The impact of microbial biotransformation of catechin in enhancing the allelopathic effects of Rhododendron formosanum

Abstract

Rhododendron formosanum is distributed widely in the central mountains in Taiwan and the major allelopathic compound in the leaves has been identified as (-)-catechin, which is also a major allelochemical of an invasive spotted knapweed in North America. Soil microorganisms play key roles in ecosystems and influence various important processes, including allelopathy. However, no microorganism has been identified as an allelochemical mediator. This study focused on the role of microorganisms in the allelopathic effects of R. formosanum. The microorganism population in the rhizosphere of R. formosanum was investigated and genetic analysis revealed that the predominant genera of microorganisms in the rhizosphere of R. formosanum were Pseudomonas, Herbaspirillum, and Burkholderia. The dominant genera Pseudomonas utilized (-)-catechin as the carbon source and catalyzed the conversion of (-)-catechin into protocatechuic acid in vitro. The concentrations of allelochemicals in the soil were quantified by liquid chromatography-electrospray ionization/tandem mass spectrometry. The concentration of (-)-catechin in the soil increased significantly during the extreme rainfall in the summer season and suppressed total bacterial populations. Protocatechuic acid accumulation was observed while total bacterial populations increased abundantly in both laboratory and field studies. Allelopathic interactions were tested by evaluating the effects of different allelochemicals on the seed germination, radicle growth, and photosynthesis system II of lettuce. Protocatechuic acid exhibited higher phytotoxicity than (-)-catechin did and the effect of (-)-catechin on the inhibition of seed germination was enhanced by combining it with protocatechuic acid at a low concentration. This study revealed the significance of the allelopathic interactions between R. formosanum and microorganisms in the rhizosphere. These findings demonstrate that knowledge regarding the precise biotransformation process of (-)-catechin by microorganisms in the environment is necessary to increase our understanding of allelopathy.

Figure 1. Pairwise correlations between (-)-catechin concentration, monthly precipitation, and bacterial populations.

(A) Correlation between the concentration of (-)-catechin in soil of R. formosanum and monthly precipitation (r = 0.919, P < 0.0001, n = 14). (B) Correlation between the concentration of (-)-catechin and bacterial populations in soil of R. formosanum (r = -0.714, P = 0.0041, n = 14).

Figure 2. Bacterial flora (A) and catechin utilizing bacteria (B) in the rhizosphere of a Rhododendron formosanum plantation.

(-)-Catechin was the only carbon source added to the medium that was used for microbial isolation. After 3 months of incubation, Pseudomonas spp., Burkholderia spp., Variovorax spp. Stenotrophomonas spp., and Pandoraea spp. were isolated and identified as dominant catechin-utilizing bacteria.

Figure 3. Metabolic pathway for (-)-catechin transformed by Pseudomonas sp. CRF3-Ps-1 was analysed by the LC-ESI-MS/MS method (A).

(-)-Catechin (CAT) was transformed into taxifolin (Tax) via ketone formation during the first 24 h. Subsequently, C-ring hydrolysis occurred and generated protocatechuic acid (PCA) and glycerol (Gly). Finally, (-)-catechin was transformed into glycerol 72 h after incubation. The possible transformation hypothesis is also illustrated (B).

Figure 4. Relative concentrations of (-)-catechin and protocatechuic acid, and the bacterial population in the medium, during 120 h incubation with Pseudomonas sp. CRF3-Ps-1.

(●) Relative concentration of catechin (r = -0.958, P = 0.0025, n = 6); (○) Bacterial population of Pseudomonas CRF3-Ps-1 (r = 0.974, P = 0.001, n = 6); (△) Relative concentration of protocatechuic acid (r = 0.874, P = 0.0226, n = 6). (B) Correlations between the concentration of protocatechuic acid and bacterial populations in the soil of R. formosanum (r = 0.734, P = 0.0066, n = 12).

Figure 5. The phytotoxic effects of (-)-catechin on the seed germination (A) and radicle growth (B) of Lactuca sativa at different concentrations in combination with 0 µg, 10 µg and 50 µg protocatechuic acid.

Error bars represent the standard errors of the mean.

Figure 6. Hypothetical scheme of allelopathic interactions between Rhododendron formosanum and dominant Pseudomonas species.

Initially, (-)-catechin from the leaves of R. formosanum accumulates in the soil via leaching. After a period of time, the population of catechin-utilizing Pseudomonas spp. increases and the (-)-catechin is converted into protocatechuic acid through biotransformation. The protocatechuic acid exhibited synergistic inhibitory effects with the original (-)-catechin on the seed germination of the plant. Finally, (-)-catechin is transformed into glycerol and utilized by microorganisms as a carbon source. Thus, interactions between R. formosanum and the dominant Pseudomonas species are established.