JOURNAL OF BACTERIOLOGY, June 1991, p. 3432-3439 Vol. 173, No. 11 0021-9193/91/113432-08$02.00/0 Copyright X 1991, American Society for Microbiology Isoflavonoid-Inducible Resistance to the Phytoalexin Glyceollin in Soybean Rhizobia MARTIN PARNISKE,* BETTINA AHLBORN, AND DIETRICH WERNER Fachbereich Biologie der Philipps-Universitat, Karl-von-Frisch-Strasse, D-3550 MarburglLahn, Federal Republic of Germany Received 13 August 1990/Accepted 25 March 1991 The antibacterial effect of the soybean phytoalexin glyceollin was assayed using a liquid microculture technique. Log-phase cells of Bradyrhizobium japonicum and Sinorhizobium fredii were sensitive to glyceollin. As revealed by growth rates and survival tests, these species were able to tolerate glyceollin after adaptation. Incubation in low concentrations of the isoflavones genistein and daidzein induced resistance to potentially bactericidal concentrations of glyceollin. This inducible resistance is not due to degradation or detoxification of the phytoalexin. The inducible resistance could be detected in B. japonicum 110spc4 and 61A101, representing the two taxonomically divergent groups of this species, as well as in S. fredii HH103, suggesting that this trait is a feature of all soybean-nodulating rhizobia. Glyceollin resistance was also inducible in a nodDlD2YABC deletion mutant of B. japonicum 110spc4, suggesting that there exists another recognition site for flavonoids besides the nodD genes identified so far. Exudate preparations from roots infected with Phytophthora megasperma f. sp. glycinea exhibited a strong bactericidal effect toward glyceollin-sensitive cells of B. japonicum. This killing effect was not solely due to glyceollin since purified glyceollin at concentrations similar to those present in exudate preparations had a much lower toxicity. However, glyceollin-resistant cells were also more resistant to exudate preparations than glyceollin-sensitive cells. Isoflavonoid-inducible resistance must therefore be ascribed an important role for survival of rhizobia in the rhizosphere of soybean roots. The gram-negative, slow-growing soil bacterium Brady- rhizobia, which may have consequences for the symbiotic rhizobium japonicum as well as the fast-growing Sinorhizo- interaction. bium fredii (3) are able to form effective nitrogen-fixing Glyceollin, a phytoalexin of soybean (Fig. 1), is strongly symbioses with the host plant Glycine max. During the early induced in response to a number of biological, chemical, and stages of interaction between host and microsymbiont, physical stresses, e.g., infection with pathogenic fungi, the rhizobia act very much like plant pathogens: they have to presence of heavy metals, or UV radiation (10, 19, 45). In invade the root hairs of the host, thereby overcoming symbiotic interactions, the accumulation of glyceollin has possible plant defense responses. Analogies between the also been observed. Morandi et al. (26) found higher con- early stages of the Rhizobium-legume symbiosis and a patho- centrations of glyceollin in mycorrhiza-infected roots of G. genic interaction have been pointed out by several investi- max than in axenic roots. Glyceollin accumulation may also gators (8, 9, 21, 38, 42). take place in later stages of the Glycine-Bradyrhizobium In a number of plant-pathogen interactions, the induction interaction: nodule cells of Glycine sp. display a hypersen- of phytoalexins, plant-derived antimicrobial low-molecular- sitive reaction when infected with incompatible or mutant weight molecules, is believed to play an essential part in host strains of Bradyrhizobium sp. (32, 34, 46). The work of resistance (10, 43). In several legumes, phytoalexins belong several laboratories confirms the assumption that, even in to the isoflavonoid type (e.g., pisatin in Pisum sp.; glyceollin very early stages of the interaction, rhizobial infection in Glycine sp.; medicarpin in Medicago sp.). This is of contributes to enhanced release of flavonoids and phytoalex- special interest because flavonoids have been shown to be ins by the host plant. Recourt et al. (37) found an increase in regulators of rhizobial nod gene activity (see reference 25 for the number of flavonoids exuded by Vicia sativa in response a review). Considering the microsymbionts of soybean, the to infection with a compatible Rhizobium leguminosarum most active inducing compounds are isoflavonoids (1, 14, strain. Enhanced levels of glyceollin could be detected in the 22). Despite this intriguing bifunctionality of isoflavonoids, rhizosphere of G. max 20 h after infection with B. japonicum i.e., regulatory substances and phytoalexins, only a few data (33, 39a). are available concerning the significance of phytoalexins in It is very likely that soybean roots produce considerable the interaction between rhizobia and their legume host. amounts of glyceollin under field conditions due to the Pankhurst and co-workers studied the inhibition of rhizobia widespread presence of stress factors that induce defense by isoflavonoids (28) as well as by flavolans (31). They found reactions. High glyceollin levels are a challenge for micro- an ineffective Rhizobium strain to be more sensitive to the organisms living in the rhizosphere, including symbiotic root flavolan of the host plant Lotus corniculatus than its bacteria. In this study, we analyzed the effect of a phyto- effective counterpart (30). In addition, the ineffective strain alexin of the host plant on the microsymbionts of soybean. induced the synthesis of higher flavolan concentrations in the nodules of this plant (29). Taken together, these results indicate the importance of flavonoids as toxic compounds for MATERIALS AND METHODS Microbial strains. B. japonicum USDA 110spc4 (17) and 61A101 (Nitragin Co., Milwaukee, Wis.) and S.fredii HH103 * Corresponding author. (B. Bohlool, Paia, Hawaii) were used as wild-type strains. 3432 VOL. 173, 1991 INDUCIBLE PHYTOALEXIN RESISTANCE IN RHIZOBIA 3433 density at 600 nm (OD6.) of the suspension in the vials. Measurements were corrected for the optical density of vials 0 containing only medium, giving the AOD6.J value. Aliquots (50 Ru) were taken at varying intervals, bacteria were pel- 1 leted by centrifugation, and the glyceollin concentration in the supematant was analyzed by HPLC. Alternatively, the degree of resistance ofbacterial cultures was tested by using viability tests. Aliquots of the cultures were adjusted to a AOD6. of 0.1. Samples of 10 ,u were mixed with 90 ,ul of 20E containing glyceollin. When indi- HO 0 cated, 20E was replaced by 5 mM MES buffer (pH 6.5) 2 (MES). The final concentration of glyceollin was 90 to 300 ,uM. After 3 h of incubation, appropriate dilutions were OH plated on 20E agar to estimate the number of surviving CFU. In some experiments, a mixed inoculum was used contain- FIG. 1. Structures of the soybean phytoalexin glyceollin I (1) and ing equal numbers of cells of B. japonicum 110spc4 and A3. the isoflavones daidzein (R = H) and genistein (R = OH) (2). To achieve this, cultures of both strains were diluted to a AOD6. of 0.1 and mixed in a 1:1 ratio. A 10-,ul sample of the resulting suspension was mixed with 90 RI of MES contain- The nodDlD2YABC deletion mutant A1240 and the ing either glyceollin or root exudate at different concentra- nijD::TnS insertion mutant A3 (17) of B. japonicum 110spc4 tions. After 3 h of incubation, appropriate dilutions were were supplied by M. Gottfert and H. Hennecke, Zurich, plated in parallel on 20E agar and on 20E agar supplemented Switzerland. Phytophthora megasperma f. sp. glycinea race with kanamycin and streptomycin (100 mg of each per liter). 1 was obtained from T. Waldmuller and J. Ebel, Freiburg, B. japonicum A3 could be differentiated from its parent Federal Republic of Germany. strain B. japonicum 110spc4 by its ability to grow at these Isolation and analysis of glyceollin. Glyceollin was isolated concentrations of antibiotics. by the method of Grisebach (16), with the modification that Growth of seedlings, zoospore infection, and exudate prep- glyceollin isomers I to III were collected after high-pressure aration. Culture and zoospore induction ofP. megasperma f. liquid chromatographic (HPLC) separation from other fla- sp. glycinea were performed as previously described (19). vonoids. The identity ofglyceollin isomers was confirmed by Soybean seeds (G. max cv. Kenwood) were surface steril- the UV spectra and mass spectra of their trimethylsilyl ized by immersion in 30% H202 for 10 min, washed 10 times derivatives. with water, soaked for 6 h, and washed again. The seeds HPLC analysis of media and culture supematants contain- were then placed on nitrogen-free nutrient agar (47) and ing glyceollin was performed under isocratic conditions grown for 2 days in a growth chamber at 25°C (16 h light/8 h using a 5-,um RP-18 column (Serva, Heidelberg, Federal dark). The seedlings were transferred to 2.5-ml test tubes Republic of Germany) with methanol-water (50:50, vol/vol) with their roots submerged in 2.3 ml of a zoospore suspen- as the solvent system. The analysis of root exudates was sion of P. megasperma f. sp. glycinea race 1 (approximately done with a linear gradient from 100% water to 70% aceto- 104 spores per plant). After 5 days of incubation in the nitrile over 32 min. After HPLC separation, glyceollin con- growth chamber, the root exudates of 100 seedlings were centration was determined with a UV detector and an pooled, adjusted to 10% methanol, and filtered successively integrator, using an 8280 of 10,000. through glass fiber and 0.2-pum-pore-size filters. The exudate Glyceollin isomers were separated by two-dimensional was evaporated to dryness and taken up in 50% ethanol. A thin-layer chromatography, using Silica Gel 60 nano-HPTLC white, ethanol-water-insoluble precipitate was discarded. plates (10 by 10 cm) with fluorescent indicator (Macherey- Aliquots were evaporated to dryness and redissolved in 5 Nagel, Duren, Federal Republic of Germany). The solvent mM MES buffer (pH 6.5) containing 1% ethanol, giving final system for the first dimension was chloroform-methanol- test concentrations of glyceollin from 90 to 270 ,uM, as formic acid (93:6:1, vol/vol), and that for the second dimen- determined by HPLC.