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Effect of cinnamic acid on soil microbial characteristics in the cucumber rhizosphere
European Journal of Soil Biology 45 (2009) 356–362
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Effect of cinnamic acid on soil microbial characteristics in the cucumber rhizosphere Fengzhi Wu*, Xuezheng Wang, Chengyu Xue Horticultural College, Northeast Agricultural University, No. 59, Mucai Street, Xiangfang District, Harbin 150030, Heilongjiang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2008 Received in revised form 25 March 2009 Accepted 2 April 2009 Available online 17 April 2009 Handling editor: Petra Marschner
In order to elucidate the effect of allelochemicals on soil microbial characteristics in the cucumber rhizosphere, the soil microbial biomass and respiration, community functional diversity and RAPD marker diversity as affected by exogenous cinnamic acid were studied. Exogenous cinnamic acid increased soil microbial respiration and the metabolic quotient, but decreased soil microbial biomass-C. Soil microbial community functional diversity and genetic diversity (as indicated by RAPD markers) were also significantly altered by exogenous cinnamic acid. These results suggest that allelochemicals can change soil microbial genetic diversity, biological activity and microbial metabolic activity, which alter soil microbial ecology and accordingly affect the growth of cucumber with accumulation in the soil of allelochemicals. Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
Keywords: Allelochemical Cucumber Soil microbial ecology
1. Introduction It is well known that continuous cropping can affect crop growth and decrease yield, quality and disease resistance [41,40]. Continuous cropping obstacles are usually found in agricultural crops, especially in horticultural crops [16,39,50], and are attributed to the buildup of pests, soil physiochemical property disorders, autotoxicity and soil microbes [50,20,52,40]. Hu et al. [22,21] observed that continuous cropping of cucumber disturbed the soil ecological balance and decreased soil microbial community diversity. Studies on agricultural soils have reported that monoculture systems, compared to multi-cropping systems, have decreased soil microbial biomass-C and N, potential cumulative N mineralization, enzyme activities, microbial community functional diversity and genetic diversity [17,29,1,49,47]. Similarly, the microbial diversity in mixed forests soil was higher than that in simple forests [12,56]. The negative effect of continuous cropping may be due to allelochemical effects on soil microbial ecology [27]. Some plant species have allelopathic potential by releasing allelochemicals into the rhizosphere [36,52]. Allelochemicals affect plant metabolism such as photosynthesis, respiration, and ion uptake [4,13], and change the chemical and biological characteristics of the rhizosphere [9]. Previous studies reported that cucumber
* Corresponding author. Tel.: þ86 451 55190278; fax: þ86 451 55190443. E-mail address: [email protected] (F. Wu).
plants possess allelopathic potential by exuding allelochemicals such as benzoic and cinnamic acids, among which cinnamic acid is a widespread and common example [52]. The released allelochemicals also serve as an important carbon and energy source for microorganisms in the rhizosphere [10,9]. However, the influence of allelochemicals on microbial community structure remains largely unknown. In the present study, we applied a single allelochemical to soil, which clearly differs from the effects of the commercial practice of continuous cropping of cucumber, and due to restrictions in the method of detecting allelochemicals in soil, we didn’t analyze cinnamic acid content in soil during the course of the experiment. Allelochemical interactions are complex and integrate biochemical and ecological processes so future investigations will require a multidisciplinary approach. Many previous studies of autotoxic effects have mainly focused on the plant itself. There is little information about the effect on soil microbes, even though soil microbes play a key role in soil–plant interactions. One possible reason is the difficulties involved in the separation and identification of soil microbial community. It is estimated that the microbes that have been identified so far comprise no more than 10% of the actual number. Moreover, the methods of separation and identification lack comparability [18,38]. The interaction between microbes and soil granules has also limited quantitative analyses [2,37]. Cucumber is one of the major greenhouse vegetables in China and its autotoxic effect is very strong [51]. The allelopathic potential of cucumber was reported based on the suppressive effect on the
1164-5563/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2009.04.001
F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
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Table 1 Basic physical and chemical properties of test soil. O.M. (%)
Total N (%)
Alkaline N (mg kg1)
Total P (%)
Avai. P (mg kg1)
Slowly Avai. K (mg kg1)
Avai. K (mg kg1)
pH
EC (Ms cm1)
3.67
0.239
141.1
0.138
260.7
901.5
150.3
6.99
293
growth of some weed species [35,3]. Yu and Matsui [54] found that and Mg2þ was significantly inhibited even at uptake of SO2 4 þ 0.01 mmol mL1 cinnamic acid, whereas the uptake of NO 3 , K and Ca2þ was significantly inhibited at a high cinnamic acid concentration (0.1 mmol mL1). Lockerman and Putnum [31] confirmed that certain cucumber accessions strongly inhibited the growth of some weeds under controlled conditions. Autotoxicity in cucumber plants was regarded as the result of the accumulation of phytotoxic compounds [34]. In this study, soil microbial biomass and respiration, community functional diversity and genetic diversity in the cucumber rhizosphere, as affected by exogenous cinnamic acid, were analyzed. The objective was to determine the effect of cinnamic acid on different aspects of soil microbial ecology, and to understand the mechanism that makes cucumber continuous cropping problematic.
community functional diversity. The remaining soil was stored at 70 C and was used to determine the soil microbial community DNA sequence diversity. 2.3. Soil microbial biomass-C and basal respiration
2. Materials and methods
The fresh soil samples were sieved (pore size < 2 mm) and large pieces of plant roots were removed. The soil samples were incubated for 7 d at 25 C and the moisture contents were adjusted to 50% of their water-holding capacity prior to microbial biomass and respiration measurements. A chloroform fumigation-extraction method was used to determine the soil microbial biomass-C. The content of organic C was determined by an automated TOC analyzer (Shimazu, TOC-500) [19]. The basal respiration (CO2 evolution) was measured in 500 cm3 soil jars using gas chromatography to measure the headspace CO2 that accumulated over 24 h at 25 C from 50 g fresh soil.
2.1. Plant material and soil
2.4. Extraction and purification of soil microbial DNA
Seeds of cucumber cv. ‘Changchun Mici’ (obtained from the Seed Company of Northeast Agricultural University) were washed with sterilized water. The seeds were germinated in the dark at 28 C for 2 d. After germination the seedlings were grown in a 9.5 h light/ 14.5 h dark photoperiod. The seedlings were transplanted into pots (25 cm long 25 cm wide 30 cm high) containing soil in which cucumber had never been planted. The soil was obtained from the experimental station of Northeast Agricultural University, Harbin, Heilongjiang province (45 410 N, 126 370 E). The basic physical and chemical properties of the tested soil are presented in Table 1 [25].
Microbial community DNA in the soil was extracted using the SDS/CTAB method described by Jiao et al. [26]. Extracted DNA was purified with the Promega Wizard Kit (Promega Co.).
2.2. Treatment with cinnamic acid In a previous report, under solar greenhouse conditions, the levels of p-hydroxybenzoic acid, ferulic acid and benzoic acid in the soil increased with increasing duration of continuous cropping and were obviously higher after continuously cropping for 5, 7 and 9 years than for 1 and 3 years. The level of allelochemicals in soil after 9 years of continuous cropping reached 47.93 mg g1 soil [32]. In commercial practice, continuous cropping usually exceeds 20 years and due to restrictions in the method of detecting allelochemicals in soil, the actual content of allelochemicals in soil may be higher than the above concentration. So, in the test, we used cinnamic acid with the concentrations of 25, 50, 100 and 200 mg kg1 soil. Cinnamic acid (Wantai Biological Technology Company) was dissolved in 95% ethanol. Solutions comprising four concentrations (25, 50, 100 and 200 mg kg1 soil) of cinnamic acid were prepared using the same volume of ethanol. Seven days after transplantation of the cucumber seedlings, 25 mL cinnamic acid solution was applied to the soil in each pot (except the control). Three replicates of each treatment were used. The seedlings were placed in a greenhouse (25 C, 4000 lux, with a 9.5 h day/14.5 h night cycle, and 75% relative humidity) using a completely randomized design. Soil without cinnamic acid was used as a control. The cucumber seedlings were watered by drip irrigation and at the bottom of each pot, plastic film was used to prevent leaching. After 30 d cultivation, the soil in each pot was harvested. One part of each sample was used to determine the soil microbial biomass-C and soil microbial
2.5. Amplification and RAPD analysis of soil microbial DNA sequence diversity The DNA sequence diversity of the soil microbial community was evaluated by Random Amplified Polymorphic DNA (RAPD) analysis. We used random primers to amplify the microbial community DNA from the soil samples [48]. Since primers sequences were random and non-selective to DNA samples, amplification for one primer was equal to one random sampling from the whole microbial DNA sequences. The number of RAPD fragments was considered to represent the RAPD fragment richness of the whole DNA sequences. Ten random primers (supplied by Beijing Baotaike Co. and Shanghai Sangon Co.), which were selected from 100 primers and that amplified fragments clearly, were used to amplify soil microbial DNA in the soil samples. The selected primers and the nucleotide sequence of each are listed in Table 2. The most suitable RAPD conditions were determined using the method of Jiao et al. [26]. Amplification using the polymerase chain reaction (PCR) was performed in a 25 ml total volume containing 1 PCR buffer, 7 ng target DNA, 20 pmol random primers, 1.5 unit Taq DNA polymerase, 3.0 mM MgCl2 and 0.2 mM dNTPs. DNA amplification was carried out in a Perkin–Elmer 9600 thermocycler with the following procedure: an initial denaturing step at 94 C for 3 min; 40 cycles for 1 min at 94 C (denaturation), 40 s at 37 C
Table 2 The selected primers and their nucleotide sequence. Code
Sequence 50 / 30
Content of GC (%)
Code
Sequence 50 / 30
Content of GC (%)
1215 1268 1508 236 261
ACACTCTGCC CACCGATCCA AAGAGCCCTC ACACCCCACA CTCAGTGTCC
60 60 60 60 60
Q8 V3 A12 M12 1387
CCTCCAGTGT CTCCCTGCAA TCGGCGATAG GGGACGTTGG CTACGCTCAC
60 60 60 70 60
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F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
Table 3 Effect of cinnamic acid on soil microbial biomass-C, basal respiration and metabolic quotient. The concentration of cinnamic acid (mg kg1 soil)
Microbial parameters
Basal respiration (CO2–C mg g1 h1) Microbial biomass-C (mg g1) Metabolic quotient (h1)
0
25
50
100
200
1.61 0.12c 541 13a 0.002
1.78 0.12c 532 16a 0.003
2.86 0.11b 522 12a 0.005
2.88 0.20b 455 11b 0.006
3.21 0.23a 4117b 0.008
Notes: Numbers in the same column with different letters indicated a significant difference. The minuscule represents P 0.05 (the treatment means were tested separately for least significant difference (LSD) at a 5% level of probability).
(annealing), 90 s at 72 C (extension) and a final elongation step at 72 C for 7 min. The PCR products were separated by electrophoresis in a 1.8% agarose gel containing 0.5 mg mL1 ethidium bromide, and photographed. Each DNA template was amplified several times using the optimized RAPD conditions and yielded identical results. The photographic plates were digitally scanned and analyzed using a Computer Image Analysis System. The presence and absence of amplified fragments were scored. The gray scales of the fragments were also measured (range of gray degree 0–255). The net gray scales were used for further analysis by subtracting the background gray degree of a gel from the measured gray degrees of the fragments. By comparing the net gray scales of the RAPD fragments, the quantity of RAPD fragments was calculated. 2.6. Soil microbial community functional diversity We used the BIOLOG carbon utilization method [7] to analyze soil microbial community functional diversity. Fresh soil (10 g) was added to 100 mL distilled water in a flask and shaken for 10 min. Ten-fold serial dilutions were made and 15 ml of the 103 dilution was used to inoculate the BIOLOG GN plates (BIOLOG, Inc., Hayward, U.S.A.). Plates were incubated at 25 C for 7 d and color development was measured as absorbance (A) using an automated plate reader at 590 nm. The average well color development (AWCD) was calculated as one of valid indices of total activity. The number of substrates utilized was considered to represent substrate richness. 2.7. Data analysis The Shannon index, evenness and McIntosh index were used to measure the species diversity of soil microbial community DNA sequences. The three parameters were estimated using the equations below: S X
Dsh ¼
Pi ln Pi ¼
i¼1
S X
ðNi =NÞlnðNi =NÞ
i¼1
The difference in genetic diversity between soil microbial communities was calculated by the coefficient of DNA sequence similarity. The coefficient was calculated with the formula:
SXY ¼ 2NXY =ðNX þ NY Þ Where NXY denotes mutual RAPD fragments of soil X and soil Y, NX is the number of RAPD fragments for soil X, and NY is the number of RAPD fragments of soil Y. The data for the BIOLOG analysis were compiled using Microlog 4.01 software (BIOLOG, Hayward, CA, U.S.A.). Analysis of variance and regression analysis were performed with Genstart 5.3 software (NAG, Oxford, UK). In order to avoid bias between samples with different inoculum densities [15], the absorbance values at an equivalent AWCD from different times of incubation were compared and were also transformed by dividing by the AWCD. The data were analyzed by canonical variate analysis after first reducing the dimensionality by principal component analysis [46]. Least significant differences (LSD) of the means at the 5% level were calculated by one-way ANOVA using SPSS software. We used the means of the three replicates of the BIOLOG and RAPD data sets, so that differences between soil microbial communities could be more easily ascertained.
3. Results 3.1. Effect of cinnamic acid on microbial biomass-C and basal respiration Cinnamic acid promoted (P 0.05) soil microbial basal respiration when the concentration was higher than 50 mg kg1 soil (Table 3). Cinnamic acid inhibited (P 0.05) soil microbial biomassC when the concentration exceeded 100 mg kg1 soil. The metabolic quotient, which is the ratio of basal respiration to microbial biomass-C, can serve as an important index reflecting soil quality and environmental stress. In the present study the metabolic
Table 4 Ten primers amplified outputs to microbial community DNA of five samples. Primer
Jsh ¼ Dsh =ln S " Dmc ¼
N
s X
!1=2 # Ni2
=½NðN 1Þ
i¼1
where Dsh is the Shannon index, Jsh is the evenness index, Dmc is the McIntosh index, Pi is the percentage of the ith RAPD fragment gray degree of each DNA sample, Ni is the net gray degree value (subtracted from the background gray degree of a gel) of the ith RAPD fragment of each DNA sample, N is the total net gray degree quality of all RAPD fragments examined in each DNA sample, and S is the number of RAPD fragments for each DNA sample.
Amplified fragments
Nonpolymorphic fragments
Polymorphic fragments
Ratio of polymorphic fragments to total fragments (%)
1215 1268 1387 1508 236 261 Q8 V3 A12 M12
29 24 26 23 28 26 29 32 31 28
3 2 4 3 4 3 3 3 3 3
26 22 22 20 24 23 26 29 28 25
89 91 84 86 85 88 89 90 93 89
Total
276
31
245
89
Richness index
F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
10 9 8 7 6 5 4 3 2 1 0
b a
c c d
0
25
50
Cinnammic acid
100
200
(mg.kg-1soil)
Fig. 1. Effect of cinnamic acid on richness index of DNA sequence of soil microbial community in cucumber rhizosphere.
quotient increased markedly with increasing cinnamic acid concentrations. 3.2. RAPD fingerprints profiles The ten random primers used to amplify soil microbial community DNA generated a total of 276 RAPD fragments. The number of fragments scored per primer varied from 23 (1508) to 32 (V3) (Table 4). Of the total number of fragments, 245 (89%) were polymorphic and could not be amplified in all five samples. The other 31 fragments (11%) were nonpolymorphic and could be amplified in all five samples. An electrophoretogram depicting fragments amplified by two of the primers for each cinnamic acid treatment is shown in Fig. 1. 3.3. Genetic richness of the soil microbial community Cinnamic acid promoted microbial community genetic richness in the cucumber rhizosphere at a concentration of 25 mg kg1 soil, but microbial genetic diversity declined at cinnamic acid concentrations of 50 mg kg1 soil and above (Fig. 1). The genetic richness of the 50, 100 and 200 mg kg1 treatments were lower that of the 25 mg kg1 treatment (P 0.05). The effect of cinnamic acid on RAPD marker diversity in the soil microbial community, as measured by the Shannon and evenness indices is shown in Table 5. The 25 mg kg1 soil cinnamic acid treatment had the highest Shannon–Weaver index and evenness index within the soil microbial community. The RAPD marker diversity decreased with increasing cinnamic acid concentrations. The RAPD markers of the soil microbial community at cinnamic acid concentrations of 100 and 200 mg kg1 soil differed considerably from those of the 25 mg kg1 treatment and the control (Table 6). The similarity coefficient was highest (0.55) between the control and 25 mg kg1 soil treatment, whereas the similarity coefficient between the 25 mg kg1 soil and 200 mg kg1 soil treatments was lowest (0.23). 3.4. Functional diversity indices of the soil microbial community Except for the McIntosh index, all of the other indices indicated that the functional diversity of the soil microbial community in the
359
cucumber rhizosphere decreased with increasing cinnamic acid concentration (Table 7). Among these indices, the Shannon index and Shannon evenness for the 50 mg kg1 soil treatment and higher cinnamic acid concentrations were significantly different from those of the control (P 0.05). Values for the Simpson index were significantly reduced in the cinnamic acid treatments compared to the control.
3.5. Effect of cinnamic acid on soil microbial community structure in cucumber rhizosphere The mean absorbance at different cinnamic acid concentrations with increasing incubation time is shown in Fig. 2. When the incubation time exceeded 24 h, mean absorbance in the 200 mg kg1 soil treatment was significantly different from the other treatments. After incubation for 72 h, mean absorbance in the 100 mg kg1 soil treatment differed significantly from the other cinnamic acid concentrations. The mean absorbance did not differ significantly among the control and the 25 mg kg1 soil and 50 mg kg1 soil treatments. Multivariate analysis of the sole carbon source data distinguished differences in soil microbial functional diversity between the control and cinnamic acid treatments (Fig. 3). The first canonical variate (CV1) of the BIOLOG data accounted for 53.38% of the variance (P < 0.05), and the second (CV2) accounted for 23.94% of the variance (P < 0.05). All cinnamic acid treatments were clearly discriminated from the control on CV1. These results implicated that cinnamic acid affected soil microbial community structure and resulted in formation of a specific soil microbial community. The variances for CV1 and CV2 in the control were very small. This result may suggest that the soil microbial community structure in the control was relatively stable. However, the variance in the functional diversity of the microbial community increased markedly following cinnamic acid addition. Analysis of the loadings of the most influential C sources indicated that there was a negative relationship between cyclodextrin, glycogen, 2-hydroxy benzoic acid and CV1, and there was positive relationship between hydrobutyric acid, L-asparagine, D-galacturonic acid and CV1. These results suggested that exogenous cinnamic acid may promote the soil microbial community’s ability to utilize cyclodextrin, glycogen and 2-hydroxy benzoic acid, and inhibit soil microbial utilization of hydrobutyric acid, L-asparagine, and D-galacturonic acid.
4. Discussion Negative impacts of continuous cropping often occur when the same crop or its relatives are cultivated on the same soil successively, which can affect the yield and quality of the crops severely [6,11,36,39]. This is a problem in the sustainable development of vegetable plants. Continuous cropping can have many effects, such as reduction in soil fertility, disruption of soil structure, adverse effects on soil properties, occurrence of soil-borne disease, alteration of the microflora, as well as allelopathy [14,57]. When the previous crop residues contain allelochemicals, such as cinnamic acid, under continuous cropping these allelochemicals will inhibit
Table 5 Effects of cinnamic acid on diversity indices of soil microbial community DNA. Index
Shannon–Weaver index Evenness index
The concentration of cinnamic acid (mg kg1 soil) 0
25
50
100
200
1.81 0.02a 0.99 0.05a
1.91 0.02a 1.00 0.05a
1.40 0.08b 0.95 0.06a
1.33 0.05b 0.93 0.06b
1.10 0.09c 0.91 0.02b
Notes: Numbers in the same column with different letters indicated a significant difference (P 0.05).
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Table 6 Similarity coefficient of soil microbial community DNA sequence.
0 25 50 100 200
0
25 0.55
65 51 47 32
47 38 27
50
100
0.42 0.31
0.37 0.28 0.43
44 30
200
1.000
0.29 0.23 0.34 0.30
0.800
25
Note: The data above diagonal are coefficients of community similarity and those down diagonal are number of common amplified fragments.
AWCD
The concentration of cinnamic acid (mg kg1 soil)
1.200
0.600 control 25mg.kg-1soil 50mg.kg-1soil 100mg.kg-1soil 200mg.kg-1soil
0.400 0.200
Table 7 Effect of cinnamic acid on function diversity of soil microbial community (168 h). The concentration of cinnamic acid (mg kg1 soil)
Shannon index
Shannon evenness
Simpson index
McIntosh index
0 25 50 100 200
3.36 3.33 3.15 2.22 2.28
1.06 0.95 1.02 0.53 0.59
54.86 41.33 43.24 43.45 42.34
4.62 4.99 4.54 5.04 4.92
0.000 0
0.29a 0.14a 0.24a 0.15b 0.31b
0.28a 0.1a 0.15a 0.15b 0.11b
1.72a 0.77b 1.85b 2.76b 2.03b
0.32a 0.13a 0.44a 0.32a 0.38a
48
72
96
120
144
168
Fig. 2. Mean absorbance (AWCD) of the BIOLOG plates at 595 nm for the tested soils with different cinnamic acid treatments.
was studied using RAPD markers. The results showed that cinnamic acid can change the microbial composition and have an impact on genetic diversity in the soil microbial community. A decrease in diversity and evenness indices for the soil microbial community with increasing cinnamic acid concentration was found. These results suggest that allelochemicals may induce the enrichment or disappearance of some microbial species. However, the application of RAPD markers may also be affected since any change less than 1% of the total community cannot be detected by the technology [44]. Consequently, it is necessary to integrate diverse approaches and perspectives to understand more precisely the changes in the diversity of microbial communities. BIOLOG analysis is based on the premise that microorganisms vary in the pattern and rate at which they utilize carbon sources. Therefore, carbon utilization patterns can be used as a measure of microbial community structure and functional potential [15]. In the present study, AWCD values decreased significantly with increasing cinnamic acid concentration. This may be due to lower microbial biomass in the treatments with high cinnamic acid concentrations. Diversity indices suggested that the composition of the soil microbial community changed significantly when the cinnamic acid concentration exceeded 50 mg kg1 soil. Normally, different diversity indices reflect different aspects of soil microbial community functional diversity. The Shannon index is affected by species richness, the Simpson index reflects the most common species in the community, and the McIntosh index is a measure of species evenness [45]. The Simpson index decreased significantly when the cinnamic acid concentration was higher than 25 mg kg1 soil. This
0.6 0.5 0 25 50 100 200
0.4 0.3 0.2 0.1 0 -0.1 -0.2 -0.3 -1
24
Time(h)
CV2
the following crop and reduce the yield once they accumulate to a certain level [53]. Exudates of cucumber roots contain phenolic allelochemicals that have an inhibitory effect on cucumber growth under continuous cropping. This is a type of autotoxic effect [54]. Cut stumps, litter and root extracts contain ferulic acid and cinnamic acid, which can accumulate and affect the following crop [23,24]. Exogenous ferulic and cinnamic acids also can inhibit the germination rate of fir seeds, their vigor and growth of seedlings [8]. Allelochemicals such as cinnamic acid can inhibit the uptake of ions 2 þ 2þ and Fe2þ) by cucumber seedlings [52]. Some (NO 3 , SO4 , K , Ca studies have found that cucumber root exudates facilitated the mycelial growth of Fusarium oxysporum f. sp. cucumerinum Owen [42]. The allelochemicals promoted the growth of soil fungi and bacteria and inhibited the growth of soil actinomycetes and F. oxysporum f. sp. cucumerinum [55,43]. Some studies reported that certain amounts of phenolic substances limit the mycelial growth of the soil pathogen burdock Rhizoctonia rot [30]. The processes of allelopathy, autotoxicity as well as allelopathic and autotoxic effects of allelochemicals are well known, but the mechanisms by which allelochemicals exert their effects are not clear. Furthermore, there is close relationship between allelochemicals and the size and activity of the soil microbial community [5]. The intensity of autotoxicity depends on the content, catabolism and the interaction of allelochemicals in soil. Rice et al. [36] compared the rate of decomposition of ferulic acid, vanillic acid, syringic acid and p-hydroxybenzoic acid and found that decomposition of p-hydroxybenzoic acid was the most rapid, whereas decomposition of syringic acid was the slowest. Zhang et al. [57] found that following addition of exogenous allelochemicals for one week, the soil residue rate was only 17.5–25%, and different allelochemicals were degraded at different rates. The study of Yu and Matsui [52] confirmed that exudates of cucumber roots contain ten different allelochemicals, including p-hydroxybenzoic acid, ferulic acid and cinnamic acid, and these occur naturally in soil. RAPD molecular marker technology was first used to identify plant gene polymorphism [28,33]. It can be used as a rapid and sensitive method to detect very small differences in the same or similar genes. Yao et al. [48] found that pesticide contamination can cause changes in the genetic diversity. In this paper, the effect of cinnamic acid on genetic diversity of the soil microbial community
-0.5
0
0.5
1
1.5
2
2.5
CV1 Fig. 3. Plot of ordination of canonical variates (CV) CV1 against CV2 generated by canonical variate analysis of sole carbon source tests at comparable time points in BIOLOG plates showing discrimination between different soils.
F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
result suggests that cinnamic acid had a significant impact on the most common species of the soil microbial community. Cinnamic acid concentrations greater than 50 mg kg1 soil had major impacts on species evenness and richness of the soil microbial community. Multivariate analysis of carbon source utilization may also reflect the change in structure of the soil microbial community. In this study, cinnamic acid increased the variation of the microbial community and significantly changed microbial metabolic profiles. However, only some metabolically active and culturable bacteria can be detected, whereas soil fungi and slow-growing bacteria cannot be assessed. The technique is also sensitive to inoculum density and cannot reflect the potential metabolic diversity in situ. Nevertheless, the BIOLOG method is useful in studying the functional diversity of microbial community and is a valuable tool especially when used in conjunction with other methods. In this study, one of the important objectives was to determine the level of cinnamic acid that has an effect on the soil microbial community. When the cinnamic acid concentration was 25 mg kg1 soil, CV1 generated by canonical variate analysis of the sole carbon source tests decreased obviously. This result may suggest that the threshold of cinnamic acid that affected the functional diversity of microorganisms is less than 25 mg kg1 soil. Moreover, most diversity indices derived from the RAPD and BIOLOG data showed that the threshold of cinnamic acid was less than 100 mg kg1 soil. It seems that the soil microbial community is very sensitive to exogenous cinnamic acid. Consequently, allelochemicals may change soil microbial genetic diversity, biological activity and microbial metabolic activity, and accordingly affect the growth of cucumber. Acknowledgement This study was supported by National ‘‘973 Project’’ (2009CB119004) and National Natural Science Foundation of China (Nos.: 30771252, 30230250). References [1] V. Acosta-Martı´nez, T.M. Zobeck, T.E. Gill, A.C. Kennedy, Enzyme activities and microbial community structure in semiarid agricultural soils, Biol. Fertil. Soils 38 (2003) 216–227. [2] M. Ahmed, J.M. Oades, Distribution of organic matter and adenosine triphosphate after fractionation of soils by physical procedures, Soil Biol. Biochem. 16 (1984) 465–470. [3] T. Asao, N. Ohtani, N. Shimizu, M. Umeyama, K. Ohta, T. Hosoki, Possible selection of cucumber cultivars suitable for a closed hydrophonic system by the bioassay with cucumber seedlings, J. Soc. High Technol. Agric. 10 (1998a) 92–95 (in Japanese with English summary). [4] N.E. Balke, Effects of allelochemicals on mineral uptake and associated physiological process, ACS Symp. Ser. 268 (1985) 161–178. [5] U. Blum, S.R. Shafer, Microbial population and phenolic acids in soils, Soil Biol. Biochem. 20( (6) (1988) 793–800. [6] J. Bonner, A.W. Galson, Toxic substances from the culture media of guayule which may inhibit growth, Bot. Gaz. 106 (1944) 185–198. [7] C.D. Campbell, S.J. Grayston, D.J. Hirst, Use of rhizosphere carbon sources in sole carbon source tests to determinate soil microbial communities, J. Microbial. Methods 30 (1997) 33–41. [8] G.Q. Cao, S.Z. Lin, S.G. Huang, Effect of the ferulic acid and cinnamic acid on the germination of Chinese-fir seeds, J. Plant Resour. Environ. 10 (2) (2001) 63–64 (in Chinese). [9] Cecile Bertin, X.H. Yang, A. Leslie, Weston, the role of root exudates and allelochemicals in the rhizosphere, Plant Soil 256 (2003) 67–83. [10] W. Cheng, Q. Zhang, D.C. Coleman, C.R. Caroll, C.A. Hoffman, Is available carbon limiting microbial respiration in the rhizosphere? Soil Biol. Biochem. 2 (1996) 1283–1288. [11] E.F. Davis, The toxic principle of Juglans nigra as identified with synthetic juglone and its toxic effect on tomato and alfalfa plants, Am. J. Bot. 15 (1928) 620–629. [12] G.J. Du, T.P. Huang, Q.R. Zhang, P.S. Zhang, R.L. Cheng, Studies on soil microorganisms and biochemical properties in mixed forests of Chinese fir, J. Zhejiang For. Coll. 12 (4) (1995) 347–352 (in Chinese). [13] F.A. Einhellig, Mechanisms and modes of action of allelochemicals, in: A.R. Putnam, C.S. Tang (Eds.), The Sciences of Allelopathy, Wiley Interscience, New York, 1986, pp. 171–188.
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Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Effect of cinnamic acid on soil microbial characteristics in the cucumber rhizosphere Fengzhi Wu*, Xuezheng Wang, Chengyu Xue Horticultural College, Northeast Agricultural University, No. 59, Mucai Street, Xiangfang District, Harbin 150030, Heilongjiang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2008 Received in revised form 25 March 2009 Accepted 2 April 2009 Available online 17 April 2009 Handling editor: Petra Marschner
In order to elucidate the effect of allelochemicals on soil microbial characteristics in the cucumber rhizosphere, the soil microbial biomass and respiration, community functional diversity and RAPD marker diversity as affected by exogenous cinnamic acid were studied. Exogenous cinnamic acid increased soil microbial respiration and the metabolic quotient, but decreased soil microbial biomass-C. Soil microbial community functional diversity and genetic diversity (as indicated by RAPD markers) were also significantly altered by exogenous cinnamic acid. These results suggest that allelochemicals can change soil microbial genetic diversity, biological activity and microbial metabolic activity, which alter soil microbial ecology and accordingly affect the growth of cucumber with accumulation in the soil of allelochemicals. Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
Keywords: Allelochemical Cucumber Soil microbial ecology
1. Introduction It is well known that continuous cropping can affect crop growth and decrease yield, quality and disease resistance [41,40]. Continuous cropping obstacles are usually found in agricultural crops, especially in horticultural crops [16,39,50], and are attributed to the buildup of pests, soil physiochemical property disorders, autotoxicity and soil microbes [50,20,52,40]. Hu et al. [22,21] observed that continuous cropping of cucumber disturbed the soil ecological balance and decreased soil microbial community diversity. Studies on agricultural soils have reported that monoculture systems, compared to multi-cropping systems, have decreased soil microbial biomass-C and N, potential cumulative N mineralization, enzyme activities, microbial community functional diversity and genetic diversity [17,29,1,49,47]. Similarly, the microbial diversity in mixed forests soil was higher than that in simple forests [12,56]. The negative effect of continuous cropping may be due to allelochemical effects on soil microbial ecology [27]. Some plant species have allelopathic potential by releasing allelochemicals into the rhizosphere [36,52]. Allelochemicals affect plant metabolism such as photosynthesis, respiration, and ion uptake [4,13], and change the chemical and biological characteristics of the rhizosphere [9]. Previous studies reported that cucumber
* Corresponding author. Tel.: þ86 451 55190278; fax: þ86 451 55190443. E-mail address: [email protected] (F. Wu).
plants possess allelopathic potential by exuding allelochemicals such as benzoic and cinnamic acids, among which cinnamic acid is a widespread and common example [52]. The released allelochemicals also serve as an important carbon and energy source for microorganisms in the rhizosphere [10,9]. However, the influence of allelochemicals on microbial community structure remains largely unknown. In the present study, we applied a single allelochemical to soil, which clearly differs from the effects of the commercial practice of continuous cropping of cucumber, and due to restrictions in the method of detecting allelochemicals in soil, we didn’t analyze cinnamic acid content in soil during the course of the experiment. Allelochemical interactions are complex and integrate biochemical and ecological processes so future investigations will require a multidisciplinary approach. Many previous studies of autotoxic effects have mainly focused on the plant itself. There is little information about the effect on soil microbes, even though soil microbes play a key role in soil–plant interactions. One possible reason is the difficulties involved in the separation and identification of soil microbial community. It is estimated that the microbes that have been identified so far comprise no more than 10% of the actual number. Moreover, the methods of separation and identification lack comparability [18,38]. The interaction between microbes and soil granules has also limited quantitative analyses [2,37]. Cucumber is one of the major greenhouse vegetables in China and its autotoxic effect is very strong [51]. The allelopathic potential of cucumber was reported based on the suppressive effect on the
1164-5563/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2009.04.001
F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
357
Table 1 Basic physical and chemical properties of test soil. O.M. (%)
Total N (%)
Alkaline N (mg kg1)
Total P (%)
Avai. P (mg kg1)
Slowly Avai. K (mg kg1)
Avai. K (mg kg1)
pH
EC (Ms cm1)
3.67
0.239
141.1
0.138
260.7
901.5
150.3
6.99
293
growth of some weed species [35,3]. Yu and Matsui [54] found that and Mg2þ was significantly inhibited even at uptake of SO2 4 þ 0.01 mmol mL1 cinnamic acid, whereas the uptake of NO 3 , K and Ca2þ was significantly inhibited at a high cinnamic acid concentration (0.1 mmol mL1). Lockerman and Putnum [31] confirmed that certain cucumber accessions strongly inhibited the growth of some weeds under controlled conditions. Autotoxicity in cucumber plants was regarded as the result of the accumulation of phytotoxic compounds [34]. In this study, soil microbial biomass and respiration, community functional diversity and genetic diversity in the cucumber rhizosphere, as affected by exogenous cinnamic acid, were analyzed. The objective was to determine the effect of cinnamic acid on different aspects of soil microbial ecology, and to understand the mechanism that makes cucumber continuous cropping problematic.
community functional diversity. The remaining soil was stored at 70 C and was used to determine the soil microbial community DNA sequence diversity. 2.3. Soil microbial biomass-C and basal respiration
2. Materials and methods
The fresh soil samples were sieved (pore size < 2 mm) and large pieces of plant roots were removed. The soil samples were incubated for 7 d at 25 C and the moisture contents were adjusted to 50% of their water-holding capacity prior to microbial biomass and respiration measurements. A chloroform fumigation-extraction method was used to determine the soil microbial biomass-C. The content of organic C was determined by an automated TOC analyzer (Shimazu, TOC-500) [19]. The basal respiration (CO2 evolution) was measured in 500 cm3 soil jars using gas chromatography to measure the headspace CO2 that accumulated over 24 h at 25 C from 50 g fresh soil.
2.1. Plant material and soil
2.4. Extraction and purification of soil microbial DNA
Seeds of cucumber cv. ‘Changchun Mici’ (obtained from the Seed Company of Northeast Agricultural University) were washed with sterilized water. The seeds were germinated in the dark at 28 C for 2 d. After germination the seedlings were grown in a 9.5 h light/ 14.5 h dark photoperiod. The seedlings were transplanted into pots (25 cm long 25 cm wide 30 cm high) containing soil in which cucumber had never been planted. The soil was obtained from the experimental station of Northeast Agricultural University, Harbin, Heilongjiang province (45 410 N, 126 370 E). The basic physical and chemical properties of the tested soil are presented in Table 1 [25].
Microbial community DNA in the soil was extracted using the SDS/CTAB method described by Jiao et al. [26]. Extracted DNA was purified with the Promega Wizard Kit (Promega Co.).
2.2. Treatment with cinnamic acid In a previous report, under solar greenhouse conditions, the levels of p-hydroxybenzoic acid, ferulic acid and benzoic acid in the soil increased with increasing duration of continuous cropping and were obviously higher after continuously cropping for 5, 7 and 9 years than for 1 and 3 years. The level of allelochemicals in soil after 9 years of continuous cropping reached 47.93 mg g1 soil [32]. In commercial practice, continuous cropping usually exceeds 20 years and due to restrictions in the method of detecting allelochemicals in soil, the actual content of allelochemicals in soil may be higher than the above concentration. So, in the test, we used cinnamic acid with the concentrations of 25, 50, 100 and 200 mg kg1 soil. Cinnamic acid (Wantai Biological Technology Company) was dissolved in 95% ethanol. Solutions comprising four concentrations (25, 50, 100 and 200 mg kg1 soil) of cinnamic acid were prepared using the same volume of ethanol. Seven days after transplantation of the cucumber seedlings, 25 mL cinnamic acid solution was applied to the soil in each pot (except the control). Three replicates of each treatment were used. The seedlings were placed in a greenhouse (25 C, 4000 lux, with a 9.5 h day/14.5 h night cycle, and 75% relative humidity) using a completely randomized design. Soil without cinnamic acid was used as a control. The cucumber seedlings were watered by drip irrigation and at the bottom of each pot, plastic film was used to prevent leaching. After 30 d cultivation, the soil in each pot was harvested. One part of each sample was used to determine the soil microbial biomass-C and soil microbial
2.5. Amplification and RAPD analysis of soil microbial DNA sequence diversity The DNA sequence diversity of the soil microbial community was evaluated by Random Amplified Polymorphic DNA (RAPD) analysis. We used random primers to amplify the microbial community DNA from the soil samples [48]. Since primers sequences were random and non-selective to DNA samples, amplification for one primer was equal to one random sampling from the whole microbial DNA sequences. The number of RAPD fragments was considered to represent the RAPD fragment richness of the whole DNA sequences. Ten random primers (supplied by Beijing Baotaike Co. and Shanghai Sangon Co.), which were selected from 100 primers and that amplified fragments clearly, were used to amplify soil microbial DNA in the soil samples. The selected primers and the nucleotide sequence of each are listed in Table 2. The most suitable RAPD conditions were determined using the method of Jiao et al. [26]. Amplification using the polymerase chain reaction (PCR) was performed in a 25 ml total volume containing 1 PCR buffer, 7 ng target DNA, 20 pmol random primers, 1.5 unit Taq DNA polymerase, 3.0 mM MgCl2 and 0.2 mM dNTPs. DNA amplification was carried out in a Perkin–Elmer 9600 thermocycler with the following procedure: an initial denaturing step at 94 C for 3 min; 40 cycles for 1 min at 94 C (denaturation), 40 s at 37 C
Table 2 The selected primers and their nucleotide sequence. Code
Sequence 50 / 30
Content of GC (%)
Code
Sequence 50 / 30
Content of GC (%)
1215 1268 1508 236 261
ACACTCTGCC CACCGATCCA AAGAGCCCTC ACACCCCACA CTCAGTGTCC
60 60 60 60 60
Q8 V3 A12 M12 1387
CCTCCAGTGT CTCCCTGCAA TCGGCGATAG GGGACGTTGG CTACGCTCAC
60 60 60 70 60
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Table 3 Effect of cinnamic acid on soil microbial biomass-C, basal respiration and metabolic quotient. The concentration of cinnamic acid (mg kg1 soil)
Microbial parameters
Basal respiration (CO2–C mg g1 h1) Microbial biomass-C (mg g1) Metabolic quotient (h1)
0
25
50
100
200
1.61 0.12c 541 13a 0.002
1.78 0.12c 532 16a 0.003
2.86 0.11b 522 12a 0.005
2.88 0.20b 455 11b 0.006
3.21 0.23a 4117b 0.008
Notes: Numbers in the same column with different letters indicated a significant difference. The minuscule represents P 0.05 (the treatment means were tested separately for least significant difference (LSD) at a 5% level of probability).
(annealing), 90 s at 72 C (extension) and a final elongation step at 72 C for 7 min. The PCR products were separated by electrophoresis in a 1.8% agarose gel containing 0.5 mg mL1 ethidium bromide, and photographed. Each DNA template was amplified several times using the optimized RAPD conditions and yielded identical results. The photographic plates were digitally scanned and analyzed using a Computer Image Analysis System. The presence and absence of amplified fragments were scored. The gray scales of the fragments were also measured (range of gray degree 0–255). The net gray scales were used for further analysis by subtracting the background gray degree of a gel from the measured gray degrees of the fragments. By comparing the net gray scales of the RAPD fragments, the quantity of RAPD fragments was calculated. 2.6. Soil microbial community functional diversity We used the BIOLOG carbon utilization method [7] to analyze soil microbial community functional diversity. Fresh soil (10 g) was added to 100 mL distilled water in a flask and shaken for 10 min. Ten-fold serial dilutions were made and 15 ml of the 103 dilution was used to inoculate the BIOLOG GN plates (BIOLOG, Inc., Hayward, U.S.A.). Plates were incubated at 25 C for 7 d and color development was measured as absorbance (A) using an automated plate reader at 590 nm. The average well color development (AWCD) was calculated as one of valid indices of total activity. The number of substrates utilized was considered to represent substrate richness. 2.7. Data analysis The Shannon index, evenness and McIntosh index were used to measure the species diversity of soil microbial community DNA sequences. The three parameters were estimated using the equations below: S X
Dsh ¼
Pi ln Pi ¼
i¼1
S X
ðNi =NÞlnðNi =NÞ
i¼1
The difference in genetic diversity between soil microbial communities was calculated by the coefficient of DNA sequence similarity. The coefficient was calculated with the formula:
SXY ¼ 2NXY =ðNX þ NY Þ Where NXY denotes mutual RAPD fragments of soil X and soil Y, NX is the number of RAPD fragments for soil X, and NY is the number of RAPD fragments of soil Y. The data for the BIOLOG analysis were compiled using Microlog 4.01 software (BIOLOG, Hayward, CA, U.S.A.). Analysis of variance and regression analysis were performed with Genstart 5.3 software (NAG, Oxford, UK). In order to avoid bias between samples with different inoculum densities [15], the absorbance values at an equivalent AWCD from different times of incubation were compared and were also transformed by dividing by the AWCD. The data were analyzed by canonical variate analysis after first reducing the dimensionality by principal component analysis [46]. Least significant differences (LSD) of the means at the 5% level were calculated by one-way ANOVA using SPSS software. We used the means of the three replicates of the BIOLOG and RAPD data sets, so that differences between soil microbial communities could be more easily ascertained.
3. Results 3.1. Effect of cinnamic acid on microbial biomass-C and basal respiration Cinnamic acid promoted (P 0.05) soil microbial basal respiration when the concentration was higher than 50 mg kg1 soil (Table 3). Cinnamic acid inhibited (P 0.05) soil microbial biomassC when the concentration exceeded 100 mg kg1 soil. The metabolic quotient, which is the ratio of basal respiration to microbial biomass-C, can serve as an important index reflecting soil quality and environmental stress. In the present study the metabolic
Table 4 Ten primers amplified outputs to microbial community DNA of five samples. Primer
Jsh ¼ Dsh =ln S " Dmc ¼
N
s X
!1=2 # Ni2
=½NðN 1Þ
i¼1
where Dsh is the Shannon index, Jsh is the evenness index, Dmc is the McIntosh index, Pi is the percentage of the ith RAPD fragment gray degree of each DNA sample, Ni is the net gray degree value (subtracted from the background gray degree of a gel) of the ith RAPD fragment of each DNA sample, N is the total net gray degree quality of all RAPD fragments examined in each DNA sample, and S is the number of RAPD fragments for each DNA sample.
Amplified fragments
Nonpolymorphic fragments
Polymorphic fragments
Ratio of polymorphic fragments to total fragments (%)
1215 1268 1387 1508 236 261 Q8 V3 A12 M12
29 24 26 23 28 26 29 32 31 28
3 2 4 3 4 3 3 3 3 3
26 22 22 20 24 23 26 29 28 25
89 91 84 86 85 88 89 90 93 89
Total
276
31
245
89
Richness index
F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
10 9 8 7 6 5 4 3 2 1 0
b a
c c d
0
25
50
Cinnammic acid
100
200
(mg.kg-1soil)
Fig. 1. Effect of cinnamic acid on richness index of DNA sequence of soil microbial community in cucumber rhizosphere.
quotient increased markedly with increasing cinnamic acid concentrations. 3.2. RAPD fingerprints profiles The ten random primers used to amplify soil microbial community DNA generated a total of 276 RAPD fragments. The number of fragments scored per primer varied from 23 (1508) to 32 (V3) (Table 4). Of the total number of fragments, 245 (89%) were polymorphic and could not be amplified in all five samples. The other 31 fragments (11%) were nonpolymorphic and could be amplified in all five samples. An electrophoretogram depicting fragments amplified by two of the primers for each cinnamic acid treatment is shown in Fig. 1. 3.3. Genetic richness of the soil microbial community Cinnamic acid promoted microbial community genetic richness in the cucumber rhizosphere at a concentration of 25 mg kg1 soil, but microbial genetic diversity declined at cinnamic acid concentrations of 50 mg kg1 soil and above (Fig. 1). The genetic richness of the 50, 100 and 200 mg kg1 treatments were lower that of the 25 mg kg1 treatment (P 0.05). The effect of cinnamic acid on RAPD marker diversity in the soil microbial community, as measured by the Shannon and evenness indices is shown in Table 5. The 25 mg kg1 soil cinnamic acid treatment had the highest Shannon–Weaver index and evenness index within the soil microbial community. The RAPD marker diversity decreased with increasing cinnamic acid concentrations. The RAPD markers of the soil microbial community at cinnamic acid concentrations of 100 and 200 mg kg1 soil differed considerably from those of the 25 mg kg1 treatment and the control (Table 6). The similarity coefficient was highest (0.55) between the control and 25 mg kg1 soil treatment, whereas the similarity coefficient between the 25 mg kg1 soil and 200 mg kg1 soil treatments was lowest (0.23). 3.4. Functional diversity indices of the soil microbial community Except for the McIntosh index, all of the other indices indicated that the functional diversity of the soil microbial community in the
359
cucumber rhizosphere decreased with increasing cinnamic acid concentration (Table 7). Among these indices, the Shannon index and Shannon evenness for the 50 mg kg1 soil treatment and higher cinnamic acid concentrations were significantly different from those of the control (P 0.05). Values for the Simpson index were significantly reduced in the cinnamic acid treatments compared to the control.
3.5. Effect of cinnamic acid on soil microbial community structure in cucumber rhizosphere The mean absorbance at different cinnamic acid concentrations with increasing incubation time is shown in Fig. 2. When the incubation time exceeded 24 h, mean absorbance in the 200 mg kg1 soil treatment was significantly different from the other treatments. After incubation for 72 h, mean absorbance in the 100 mg kg1 soil treatment differed significantly from the other cinnamic acid concentrations. The mean absorbance did not differ significantly among the control and the 25 mg kg1 soil and 50 mg kg1 soil treatments. Multivariate analysis of the sole carbon source data distinguished differences in soil microbial functional diversity between the control and cinnamic acid treatments (Fig. 3). The first canonical variate (CV1) of the BIOLOG data accounted for 53.38% of the variance (P < 0.05), and the second (CV2) accounted for 23.94% of the variance (P < 0.05). All cinnamic acid treatments were clearly discriminated from the control on CV1. These results implicated that cinnamic acid affected soil microbial community structure and resulted in formation of a specific soil microbial community. The variances for CV1 and CV2 in the control were very small. This result may suggest that the soil microbial community structure in the control was relatively stable. However, the variance in the functional diversity of the microbial community increased markedly following cinnamic acid addition. Analysis of the loadings of the most influential C sources indicated that there was a negative relationship between cyclodextrin, glycogen, 2-hydroxy benzoic acid and CV1, and there was positive relationship between hydrobutyric acid, L-asparagine, D-galacturonic acid and CV1. These results suggested that exogenous cinnamic acid may promote the soil microbial community’s ability to utilize cyclodextrin, glycogen and 2-hydroxy benzoic acid, and inhibit soil microbial utilization of hydrobutyric acid, L-asparagine, and D-galacturonic acid.
4. Discussion Negative impacts of continuous cropping often occur when the same crop or its relatives are cultivated on the same soil successively, which can affect the yield and quality of the crops severely [6,11,36,39]. This is a problem in the sustainable development of vegetable plants. Continuous cropping can have many effects, such as reduction in soil fertility, disruption of soil structure, adverse effects on soil properties, occurrence of soil-borne disease, alteration of the microflora, as well as allelopathy [14,57]. When the previous crop residues contain allelochemicals, such as cinnamic acid, under continuous cropping these allelochemicals will inhibit
Table 5 Effects of cinnamic acid on diversity indices of soil microbial community DNA. Index
Shannon–Weaver index Evenness index
The concentration of cinnamic acid (mg kg1 soil) 0
25
50
100
200
1.81 0.02a 0.99 0.05a
1.91 0.02a 1.00 0.05a
1.40 0.08b 0.95 0.06a
1.33 0.05b 0.93 0.06b
1.10 0.09c 0.91 0.02b
Notes: Numbers in the same column with different letters indicated a significant difference (P 0.05).
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F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
Table 6 Similarity coefficient of soil microbial community DNA sequence.
0 25 50 100 200
0
25 0.55
65 51 47 32
47 38 27
50
100
0.42 0.31
0.37 0.28 0.43
44 30
200
1.000
0.29 0.23 0.34 0.30
0.800
25
Note: The data above diagonal are coefficients of community similarity and those down diagonal are number of common amplified fragments.
AWCD
The concentration of cinnamic acid (mg kg1 soil)
1.200
0.600 control 25mg.kg-1soil 50mg.kg-1soil 100mg.kg-1soil 200mg.kg-1soil
0.400 0.200
Table 7 Effect of cinnamic acid on function diversity of soil microbial community (168 h). The concentration of cinnamic acid (mg kg1 soil)
Shannon index
Shannon evenness
Simpson index
McIntosh index
0 25 50 100 200
3.36 3.33 3.15 2.22 2.28
1.06 0.95 1.02 0.53 0.59
54.86 41.33 43.24 43.45 42.34
4.62 4.99 4.54 5.04 4.92
0.000 0
0.29a 0.14a 0.24a 0.15b 0.31b
0.28a 0.1a 0.15a 0.15b 0.11b
1.72a 0.77b 1.85b 2.76b 2.03b
0.32a 0.13a 0.44a 0.32a 0.38a
48
72
96
120
144
168
Fig. 2. Mean absorbance (AWCD) of the BIOLOG plates at 595 nm for the tested soils with different cinnamic acid treatments.
was studied using RAPD markers. The results showed that cinnamic acid can change the microbial composition and have an impact on genetic diversity in the soil microbial community. A decrease in diversity and evenness indices for the soil microbial community with increasing cinnamic acid concentration was found. These results suggest that allelochemicals may induce the enrichment or disappearance of some microbial species. However, the application of RAPD markers may also be affected since any change less than 1% of the total community cannot be detected by the technology [44]. Consequently, it is necessary to integrate diverse approaches and perspectives to understand more precisely the changes in the diversity of microbial communities. BIOLOG analysis is based on the premise that microorganisms vary in the pattern and rate at which they utilize carbon sources. Therefore, carbon utilization patterns can be used as a measure of microbial community structure and functional potential [15]. In the present study, AWCD values decreased significantly with increasing cinnamic acid concentration. This may be due to lower microbial biomass in the treatments with high cinnamic acid concentrations. Diversity indices suggested that the composition of the soil microbial community changed significantly when the cinnamic acid concentration exceeded 50 mg kg1 soil. Normally, different diversity indices reflect different aspects of soil microbial community functional diversity. The Shannon index is affected by species richness, the Simpson index reflects the most common species in the community, and the McIntosh index is a measure of species evenness [45]. The Simpson index decreased significantly when the cinnamic acid concentration was higher than 25 mg kg1 soil. This
0.6 0.5 0 25 50 100 200
0.4 0.3 0.2 0.1 0 -0.1 -0.2 -0.3 -1
24
Time(h)
CV2
the following crop and reduce the yield once they accumulate to a certain level [53]. Exudates of cucumber roots contain phenolic allelochemicals that have an inhibitory effect on cucumber growth under continuous cropping. This is a type of autotoxic effect [54]. Cut stumps, litter and root extracts contain ferulic acid and cinnamic acid, which can accumulate and affect the following crop [23,24]. Exogenous ferulic and cinnamic acids also can inhibit the germination rate of fir seeds, their vigor and growth of seedlings [8]. Allelochemicals such as cinnamic acid can inhibit the uptake of ions 2 þ 2þ and Fe2þ) by cucumber seedlings [52]. Some (NO 3 , SO4 , K , Ca studies have found that cucumber root exudates facilitated the mycelial growth of Fusarium oxysporum f. sp. cucumerinum Owen [42]. The allelochemicals promoted the growth of soil fungi and bacteria and inhibited the growth of soil actinomycetes and F. oxysporum f. sp. cucumerinum [55,43]. Some studies reported that certain amounts of phenolic substances limit the mycelial growth of the soil pathogen burdock Rhizoctonia rot [30]. The processes of allelopathy, autotoxicity as well as allelopathic and autotoxic effects of allelochemicals are well known, but the mechanisms by which allelochemicals exert their effects are not clear. Furthermore, there is close relationship between allelochemicals and the size and activity of the soil microbial community [5]. The intensity of autotoxicity depends on the content, catabolism and the interaction of allelochemicals in soil. Rice et al. [36] compared the rate of decomposition of ferulic acid, vanillic acid, syringic acid and p-hydroxybenzoic acid and found that decomposition of p-hydroxybenzoic acid was the most rapid, whereas decomposition of syringic acid was the slowest. Zhang et al. [57] found that following addition of exogenous allelochemicals for one week, the soil residue rate was only 17.5–25%, and different allelochemicals were degraded at different rates. The study of Yu and Matsui [52] confirmed that exudates of cucumber roots contain ten different allelochemicals, including p-hydroxybenzoic acid, ferulic acid and cinnamic acid, and these occur naturally in soil. RAPD molecular marker technology was first used to identify plant gene polymorphism [28,33]. It can be used as a rapid and sensitive method to detect very small differences in the same or similar genes. Yao et al. [48] found that pesticide contamination can cause changes in the genetic diversity. In this paper, the effect of cinnamic acid on genetic diversity of the soil microbial community
-0.5
0
0.5
1
1.5
2
2.5
CV1 Fig. 3. Plot of ordination of canonical variates (CV) CV1 against CV2 generated by canonical variate analysis of sole carbon source tests at comparable time points in BIOLOG plates showing discrimination between different soils.
F. Wu et al. / European Journal of Soil Biology 45 (2009) 356–362
result suggests that cinnamic acid had a significant impact on the most common species of the soil microbial community. Cinnamic acid concentrations greater than 50 mg kg1 soil had major impacts on species evenness and richness of the soil microbial community. Multivariate analysis of carbon source utilization may also reflect the change in structure of the soil microbial community. In this study, cinnamic acid increased the variation of the microbial community and significantly changed microbial metabolic profiles. However, only some metabolically active and culturable bacteria can be detected, whereas soil fungi and slow-growing bacteria cannot be assessed. The technique is also sensitive to inoculum density and cannot reflect the potential metabolic diversity in situ. Nevertheless, the BIOLOG method is useful in studying the functional diversity of microbial community and is a valuable tool especially when used in conjunction with other methods. In this study, one of the important objectives was to determine the level of cinnamic acid that has an effect on the soil microbial community. When the cinnamic acid concentration was 25 mg kg1 soil, CV1 generated by canonical variate analysis of the sole carbon source tests decreased obviously. This result may suggest that the threshold of cinnamic acid that affected the functional diversity of microorganisms is less than 25 mg kg1 soil. Moreover, most diversity indices derived from the RAPD and BIOLOG data showed that the threshold of cinnamic acid was less than 100 mg kg1 soil. It seems that the soil microbial community is very sensitive to exogenous cinnamic acid. Consequently, allelochemicals may change soil microbial genetic diversity, biological activity and microbial metabolic activity, and accordingly affect the growth of cucumber. Acknowledgement This study was supported by National ‘‘973 Project’’ (2009CB119004) and National Natural Science Foundation of China (Nos.: 30771252, 30230250). References [1] V. Acosta-Martı´nez, T.M. Zobeck, T.E. Gill, A.C. Kennedy, Enzyme activities and microbial community structure in semiarid agricultural soils, Biol. Fertil. Soils 38 (2003) 216–227. [2] M. Ahmed, J.M. Oades, Distribution of organic matter and adenosine triphosphate after fractionation of soils by physical procedures, Soil Biol. Biochem. 16 (1984) 465–470. [3] T. Asao, N. Ohtani, N. Shimizu, M. Umeyama, K. Ohta, T. Hosoki, Possible selection of cucumber cultivars suitable for a closed hydrophonic system by the bioassay with cucumber seedlings, J. Soc. High Technol. Agric. 10 (1998a) 92–95 (in Japanese with English summary). [4] N.E. Balke, Effects of allelochemicals on mineral uptake and associated physiological process, ACS Symp. Ser. 268 (1985) 161–178. [5] U. Blum, S.R. Shafer, Microbial population and phenolic acids in soils, Soil Biol. Biochem. 20( (6) (1988) 793–800. [6] J. Bonner, A.W. Galson, Toxic substances from the culture media of guayule which may inhibit growth, Bot. Gaz. 106 (1944) 185–198. [7] C.D. Campbell, S.J. Grayston, D.J. Hirst, Use of rhizosphere carbon sources in sole carbon source tests to determinate soil microbial communities, J. Microbial. Methods 30 (1997) 33–41. [8] G.Q. Cao, S.Z. Lin, S.G. Huang, Effect of the ferulic acid and cinnamic acid on the germination of Chinese-fir seeds, J. Plant Resour. Environ. 10 (2) (2001) 63–64 (in Chinese). [9] Cecile Bertin, X.H. Yang, A. Leslie, Weston, the role of root exudates and allelochemicals in the rhizosphere, Plant Soil 256 (2003) 67–83. [10] W. Cheng, Q. Zhang, D.C. Coleman, C.R. Caroll, C.A. Hoffman, Is available carbon limiting microbial respiration in the rhizosphere? Soil Biol. Biochem. 2 (1996) 1283–1288. [11] E.F. Davis, The toxic principle of Juglans nigra as identified with synthetic juglone and its toxic effect on tomato and alfalfa plants, Am. J. Bot. 15 (1928) 620–629. [12] G.J. Du, T.P. Huang, Q.R. Zhang, P.S. Zhang, R.L. Cheng, Studies on soil microorganisms and biochemical properties in mixed forests of Chinese fir, J. Zhejiang For. Coll. 12 (4) (1995) 347–352 (in Chinese). [13] F.A. Einhellig, Mechanisms and modes of action of allelochemicals, in: A.R. Putnam, C.S. Tang (Eds.), The Sciences of Allelopathy, Wiley Interscience, New York, 1986, pp. 171–188.
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