Isolation, identification and characterization of ... - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Isolation, identification and characterization of soil microbes which degrade phenolic allelochemicals Z.-Y. Zhang, L.-P. Pan and H.-H. Li Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou, China

Keywords allelochemicals, biodegradation, p-coumaric acid, phenolic compounds, soil microorganism. Correspondence Hai-Hang Li, Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631, China. E-mail: [email protected]

2009 ⁄ 0259: received 10 February 2009, revised 6 July 2009 and accepted 6 October 2009 doi:10.1111/j.1365-2672.2009.04589.x

Abstract Aims: To isolate and characterize microbes in the soils containing high contents of phenolics and to dissolve the allelopathic inhibition of plants through microbial degradation. Methods and Results: Four microbes were isolated from plant soils using a screening medium containing p-coumaric acid as sole carbon source. The isolates were identified by biochemical analysis and sequences of their 16S or 18S rDNA, and designated as Pseudomonas putida 4CD1 from rice (Oryza sativa) soil, Ps. putida 4CD3 from pine (Pinus massoniana) soil, Pseudomonas nitroreducens 4CD2 and Rhodotorula glutinis 4CD4 from bamboo (Bambusa chungii) soil. All isolates degraded 1 g l)1 of p-coumaric acid by 70–93% in inorganic and by 99% in Luria-Bertani solutions within 48 h. They also effectively degraded ferulic acid, p-hydroxybenzoic acid and p-hydroxybenzaldehyde. The microbes can degrade p-coumaric acid and reverse its inhibition on seed germination and seedling growth in culture solutions and soils. Low pHs inhibited the growth and phenolic degradation of the three bacteria. High temperature inhibited the R. glutinis. Co2+ completely inhibited the three bacteria, but not the R. glutinis. Cu2+, Al3+, Zn2+, Fe3+, Mn2+, Mg2+ and Ca2+ had varying degrees of inhibition for each of the bacteria. Conclusions: Phenolics in plant culture solutions and soils can be decomposed through application of soil microbes in laboratory or controlled conditions. However, modification of growth conditions is more important for acidic and ions-contaminated media. Significance and Impact of the Study: The four microbes were first isolated and characterized from the soils of bamboo, rice or pine. This study provides some evidence and methods for microbial control of phenolic allelochemicals.

Introduction Phenolics have been reported to play a major allelochemical role in wide range of plant species (Rice 1984; Kuiters and Sarink 1986; Seal et al. 2004; Belz 2007; Macıás et al. 2007). They can be released into soils as root exudates, leaf leachates and products of plant tissue decomposition. The degradation of phenolics from soil is a result of ionization, oxidation, polymerization, sorption onto soil particles, seed and root uptake and transformation or utilization by micro-organisms (Blum 2004). Although it was hypothesized that phenolics are readily metabolized

by soil micro-organisms, phenolic acids accumulated in high amounts in soils of many crops and forestry plants and inhibited the growth of their neighbouring plants (Chou and Patrick 1976; Kuiters 1986; Li et al. 1992; Tsai and Young 1993; Zeng and Mallik 2006). Several methods for degradation of toxic chemicals have been developed and applied in environmental and wastewater industries, including biodegradation, chemical and physical degradation, sorption or fining by adsorbents and fractionation with solvents (Cerniglia 1992; Niu et al. 2005; Polymenakou and Stephanou 2005; Yang et al. 2007). In allelochemicals decomposition, Asao et al.

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1839–1849

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(1998) reported that the decrease of cucumber fruit yields caused by its root exudates can be reversed through removal of the root exudates by activated charcoal. Nilsson (1994) found that the allelopathic inhibition of Scots pine by toxins leached from Empetrum hermaphroditum can be removed by spreading of activated carbon on the soil to adsorb the toxins. Zeng and Mallik (2006) reported that some ectomycorrhizal fungi, such as Paxillus involutus and Laccaria bicolor, are able to use phenolic compounds as sole carbon sources in noncarbon nutrient medium. The growth inhibition of black spruce (Picea mariana) by several phenolic acids of Kalmia origin can be reversed by P. involutus. They pointed out that ectomycorrhizal species can control species interactions in higher plants by changing the rhizosphere chemistry. Bamboo is one of the most widely distributed plants in the world. It is well known that in most cases, there were almost no grasses growing beneath and near bamboo forests. Tsao and Young (1993) identified eleven phenolic compounds from the soil beneath bamboo, Denrocalamus latiflorus Munro. Their total phenolic concentration reached 1% of the soils. p-Coumaric acid was the main inhibitory component in the soil. Bamboo forests are common in the south provinces of China both as economic and landscape species. We developed three methods (Pan et al. 2008), including chemical oxidation, soil microbial degradation and transformation by transgenic plants with a plant polyketide synthase gene, to investigate the decomposition of phenolic acids in the bamboo soil. Li et al. (2007) found that application of 0Æ1 or 1% of H2O2 of the Fenton’s reagent to the soil of bamboo could reduce its p-coumaric acid by 32 or 37%, respectively. In this current study, we reported our results on identification and characterization of soil microbes isolated from fields of bamboo (Bambusa chungii), pine (Pinus massoniana) and rice (Oryza sativa), as well as their possible application in decomposing phenolics and reversal of growth inhibition of plants in culture solution and bamboo soil.

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plant growth tests were sampled from the same bamboo forest mentioned earlier. Chemical compounds of p-coumaric, ferulic and p-hydroxybenzoic acids are Sigma products (Sigma, St Louis, MO). HPLC solvents are purchased from Burdick & Jackson Inc. (Muskegon, MI). Other chemical reagents are all analytical grades and are purchased from local suppliers. Seeds of rice (O. sativa L.) were gift from the Rice Research Institute of Guangdong Province. Seeds of mung bean (Vigna radiata Linn. R. Wilczak) were purchased from a local market. Isolation and purification of microbes from soil samples Inorganic culture solution (K2HPO4 0Æ5 g, KH2PO4 0Æ5 g, MgSO4Æ7H2O 0Æ2 g, NaCl 0Æ2 g, CaCl2 0Æ1 g, MnSO4 0Æ01 g, NH4NO3 1Æ0 g, FeCl3 0Æ01 g in one litre, pH 6Æ0) containing p-coumaric acid as sole carbon source was used for all experiments concerning screening, isolation and culture of microbes, unless otherwise stated. One g of each soil sample was added to 200 ml of autoclaved inorganic culture solution containing 0Æ5 g l)1 of p-coumaric acid and incubated for 5 days at 30C with shaking (150 rpm). One millilitre of the first culture solution was transferred to 100 ml of the same fresh solution and incubated for 5 days at the same conditions. One millilitre of the second culture solution was transferred to 50 ml of inorganic culture solution containing 1 g l)1 of p-coumaric acid and incubated for another 5 days. The purpose of three transfers to fresh culture solutions is to dilute and reduce possible carbon sources from soils. The third culture solution was inoculated onto agar-solidified inorganic medium containing 1 g l)1 of p-coumaric acid. After 5 days incubation at 30C, a single colony for each possible species of microbe was selected, suspended in autoclaved ddH2O, inoculated onto fresh agar-solidified plates and incubated for another 5 days at 30C. This step was repeated for one or two times. Plates with separated single colonies were stored at 4C for further experiments. Tests of microbial growth and phenolic degradation

Materials and methods Materials and reagents Soil samples for isolation of microbes were obtained from the 5 to 10-cm layers below soil surface at central areas of natural forests of bamboo (B. chungii McClure) and pine (Pi. massoniana Lamb.) at a public park near our campus in Guangzhou city or from a rice (O. sativa L.) field of the Rice Research Institute of Guangdong Province. They were used immediately for experiments after sampling. Soils for microbial treatments, phenolic degradation and 1840

Isolated colony of each strain was inoculated in 3 ml of culture solution (in a 15 ml plastic tube with cover) and incubated for 1 day at 30C and then transferred to a flask with 50 ml of culture solution and incubated for another 2 days until OD600 of the culture solution was 0Æ5. The culture solution was then used for tests of microbial growth and phenolic degradation. One millilitre of microbial solution with OD600 = 0Æ5 was added to 100 ml inorganic culture solution containing 1 g l)1 p-coumaric acid and cultured at 30C with shaking (150 rpm). After certain hours (6, 12, 24, 48, 72 and 96 h), 1 ml of the


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culture solution was taken from each culture and centrifuged at 10 000 rpm for 5 min. The pellet was resuspended in equal amount of water for detection of OD600, and the supernatant was used for analysis of phenolic contents by HPLC, as described later. To test the growth of the microbes and their degradation on p-coumaric acid in full nutrient medium, LuriaBertani (LB) medium (10 g of trypton, 5 g of yeast extracts and 10 g of NaCl in one litre) was used to replace the inorganic culture me´dium. Tests for the ability of the cultures to degrade ferulic acid, p-hydroxybenzoic acid or p-hydroxybenzaldehyde were made by replacing these acids in place of p-coumaric acid in the culture solutions. The initial pH values of culture solutions were adjusted to 3Æ0, 4Æ0, 5Æ0, 6Æ0 or 7Æ0 with HCl or NaOH for growth tests at different pHs. For temperature tests, 20C, 25C, 30C or 35C were used. One mmol l)1 of CaCl2, MgCl2, CuSO4, MnSO4, CoCl2, AlCl3, ZnCl2, or FeCl3 was added to a set of culture solutions to test the effect of selected metal ions on growth of the cultured microbes. Soil treatment with microbes and analysis of phenolic degradation Soil from a bamboo forest was dried in an electric dryer at 60C for 6 h and was passed through a 0Æ84-mm sieve. p-Coumaric acid, dissolved in water, was added to the dried soil. The soil was dried again at 60C for 2 h. Twenty millilitre with OD600 = 1 of microbial solutions was added to 20 g of soil, and the soil was kept in an incubator in dark at 30C, and ddH2O was added to keep the soil moist. Soil phenolic compounds were extracted from soils based on our previous method (Li et al. 2007) and determined by HPLC. A Shimadzu LC-20AT HPLC system with SPD-20A UV detector and Shimadzu Chromatographic Workstation Software was used for quantitative and qualitative analysis of phenolics. HPLC conditions are column: YMC-packed Hypersil ODS column (4Æ6 · 200 mm, 5 lm); detection wavelengths: 285 nm for p-coumaric and ferulic acids, 254 nm for p-hydroxybenzoic acid and 310 nm for p-hydroxybenzaldehyde; mobile phase: Methanol : H2O : acetic acid (20 : 80 : 1, v ⁄ v ⁄ v); flow rate: 1Æ0 ml min)1; injection amount: 10 ll. Samples were filtered through 0Æ45-lm nylon filters before injected into HPLC and were diluted to proper concentrations within their corresponding standard curves. Contents of the phenolics were determined by their corresponding peak areas. Phenolic compounds in culture solution and soil extracts were identified by its retention times and co-injection tests with their corresponding standard compounds. Recovery tests were per-


formed by adding 0Æ2 mg of standard compound of p-coumaric acid to 1 g of soil, followed by extraction and HPLC analysis. An average recovery rate of 83Æ4% ± 2Æ5 was detected in three independent repeats. Microbial identification by biochemical and molecular analysis of rDNA sequences Biochemical identification of bacteria is based on the methods of Holt et al. (1994) and Dong and Cai (2001). For the analysis of the conserved region of bacterial 16S rDNA, single colonies of the bacterial strains from agarsolidified MS medium were suspended in 100 ll of ddH2O by vortexing and treated in boiling water for 2 min. After centrifugation at 13 400 g for 5 min, the supernatant was used for PCR amplification of 16S rDNA. Primers for bacterial 16S rDNA amplification are forward: 5¢-AGA GTT TGA TCC TGG CTC AG-3¢ and reverse: 5¢-AAG GAG GTG ATC CAG CCG CA-3¢. PCR solution consists of 10 · LA PCR buffer 5 ll, dNTP mixture 8 ll, MgCl2 5 ll, Primer 1 1Æ0 lm, Primer 2 1Æ0 lm, DNA template 1 lg, Takara LA Taq 0Æ5 ll, and ddH2O was added to a total of 50 ll. PCR conditions are 95C for 5 min, and then 94C 1 min, 55C 1 min, 72C 2 min for 29 cycles. The PCR products were used for sequencing after verified by agarose gel electrophoresis. All reagents, primers and sequencing services are provided by Takara Co. (Shanghai, China). Biochemical and molecular identification of Rhodotorula glutinis is based on the methods of Barnett et al. (1990). The 18S rDNA was amplified by PCR with primers of GEOA2: 5¢-CCA GTA GTC ATA TGC TTG TCT C-3¢ and GEO11: 5¢-ACC TTG TTA CGA CTT TTA CTT CC-3¢, using purified DNA as templates. PCR reagents and conditions and sequencing of PCR products are the same as for bacterial 16S rDNA described earlier. Sequences of 16S or 18S rDNA were aligned and analysed with sequences registered in GenBank using the Blast programs in NCBI’s website. Tests of seed germination and seedling growth For seed germination tests in culture solutions, 20 seeds of rice for each test were sown on filter paper in Petri dishes containing 10 ml of p-coumaric acid solution (1 g l)1 in ddH2O) or ddH2O as controls. Germinated seeds were counted after 5 days in dark. For seedling growth tests in solutions, seeds of rice were germinated in ddH2O for 48 h. Uniform germinated seeds were transferred to filter paper in Petri dishes containing 1 g l)1 of p-coumaric acid or ddH2O as controls. After 7 days of growing at room temperature (25–30C), lengths of leaves and roots were measured. For treatments with microbes


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in culture solutions, p-coumaric acid solution (1 g l)1) was inoculated with one of the microbes and incubated at 30C for 5 days before used for tests of seed germination and seedling growth. For germination and growth tests in soils, culture solutions with OD600 = 1 for each of the four microbes were mixed together, washed twice with distilled water by centrifugation and resuspended in equal volume of distilled water before added to soils. Bamboo soils were dried at 60C for 6 h, treated with or without p-coumaric acid (1 mg g)1) and microbes (1 ml g)1) and incubated at 30C for 45 days before used for germination and growth tests. Three seeds of mung bean were sowed in soils in each plastic cup, with three repeats for each treatment, and germinated in a growth chamber with 15-h

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photoperiod under a 15Æ4 lmol m)2 s)1 photon flux at 30C for 15 days. All experiments, except for the isolation and identification of microbes, were repeated at least three times, each with three parallel treatments. Presented data are the means of three repeats with standard errors. Results Isolation and identification of soil microbes degrading p-coumaric acid After three subculture in the screening solution and three times of colony purification on agar-solidified medium, four strains of microbes with relatively high growth in the

Table 1 Biochemical analysis of the microbial strains isolated from plant soils* Test

Strain 1

Strain 2

Strain 3

Gram stain Crystal blue stain Anaerobic growth Motility Glucose oxidation-fermentation Ethanol oxidation Gelatin hydrolysis Catalase Oxidase Manitol fermentation Salt tolerance Growth at 4C Growth at 41C Pyocyanine production Starch hydrolysis Nitrate reduction Methyl red test Indole test V-P test Arginase Ornithine decarboxylase Phenylalanine deaminase Hydrogen sulfide test Lecithin hydrolysis Denitrification Citrate utilization Malonate utilization Urease test Fermentation with fructose and glycerol Fermentation with arabinose, D-mannose, dulcitose, ribose, D-xylose and galactose Species identified Designated name

) No spore Strict aerobe + + + ) + + ) + Weak ) ) ) + ) ) ) + ) ) ) ) ) NT NT NT NT NT

) No spore Facultative aerobes + + + ) + + ) + ) ) ) ) + ) ) ) + ) ) ) ) ) + ) + + )

) No spore Strick aerobe + + + ) + + ) + Weak ) ) ) + ) ) ) + ) ) ) ) ) NT NT NT NT NT

Pseudomonas Putida Ps. putida 4CD1

Pseudomonas nitroreducens Ps. nitroreducens 4CD2

Ps. putida Ps. putida 4CD3

*Strain 4 is best matches with Rhodotorula glutinis, as it showed positive in nitrate test, urea decomposition, growth at 37C and assimilation with glucose, sucrose, fructose, galactose, manitol and ethanol; negative in starch formation, fermentation with glucose, galactose, maltose, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose and mannose, and assimilation with raffinose, lactose, xylose, arabinose, cellobiose, sorbose, inositol and maltose. Note: ‘+’ means positive, ‘)’ means negative and NT means not tested.

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Table 2 Analysis of the partial sequences of 16S or 18S rDNA amplified by PCR from the four microbial strains isolated from plant soils*

Sequenced bps Identity level (%) Total hits blasted Same species Same Genus Other species of the Genus Other matches

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Strain 2

Strain 3

Strain 4

1472 >98 117 Pseudomonas putida 31 Pseudomonas 48 Pseudomonas 7 Unknown 31

1469 >98 100 Pseudomonas nitroreducens 5 Pseudomonas 74 Pseudomonas 11 Chryseobacterium 1 Unknown 9

735 97 123 Ps. putida 27 Pseudomonas 56 Pseudomonas 9 Unknown 31

1627 >98 38 Rhodotorula glutinis 8 Rhodotorula 2 Rhodotorula 12 Rhodosporidium 11 Sporidiobolus 4 Cystoisospora 1

Contents of P-coumaric acid (%)

*Sequences were aligned with GenBank sequences using the BLAST program. Same species means GenBank sequences identified to be the same species as our biochemical identification. Same Genus means GenBank sequences identified to be the same genus as our biochemical identification, but unknown species. Other species of the Genus means GenBank sequences identified to be the same genus as our biochemical identification, but different species.

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Figure 1 Degradation of p-coumaric acid in inorganic culture solution (a), in Luria-Bertani (LB) solution (b) and in bamboo soil (c), and degradation of ferulic acid (d), p-hydroxybenzoic acid (e) and p-hydroxybenzaldehyde (f), by the microbes isolated from plant soils. For a, b, d, e and f, all culture solutions contain 1 g l)1 of the phenolic compounds; ( ) Strain 1; (h) Strain 2; ( ) Strain 3 and (·) Strain 4. For C, (s) Control; (X) Strain 1; (h) Strain 2; ( ) Strain 3 and (+) Strain 4.

medium were isolated. Biochemical analysis indicated that strain 1 and strain 3 matched best with Pseudomonas putida, strain 2 matched best with Pseudomonas nitroreducens. Strain 4 was best matched with R. glutinis (Table 1). Sequence alignment and analysis of the 16S or 18S rDNA matched and supported the biochemical identifications (Table 2). Both biochemical and ribosomal rDNA sequence analysis indicated that strains 1 and 3 are belongs to the same species of Ps. putida. But, they are not the same strain for the following two reasons. First, the changes of medium colours of the two strains were different during a 5-d culture. Both of them were colourless and transparent at

the beginning of incubation. Strain 1 changed to brown via a green stage, while strain 3 changed to milky white. Second, the reactions of the two strains to metal ions, were different. Strain 1 was more sensitive to Mn2+, while strain 3 was more sensitive to Cu2+ compared each other. Based on these results, the four strains were identified and designated as Ps. putida 4CD1 (strain 1) from rice soil, Ps. nitroreducens 4CD2 (strain 2) from bamboo soil, Ps. putida 4CD3 (strain 3) from pine soil, and R. glutinis 4CD4 (strain 4) from bamboo soil. The four strains of microbes were deposited to the Center for Species Reservation and Identification of Microorganisms, The Institute of Microbiology of Guangdong Province.


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As shown in Fig. 1(c), contents of p-coumaric acid decreased only 10% in the control soils without the addition of isolates, but decreased 70–80% in treatments with each of the four strains of the isolates within 30-d incubation. Although the four strains had similar levels of decomposition after 30 days, R. glutinis 4CD4 showed relatively faster in the decomposition on p-coumaric acid than the others. Figure 1(d–f) shown that all four strains of microbes can efficiently decompose ferulic and p-hydroxybenzoic acids, and p-hydroxybenzaldehyde in inorganic culture solutions. They completely degrade ferulic acid within 48 h, with R. glutinis 4CD4 being more efficient than the others (Fig. 1d). They completely degrade p-hydroxybenzaldehyde within 72 h, with Ps. putida 4CD1 and R. glutinis 4CD4 being more active than the other two strains (Fig. 1f). In case of p-hydroxybenzoic acid, the three strains of Pseudomonas can completely degrade it within 48 h, while R. glutinis 4CD4 was less active than the others, with only a 59% decrease in 48 h (Fig. 1e).

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Figure 2 Effects of microbes isolated from plant soils on the reversal of inhibitions of p-coumaric acid on seed germination and seedling growth of rice. 1: Control, 2: p-Coumaric acid, 3, 4, 5 and 6 are p-coumaric acid solutions treated with Strain 1, 2, 3 and 4, respectively, for 5 days.

Degradation of p-coumaric acid and related phenolics in culture solutions and bamboo soil by microbes isolated from plant soils As shown in Fig. 1(a), all the four strains showed high efficiencies in utilizing p-coumaric acid. The residues of p-coumaric acid decreased 93% or more in treatments with the three Pseudomonas strains, and 71% in that of R. glutinis 4CD4 within 48 h. The decrease of p-coumaric acid content was fast in Ps. nitroreducens 4CD3, and relatively slow in R. glutinis 4CD4, with the other two strains in between them. Figure 1(b) shows that residues of p-coumaric acid contents decreased even faster in LB solutions than in the inorganic solutions, with a 99% decomposed in all treatments of the four strains of microbes. R. glutinis 4CD4, the weakest strain in inorganic solution, showed the highest activity in the four species in LB solution, with a 99% decomposition within 24 h. Results indicated that all the four strains of microbes can effectively utilize and degrade p-coumaric acid in the existence of other carbon sources. 1844

p-Coumaric acid could completely inhibit seed germination and early growth of seedling of rice at concentrations of 1 and 0Æ5 g l)1 (data not shown). As shown in Fig. 2, the germination of rice seeds was 95% in controls. It was completely inhibited by 1 g l)1 of p-coumaric acid. Treatments of p-coumaric acid solutions with microbes could completely reverse the inhibition of p-coumaric acid on seed germination. In seedling growth tests, the inhibitions of p-coumaric acid on the elongations of leaves and roots could be largely, but not completely, reversed by microbial treatments. The three strains of Pseudomonas 4CD1, 4CD2 and 4CD3 reversed the inhibition on leaf elongation by 70–90%, and the inhibition on root elongation by 60–75%. R. glutinis 4CD4 was less effective than the other three strains and could reverse the inhibitions on elongations of leave and roots by 60 and 35%, respectively. Seeds of mung bean cannot germinate and grow in bamboo soils with or without the addition of 1 mg g)1 of p-coumaric acid. Soil treatments with a mixture of the four strains of microbes for 45 days could reverse the inhibitions of bamboo soil and p-coumaric acid on seed germination and growth of mung bean (Fig. 3). However, results varied among three independent repeats (data not shown). Effects of temperatures and pHs on the growth of the microbes isolated from plant soils As shown in Fig. 4(a), all four strains could grow well in all temperatures tested. The growth of the two strains of Ps. putida 4CD1 and 4CD3 increased with the


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1

2

3

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Figure 3 Effects of bamboo soil and p-coumaric acid on seed germination and seedling growth of mung bean and its reversal by the application of microbes isolated from soils. 1: Bamboo soil, 2: Bamboo soil + p-coumaric acid (1 mg g)1), 3: Bamboo soil + microbes, 4: Bamboo soil + p-coumaric acid (1 mg g)1) + microbes. Culture solutions with OD600 = 1 for each of the four microbes were mixed together, washed twice with distilled water by centrifugation and resuspended in distilled water before added to soils. Bamboo soils were dried in at 60C for 6 h, treated with or without p-coumaric acid and microbes (1 ml g)1) and incubated at 30C for 45 days before used for germination and growth tests. Three mung seeds were sowed in soils of each plastic cup, germinated and grown in a growth chamber with 15-h photoperiod under a 15Æ4 lmol m)2 s)1 photon flux at 30C for 15 days.

temperatures from 20C to 30C and reached the highest levels at 30C, but no more increases when temperature increased to 35C. The growth of Ps. nitroreducens 4CD2 increased with temperatures from 20C to 35. However, the growth of R. glutinis 4CD4 increased when temperatures changed from 20C to 30C and reached maximum at 30C, but decreased dramatically when temperature was higher than 30C. Its growth was even lower at 35C than at 20C. Soil acidification is a serious problem in our area of south China. Effects of pH values, with emphasis of low pHs from 3Æ0 to 7Æ0, on the growth of microbes were studied. The two strains of Ps. putida 4CD1 and 4CD3 could grow at pH 5Æ0–7Æ0, with the best growth at pH 6Æ0. They could not grow at pH 4Æ0 and pH 3Æ0. Ps. nitroreducens 4CD2 could grow in all pH conditions tested, but better at pH 5Æ0 or above and best at pH 6Æ0. R. glutinis 4CD4 grown well in all pH conditions tested and showed almost the same growth levels in different pH conditions from 3Æ0 to 7Æ0 (Fig. 4b). Effects of metal ions on the growth of microbes and p-coumaric acid degradation As shown in Fig. 5, after a 96-h incubation with different metal ions, CoCl2 completely inhibited, while CaCl2 and MgCl2 did not inhibit the growth and p-coumaric acid decomposition of Ps. putida 4CD1. Other metal ions, Al3+, Zn2+, Mn2+, Cu2+ and Fe3+ had moderate of 27– 50% inhibitions on its growth and 10–30% inhibition on p-coumaric acid decomposition. However, Ps. putida

4CD3, the same species with Ps. putida 4CD1, was completely inhibited by CoCl2 and CuSO4, not inhibited by CaCl2, MnSO4 and MgCl2 for both growth and p-coumaric acid degradation. AlCl3, ZnCl2 and FeCl3 inhibited its growth by 65, 57 and 46%, respectively, and inhibited p-coumaric acid degradation by 29, 41 and 70%, respectively. The difference between the two strains of Ps. putida was that Cu2+ inhibited Ps. putida 4CD3 (100%) more than Ps. putida 4CD1 (30%), while Mn2+ inhibited Ps. putida 4CD1 (30%), but not Ps. putida 4CD3. These means Ps. putida 4CD1 is sensitive to Mn2+, while Ps. putida 4CD3 is more sensitive to Cu2+. Pseudomonas nitroreducens 4CD2 was completely inhibited by CoCl2, not inhibited by CaCl2, MnSO4 and MgCl2. Other ions of CuSO4, AlCl3, ZnCl2 and FeCl3 inhibited its growth by 88, 71, 69 and 35%, respectively (Fig. 5a), and inhibited p-coumaric acid degradation by 90, 50, 46 and 3%, respectively (Fig. 5b). The growth of R. glutinis 4CD4 was strongly inhibited by AlCl3 (70%), CuSO4 (43%) and CaCl2 (36%), slightly inhibited by ZnCl2 (17%) and FeCl3 (12%) and not inhibited by MnSO4, MgCl2 and CoCl2 (Fig. 5a). Similar inhibitions were also observed on the decomposition of p-coumaric acid (Fig. 5b). Unlike the other three strains, R. glutinis 4CD4 was not inhibited by CoCl2, but was inhibited by CaCl2. Discussion Microbial metabolism is an important factor in determining the magnitude and duration of decomposition and


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concentrations of phenolic allelochemicals in soils. In this study, we isolated microbes that can effectively decompose phenolics from soils of all three species of plants, bamboo, pine and rice, which indicates that all these plant soils have potential to decompose phenolic compounds through microbes. Our results also showed that inoculation of soil microbes into culture solutions and bamboo soils effectively decomposed p-coumaric acid and related phenolics. These microbes can be used for decomposition and recovery of soils contaminated with high amount of phenolics. Compared to our early reports (Li et al. 2007) on chemical oxidation of phenolic allelochemicals in bamboo soils, in which p-coumaric acid in bamboo soil was decomposed by 32 or 37% when treated with 0Æ1 or 1% H2O2 of the Fenton’s reagent, microbial treatments showed 70–80% decomposition of p-coumaric 1846

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Figure 4 Growth of the four strains of microbes isolated from plant soils at different temperatures (a) and pHs (b). Left: ( h ) 35C; ( ) 30C; ( ) 25C and ( · ) 20C. Right: ( ) pH 7Æ0; ( h ) pH 6Æ0; ( ) pH 5Æ0; (·) pH 4Æ0 and (s) pH 3Æ0.

acid (Fig. 1c). However, microbial decomposition needs a much longer period of time (30 days). Our results reveal that soil conditions are important factors for the growth and metabolism of microbes, as well as for microbial decomposition of soil phenolics. The growth and functions of all the four isolates from soils are largely dependent on medium conditions of pHs, temperatures and metal ions. For example, the two strains of Ps. putida 4CD1 and 4CD3 could not grow at pH 4Æ0 and 3Æ0 conditions (Fig. 4a), which are common in most acidified soils in our area; R. glutinis 4CD4 could not grow well at temperatures higher than 30C (Fig. 4b); while all three strains of Pseudomonas could not grow in soils contaminated with Co2+ (Fig. 5). This may explain the accumulation of high concentrations of phenolics in the bamboo soils from which Ps. nitroreducens 4CD2 and


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Figure 5 Effects of metal ions on the growth (a) and degradation of 4-coumaric acid (b) by microbes isolated from plant soils. ( ) CaCl2; ( r ) MnSO4; ( h ) MgCl2; ( ) FeCl3; ( s ) CuSO4; ( d ) ZnCl2; ( h ) AlCl3 and ( ) CoCl2.

1·0 0·8 0·6 0·4 0·2 0·0 1·0 0·8 0·6 0·4 0·2 0·0 1·0 0·8 0·6 0·4 0·2 0·0

(b) Strain 1

1·0 0·8 0·6 0·4 0·2 0·0

Strain 1

1·0

Strain 2

0·8 4-Coumarate content (g l–1)

Microbe concentration (OD600)

(a) 1·0 0·8 0·6 0·4 0·2 0·0

Strain 3

0·6 0·4

Strain 2

0·2 0·0 1·0 0·8 0·6 0·4 0·2

Strain 3

0·0 1·0

Strain 4

0·8 0·6 0·4 0·2 0 h 24 h 48 h 72 h 96 h Incubation hours

R. glutinis 4CD4 were isolated. The common problem of soil acidification in our region may have restricted the growth of Ps. nitroreducens 4CD2, and the long period of high temperature of more than 35C could inhibited the growth of R. glutinis 4CD4. These results indicated that, in laboratory or controlled conditions (at 30C and with initial pH of 6Æ0), inoculation of microbes can effectively decompose p-coumaric acid in culture solutions (Fig. 1a,b) and bamboo soils (Fig. 1c) and reverse its inhibitions on seed germination and seedling growth (Figs 2 and 3). To improve microbial growth and decomposition of phenolics in soils, modification of soil conditions, such as pH and toxic metal ions seems to be more important than inoculation of microbes into soils. Degradation of toxic contaminants by soil microbes has been studied extensively, such as degradation of 2,4-dichlorophenol (Chen et al. 1999), 2,4-dichlorophenoxyacetic acid (Michel et al. 1995), 2,4,6-trichlorophenol (Godoy et al. 1999), 2,4,6-trinitrotoluene (Esteve-Nuń˜ez et al. 2001), atrazine (Radosevich and Tuovinen 2004), benzoxazolinone and benzoxazinone (Fomsgaard et al. 2004), dichloro-diphenyl-trichloroethane (DDT, Aislabie et al. 1997), naphthalene (Tuleva et al. 2005), phenanthrene (Tao et al. 2007), polyhydroxyalkanoates (Jendros-

Strain 4

0·0 0h

24 h 48 h 72 h 96 h Incubation hours

sek and Handrick 2002) and other compounds (Healy and Young 1979; Song et al. 2000; Manickam et al. 2008). The three bacterial species identified as Ps. putida, Ps. nitroreducens and R. glutinis are common microbes in the environments and have been demonstrated to be effective in decomposing and transforming toxic organic compounds (Hinteregger et al. 1992; Delneri et al. 1995; Heinaru et al. 2000; Gonzalez et al. 2001; Kwon et al. 2003; Margesin et al. 2004; Qualls 2005; Sampaio et al. 2007; Unno et al. 2007). However, the degradation of phenolic allelochemicals in soils by the microbes has not been investigated to a great extent. The three microbes were first isolated from the soils of bamboo, pine and rice and demonstrated to be effective in decomposing phenolic allelochemicals in the soils. Zeng and Mallik (2006) pointed that ectomycorrhizal species can control species interactions in higher plants by changing the rhizosphere chemistry. Our results indicated that more soil microbes have this function. Microbial utilization of cinnamic acid derivatives (e.g. p-coumaric and ferulic acids) involves multistep transformations and leads to production of other phenolic compounds, such as p-hydroxybenzaldehyde, p-hydroxybenzoic acid and catechol or vanillin, vanillic acid and protocatechuic acid, before cleavage of the aromatic ring


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and further oxidation into components of tricarboxylic acid cycle (Venturi et al. 1998; Priefert et al. 2001). In many species of microbes and plants, this process is initiated by coumarate-CoA ligase or feruloyl-CoA synthetase which uses several phenolics as substrates, such as p-coumaric, ferulic and caffeic acids. The complete set of genes responsible for conversion of coumaric or ferulic acids to the cleavage of aromatic ring has been identified from Pseudomonas sp. strain HR199 (Priefert et al. 2001). This explains that the Pseudomonas sp. we isolated from soils can utilize p-coumaric acid, ferulic acid, p-hydroxybenzoic acid and p-hydroxybenzaldehyde. Our results also show that R. glutinis can utilize the four phenolic compounds as sole carbon source, which indicates that it probably has similar metabolic processes for the decomposition of phenolic compounds. In summary, four strains of microbes with high efficiency in utilizing p-coumaric acid were first isolated from the growth soils of bamboo (B. chungii), pine (Pi. massoniana) and rice (O. sativa) using an inorganic screening medium with p-coumaric acid as sole carbon source. They were identified and designated as Ps. putida 4CD1, Ps. nitroreducens 4CD2, Ps. putida 4CD3 and R. glutinis 4CD4, by morphological, biochemical and ribosomal rDNA sequence analysis. The four strains can effectively grow and decompose p-coumaric acid in both inorganic or LB solutions containing 1 g l)1 p-coumaric acid as sole or cocarbon source. They can also effectively decompose ferulic acid, p-hydroxybenzoic acid and p-hydroxybenzaldehyde. Treatments with the four microbes can effectively decompose p-coumaric acid and reverse its inhibition on seed germination and seedling growth of plants in culture solution and plant soils in laboratory conditions. However, the growth of the microbes is largely dependent on culture pHs, temperatures and metal ions. Results indicated that high contents of phenolics in plant culture solutions and bamboo soils can be effectively decomposed through the application of microbes in favoured conditions. But, modification of growth environments for microbes is important in soils that are acidic or contaminated with metal ions. Acknowledgements Special thanks to the Center for Species Reservation and Identification of Microorganisms, The Institute of Microbiology of Guangdong Province, for biochemical analysis and identification of the microbes isolated from plant soils. This work was supported by the Science and Technology Programs of Guangdong Province (program number 2006B20101005) and the Science and Technology Supporting Programs of the Guangzhou Municipal Government (program number 2008Z1-E591). 1848

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