Effects of Cinnamic Acid on Bacterial Community Diversity in Rhizosphere Soil of Cucumber Seedlings Under Salt Stress

Agricultural Sciences in China

February 2010

2010, 9(2): 266-274

Effects of Cinnamic Acid on Bacterial Community Diversity in Rhizosphere Soil of Cucumber Seedlings Under Salt Stress LIU Jing, WU Feng-zhi and YANG Yang Horticulture College, Northeast Agricultural University, Harbin 150030, P.R.China

Abstract To investigate the effects of a plant autotoxin, cinnamic acid, on bacterial communities in the rhizosphere soil of cucumber seedlings under salt stress, we used cucumber as the experimental material, cinnamic acid as the autotoxin, and NaCl to apply salt stress. Bacterial communities in the rhizosphere soil were analyzed using polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and clone sequencing. Salt stress decreased the diversity of bacterial species in rhizosphere soil of cucumber seedlings at several growth stages. Cinnamic acid exacerbated the effects of salt stress at high concentrations, but alleviated its effects at low concentrations. Cloning and sequencing results indicated that DGGE bands amplified from soil samples showed high homology to uncultured bacterial species. Cinnamic acid at 50 mg kg-1 soil improved cucumber growth and was the most effective treatment to alleviate the effects of salt stress on bacterial communities. Key words: bacteria, cinnamic acid, cucumber, salt stress, PCR-DGGE

INTRODUCTION China has the largest area of protected horticulture in the world. In recent years, high yields of vegetable crops have been achieved using large-scale, industrialized, and specialized production methods. Problems that arise in continuous cropping affect the sustainable production of vegetable crops (Wu et al. 2000). Some of the major problems in continuous cropping include soil-borne pests (Yu and Du 2000), secondary salinization of soil (Davis et al. 2003; Sun et al. 2005), low diversity of soil microorganisms (Meyer and Shew 1991; Sun et al. 2005), and autotoxicosis (Sun et al. 2005; Zheng et al. 2004). Autotoxicosis results from the accumulation of root exudates and the decomposition of organic matter (Yao et al. 2006). However, the interactions among autotoxic Received 9 June, 2009

compounds, soil bacteria, and salt-stress have not been investigated. In this paper, we studied the effects of an autotoxin on rhizosphere bacteria of plants under salt stress. Yu et al. (2005) reported that secondary salinization of soil not only inhibited crop growth, but also directly affected the activity of soil microorganisms. Salinization indirectly influences the environment of soil microorganisms by altering physiochemical properties, resulting in changes in the quantities and activities of soil microbes (Yu et al. 2005). Soil salinity also alters the availability of soil nutrients, thus indirectly influences the supply of soil nutrient to crops (Zhang et al. 2002). Soil microbial communities are widely recognized as an integral component of soil quality because they are involved in many ecosystem processes, such as energy flow, nutrient cycling, and organic matter turnover (Li et al. 2008). Therefore, analyses of soil

Accepted 14 November, 2009

Correspondence WU Feng-zhi, Professor, Ph D, Tel: +86-451-55190278, Fax: +86-451-55190443, E-mail: [email protected]

© 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60092-4

Effects of Cinnamic Acid on Bacterial Community Diversity in Rhizosphere Soil of Cucumber Seedlings Under Salt Stress

microbial diversity and community structures are essential to prevent environmental degradation of soil. Low concentrations of p-hydroxybenzoic acid and cinnamic acid have been shown to promote growth of cucumber seedlings, whereas high concentrations can inhibit growth (Lü et al. 2002; Wu et al. 2002; Wu et al. 2005). When added to growth media, phenolic acids inhibited microbial production of gas and volatile fatty acids, and reduced the consumption of growth media (Vaughn et al. 1983). Ma et al. (2005) reported that phenolic acids not only decreased the amount and diversity of microbial communities in soil, but also stimulated growth of pathogenic soil fungi such as Fusarium oxysporum and Phytophthora. When purple basil is attacked by Pythium ultimum, the root system secretes massive quantities of rosmarinic acid, which eliminates several types of soil bacteria and pathogens. This demonstrates that rosmarinic acid has substantial bacteriostasis activity (Bais et al. 2002). Wu et al. (2001a, b, c) showed that the phenolic acid present in wheat root exudates had both antifungal and antibacterial activities. Low concentrations of phenolic acids in the soil can promote microbial growth, while high concentrations can inhibit it (Yuan et al. 2004). At some concentrations, salicylic acid can alleviate salt stress during seed germination and subsequent seedling growth (Zhang et al. 1992, 1998; She et al. 2002; Peng et al. 2003; Wang et al. 2006). Cinnamic acid can also alleviate salt damage at the seedling stage (Zhang and Wu 2007). In summary, although there are researches on the effects of autotoxins on microbes, and on plants under salt stress, interactions among these three factors have never been reported. In this study, we determined the effects of an autotoxin, cinnamic acid, on soil microorganisms in the rhizosphere of cucumber under salt stress. Cucumber is one of the major greenhouse vegetables in China, and many problems occur when it is cultivated in continuously protected systems. In this study, we used cucumber as the experimental material, cinnamic acid as the autotoxin, and added sodium chloride (NaCl) to soil to apply salt stress. The soil bacterial community was analyzed using polymerase chain reaction (PCR) to amplify bacterial 16S rDNA and denaturing gradient gel electrophoresis (DGGE). The aim was to understand the relationship between

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autotoxicosis and soil microorganisms in a continuous cropping system under salt stress. Increasing our understanding of such interactions will help to identify and address problems that arise in continuous cropping systems.

MATERIALS AND METHODS Plant materials and soil Cucumber (Cucumis sativus L.) Jinchun 4 was used in these experiments. Seeds were washed for a few minutes with sterilized water, then first soaked in water at 55°C for 15 min and then at 30°C for 12 h. After rinsing several times with sterilized water, the seeds were germinated in the dark at 28°C for 2 d. After germination, the seedlings were transplanted into pots (12 cm diameter × 12 cm high) containing 400 g black soil, which was obtained from the Horticultural Experimental Station of Northeast Agricultural University, Harbin, Heilongjiang Province, China (45°41´N, 126°37´E). The physicochemical properties of the soil were as follows: organic matter, 33.16 g kg-1; alkaline N, 104.9 mg kg-1; available P, 153.9 mg kg-1; available K, 266.9 mg kg-1; total N, 2.39 g kg-1; total P, 1.79 g kg-1; slowly available K, 983.4 mg kg-1; and pH, 7.38. All measurements of soil indices were based on the method of Bao (2005).

Soil samples and seedling growth analysis Sodium chloride was dissolved in distilled water at 585 mg kg-1 soil (Zhang and Wu 2007). Cinnamic acid (CA; Beijing Yuanpinghao Biological Technology Co., China) was dissolved in 2% ethanol. Five concentrations of CA (0, 25, 50, 100, and 200 mg kg-1 soil) were prepared, each containing the same volume of ethanol (Chen et al. 2005). Seven days after transplantation of cucumber seedlings, 25 mL NaCl solution was added to the soil in each pot. After the NaCl solution was fully absorbed, 25 mL CA solution of different concentrations was added. We designed six experimental treatments, and each was replicated three times (Table 1). The six experimental treatments were as follows:soil without NaCl and CA, designated as

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Table 1 Experimental design Soil code CK1 CK2 A B C D

Concentration (mg kg soil) -1

Sodium chloride (NaCl)

Cinnamic acid (CA)

0 585 585 585 585 585

0 0 25 50 100 200

control 1 (CK1), soil with NaCl at 585 mg kg-1 soil and without CA, designated as control 2 (CK2), and soil with NaCl at 585 mg kg -1 soil and with 25, 50, 100, and 200 mg CA per kg soil (A, B, C, and D, respectively). The seedlings were placed in a greenhouse (13 h light/11 h dark photoperiod) using a completely randomized design and watered by drip irrigation. Plastic film was placed at the bottom of each pot to prevent leaching. After treatment for 7 (the 2nd euphylla stage), 14 (the 3rd euphylla stage), and 21 d (the 4th euphylla stage), the soil adhering to the root, designated as ‘rhizosphere soil’ (Fujii et al. 2005), was harvested. Soil samples were mixed, sieved through a 2-mm mesh sieve and stored at -70°C until use. After treatment for 21 d, the cucumber seedlings were harvested, and plant height, stem diameter, and fresh weight were determined.

Extraction and purification of DNA from soil DNA was extracted from soil as described by Jiao et al. (2005). Crude DNA was purified using a Wizard® Genomic DNA Purification Kit (Promega Co., USA). Purified DNA was separated by electrophoresis on 0.8% agarose gels.

Amplification of 16S rDNA To amplify soil bacterial 16S rDNA, PCR analysis was carried out using DNA purified from soil as the template. Fragments were amplified using the primers F968GC (5´-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GAA CGC GAA GAA CCT TAC3´) and R1401 (5´-GCG TGT GTA CAA GAC CC-3´). Primers were designed from the V6-V8 conserved region of the 16S rDNA gene (Xing et al. 2006) and synthesized by the Shanghai Bioengineering Co. Ltd., China. Amplification reactions were performed in a total volume

of 50 —L containing 5 —L 10 × buffer (Mg2+), 10 ng target DNA, 1 —L 10 mmol L-1 dNTPs, 0.25 —mol L-1 forward and reverse primers, 2 U Pfu DNA polymerase, and sterilized double-distilled water to complete the final volume to 50 —L. DNA amplification was carried out in a Bio-Rad PCR thermocycler. The program was as follows: 94°C for 5 min; 20 touchdown cycles consisting of 94°C for 60 s, 65-55°C for 60 s, and 72°C for 60 s, each cycle decreasing by 0.5°C; 20 cycles consisting of 94°C for 60 s, 56°C for 60 s, and 72°C for 60 s; and final extension at 72°C for 7 min. All amplification products were analyzed by electrophoresis on 1.0% agarose gels. Amplification products were all approximately 450 bp in length.

DGGE analysis DGGE was performed with the DCode Universal Mutation Detection System (Bio-Rad Co., USA). The PCR products separated on a polyacrylamide gel with a 3060% denaturant gradient [7 mol L-1 urea plus 40% (v/v) deionized formamide]. The gels were prepared from 8% (w/v) acrylamide stock solutions (acrylamide: bisacrylamide ratio = 37.5:1) containing 0 and 100% denaturant. Each well contained 30 —L PCR products, and electrophoresis was carried out in 1 × TAE buffer (4.84 g Tris base, 1.142 mL acetic acid, 2 mL 0.5 mol L-1 EDTA, pH 8.0, and dH2O to 1 L) at a constant voltage of 150 V for 300 min at 60°C. To compare the patterns of all the different treatments on one denaturing gradient gel, we loaded PCR products amplified from three replicates per treatment (each representing one composite sample) onto the gel. After silver staining of the gels as described by Li et al. (2007), the gels were photographed with a GelDoc-It Imaging Station (UVP Co., USA) and stored as TIFF files.

Isolation and sequencing of dominant DGGE bands Dominant DGGE bands were excised and eluted by incubation in 50 —L TE (pH 8.0) at a constant temperature of 37°C for 2 h. Then, a 5 —L aliquot from each sample was used as template to reamplify the band of interest with the primers F968 (without GC binder) and R1401, in the PCR conditions described

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Effects of Cinnamic Acid on Bacterial Community Diversity in Rhizosphere Soil of Cucumber Seedlings Under Salt Stress

above. The reamplified products were purified and cloned into a pUCM-T vector (Promega Co., USA) overnight, and then transformed into TG1 competent cells. Positive clones were screened and sequenced using an automatic sequencing system (Bioasia Co., Shanghai, China). Sequence analyses were performed by using the BLAST database (Bioasia Co., Shanghai, China).

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CK1. The band patterns of C and D were similar to that of CK2, but were not similar to those of other treatments.

Data analysis DGGE bands were analyzed using Quantity One Software (Bio-Rad Co., USA). Sequence homology was analyzed using BLAST procedures of the NCBI (http://www.ncbi.nlm.nih.gov/). Bacterial classification was analyzed using the Sequence Match Procedure (Ribosomal Database Project II-Release 9 website). The phylogenetic tree was constructed with a maximum-likelihood tree algorithm using MEGA 3.1 software. Seedling growth data were analyzed using the least significant difference test of the means at the 5% level by SAS 8.2 software.

RESULTS DGGE analysis of bacterial communities Rhizosphere soil from cucumber seedlings at the second euphylla stage Analyses of DGGE patterns were used to compare bacterial communities in CK1, CK2, and saline soil supplemented with 25, 50, 100, and 200 mg CA per kg soil (A, B, C, and D, respectively) as shown in Fig.1-A. Compared with bands in the CK1 sample, CK2 showed decreased intensity of band A3, and bands A1, A4, and A5 were absent. Bands A1 and A5 reappeared in saline soils supplemented with CA (lanes A, B, C, and D). Compared with CK2, the intensity of band A3 increased in A and B, but A3 was absent from C and D. Band A4 was present in A and B but absent from C and D. Band A2 was present in soil from all treatments except D. The band patterns were similar in CK1 and B. Fig.1-B shows the similarity of band patterns in the six treatments as determined by cluster analysis. The band patterns of A and B were very similar to that of

Fig. 1 A, silver-stained 16S rDNA DGGE fingerprints. B, cluster analysis profiles of rhizosphere soil from different treatments at the cucumber second euphylla stage. Bands excised and sequenced are indicated with arrows, the same below.

Rhizosphere soil from cucumber seedlings at the third euphylla stage Fig.2-A shows bands from rhizosphere soil of plants at the third euphylla stage. Compared with CK1, bands B1, B2, and B4 were absent from CK2. Bands B1, B2, and B4 reappeared in A and B, however, B4 was absent from C, and B1 and B4 were absent from C and D. A new band B5, was present in all CA treatments, and was absent from both CK1 and CK2. In addition, B3 was present in all treatments except D. The band patterns of CK1 and B (saline soil supplemented with 50 mg CA kg-1 soil) were very similar. Fig.2-B shows similarity of band patterns in the six treatments at the third euphylla stage as determined by cluster analysis. The band pattern of B was very simi-

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lar to that of CK1. Band patterns of A and C were very similar. The band pattern of D was similar to that of CK2, but both were dissimilar to those of other treatments.

LIU Jing et al.

ated the detrimental effects of salt stress on diversity of soil bacteria, but high concentrations of cinnamic acid had toxic effects. The cluster analysis shown in Fig.3-B indicated that under salt stress, the bacterial community in B was similar to that of CK1, and that the communities in A and C were similar to each other. The bacterial community in CK2 was dissimilar to those in other CA treatments.

Sequence analysis of 16S rDNA fragments The DGGE bands indicated by arrows were sequenced (Table 2). Most sequences showed high homology to uncultured bacteria, indicating that salt stress and cinnamic acid treatments strongly influenced uncultured bacteria in cucumber rhizosphere soil. Bands A3, B4,

Fig. 2 A, silver-stained 16S rDNA DGGE fingerprints. B, cluster analysis profiles of rhizosphere soil from different treatments at the cucumber third euphylla stage.

Rhizosphere soil from cucumber seedlings at the fourth euphylla stage Bands in rhizosphere soil samples collected from cucumber plants at the fourth euphylla stage are shown in Fig.3-A. Compared with CK1 and CK2 showing decreased intensities of bands C4 and C5, and bands C1, C2 and C3 were absent (lane CK2). This result indicated that salt stress reduced the number of bacterial species in rhizosphere soil. Compared with CK1, the bands in B did not change significantly. In D, bands C1, C2 and C5 were absent and a new band - C6 was present. These results indicated that cinnamic acid at low concentrations allevi-

Fig. 3 A, silver-stained 16S rDNA DGGE fingerprints. B, cluster analysis profiles of rhizosphere soil from different treatments at the cucumber fourth euphylla stage.

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Effects of Cinnamic Acid on Bacterial Community Diversity in Rhizosphere Soil of Cucumber Seedlings Under Salt Stress

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Table 2 Sequences of 16S rDNA PCR-DGGE fragments for selected bands Sequenced bands

GenBank submission no.

A1

EU339589

A2

EU339590

A3

EU339591

A4

EU339592

A5

EU339593

B1

EU339594

B2 B3

EU339595 EU339596

B4

EU339597

B5

EU339598

C1

EU339599

C2

EU339600

C3

EU339601

C4 C5

EU339602 EU339603

C6

EU339604

Strains or clones with the highest identity from NCBI Uncultured bacterium clone M0022_117 (94%) Uncultured bacterium clone adhufec17d10 (93%) Uncultured bacterium clone I4 (100%) Uncultured bacterium clone abc55e04.x1 (99%) Uncultured Proteobacterium clone CSB23 (98%) Uncultured beta Proteobacterium clone CSB27 (98%) Uncultured soil bacterium clone D44-Rv (97%) Uncultured soil bacterium clone Y9-19 (97%) Uncultured soil bacterium clone Y1-18 (98%) Uncultured bacterium clone WHEATSIP-EL38 (95%) Microbacterium sp. DQ121 (99%) Microbacterium trichotecenolyticum strain B45 (99%) Uncultured bacterium clone VE36 (99%) Microbacterium sp. SD-9 (97%) Microbacterium sp. MTR18 (98%) Uncultured beta Proteobacterium clone 100M1_G3 (98%) Uncultured beta Proteobacterium clone Soil-9 (99%) Uncultured bacterium clone HKT127 (92%) Uncultured alpha Proteobacterium clone C5T12 (90%) Pseudomonas jessenii clone PJM15 (94%) Pseudomonas fluorescens clone BC-UID4a (94%) Uncultured gamma Proteobacterium clone 871 (92%) Uncultured Pseudomonas sp. clone 20 (91%) Uncultured bacterium clone I06 (100%) Uncultured bacterium clone SMC49 Uncultured bacterium clone I12 (100%) Uncultured beta Proteobacterium clone CSB16 (98%) Beta Proteobacterium MC-5 clone (99%) Uncultured soil bacterium clone Y12-12 (99%) Uncultured soil bacterium clone Y10-5 (99%)

C2, and C5 showed 98-99% sequence homology with sequences from Proteobacteria. Bands B1 and B3 showed 97-99% sequence homology with Microbacteria sequences. Band C1 showed 94% homology to a Pseudomonas sequence. Other bands showed 90-100% homology with unclassified sequences. New sequences have been submitted to GenBank (accession no. EU339589-EU339604).

Source EF071350 AF530341 AY667747 AB200741 AB288653 DQ069182 EF455283 AY930341 EF066562 AY930130 DQ300353 DQ335103 EF681652 AY336120 DQ507207 DQ514024 AB286273 DQ439556 AY822189 AM707022 DQ862546 AY960253 AY881672 AB200711 AM183098 AB200716 DQ069185 AF190215 AY930415 AY930354

lings (CK2). When plants grown under salt stress were treated with CA at 25 or 50 mg kg-1 soil, plant height and fresh weight were greater than those of CK2 plants (P < 0.05). At CA concentrations of 100 and 200 mg kg-1 soil, plant height, stem diameter, and fresh weight were

Bioinformatic analysis of sequence data The phylogenetic tree (Fig.4) indicated that very close relationships existed between bands A3 and C5, A4 and C6, and C4, A2, B1 and C3. Bands C2 and C1 were closely related to each other, but distantly related to others.

Seedling growth Effects of cinnamic acid on cucumber seedlings under salt stress are shown in Table 3. Compared with CK1, salt stress markedly inhibited growth of cucumber seed-

Fig. 4 Phylogenetic tree of excised and sequenced bands.

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Table 3 Effect of cinnamic acid on growth of cucumber seedlings under salt stress Treatments

Plant height (cm)

Stem diameter (cm)

CK1 CK2 A B C D

24.500 ± 1.000 a 18.675 ± 1.269 d 21.775 ± 1.786 bc 22.175 ± 1.814 ab 19.433 ± 2.225 cd 14.600 ± 1.122 e

0.603 ± 0.044 a 0.555 ± 0.016 b 0.574 ± 0.016 ab 0.583 ± 0.035 ab 0.561 ± 0.024 bc 0.517 ± 0.006 c

Fresh weight (g) 13.910 ± 0.131 a 11.157 ± 0.494 c 12.183 ± 0.713 ab 12.371 ± 0.209 ab 10.321 ± 1.365 c 8.137 ± 1.027 d

The data are means ± SD. Means with different letters are significantly different at P < 0.05.

significantly lower than those of CK1 plants, but were not significantly different to those of CK2 plants (P < 0.05). These results indicate that under salt stress, low concentrations of CA can alleviate the detrimental effects of salt stress on seedling growth. This effect was particularly notable at the CA concentration of 50 mg kg-1 soil.

DISCUSSION Soil microorganisms play an important role in the soil ecosystem, and to a certain extent, soil microbial community composition and changes in types or amounts of soil microorganisms can reflect changes in soil quality (Jiao and Wu 2004). Microorganisms are also the key to overcome problems associated with continuous cropping and other agricultural practices that detrimentally affect soil health (Hu et al. 2004). Many molecular biology approaches have been used to study diversity of microbial communities. One widely used approach is PCR-DGGE, which can be used to study the structure, dynamic changes, and diversity or homology of microbial communities without culturing microorganisms (Li et al. 2007). Jurelevicius et al. (2008) used DGGE to analyze the effects of nitrate on the bacterial community in a water-oil tank system. Zhao et al. (2007) analyzed bacterial communities on aging fluecured tobacco leaves by 16S rDNA PCR-CDGGE technology. In our study, salt stress during different growth periods of cucumber seedlings led to significant differences in DGGE band patterns among rhizosphere soil samples. For example, salt stress resulted in an increase in the intensities of bands A3, B4, and C4, and a decrease in the intensities of bands A1, B1, and C1. This result indicates that salt stress during seedling growth decreases the amount and diversity of

soil microorganisms. This is consistent with the results of Hartmann and Widmer (2006). Several other researchers have reported that PCR-DGGE combined with clone and sequence analyses could yield more information about the structure and characteristics of microbial communities (Lai et al. 2006). Garbers et al. (2004) used PCR-CDGGE techniques to characterize the microbial communities on kefir grains. Sequenced bands from DGGE profiles were used to identify yeast rRNA sequences, and the homology of yeast rRNA sequences was compared using databases at NCBI. Salicylic acid and its analog can alleviate the effects of salt stress, and improve the ability of plants to adapt to salt stress (Gaffney et al. 1993). The results of our study indicate that cinnamic acid can relieve the detrimental effects of salinity on the quantity and diversity of microorganisms in rhizosphere soil of cucumber plants under salt stress. At three cucumber seedling growth stages, cinnamic acid at 50 mg kg-1 soil was the most effective concentration to alleviate the effects of salt stress on the bacterial community in rhizosphere soil. For example, cinnamic acid treatment led to an increase in the intensities of bands A2, B4, and C4, and an increase in the total number of bands, compared with rhizosphere soil of plants under salt stress. Cinnamic acid is a type of root exudate, and phenolic acids secreted by plants can influence the amount and activity of microorganisms in the rhizosphere soil (Xue et al. 2005). We found that cinnamic acid at 25 mg kg-1 soil alleviated salt stress; however, the effects were less significant than in the 50 mg kg-1 treatment. The result that cinnamic acid can relieve salt stress is similar to the results of Wang et al. (2006). At 200 mg kg-1 soil, cinnamic acid did not alleviate the effects of salt stress. As well, the absence of bands from the DGGE profile of this treatment (Figs.1-A, 2-A and 3-A) showed that bacterial species diversity decreased in rhizosphere soil. This is consistent with the findings of Yuan et al. (2004). Furthermore, saline soil supplemented with high concentrations of cinnamic acid showed two new bands, B5 and C6, which were not present in controls. Cloning and sequencing data showed that new bacterial species, e.g., uncultured bacteria and Proteobacteria genus were present in bacterial communities in the presence of cinnamic acid. However, further research is required to determine whether the uncultured bacteria

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Effects of Cinnamic Acid on Bacterial Community Diversity in Rhizosphere Soil of Cucumber Seedlings Under Salt Stress

Microbacterium and Proteobacteria are beneficial or harmful for growth of cucumber seedlings. Our results indicated that salt stress decreased the amount and diversity of bacterial species in rhizosphere soil of cucumber seedlings. Exogenous application of cinnamic acid to saline soils increased the amount and diversity of soil microorganisms, and improved the growth of cucumber seedlings (Table 3). Together, our results suggest that a low concentration of cinnamic acid (50 mg kg-1 soil) can efficiently alleviate damage caused by salinity to soil bacterial communities, increase the amount and diversity of soil microbes, and stimulate seedling growth.

Acknowledgements This research was funded by the National 973 Program of China (2009CB119004-05), the National Natural Science Foundation of China (30771252) and the Education Department Project of Heilongjiang Province, China (11531018).

References Bais H P, Walker T S, Schweizer H P, Vivanco J M. 2002. Root specific elicitation and antimicrobial activity of rosmarinic acid in hairy root cultures of Ocimum basilicum. Plant Physiology and Biochemistry, 40, 983-995. Bao S D. 2005. Soil Analysis in Agricultural Chemistry. 3rd ed. vol. 1. China Agriculture Press, Beijing. pp. 20-109. (in Chinese) Chen L X, Wu F Z, Ma F M. 2005. Effects of exogenous cinnamic acids on the growth and physiological characteristics of cucumber seedlings. Agricultural Sciences in China, 4, 747751. Davis J A, McGuire M, Halse S A, Hamilton D, Horwitz P, McComb A J, Froend R H, Lyons M, Sim L. 2003. What happens when you add salt: predicting impacts of secondary salinisation on shallow aquatic ecosystems by using an alternative-states model. Australian Journal of Botany, 51, 715-724. Fujii Y, Furubayashi A, Hiradate S. 2005. Rhizosphere soil method: a new bioassay to evaluate allelopathy in the field. In: Haper J D I, An M, Kent J H, eds, Proceedings of the Fourth World Congress on Allelopathy ‘Establishing the Scientific Base’. Charles Sturt University, Wagga, NSW, Australia. pp. 490-492. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J. 1993. Requirement of salicylic acid for the induction of systemic acquired resistance,

273

Science, 261, 754-751. Garbers I M, Britz T J, Witthuhn R C. 2004. PCR-based denaturing gradient gel electrophoretic typification and identification of the microbial consortium present in kefir grains. World Journal of Microbiology Biotechnology, 20, 687-693. Hartmann M, Widmer F. 2006. Community structure analyses are more sensitive to differences in soil bacterial communities than anonymous diversity indices. Applied and Environmental Microbiolog, 72, 7804-7812. Hu Y S, Wu K, Liu N, Chen H G, Jia X C. 2004. Dynamics of microbial communities in bulk and developing cucumber rhizosphere soil. Agricultural Sciences in China, 5, 376-383. Jiao X D, Wu F Z. 2004. Progress of the methods for studying soil microbial diversity. Chinese Journal of Soil Science, 35, 789-793. (in Chinese) Jiao X D, Wu F Z, Gao H J. 2005. Optimization of RAPD conditions for soil microbes. Chinese Journal of Ecology, 24, 921-924. (in Chinese) Jurelevicius D, von der Weid I, Korenblum E, Valoni E, Penna M, Seldin L. 2008. Effect of nitrate injection on the bacterial community in a water-oil tank system analyzed by PCRDGGE. Journal of Industrial Microbiology Biotechnology, 35, 251-255. Lai X T, Zeng X F, Fang S, Huang Y L, Cao L X, Zhou S N. 2006. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea. World Journal of Microbiology and Biotechnology, 22, 1337-1345. Li Q H, Wu F Z, Yang Y. 2008. Effects of rotation and interplanting on soil bacterial communities and cucumber yield. Acta Agricultura Scandinavica (Section B Soil and Plant Science). doi: 10.1080/09064710802342319 Li Z Y, He L M, Miao X L. 2007. Cultivable bacterial community from South China Sea Sponge as revealed by DGGE fingerprinting and 16S rDNA phylogenetic analysis. Current Microbiology, 55, 465-472. Lü W G, Lü F, Zhang C L, Yu P. 2002. Mechanism of allelochemicals inhibiting continuous cropping cucumber growth. Scientia Agricultura Sinica, 35, 106-109. (in Chinese) Ma Y H, Wei M, Wang X F. 2005. Dynamics of soil phenols in continuous cropping solar greenhouse and their effects on disease resistance-related enzyme activities in cucumber root. Chinese Journal of Applied Ecology, 16, 79-82. (in Chinese) Meyer J R, Shew H D. 1991. Soils suppressive to black root rot of burley tobacco caused by Thielaviopsis basicola. Phytopathology, 81, 946-954. Peng Y, Zhang C L, Shen Q R Zhou H J, Yang G C. 2003. Effects of two phenolic acids on seed germination and activities of some enzymes in cucumber seedlings under salt stress. Journal

© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.

274

LIU Jing et al.

of Nanjing Agricultural University, 26, 33-36. (in Chinese) She X P, He J M, Zhang J, Zou Q C. 2002. Mitigative effect of

Environmental Science, 7, 1425-1428. (in Chinese) Xue C Y, Wu F Z, Wang H C, Li J. 2005. The summary of

salicylic acid on salt stress-induced growth inhibition in cucumber seedling. Acta Botanica Boreali-occidentalia Sinica,

reciprocity between phenolic acids and soil microorganism. Heilongjiang Agricultural Sciences, (3), 45-47. (in Chinese)

22, 401-405. Sun G W, Chen R Y, Liu H C. 2005. Causes and control measures

Yao H Y, Jiao X D, Wu F Z. 2006. Effects of continuous cucumber cropping and alternative rotations under protected

for continuous cropping obstacles in protected vegetable cultivation. Transactions of the CSAE, 21(Suppl.), 184-188.

cultivation on soil microbial community diversity. Plant and Soil, 284, 195-203.

(in Chinese) Vaughn D, Sparling G P, Ord B G. 1983. Amelioration of the

Yu H Y, Li T X, Zhou J M. 2005. Secondary salinization of greenhouse soil and its effects on soil properties. Soils, 37,

phytotoxicity of phenolic acids by some soil microbes. Soil Biology & Biochemistry, 5, 613-614.

581-586. (in Chinese) Yu J Q, Du Y S. 2000. Soil-sickness problem in the sustainable

Wang X Z, Wang H, Wu F Z, Bo L. 2006. Effects of cinnamic acid on the physiological characteristics of cucumber seedling

development for the protected production of vegetables. Journal of Shenyang Agricultural University, 31, 124-126.

under salt stress. Journal of Shenyang Agricultural University, 37, 352-355. (in Chinese)

(in Chinese) Yuan F, Zhang C L, Shen Q R. 2004. Effect and mechanism of

Wu F Z, Huang C H, Zhao F Y. 2002. Effects of phenolic acids on growth and activities of membrane protective enzymes of

phenol compounds in alleviating cucumber fusarium wilt. Scientia Agricultura Sinica, 37, 545-551. (in Chinese)

cucumber seedlings. Agricultural Sciences in China, 1, 900904.

Zhang J E, Liu W G, Hu G. 2002. The relationship between quantity index of soil microorganisms and soil fertility of

Wu F Z, Pan K, Zhou X Y. 2005. Effects of cinnamic acid on physiological characteristics of Cucumis sativus seedling.

different land use systems. Soil and Environmental Sciences, 11, 140-143. (in Chinese)

Chinese Journal of Applied Ecology, 16, 915-918. (in Chinese) Wu F Z, Zhao F Y, Liu Y Y. 2000. On the reasons of continuous

Zhang J Y, Wu F Z. 2007. Effects of salt stress on membrane lipid peroxidation and proline content in cucumber cultivars.

cropping obstacles in vegetable facility gardening. Journal of Northeast Agricultural University, 31, 241-247. (in Chinese)

China Vegetables, (7), 12-15. (in Chinese) Zhang S G, Gao J Y, Song J Z. 1992. Mitigative effects of salicylic

Wu H W, Haig T, Pratley J, Lemerle D, An M. 2001a. Allelochemicals in wheat (Triticum aestivum L.): cultivar

acid and aspirin on salt stress induced injuries in wheat (Triticum aestivum L.). Acta Photophysiol Sinica, 25, 159-

difference in the exudation of phenolic acids. Journal of Agricultural and Food Chemistry, 49, 3742-3745.

164. Zhang S G, Gao J Y, Song J Z, Weng Y J. 1998. Mitigative effects

Wu H W, Haig T, Pratley J, Lemerle D, An M. 2001b. Allelochemicals in wheat (Triticum aestivum L.): production

of salicylic acid and aspirin on salt stress induced injuries in wheat (Triticum aestivum L.) during the stage of seeding

and exudation of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin3-one. Journal of Chemical Ecology, 27, 1691-1700.

growth. Acta Botanica Boreali-occidentalia Sinica, 18, 549554.

Wu H W, Haig T, Pratley J, Lemerle D, An M. 2001c. Allelochemicals in wheat (Triticum aestivum L.): variation of

Zhao M Q, Wang B X, Li F X, Qiu L Y, Li F F, Wang S M, Cui J K. 2007. Analysis of bacterial communities on aging flue-

phenolic acids in shoot tissues. Journal of Chemical Ecology, 27, 125-135.

cured tobacco leaves by 16S rDNA PCR-DGGE technology. Applied Microbiology and Biotechnology, 73, 1435-1440.

Xing D F, Ren N Q, Song J X, Qu M, Xu X L. 2006. Community of activated sludge based on different targeted sequence of

Zheng J H, Ye S F, Yu J Q. 2004. The reason and biological defense of vegetable crops continuous cropping obstacles.

16S rDNA by denaturing gradient gel electrophoresis.

China Vegetables, (3), 56-58. (in Chinese) (Managing editor WANG Ning)

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