infiltrated with P. pisi, at a concentration of lo6 cells/ml or higher, develop a typical HR within 12 h and the infiltrated areas usually dry within 1 to 2 days after infiltration. P. tabaci and P. jluorescenSdo not induce symptoms during this period, although the leaves infiltrated with P. tabaci develop wildfire disease 4 to 5 days later. P. tabaci multiplies in the infiltrated tobacco leaf tissue at a rapid rate, whereas P. pisi mutiplies only for a short period of time, followed by an abrupt inhibition of bacterial multiplication; this inhibition coincides with a rapid breakdown of host cellular organization. P. jluorescenris unable to multiply in the tobacco leaf [IO]. In the dark and at high relative humidity (r.h.), the tobacco leaves and the bacteria interacted differently from observations under ordinary conditions. P. pi.ri did not evoke typical hypersensitive symptoms even several days after inoculation. Both P. pisi and P. jluorescem multiplied for several days in infiltrated tobacco leaf tissues at rates lower than the one for P. tabaci. The observations, however, are in agreement with those reported previously by other workers [Z, II]. Goodman [,?I reported that P. @ i did not induce the HR on tobacco leaves and that the bacteria multiplied for 48 h if the inoculated leaves were kept in a humid chamber. Lovrekovich & Lovrekovich [II] also reported that P. jluorescens multiplied in tobacco leaves if the leaves were kept in the dark and at high r.h. Based on the population trends of P. tabaci, P. pisi and P. juorescens in tobacco callus and in leaves kept in the dark and at high r.h. (Figs 1 and 2), the reaction of tobacco callus with these three pseudomonads is similar to that of tobacco leaf tissue. Goodman [Z] reported that membrane damage and electrolyte leakage took place in tissue infiltrated with an incompatible bacterium whether it was kept
Tobacco callus tissue with Pseudomonas spp.
71
under ordinary, or dark and high r.h. conditions. Whether these ultrastructural and physiological changes occur in P. @ i-inoculated callus tissue has not been investigated. Cells of P. tubaci covered most of the surfaces of inoculated callus within 36 h after inoculation; bacterial cells retained their structural integrity throughout the entire experimental period. Fine filaments were observed in many areas, but they did not restrict bacterial growth. In observations of intact tissue, Goodman et al. [3] also reported that P. tabaci did not induce the formation of a bacteria-limiting wall cuticle in tobacco leaves, and the bacteria were not immobilized. The predominant characteristic of P. @ i callus cell interactions was the formation of thick fibrillae which entrapped the bacterial cells. It is apparent that the entrapment of P. pisi cells by the fibrillae may be one of the factors responsible for the localization of the bacteria. The origin and the chemical nature of the fibrillar material have not been determined. Many areas covered heavily by the fibrillae had no bacterial cells which suggests that fibrillae are probably of callus origin. Goodman et al. [3] examined tobacco leaves and observed that the cell wall cuticle of parenchyma cells separated from the cell wall in P. pi&inoculated tissue. The cuticle was initially filamentous but became progressively thicker and was impregnated by dense staining fibrils which eventually enveloped and immobilized P. pi& cells. Little is known about the interaction of plants with saprophytes. Misaghi & Grogan [12] reported that P. jluorescens is nutritionally more versatile and biochemically more active than many phytopathogenic pseudomonads. Klement et al. [IO] also noted that this saprophyte multiplies in juice expressed from tobacco leaves immediately following inoculation. However, it has been known for some time that P. jluorescens does not multiply in tobacco leaves under ordinary laboratory and greenhouse conditions. Although the saprophyte multiplies in tobacco leaves kept in the dark and at high r.h., it multiplies at a slower rate than for phytopathogenic pseudomonads. Wood [16] attributed the inability of saprophytes to multiply in plant tissue to the plant’s resistance, and assumed that the plant prevents the saprophyte growth. The morphological abnormality of P.Jluorescens observed in inoculated callus tissues, e.g. the formation of spherical bodies by the saprophyte (Plates 12, 13, 14 and 15), apparently is the result of an active resistance mechanism by the plant tissue to the bacteria and is not the effect of chemical constituents of SH medium, since the bacterial cells grown directly on the medium for 2 days did not produce spherical bodies (Plate 16). Induction of P. Juorescensto form spherical bodies may be one of the host resistance mechanisms which prevent bacterial multiplication. It has been known that under certain conditions, such as in the presence of penicillin and lysozyme, many bacterial cells transform to L-forms and spheroplasts. The transition from the normal to the L-form or spheroplast has been suggested to occur as the result of injury to the bacterial cell wall [a. The formation of spherical bodies is apparently a process similar to spheroplast formation. The principle responsible for the transformation, however, has not been determined. This work was supported in part by a grant to the senior author from the Faculty Research and Professional Development Fund, North Carolina State University.
12
J.-S. Huang and C. G. Van Dyke
Paper no. 5384 of the Journal Series of the North Carolina Agriculutral Station, Raleigh, N.C.
Experiment
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