Effect of different fertilization treatments on indole-3-acetic acid producing bacteria in soil
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Soil microorganisms directly affect the growth of plants. Especially, plant growth-promoting rhizobacteria (PGPR) play an important role in plant growth. There are many studies about the effects of different fertilization treatments on soil microbial community structure; however, the effects on PGPR, including indole-3-acetic acid (IAA)-producing bacteria have not been previously reported. The objective of this study is to determine the effects of different types of fertilizers on IAA-producing bacteria.
Materials and methods
The field trial was completed in the North China with a winter wheat and summer corn rotation system. IAA-producing bacteria were screened from soil treated with different fertilizer (non-nitrogen fertilizer (CK), controlled-release fertilizer (CR), chemical fertilizer (CF), and organic fertilizer (OF)) which was established in September 2005. Quantity of IAA produced by bacteria was determined by spectrophotometer. IAA-producing bacteria were identified based on 16S rDNA sequence. Community structures and phylogenetic relationships of IAA-producing bacteria were analyzed by online Basic Local Alignment Search Tool search engine, biosoftware of DNAMAN and Molecular Evolutionary Genetics Analysis.
Results and discussion
Compared with CK treatment, CF and CR treatment increased soil pH values, while OF treatment decreased pH. The three types of fertilizers all increased soil organic carbon and total nitrogen, with OF treatment causing the significant increase. Soils treated with OF or CR fertilizer could significantly increase the number of culturable bacteria compared with CF or CK treatment. Fifty-three IAA-producing bacteria (14 strains from CK, nine from CF, eight from CR, and 22 from OF) were identified based on 16S rDNA sequence. The Shannon–Weiner index of IAA-producing bacteria isolated from CK and OF (2.06 and 2.45, respectively) was significantly higher than those from CF and CR (0.50 and 0.95, respectively). Arthrobacter sp. was the most prevalent group of IAA-producing bacteria.
The fertilizers increased soil organic carbon and total nitrogen, particularly the organic fertilizers. Controlled-release fertilizers and organic fertilizers can promote growth of soil-culturable bacteria and IAA-producing bacteria. These may be reasons why organic fertilizers and controlled-release fertilizers can promote crop growth. Different fertilization treatments affected IAA yield mainly through modifying the quantities of microorganisms, rather than changing the IAA-producing ability of the same microorganisms. Pedobacter sp. which can produce IAA has not been described previously.
KeywordsBiodiversity Fertilization treatments IAA-producing bacteria PGPR
There is continuous interaction between plants and soil microorganisms. Plant growth promoting rizobacteria (PGPR) are naturally occurring, rhizosphere-inhabiting bacteria, and have been isolated from a wide variety of wild and cultivated plant species including thale cress (Arabidopsis thaliana), barley (Hordeum vulgare), rice (Oryza), canola (Brassica campestris), and bean (Fabaceae; Persello-Cartieaux et al. 2003). PGPR can increase plant growth and productivity through direct and indirect mechanisms. The direct mechanisms include contributions to root system architecture modulation and increased shoot growth by production of phytohormones such as auxins and cytokinins, and the indirect mechanisms include the effects of products such as antibiotics and hydrogen cyanide inhibiting the growth of deleterious microorganisms in the rhizosphere (Ortiz-Castro et al. 2009).
Diverse bacterial species are capable of producing the auxin phytohormone, indole-3-acetic acid (IAA), such as Pseudomonas sp. (Patten and Glick 2002b), Arthrobacter sp. (Forni et al. 1992), Acinetobacter sp. (Huddedar et al. 2002), Ensifer/Sinorhizobium sp. (Bianco and Defez 2009; Appunu et al. 2009), Alcaligenes sp. (Li et al. 2000), Enterobacter sp. (Slininger et al. 2004), Bacillus sp. (Idris et al. 2007), Agrobacterium sp. (Liu and Kado 1979), Kocuria sp. (Egamberdieva 2008), Flavobacterium sp. (Loper and Schroth 1986), among others. IAA which is recognized as the key auxin in most plants is a multivalent signaling molecule which transmits its effects within plants and among plants and bacterial cells (Woodward and Bartel, 2005). Many experiments have demonstrated that IAA is a regulator of the modulation of root and shoot architecture, and it has an effect on numbers of lateral roots and root hairs and growth of shoots and leaves (Ali et al. 2010; Ortiz-Castro et al. 2009). Bacteria use IAA to interact with plants as part of their colonization strategy, including phytostimulation and circumvention of basal plant defense mechanisms (Spaepen et al. 2007).
Fertilization treatments have an impact on soil microbial communities. Soil bacterial and fungal communities were great affected by chemical and organic fertilization treatments, and organic fertilizer applications induced the least culturable bacterial colony-forming units but the highest bacterial diversity, while chemical fertilizer applications had less impact on soil bacterial community (He et al. 2008). Some reports indicated that fertilization impacted special bacteria communities, for example manure could enhance number of denitrifying and aerobic N-fixing bacteria (Ahamadou et al. 2009), while it did not result in changes in the soil sulfate reducing prokaryotes community structure (Liu et al. 2009). However, the effects of different fertilization treatments on PGPR bacteria have not been previously reported. In this paper, we analyzed the abundance, community structure of IAA-producing PGPR bacteria treated with different nitrogen fertilizers. The objective of this study was to determine the effects of different types of fertilizers on PGPR bacteria.
2 Materials and methods
2.1 Study site and sampling
The field trial was completed in the North China Intensive Agro-ecosystem Experimental Station (35°00′N, 114°24′E), Huantai County, Shandong Province. The area had a mean annual temperature of 12.5°C and an annual precipitation of 586.4 mm. The soil texture is sandy loam (70.8% sand, 26.9% silt, and 2.3% clay). The soil type is a calcaric cambisol according to the FAO/UNESCO soil map of the world (FAO/UNESCO 1988). The fertilization experiment was established in September 2005 with a winter wheat and summer corn rotation system, including four treatments with four replicates (plot area 9.5 × 5 m2) for each treatment in a randomized plot design. The four treatments were control (non-nitrogen fertilizer, CK) with 0 kg N ha−1 year−1; controlled-release fertilizer (CR) with 400 kg N ha−1 year−1; chemical fertilizer (CF) with 400 kg N ha−1 year−1; organic fertilizer (OF) with 400 kg N ha−1 year−1. Amounts of P and K were the same in all fertilizers with 120 kg P2O5 ha−1 year−1 and 90 kg K2O ha−1 year−1 being applied. Soil samples were collected from 0 to 20 cm surface soil in April 2009 by taking four soil cores from each plot.
2.2 Soil chemical analysis
Soil samples were passed through a 2.0-mm sieve and were analyzed for total nitrogen (TN), total organic carbon (TOC), and pH. TN was determined by high-temperature combustion with chemiluminescence detection with an Antec TN analyzer, and TOC was quantified using high temperature combustion with a Shimadzu TOC-5000.
2.3 Isolation and screening of IAA-producing bacteria
Soil bacteria were isolated and quantified according to Mew et al. (1976). Each IAA-producing colony was identified using the method of Patten and Glick (2002a) with the following modifications: each strains was grown in a shaker at 150 rpm at 28°C for 7 days in DF+ medium (peptone 5.0 g, yeast extract 1.5 g, beef extract 1.5 g, NaCl 5.0 g, tryptophan 0.5 g, per liter) and DF− medium (DF+ medium without tryptophan). Five-milliliter culture was removed from each flask and centrifuged at 12,000 rpm for 10 min and 1 ml supernatant was mixed with 50 μl of 10 mM orthophosphoric acid and 2 ml of reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35% HClO4). Samples were incubated at room temperature for 25 min. Absorbance at 530 nm was determined using ultraviolet spectrophotometer. The IAA concentration in each culture was determined with a calibration curve of pure IAA as a standard following linear regression analysis.
2.4 Identification of bacteria strains
Strains of IAA-producing bacteria were identified based on the sequence of 16S rDNA. The 16S rDNA was amplified from the bacteria using primers 530 F: 5′-GTGCCAGCMGCCGCGG-3′, and 1492R: 5′-GGYTACCTTGTTACGACTT-3′ (Cho et al. 2003). Polymerase chain reaction (PCR) amplification was carried out in a final volume of 50 μl. Briefly, the amplification reaction contained 10 μmol of each primer, 0.2 mM dNTP, 1.5 mM MgCl2, 2.5 U Taq polymerase (TaKaRa, Japan) in 1× PCR buffer (TaKaRa, Japan) and a 24-h incubation colony as template. PCR was performed as follows: 94°C for 10 min, followed by 34 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, and 72°C for 10 min. Amplified products were analyzed by 1% (w/v) agarose gel in 1× TAE buffer, and then the products were purified with Gel Extraction Kit (OMEGA, USA). Clean 16S rDNA fragments were sequenced with the ABI 3730 DNA sequencer at Invitrogen Corporation in Beijing. DNA sequence similarity searches were performed with the online Basic Local Alignment Search Tool (BLAST) search engine in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST).
2.5 Data analysis
Sequences were aligned with a BLAST search program and similarity analysis was performed with DNAMAN DNA analysis software package (DNAMAN version 5.22; Lynnon Biosoft, Montreal, Canada). Phylogenetic analyses were conducted using Molecular Evolutionary Genetics Analysis version 4 (Tamura et al. 2007).
Where, S is the number of species, Pi is the proportion of individuals belonging to species i in the total sample, and H represents the species diversity index. In this study, we classified strains in the same genus as an identical class, so “species” was substituted by “genus”.
Statistic analyses of Student’s t test, analysis of variance (ANOVA), and multiple comparisons were performed by SPSS version 13.0 (SPSS Inc., USA).
2.6 Nucleotide sequences accession number
The 16s rDNA sequences of IAA-producing bacteria have been deposited in GenBank under accession No. HM626408 to HM626460.
3.1 Soil chemical properties
Chemical properties of the soil under different fertilization treatments
Organic carbon (g kg−1)
Total nitrogen (g kg−1)
7.43 ± 0.01ba
14.2 ± 3.3 b
1.27 ± 0.25 b
7.89 ± 0.01 a
17.6 ± 0.7 ab
1.56 ± 0.08 ab
7.95 ± 0.02 a
19.4 ± 3.7 ab
1.76 ± 0.33 ab
7.40 ± 0.03 b
23.7 ± 4.3 a
2.13 ± 0.34 a
3.2 Isolation and screening of IAA-producing bacteria
IAA-producing bacteria from soil samples of different fertilization treatments
IAA producing bacteriaa
Proportion of IAA producing bacteria
Shannon-Wiener index of IAA producing bacteria
3.3 Identification of IAA-producing bacteria
IAA production of different genera
Mean of DF+ (μg ml−1)
Mean of DF− (μg ml−1)
The proportions of Arthrobacter sp. in IAA-producing bacteria, which were 50%, 89%, 63%, and 41% in CK, CF, CR, and OF, respectively, differed between fertilization treatments (see Fig. 2). Despite the dominance of Arthrobacter sp. in all four treatments, its proportion in OF was clearly lower than in others. Other genera were not evenly distributed either, Acinetobacter sp. and some other genera were only found in CK, while Kocuria sp. was observed only in CR, Ensifer/Sinorhizobium sp. and some other genera were only found in OF. This reflected the different community structures of IAA-producing bacteria under different fertilization treatments.
3.4 Biodiversity of IAA-producing bacteria in the soil
The Shannon–Weiner index of IAA-producing bacteria isolated from CK and OF (2.06 and 2.45, respectively) was significantly higher than those from CF and CR (0.50 and 0.95, respectively; see Table 2). This result indicated that not applying nitrogen fertilizer or applying organic fertilizer favored the biodiversity of IAA-producing bacteria in soil, and that controlled-release fertilizers were better than chemical fertilizers as well. Additionally, as mentioned previously, both quantities of culturable bacteria and IAA-producing bacteria were higher in OF and CR, which indicates that organic fertilizer and controlled-release fertilizers may promote IAA-producing bacteria growth in soil.
3.5 Detection of IAA production from soil bacteria
IAA production differed among the fertilization treatments. IAA yields ranged from 0.7 to 290.0 μg/ml when bacteria were cultured in DF+ medium, and the average was 65.7 μg/ml. The number of strains that yielded 0–100, 100–200, and 200–300 μg/ml IAA was 37, 14, and 2, respectively. A t test showed that the average IAA production of IAA-producing bacteria from soil samples CK, CF, and CR were significantly higher than those from OF, and the average yields of the four treatments were: 72.2, 110.0, 116.0, and 25.3 μg/ml for CK, CF, CR, and OF, respectively. When cultured in DF− medium, IAA productions ranged from 0.5 to 39.2 μg/ml, with an average of 7.5 μg/ml. The number of strains which yielded 0–10, 10–20, 20–30, and over 30 μg/ml IAA was 38, 11, 2, and 2, respectively. The average yields of CK, CF, CR, and OF were 9.0, 9.4, 14.8, and 3.1 μg/ml, respectively. When cultured in DF− medium, the average IAA production of IAA-producing bacteria from soil samples CK, CF, and CR were also significantly higher than those from OF.
As shown in Table 3 whether cultured in DF+ or DF− medium, the highest average yields of IAA were obtained from Arthrobacter sp., Enterobacter sp., and Kocuria sp. When cultured with DF+ medium, the five highest yielding strains (10% of the total) were distributed in all soil samples, and were all Arthrobacter sp. In DF− medium, the five highest yielding strains were distributed in all soil samples except OF, of which only two were Arthrobacter sp., plus two Kocuria sp. and one Enterobacter sp. Table 3 also shows that the ratio of average IAA production in DF+ to DF− medium of Arthrobacter sp. (10.9) was larger than those of Kocuria sp. (5.3) and Enterobacter sp. (3.5). When cultured in medium without tryptophan (Trp), either the absolute or relative yields of IAA significantly decreased which indicated that Arthrobacter sp. was more sensitive to Trp. Kocuria sp. and Enterobacter sp. strains can produce more IAA compared with other strains in medium without Trp.
There were significant differences in IAA production when strains were cultured in media with and without Trp. When Trp was present, IAA production was averagely about ten times (average of ratios) higher than the absence of Trp. There was a significant regression relationship between DF+ IAA production and DF− IAA production (P = 4.12 × 10−8), indicating that strains having high yields in DF+ medium also had high yields in DF− medium.
The results described in this study demonstrated that different fertilization treatments affected soil bacteria, in particular the IAA-producing bacteria, which are important PGPR bacteria. Quantities of culturable bacteria in organic fertilizer and controlled-release fertilizer treatments were significantly increased, the corresponding IAA-producing bacteria were greater in number and their biodiversity was higher. This indicated that controlled-release fertilizer and organic fertilizer could both promote growth of bacteria and IAA-producing bacteria in soil, while no fertilizer and chemical fertilizer were less effective. All types of fertilizers used in this study increased soil organic carbon and soil total nitrogen, particularly the organic fertilizer. These may be reasons why organic fertilizers and controlled-release fertilizer can promote crop growth (data not shown). These results were in accordance with other studies showing that different fertilizers could affect soil microorganisms and changes in microbial activity and composition could, in turn, influence plant growth (Marschner et al. 2003). Many reports indicate that organic fertilizers, such as swine or cow manure, and even biowaste compost, can promote microbial activities (Šimek et al. 1999; Enwall et al. 2005; Palmroth et al. 2006). Cai et al. (2003) reported that ecological organic fertilizer improved the soil microbial community structure and promoted growth of beneficial microorganisms in soil, increased soil microbial diversity, and improved soil quality.
Nevertheless, the effects of different soil microorganisms on crops are diverse, and undoubtedly PGPR play a significant role. However, there are very few reports discussing the effects of fertilization on PGPR, including IAA-producing bacteria. Although controlled-release fertilizer has a significant advantage over ordinary chemical fertilizer (Xu et al. 2007), its impacts on soil microbes or PGPR have rarely been reported. We calculated quantities of culturable bacteria and studied communities of IAA-producing bacteria treated with organic fertilizer, controlled-release fertilizer, chemical fertilizer, and no nitrogen fertilizer, but a more detailed study on how fertilizers impact soil microbes is warranted.
Soil contains abundant in microbial resources and changing its community structure lead to alteration in community function. Fifty-three strains producing IAA were identified from soil treated with different fertilizers, although the proportion of IAA producer to culturable bacteria was approximately 8%, which was relatively lower than other reports. Lottmann et al. (1999) found that the percentage of IAA producer in the transgenic potato (Solanum tuberosum) plants rhizosphere ranged from 8.4 to 67.7. And in the study of Weisskopf et al. (2005), the frequency of bacteria producing auxin in total isolated bacteria associated with white lupin (Lupinus albus L. cv. Amiga) roots was from 4.5% to 33.8%. The variation of the ratio of IAA producing-bacteria to the total culturable bacteria can be influenced by the kind of plant and its growth stage (Vestergård et al. 2007), and other factors such as fertilizer application in this study.
Tryptophan has been identified as a main precursor for IAA biosynthesis pathway in bacteria such as indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway, tryptophan side-chain oxidase pathway and indole-3-acetonitrile pathway (Spaepen et al. 2007). However, some bacteria have the tryptophan-independent pathway to produce IAA, such as Azospirillum brasilense (Prinsen et al. 1993). Two types of media DF+ (with tryptophan) and DF− (without tryptophan) were used to screen IAA-producing bacteria and to analyze IAA producing ability of bacteria. The maximum IAA production after 7 days was 290.0 and 39.2 μg/ml when cultured in DF+ and DF− medium, respectively. When Trp was added to the medium, IAA production increased about ten times (average of ratios). The presence of Trp had a significant effect on IAA production, indicating that Trp may be the precursor for IAA synthesis in these organisms (Patten and Glick 1996). When cultured in DF+ medium, total IAA yields of strains in CK, CF, CR, and OF were 1,010.3, 990.2, 927.7, and 556.4 μg/ml, respectively; while when cultured in DF− medium, total IAA yields of strains in CK, CF, CR, and OF were 125.3, 84.2, 118.4, and 69.2 μg/ml, respectively. However, it should be noted that CR and OF samples were diluted 106 times, while CK and CF were diluted 105 times. Therefore, IAA production in CR and OF soil should be higher. Furthermore, when cultured with DF+ medium, average IAA production of Arthrobacter sp. in CF, CK, CR, and OF was 112.3, 107.4, 102.4, and 42.4 μg/ml, respectively; while when cultured in DF− medium, average IAA production of Arthrobacter sp. in CF, CK, CR, and OF was 9.5, 10.8, 8.1, and 4.5 μg/ml, respectively. Although values for OF were rather lower than for other treatments, ANOVA showed that, whether cultured in DF+ or DF− medium, the four treatments were not significantly different (P = 0.22 and 0.15, respectively). This may indicate that different fertilization treatments affected IAA yield mainly through modifying the quantities of microorganisms, rather than changing the IAA-producing ability of the same microorganisms.
Arthrobacter sp. was the main species identified in all fertilizer treatments. Arthrobacter sp. are ubiquitous and have been found in common soils and in extreme environments, for instance the deep subsurface, arctic ice, chemically contaminated sites, and radioactive environments (Mongodin et al. 2006). These bacteria have been shown to be able to biodegrade environmental pollutants (Ferreira et al. 2008) and promote plant growth (Patten and Glick 2002b). Some genera of IAA-producing bacteria occurred only in certain samples. For example, Ensifer/Sinorhizobium sp. was only found in OF. Ensifer/Sinorhizobium sp. is a genus of nitrogen-fixing bacteria (rhizobia), and it was probably the N demand during the mineralization of organic matter that stimulated the growth of this group (Chapin et al. 2002). Some strains that are capable of producing IAA, have not been described previously. For instance, Pedobacter sp. which was isolated from soil treated with organic fertilizer, produced 22.2 and 5.8 μg ml−1 IAA in medium with and without tryptophan, respectively.
This work was supported by the Specialized Research Fund for Doctor Program in University (20090008120043), China Agricultural University Basic Research Fund (2009JS108), Special Fund for Agro-scientific Research in the Public Interest (200803033), and China International Science and Technology Partnership Program (2009DFA91790).
- Cai Y, Liao Z, Je H, Kong W, He C (2003) Effect of ecological organic fertilizer on tomato bacterial wilt and soil microbial diversities (in Chinese). J Appl Ecol Sin 14(3):349–353Google Scholar
- Chapin FS, Matson PA, Mooney HA (2002) Principles of terrestrial ecosystem ecology. Springer, New York, pp 200Google Scholar
- Egamberdieva D (2008) Plant growth promoting properties of rhizobacteria isolated from wheat and pea grown in loamy sand soil. Turk J Biol 32:9–15Google Scholar
- FAO/UNESCO (1988) Soil map of the world. Revised legend. FAO, RomeGoogle Scholar
- Forni C, Riov J, Grilli Caiola M, Tel-Or E (1992) Indole-3-acetic acid (IAA) production by Arthrobacter species isolated from azolla. J Gen Microbiol 138(2):377–381Google Scholar
- Jones D, Keddie R (2006) The genus Arthrobacter. In: The prokaryotes. Springer: New York, pp 945–960Google Scholar
- Li F, Ping S, Su B, Lin M (2000) Tn5 mutagenesis and the characteristics of indole-3-acetic acid biosynthesis in Alcaligenes faecalis A1501 (in Chinese). Acta Microbiol Sin 40(5):551–555Google Scholar
- Loper JE, Schroth MN (1986) Influence of bacterial sources of indole-3-acetic acid on root elongation of sugar beet. Physiol Biochem 76:386–389Google Scholar
- Mongodin EF, Shapir N, Daugherty SC, DeBoy RT, Emerson JB, Shvartzbeyn A, Radune D, Vamathevan J, Riggs F, Grinberg V, Khouri H, Wackett LP, Nelson KE, Sadowsky MJ (2006) Secrets of soil survival revealed by the genome sequence of Arthrobacter aurescens TC1. PLoS Genet 2(12):e214CrossRefGoogle Scholar
- Sun R (2002) Basic ecology (in Chinese). Higher Education Press, Beijing, p 144Google Scholar
- Xu Y, Zhao Z, Zhang F, Liu J (2007) Advances in development and application of controlled-release fertilizers (in Chinese). Acta Agriculturae Boreali-Sinica 22(supplement):190–194Google Scholar