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Invasion by Pseudomonas syringae pv. coriandricola is responsible for bacterial blight of coriander
Plant Science 161 (2001) 621– 625 www.elsevier.com/locate/plantsci
Invasion by Pseudomonas syringae pv. coriandricola is responsible for bacterial blight of coriander S.J. Refshauge, M. Nayudu * School of Botany and Zoology, Australian National Uni6ersity, Canberra ACT 0200, Australia Received 25 January 2001; received in revised form 14 May 2001; accepted 15 May 2001
Abstract Bacterial blight is a major disease of coriander caused by Pseudomonas syringae pv. coriandricola. The nature of this disease was studied by inoculating coriander plants with rifampicin resistant P. s. pv. coriandricola. All inoculated plants showed disease symptoms and the level of mortality was significant. The pathogen was isolated from different parts of inoculated coriander plants implying internal infection. Macroscopic inspection of diseased plants showed blackening of leaf veins and surrounding tissues. Closer examination confirmed the presence of large numbers of bacterial cells within leaf veins. Examination using light and electron microscopy showed infected coriander stem tissue contains large numbers of bacteria within xylem vessels and neighbouring pith cells. The interstitium of infected plants also contained large bacterial populations. Isolation and culture of bacteria from infected tissues confirmed the identity of the pathogen observed. No bacterial cells were seen within phloem vessels or below the inoculation site. These results suggest that the coriander pathogen utilizes the vascular system of the host for multiplication and transport, leading to infection of many tissues and, potentially, host death. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pseudomonas syringae pv. coriandricola; Coriander; Bacterial blight
1. Introduction Pathogenic variants (pathovars) of the bacterium Pseudomonas syringae are responsible for bacterial blights in stone fruits, legumes and many grain crops world wide [1]. Several of these diseases have been shown to involve invasive bacteria. Pathovars of P. syringae have been examined in a number of plants demonstrating that spread of bacteria throughout the plant is usually due to invasion of the vascular system [1 – 6]. Infection typically is initiated by multiplication within stomata followed by invasion of sub-stomatal tissues [5–8]. Bacterial blights of bean and pea (caused by P. s. pv. phaseolicola and P. s. pv. pisi, respectively) are well studied models for understanding pathogenesis in simiAbbre6iations: TEM, transmission electron microscopy; pv., pathovar. * Corresponding author. Tel.: +61-2-612-53643; fax: + 61-2-61255573. E-mail address: [email protected] (M. Nayudu).
lar systems. Both pathogens are seed-borne and reside epiphytically until an opportunity for invasion occurs [9–11]. Each pathogen develops in the seed coat of the germinating seedling and may contaminate the growing cotyledons [2,12]. Once within the vascular system these pathogens spread throughout the plant causing lesions, cankers and death [2,11]. Bacterial blight is a major disease of coriander (Coriandrum sati6um) caused by the non-fluorescent bacterium P. s. pv. coriandricola. The disease is responsible for large production losses in Australia, Europe and America [13,14]. The pathogen, like P. s. pv. phaseolicola and P. s. pv. pisi, is seed-borne and typically affects plants during flowering [14]. Initial symptoms include lesion formation, blackening of leaf veins and wilting. Extended infection reduces total plant biomass [15] leading to yield losses and, in severe cases, plant death. P. s. pv. coriandricola has been isolated from infected coriander tissues using selective media [16], suggesting that the pathogen may invade host tissues. In this study we demonstrate that P. s. pv. coriandricola is an invasive pathogen of coriander with the ability to penetrate cell walls and multiply intracellularly.
0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 4 6 1 - 7
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S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625
2. Materials and methods
2.1. Bacterial strains and media P. s. pv. coriandricola strain 1088b (supplied by J. Dennis, South Australian Research and Development Institute, Adelaide, Australia) was cultured on Kings B media [17] at 25 °C for 48 h. A rifampicin resistant strain was obtained by isolating spontaneous mutants on nutrient agar containing rifampicin (200 mg/ml). This strain was observed to retain normal levels of pathogenicity [15]. Detection of P. s. pv. coriandricola in infected coriander tissues was achieved by crushing surface sterilized samples in nutrient broth and spreading 100 ml of the suspension onto agar containing rifampicin (200 mg/ ml). Plates were incubated at 25 °C for 24 h and inspected for growth of P. s. pv. coriandricola.
2.2. Plant culture Sterilized coriander seeds were germinated at 20 °C on Herridges medium [18] containing 2% agar. Seedlings were planted into soil and grown in a temper-
ature controlled glasshouse. One-month-old plants were inoculated by inserting a sterile 25 gauge needle coated with P. s. pv. coriandricola through the crown region. Infection proceeded for 10 days, whereupon tissue samples were prepared for electron microscopy. Sampling of untreated plants provided the control.
2.3. Light and electron microscopy Samples of coriander leaves with symptoms visible with the unaided eye were externally examined using a low power light microscope. The following method of section preparation for high power microscopy was adapted from Bender et al. [19]. Resin fixing of samples to enable sectioning was achieved by immersing tissue samples from several different regions of infected plants in primary fixative consisting of 2% paraformaldehyde and 2.5% glutaraldehyde in 30 mM PIPES buffer (pH 7) for 3 h. Samples were post-fixed in 1% osmium tetroxide, dehydrated with serial ethanol rinses and embedded in Epon araldite resin. Sections were cut using a Reichert–Jung Ultracut microtome. Light microscope sections were stained using toluidine blue while silver ultrathin sections for transmission electron
Fig. 1. Coriander leaves 10 days after inoculation with P. s. pv. coriandricola: (A) blackening of tissues along veins on artificially infected coriander leaves (scale in millimetres); (B) higher magnification of inset (A) showing spread of symptoms from leaf veins to surrounding tissues (mag× 10); (C) cross section of infected coriander leaf tissue showing bacteria (arrows) within the vascular system and several neighbouring cells (mag×50).
S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625
microscopy (TEM) were stained using 6% aqueous uranyl acetate and lead citrate. TEM sections were visualized at 75 kV using an Hitachi 7100 electron microscope.
3. Results and discussion The high mortality rate observed among inoculated plants (approximately 90%) demonstrated the potential severity of the coriander blight disease. P. s. pv. coriandricola was isolated on selective media from infected roots, stems, petioles and leaves. No bacterial cells were recovered from uninoculated control plants. Light microscopy of infected leaves showed discolouration of the vascular system and surrounding tissues (Fig. 1A and B). These symptoms are typical of heavily infected tissue and suggest that P. s. pv. coriandricola spreads by following the path of the vascular system and penetrating surrounding tissues. The observation of large populations of P. s. pv. coriandricola
623
within the vessels of infected leaf tissue (Fig. 1C) provides evidence that the pathogen may spread via this route. The absence of the pathogen from surrounding tissues may simply be an artefact of sampling, as the pathogen may have penetrated these tissues given adequate time. This is the first visual evidence that P. s. pv. coriandricola invades the vascular system of coriander plants. Ultrastructural studies showed large populations of the pathogen within the xylem vessels of coriander stem tissue (Fig. 2A), suggesting that blockage may occur and cause wilting. Such blockage has been observed in plum trees infected with P. s. pv. syringae [5]. Nutrient depletion caused by the growing P. s. pv. coriandricola population also may contribute to degeneration of host tissues. Pathogen transmission within the plant appears to be primarily through xylem vessels. This may be an active process, since the bacterium is motile, or passive, induced by fluid flow and expansion of the bacterial population. The number of bacteria in stem tissues was
Fig. 2. Cross sections of coriander stem tissue 10 days after artificial infection with P. s. pv. coriandricola: (A) xylem vessels (arrow) heavily infected with the pathogen are surrounded by apparently uninfected interfascicular cambium (ic) and pith cells (p); (B) heavily infected pith cells (arrow) between apparently uninfected vascular bundles and interfascicular cambium (ic); (C) TEM image of pith cells infected with the bacterium, bound by the cell wall (cw) and neighbouring uninfected pith cells (mag × 4000); (D) TEM image clearly showing bacteria in the interstitium between three apparently uninfected pith cells (mag × 8000).
624
S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625
noted to diminish with distance from the infection site, possibly due to the slow rate of bacterial spread and narrowing of the vessels. Interestingly, the pathogen was not observed in phloem vessels. Pith cells neighbouring xylem vessels also were observed to be infected with large numbers of bacteria (Fig. 2B). Examination of stem tissues using TEM revealed varied numbers of P. s. pv. coriandricola within pith cells (Fig. 2C) and interstitial spaces (Fig. 2D). While pith cells close to the xylem vessels were frequently infected, the number of infected cells diminished with distance from the vessels. No bacteria were observed within the untreated control plants. The ability of P. s. pv. coriandricola to enter surrounding tissues from xylem vessels in the stem may allow the organism to exit veins and spread locally within leaves. Here the pathogen may have a powerful effect on its host by destroying large numbers of chloroplasts and reducing productivity. This effect has been observed in bacterial blight of bean caused by Xanthomonas campestris pv. phaseoli [20,21]. The presence of bacteria within the interstitial spaces close to xylem vessels is indicative of the pathogen’s ability to exit between or through xylem cells. This is similar to the nature of P. s. pv. pisi, the closely related causative agent of bacterial blight of bean [2]. Further, the observation of bacteria within cells is evidence of the ability of P. s. pv. coriandricola to penetrate cell walls. This suggests that P. s. pv. coriandricola produces cell wall degrading enzymes or pectinases as observed in related pathovars [1,7]. Infection of plant organs is likely hastened by infection of the interstitium, which may allow bacteria to move freely throughout the host. Bacteria were not observed during microscopic examination of root tissue. However, bacteria in low numbers were isolated repeatedly from root tissues on selective media. This discrepancy could be due to low bacterial populations in the root tissue and may be an artefact of sampling time. Since the pathogen is motile, some cells may move against the fluid flow within xylem vessels, and hence would be detected by isolation from root tissues onto selective media. This ultrastructural examination of infected coriander tissues has demonstrated for the first time that P. s. pv. coriandricola penetrates into tissues and cells of coriander. The findings of this study contribute to the current understanding of related P. s. pathovars and, through an improved understanding of the mechanism of pathogenesis, will assist future research on control strategies for coriander blight.
Acknowledgements This research was only possible with the assistance and support of D. Jacobs (AgSearch Australia Pty. Ltd., Harden, Australia) through a research grant. J. Goodan
and J. Dennis (South Australian Research and Development Institute, Adelaide, Australia) are thanked for provision of P. s. pv. coriandricola strain 1088b. S. Stowe and L. Shen (Electron Microscopy Unit, Research School of Biological Sciences, ANU, Canberra, Australia) are thanked for their assistance with electron microscopy. References [1] K.W.E. Rudolph, Pseudomonas syringae pathovars, in: U.S. Singh, R.P. Singh, K. Kohmoto (Eds.), Prokaryotes, vol. I, (3volume series: Pathogenesis and Host Specificity in Plant Diseases), Elsevier Science Ltd, 1995, pp. 47 – 138. [2] J.C. Walker, Plant Pathology, McGraw Hill Inc., Sydney, 1969, pp. 126 – 133. [3] I.M.M. Roos, M.J. Hattingh, Systemic invasion of plum leaves and shoots by Pseudomonas syringae pv. syringae introduced into petioles, Phytopathology 77 (1987) 1253 – 1257. [4] I.M.M. Roos, M.J. Hattingh, Systemic invasion of cherry leaves and petioles by Pseudomonas syringae pv. morsprunorum, Phytopathology 77 (1987) 1246 – 1252. [5] M.J. Hattingh, I.M.M. Roos, E.L. Mansvelt, Infection and systemic invasion of deciduous fruit trees by Pseudomonas syringae in South Africa, Plant Disease 73 (1989) 784 – 789. [6] E.L. Mansvelt, M.J. Hattingh, Scanning electron microscopy of invasion of apple leaves and blossoms by Pseudomonas syringae pv. syringae, Applied and Environmental Microbiology 55 (1989) 533 – 538. [7] J. Hallmann, A. Quadt – Hallmann, W.F. Mahaffe, J.W. Kloepper, Bacterial endophytes in agricultural crops, Journal of Microbiology 43 (1997) 895 – 914. [8] G.A. Beattie, S.E. Lindow, Bacterial colonisation of leaves: a spectrum of strategies, Phytopathology 89 (1999) 353 – 359. [9] D.E. Legard, J.E. Hunter, Pathogenicity on bean of Pseudomonas syringae pv. syringae recovered from the phylloplane of weeds and from bean crop residue, Phytopathology 80 (1990) 938 –942. [10] C. Grondeau, A. Mabiala, R. Ait-Oumeziane, R. Samson, Epiphytic life is the main characteristic of the life cycle of Pseudomonas syringae pv. pisi, pea bacterial blight agent, European Journal of Plant Pathology 102 (1996) 353 – 363. [11] W.J. Rennie, Seedborne diseases, in: D.G. Jones (Ed.), The Epidemiology of Plant Diseases, Kulwer Academic Publishers, 1998, pp. 295 – 305. [12] S.J. Roberts, M.S. Ridout, L. Peach, J. Brough, Transmission of pea bacterial blight (Pseudomonas syringae pv. pisi ) from seed to seedling: effects of inoculum dose, inoculation method, temperature and soil moisture, Journal of Applied Bacteriology 81 (1996) 65 – 72. [13] J.D. Taylor, C.L. Dudley, Bacterial disease of coriander, Plant Pathology 29 (1980) 117 – 121. [14] H.-M. Toben, K. Rudolph, Pseudomonas syringae pv. coriandricola, incitant of bacterial umbel blight and seed decay of coriander (Coriandrum sati6um L.) in Germany, Journal of Phytopathology 144 (1996) 169 – 178. [15] S.J. Refshauge, Bacterial blight of coriander, Honours thesis, School of Botany and Zoology, Australian National University, 2000. [16] J. Dennis, J. Wilson, Disease control in coriander and other spice seeds, Rural Industries Research and Development Institute report, (1999). Available online: http://www.rirdc.gov.au/reports/ NPP/DAS-40A.doc, 1997. [17] E.O. King, M.K. Ward, D.E. Raney, Two simple media for the demonstration of pyocyanin and fluorescin, Journal of Laboratory and Clinical Medicine 44 (1954) 301 – 307.
S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625 [18] A.C. Delves, A. Matthews, D.A. Day, A.S. Carter, B.J. Carroll, P.M. Gresshoff, Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors, Plant Physiology 82 (1986) 588 – 590. [19] G.L. Bender, M. Nayudu, W. Goydych, B.G. Rolfe, Early infection events in the nodulation of the non-legume Parasponia andersonii by Bradyrhizobium, Plant Science 51 (1987) 285 – 293.
.
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[20] P.H. Goodwin, Effect of common bacterial blight on leaf photosynthesis of bean, Canadian Journal of Plant Pathology 14 (1992) 203 – 206. [21] J. Jiao, P. Goodwin, B. Grodzinski, Photosynthesis and export during steady-state photosynthesis in bean leaves infected with the bacterium Xanthomonas campestris pv. phaseoli, Canadian Journal of Botany 74 (1996) 1 – 9.
Invasion by Pseudomonas syringae pv. coriandricola is responsible for bacterial blight of coriander S.J. Refshauge, M. Nayudu * School of Botany and Zoology, Australian National Uni6ersity, Canberra ACT 0200, Australia Received 25 January 2001; received in revised form 14 May 2001; accepted 15 May 2001
Abstract Bacterial blight is a major disease of coriander caused by Pseudomonas syringae pv. coriandricola. The nature of this disease was studied by inoculating coriander plants with rifampicin resistant P. s. pv. coriandricola. All inoculated plants showed disease symptoms and the level of mortality was significant. The pathogen was isolated from different parts of inoculated coriander plants implying internal infection. Macroscopic inspection of diseased plants showed blackening of leaf veins and surrounding tissues. Closer examination confirmed the presence of large numbers of bacterial cells within leaf veins. Examination using light and electron microscopy showed infected coriander stem tissue contains large numbers of bacteria within xylem vessels and neighbouring pith cells. The interstitium of infected plants also contained large bacterial populations. Isolation and culture of bacteria from infected tissues confirmed the identity of the pathogen observed. No bacterial cells were seen within phloem vessels or below the inoculation site. These results suggest that the coriander pathogen utilizes the vascular system of the host for multiplication and transport, leading to infection of many tissues and, potentially, host death. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pseudomonas syringae pv. coriandricola; Coriander; Bacterial blight
1. Introduction Pathogenic variants (pathovars) of the bacterium Pseudomonas syringae are responsible for bacterial blights in stone fruits, legumes and many grain crops world wide [1]. Several of these diseases have been shown to involve invasive bacteria. Pathovars of P. syringae have been examined in a number of plants demonstrating that spread of bacteria throughout the plant is usually due to invasion of the vascular system [1 – 6]. Infection typically is initiated by multiplication within stomata followed by invasion of sub-stomatal tissues [5–8]. Bacterial blights of bean and pea (caused by P. s. pv. phaseolicola and P. s. pv. pisi, respectively) are well studied models for understanding pathogenesis in simiAbbre6iations: TEM, transmission electron microscopy; pv., pathovar. * Corresponding author. Tel.: +61-2-612-53643; fax: + 61-2-61255573. E-mail address: [email protected] (M. Nayudu).
lar systems. Both pathogens are seed-borne and reside epiphytically until an opportunity for invasion occurs [9–11]. Each pathogen develops in the seed coat of the germinating seedling and may contaminate the growing cotyledons [2,12]. Once within the vascular system these pathogens spread throughout the plant causing lesions, cankers and death [2,11]. Bacterial blight is a major disease of coriander (Coriandrum sati6um) caused by the non-fluorescent bacterium P. s. pv. coriandricola. The disease is responsible for large production losses in Australia, Europe and America [13,14]. The pathogen, like P. s. pv. phaseolicola and P. s. pv. pisi, is seed-borne and typically affects plants during flowering [14]. Initial symptoms include lesion formation, blackening of leaf veins and wilting. Extended infection reduces total plant biomass [15] leading to yield losses and, in severe cases, plant death. P. s. pv. coriandricola has been isolated from infected coriander tissues using selective media [16], suggesting that the pathogen may invade host tissues. In this study we demonstrate that P. s. pv. coriandricola is an invasive pathogen of coriander with the ability to penetrate cell walls and multiply intracellularly.
0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 4 6 1 - 7
622
S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625
2. Materials and methods
2.1. Bacterial strains and media P. s. pv. coriandricola strain 1088b (supplied by J. Dennis, South Australian Research and Development Institute, Adelaide, Australia) was cultured on Kings B media [17] at 25 °C for 48 h. A rifampicin resistant strain was obtained by isolating spontaneous mutants on nutrient agar containing rifampicin (200 mg/ml). This strain was observed to retain normal levels of pathogenicity [15]. Detection of P. s. pv. coriandricola in infected coriander tissues was achieved by crushing surface sterilized samples in nutrient broth and spreading 100 ml of the suspension onto agar containing rifampicin (200 mg/ ml). Plates were incubated at 25 °C for 24 h and inspected for growth of P. s. pv. coriandricola.
2.2. Plant culture Sterilized coriander seeds were germinated at 20 °C on Herridges medium [18] containing 2% agar. Seedlings were planted into soil and grown in a temper-
ature controlled glasshouse. One-month-old plants were inoculated by inserting a sterile 25 gauge needle coated with P. s. pv. coriandricola through the crown region. Infection proceeded for 10 days, whereupon tissue samples were prepared for electron microscopy. Sampling of untreated plants provided the control.
2.3. Light and electron microscopy Samples of coriander leaves with symptoms visible with the unaided eye were externally examined using a low power light microscope. The following method of section preparation for high power microscopy was adapted from Bender et al. [19]. Resin fixing of samples to enable sectioning was achieved by immersing tissue samples from several different regions of infected plants in primary fixative consisting of 2% paraformaldehyde and 2.5% glutaraldehyde in 30 mM PIPES buffer (pH 7) for 3 h. Samples were post-fixed in 1% osmium tetroxide, dehydrated with serial ethanol rinses and embedded in Epon araldite resin. Sections were cut using a Reichert–Jung Ultracut microtome. Light microscope sections were stained using toluidine blue while silver ultrathin sections for transmission electron
Fig. 1. Coriander leaves 10 days after inoculation with P. s. pv. coriandricola: (A) blackening of tissues along veins on artificially infected coriander leaves (scale in millimetres); (B) higher magnification of inset (A) showing spread of symptoms from leaf veins to surrounding tissues (mag× 10); (C) cross section of infected coriander leaf tissue showing bacteria (arrows) within the vascular system and several neighbouring cells (mag×50).
S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625
microscopy (TEM) were stained using 6% aqueous uranyl acetate and lead citrate. TEM sections were visualized at 75 kV using an Hitachi 7100 electron microscope.
3. Results and discussion The high mortality rate observed among inoculated plants (approximately 90%) demonstrated the potential severity of the coriander blight disease. P. s. pv. coriandricola was isolated on selective media from infected roots, stems, petioles and leaves. No bacterial cells were recovered from uninoculated control plants. Light microscopy of infected leaves showed discolouration of the vascular system and surrounding tissues (Fig. 1A and B). These symptoms are typical of heavily infected tissue and suggest that P. s. pv. coriandricola spreads by following the path of the vascular system and penetrating surrounding tissues. The observation of large populations of P. s. pv. coriandricola
623
within the vessels of infected leaf tissue (Fig. 1C) provides evidence that the pathogen may spread via this route. The absence of the pathogen from surrounding tissues may simply be an artefact of sampling, as the pathogen may have penetrated these tissues given adequate time. This is the first visual evidence that P. s. pv. coriandricola invades the vascular system of coriander plants. Ultrastructural studies showed large populations of the pathogen within the xylem vessels of coriander stem tissue (Fig. 2A), suggesting that blockage may occur and cause wilting. Such blockage has been observed in plum trees infected with P. s. pv. syringae [5]. Nutrient depletion caused by the growing P. s. pv. coriandricola population also may contribute to degeneration of host tissues. Pathogen transmission within the plant appears to be primarily through xylem vessels. This may be an active process, since the bacterium is motile, or passive, induced by fluid flow and expansion of the bacterial population. The number of bacteria in stem tissues was
Fig. 2. Cross sections of coriander stem tissue 10 days after artificial infection with P. s. pv. coriandricola: (A) xylem vessels (arrow) heavily infected with the pathogen are surrounded by apparently uninfected interfascicular cambium (ic) and pith cells (p); (B) heavily infected pith cells (arrow) between apparently uninfected vascular bundles and interfascicular cambium (ic); (C) TEM image of pith cells infected with the bacterium, bound by the cell wall (cw) and neighbouring uninfected pith cells (mag × 4000); (D) TEM image clearly showing bacteria in the interstitium between three apparently uninfected pith cells (mag × 8000).
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S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625
noted to diminish with distance from the infection site, possibly due to the slow rate of bacterial spread and narrowing of the vessels. Interestingly, the pathogen was not observed in phloem vessels. Pith cells neighbouring xylem vessels also were observed to be infected with large numbers of bacteria (Fig. 2B). Examination of stem tissues using TEM revealed varied numbers of P. s. pv. coriandricola within pith cells (Fig. 2C) and interstitial spaces (Fig. 2D). While pith cells close to the xylem vessels were frequently infected, the number of infected cells diminished with distance from the vessels. No bacteria were observed within the untreated control plants. The ability of P. s. pv. coriandricola to enter surrounding tissues from xylem vessels in the stem may allow the organism to exit veins and spread locally within leaves. Here the pathogen may have a powerful effect on its host by destroying large numbers of chloroplasts and reducing productivity. This effect has been observed in bacterial blight of bean caused by Xanthomonas campestris pv. phaseoli [20,21]. The presence of bacteria within the interstitial spaces close to xylem vessels is indicative of the pathogen’s ability to exit between or through xylem cells. This is similar to the nature of P. s. pv. pisi, the closely related causative agent of bacterial blight of bean [2]. Further, the observation of bacteria within cells is evidence of the ability of P. s. pv. coriandricola to penetrate cell walls. This suggests that P. s. pv. coriandricola produces cell wall degrading enzymes or pectinases as observed in related pathovars [1,7]. Infection of plant organs is likely hastened by infection of the interstitium, which may allow bacteria to move freely throughout the host. Bacteria were not observed during microscopic examination of root tissue. However, bacteria in low numbers were isolated repeatedly from root tissues on selective media. This discrepancy could be due to low bacterial populations in the root tissue and may be an artefact of sampling time. Since the pathogen is motile, some cells may move against the fluid flow within xylem vessels, and hence would be detected by isolation from root tissues onto selective media. This ultrastructural examination of infected coriander tissues has demonstrated for the first time that P. s. pv. coriandricola penetrates into tissues and cells of coriander. The findings of this study contribute to the current understanding of related P. s. pathovars and, through an improved understanding of the mechanism of pathogenesis, will assist future research on control strategies for coriander blight.
Acknowledgements This research was only possible with the assistance and support of D. Jacobs (AgSearch Australia Pty. Ltd., Harden, Australia) through a research grant. J. Goodan
and J. Dennis (South Australian Research and Development Institute, Adelaide, Australia) are thanked for provision of P. s. pv. coriandricola strain 1088b. S. Stowe and L. Shen (Electron Microscopy Unit, Research School of Biological Sciences, ANU, Canberra, Australia) are thanked for their assistance with electron microscopy. References [1] K.W.E. Rudolph, Pseudomonas syringae pathovars, in: U.S. Singh, R.P. Singh, K. Kohmoto (Eds.), Prokaryotes, vol. I, (3volume series: Pathogenesis and Host Specificity in Plant Diseases), Elsevier Science Ltd, 1995, pp. 47 – 138. [2] J.C. Walker, Plant Pathology, McGraw Hill Inc., Sydney, 1969, pp. 126 – 133. [3] I.M.M. Roos, M.J. Hattingh, Systemic invasion of plum leaves and shoots by Pseudomonas syringae pv. syringae introduced into petioles, Phytopathology 77 (1987) 1253 – 1257. [4] I.M.M. Roos, M.J. Hattingh, Systemic invasion of cherry leaves and petioles by Pseudomonas syringae pv. morsprunorum, Phytopathology 77 (1987) 1246 – 1252. [5] M.J. Hattingh, I.M.M. Roos, E.L. Mansvelt, Infection and systemic invasion of deciduous fruit trees by Pseudomonas syringae in South Africa, Plant Disease 73 (1989) 784 – 789. [6] E.L. Mansvelt, M.J. Hattingh, Scanning electron microscopy of invasion of apple leaves and blossoms by Pseudomonas syringae pv. syringae, Applied and Environmental Microbiology 55 (1989) 533 – 538. [7] J. Hallmann, A. Quadt – Hallmann, W.F. Mahaffe, J.W. Kloepper, Bacterial endophytes in agricultural crops, Journal of Microbiology 43 (1997) 895 – 914. [8] G.A. Beattie, S.E. Lindow, Bacterial colonisation of leaves: a spectrum of strategies, Phytopathology 89 (1999) 353 – 359. [9] D.E. Legard, J.E. Hunter, Pathogenicity on bean of Pseudomonas syringae pv. syringae recovered from the phylloplane of weeds and from bean crop residue, Phytopathology 80 (1990) 938 –942. [10] C. Grondeau, A. Mabiala, R. Ait-Oumeziane, R. Samson, Epiphytic life is the main characteristic of the life cycle of Pseudomonas syringae pv. pisi, pea bacterial blight agent, European Journal of Plant Pathology 102 (1996) 353 – 363. [11] W.J. Rennie, Seedborne diseases, in: D.G. Jones (Ed.), The Epidemiology of Plant Diseases, Kulwer Academic Publishers, 1998, pp. 295 – 305. [12] S.J. Roberts, M.S. Ridout, L. Peach, J. Brough, Transmission of pea bacterial blight (Pseudomonas syringae pv. pisi ) from seed to seedling: effects of inoculum dose, inoculation method, temperature and soil moisture, Journal of Applied Bacteriology 81 (1996) 65 – 72. [13] J.D. Taylor, C.L. Dudley, Bacterial disease of coriander, Plant Pathology 29 (1980) 117 – 121. [14] H.-M. Toben, K. Rudolph, Pseudomonas syringae pv. coriandricola, incitant of bacterial umbel blight and seed decay of coriander (Coriandrum sati6um L.) in Germany, Journal of Phytopathology 144 (1996) 169 – 178. [15] S.J. Refshauge, Bacterial blight of coriander, Honours thesis, School of Botany and Zoology, Australian National University, 2000. [16] J. Dennis, J. Wilson, Disease control in coriander and other spice seeds, Rural Industries Research and Development Institute report, (1999). Available online: http://www.rirdc.gov.au/reports/ NPP/DAS-40A.doc, 1997. [17] E.O. King, M.K. Ward, D.E. Raney, Two simple media for the demonstration of pyocyanin and fluorescin, Journal of Laboratory and Clinical Medicine 44 (1954) 301 – 307.
S.J. Refshauge, M. Nayudu / Plant Science 161 (2001) 621–625 [18] A.C. Delves, A. Matthews, D.A. Day, A.S. Carter, B.J. Carroll, P.M. Gresshoff, Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors, Plant Physiology 82 (1986) 588 – 590. [19] G.L. Bender, M. Nayudu, W. Goydych, B.G. Rolfe, Early infection events in the nodulation of the non-legume Parasponia andersonii by Bradyrhizobium, Plant Science 51 (1987) 285 – 293.
.
625
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