Effects of glyphosate on compatible and incompatible plant-bacterium interactions

Acta Phytopathologica et Entomologica Hungarica 29 (3-4), pp. 353-359 (1994)

Effects of Glyphosate on Compatible and Incompatible Plant-Bacterium Interactions*

MARJA UOTILA 1, T. KOMIVES 2, P. OTT 2 and Z. KLEMENT 2

'Department of Botany, University of Helsinki, P.O. Box 7, SF-00014 Helsinki, Finland !Plant Protection Institute, Hungarian Academy of Science, H-1525 Budapest, P.O. Box 102, Hungary

Effects of the herbicide glyphosate on the infection by Pseudomonas bacteria of susceptible and resistant bean varieties were investigated. Glyphosate, up to 250 millimolar concentrations had no effect on the growth of Pseudomonas syringae pv. phaseolicola bacteria in vitro. Subphytotoxic levels of glyphosate did not influence the compatible interaction between the bacteria and the sus-ceptible trifolia of Red Kidney bean plants. Following treatment with glyphosate, the type of the in-compatible interaction between the bacteria and the trifolia and primary leaves of the resistant bean variety 0 2 , and primary leaves of older Red Kidney bean plants was shifted towards the compatible one,

Phytotoxicity of the herbicide glyphosate is due to its ability to block the biosyn-thesis of aromatic amino acids at the stage of 5-enolpyruvylshikimate 3-phosphate syn-thase (Amrhein et al., 1980). As a result, biochemical alteration of several processes in plants take place. In addition to aromatic amino acid depletion leading to dramatic changes in protein synthesis (Duke and Hoagland, 1985; Uotila et al., 1995) glyphosate also disrupts biosynthesis of plant defense compounds (phytoalexins) derived from phenylalanine. Thus, glyphosate inhibits glyceollin accumulation in soybean and leads to a concomitant loss of resistance against an incompatible race of Phytophthora mega-sperma (Keen et al., 1982). Sublethal concentrations of glyphosate increase the activity of the mycoherbicide Alternaria crassicae to Cassia obtusifolia (Sharon et al., 1992). In fact, the apparent herbicidal activity of glyphosate on bean seedlings has been partly attributed to parasitization of seedlings by soilborne fungi (Johal and Rahe, 1984). Glyphosate was shown to interfere with the expression of defence in beans against Colletotrichum lindemuthianum by suppressing the plant's ability to produce phytoalex-ins (Johal and Rahe, 1988). Interestingly, according to a recent study, glyphosate has no effect on the pathogenicity of Fusarium solani f. sp. pisi to pea seedlings (Kawate et al., 1992), although levels of pisatin, the main phytoalexin of this plant are also dependent on the activity of the phenolic biosynthesis pathway (Kőmíves and Casida, 1983).

The objective of our investigation was to determine the effect of glyphosate on the infection of bean plants by Pseudomonas syringae pv. phaseolicola bacterium using bacterial count determinations to characterize compatible and incompatible plant-patho-gen interactions (Klement and Lovrekovich, 1962).

•Presented in part at the 8th IUPAC Congress of Pesticide Chemistry, 4 - 9 July, 1994, Washington, DC, USA

0238-1249/94/$ 4.00© 1994, Akadémiai Kiadó, Budapest

354 JJotila et at.: Effects of glyphosate on plant-bacterium interactions 354

Materials and Methods

Chemicals

Glyphosate (96.7%) was obtained from Monsanto Agricultural Co., St. Louis, Mo. Phaseollin was a gift of Dr. J. A. Bailey (University of London, UK). Other chemicals (buffers, solvents, etc.), were from commercial sources, and used without further purification.

Bacteria

Pseudomonas syringae pv. phaseolicola (Burkholder) Young et al., strain S2/IP038 (smooth type, race 2; a generous gift from Dr. Klaus Rudolph, University of Gottingen, FRG), has been cultivated on King B medium for two days, then diluted with distilled water to 106 bacteria/ml for use as inoculum.

Plant material

Surface disinfected seeds of Phaseolus vulgaris L. var. nanus cv. Red Kidney (sensitive strain) and resistant white dwarf bean (Phaseolus vulgaris L. breeding line 02) obtained from Hajdúsági Seed Production Ltd. (Hungary) and from Dr. Klaus Rudolph (University of Gottingen, FRG), respectively, were sown in soil in plastic pots. Plants were cultivated in the greenhouse, and at the age of 14 days, transferred to the phytotron. 21 days old plants were treated with glyphosate (applied as spray to the leaves), and, after 3 days, inoculated with the bacterial suspension. From each plant one young, half developed trifoliate and one primary leaf were infiltrated by the bacterial suspension using high pressure spray applied to the abaxial surface (Klement, 1990). Following inoculation leaves were thoroughly washed with running water to remove all bacteria from the leaf surface. Symptoms of bacterial infection on the leaves (appearance of water-soaked spots followed by loss of turgor and, finally, tissue death) were assayed daily.

Bacterial counts

Three 1 cm discs were taken from each treated leaf, homogenized in distilled water, and aliquots from three dilutions were assayed for bacterial numbers by the plate count method (Klement, 1990).

Acta Phytopathologica et Entomologica Hungarica 29, 1994


Results

Effect of glyphosate on bacteria in vitro

In vitro, glyphosate (up to 2.5 mM) had no effect on the multiplication of Pseu-domonas syringae pv. phaseolicola (data not shown).

Response of plants to glyphosate and to bacterial infection

Preliminary time-course and dose-response experiments were carried out to es-tablish a glyphosate concentration range resulting in no phytotoxic damage to the plants. Concentrations of glyphosate higher than 2.5 mM at exposure times longer than two weeks resulted in phytotoxic symptoms typical to glyphosate: slow, but continuous loss of chlorophyll and tissue turgor, leading to uniform yellowing and browning and finally death of the leaf tissues. No difference was observed between the sensitivity of the two bean cultivars to glyphosate.

Symptoms of bacterial infection on the leaves were distinctly different from those caused by glyphosate: first water-soaked spots appeared, these spots lost turgor and, finally, tissue death became evident. Glyphosate treatment had no influence on the symptom development caused by the bacterial infection to trifolia of Red Kidney bean plants: within one week all treated leaves had water-soaked spots that increased in size with time, and within two weeks tissues of the whole leaf have collapsed. Primary leaves (resistant) of Red Kidney and primary leaves and trifolia of resistant 0 2 beans, however, responded differently: bacterial infection of control leaves resulted in no visual symp-toms. In contrast, bacterial infection of these leaves pretreated with glyphosate resulted in symptoms described for the trifolia of Red Kidney plants.

Bacterial counts

A logarithmic curve characterizes the time-dependence of the number of bacteria in the compatible plant-parasite relation, e.g., in tissues of trifolia of Red Kidney bean plants. Following an initial lag period bacterial numbers rapidly increase, and, after about a week, they reach a maximum value of ca. 610 7 bacteria/g fresh weight of leaf tissue (Fig. 1). In the incompatible plant-parasite systems, e.g., in primary leaves of this susceptible bean variety, the bacterial counts follow a maximum curve (Fig. 2), similar to those for both the primary leaves and the trifolia of the resistant 0 2 bean plants (Figs 1 and 2).

Glyphosate has no effect on the bacterial numbers in the susceptible trifolia of Red Kidney bean plants (Fig. 1). Significant changes take place, however, in the resis-tant primary leaves of this plant following glyphosate treatment: the curve for bacterial counts becomes similar to that for the compatible one, i.e., to that for the trifolia. As a result of glyphosate treatment, maximum bacterial number in this plant tissue becomes



No. of bacteria (millions)

No. of bacteria (millions)

Fig. I. Effects of 0.25 mM glyphosate on bacterial numbers in the trifolia of Red Kidney and 0 2 bean plants



No, of bacteria (.millions)

days

No. of bacteria (thousands)

days

Fig. 2. Effects of 0.25 mM glyphosate on bacteriai numbers in the primary leaves of Red Kidney and 0 2 bean plants



three times higher and is recorded at a later time (Fig. 2). Similar changes take place in the trifolia and the primary leaves of the resistant 0 2 bean plants (Figs I and 2): glypho-sate shifts the type of the incompatible plant-parasite relationship in the direction of a compatible one.

Discussion

Several types of environmental and chemical stress influence the phenolic me-tabolism of plants: diphenyl ether (Komives and Casida, 1982) and N-phenylimide her-bicides and heavy metal pollutants (Uotila and Komives, unpublished, 1993), for exam-ple, are very efficient inducers of phytoalexin accumulation in several plants. Glyphosate may increase or decrease the contents of total phenols in plants (Duke and Hoagland, 1985). Since phenylpropanoid phytoalexins are constituents of the shikimate pathway after the site of glyphosate inhibition (Duke and Hoagland, 1985), their levels were consistently lowered in glyphosate-treated soybean (Keen et al., 1982), bean (Johal and Rahe, 1988) and Cassia obtusifolia plants (Sharon et al., 1992).

The bean - P. phaseolicola system provides an excellent model for studying pesti-cide effects on plant-microbe interactions. Young trifolia of Red Kidney beans are highly susceptible to infection by these bacteria. Primary leaves of older plants of this bean variety, as well as trifoiia and primary leaves of bean variety 02 , however, are resistant to P. phaseolicola. Symptomatology and time-course of bacterial growth (Figs 1 and 2) clearly differentiate between compatible and incompatible plant-parasite interactions.

It has previously been shown that treatment of plants with glyphosate decreased plant response to a point at which avirulent strains of pathogenic bacteria and fungi were rendered pathogenic (Sharon et al., 1992). Our data show that resistance of a bean culti-var to P. phaseolicola bacteria may be suppressed by glyphosate. A modification of the characteristic growth pattern of bacteria for the incompatible interaction provides a strong argument for the assumption that the presence of glyphosate mimics the com-patible interaction (Keen et al., 1982).

The results presented in this paper extend those of others who have reported that treatment of plants with glyphosate suppresses disease resistance. They demonstrate that resistance against bacteria is suppressed also and, in addition, that this is accompanied by suppression of phytoalexin formation. Although our findings support the possible role of phytoalexins protecting bean plants against infection by P. phaseolicola bacteria, they do not exclude the importance of other mechanisms of resistance influenced by glyphosate.

Acknowledgements

This research was supported by the Hungarian Scientific Research Fund (OTKA 1440 and F 6314). We thank Dr. Mária Hevesi, Dr. Gábor Gullner, Dr. Gyula Oros and Dr. Klaus Rudolph for helpful discus-sions.



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Effects of glyphosate on compatible and incompatible plant-bacterium interactions

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