Analysis of bacterial communities associated with potting media
© Al-Sadi et al. 2016
Received: 12 December 2015
Accepted: 15 January 2016
Published: 26 January 2016
Potting media are commonly used by growers in different parts of the world for potted plants, raising seedlings and for improving soil characteristics. This study was conducted to characterize bacterial communities occurring in 13 commercial potting media products originating from seven countries.
Bacteria were isolated using serial dilution. Identification to the species level was based on phylogenetic analysis of the 16S rRNA gene. The analysis showed the association of 13 bacterial species with the different potting media samples, namely Arthrobacter livingstonensis, Kocuria flava, Leifsonia lichenia, Bacillus vallismortis, Bacillus pumilus, Staphylococcus warneri, Burkholderia phenazinium, Burkholderia sp., Ralstonia pickettii, Rhodanobacter spathiphylli, Rhodanobacter sp., Pseudomonas thivervalensis and Chryseobacterium gallinarum. Bacterial densities in the samples ranged from 8 × 107 to 1.2 × 109 colony forming units per gram of substrate.
The study shows the isolation of some potential plant and human bacterial pathogens. However, most of the isolated species were either biocontrol species or saprophytes. The study questions the ways by which these bacterial species were introduced into potting media. To the best of our knowledge, this appears to be the first report of most of the isolated bacteria from potting media, except B. pumilus.
Soil in arid areas of the world are known be poor in fertility and structure. This motivates growers to use various biological and chemical amendments to improve soil in their fields. Potting medium is a growing medium suitable for the establishment and development of a wide range of plants in containers. Several types of potting media are imported from European countries (Al-Sadi et al. 2011). They are mainly used for growing potted plants such as citrus, mango and ornamental plants as well as for the germination of several vegetable crops before transplanting into fields.
Studies provided evidence that potting media may harbor some plant pathogenic fungi (Al-Sadi et al. 2011; Al-Sa’di et al. 2008). In addition, potting media can also be an important source of several beneficial fungal species that can be used as bicontrol agents or as decomposers of plant residue material (Al-Sadi et al. 2015; Al-Mazroui and Al-Sadi 2015). However, limited studies addressed the occurrence of bacterial communities in potting media (Whiley et al. 2011; Lindsay et al. 2012).
Using molecular technologies, the studies of microbial ecology have been made easy (Querido et al. 2013). Studies have shown that sequence analysis of the 16S rRNA gene is an important molecular tool for the identification of bacterial species (Jagielski et al. 2014). This study was carried out to characterize the bacterial communities in potting media originating from seven countries. Knowledge into this area may help understand the potential occurrence of plant pathogenic bacteria and other bacterial types in these growing media.
Results and discussion
Detection of bacteria isolates in 13 commercial potting media products originating from seven countries
GenBank acc. no.c
White peat (particle size: 0–7 mm), with 95–99 % organic matter
30 % white peat (particle size: 0–7 mm) and 70 % frozen black peat; fine structure
SQU P004, 5
High bog peat in fine structure (particle size: 0–7 mm)
20 % green compost, 40 % white peat (particle size: 0–25 mm) and 40 % frozen black peat; medium structure
White and dark sphagnum peat, fine structure
SQU P010, 11
Black peat and white peat (particle size: 0–6 mm); at least 92 % organic matter
Black and white milled peat (particle size 6–20 mm); at least 95 % organic matter
Peat moss (particle size: 1–10 mm); at least 95 % organic matter
Sphagnumfuscum peat (particle size: 0–10 mm)
Sphagnum moss peat (particle size: 0–14 mm)
Sphagnum moss peat (particle size: 0–14 mm)
White and dark sphagnum peat, fine structure
Container mix, 10 % vermiculate and 15 % perlite coarse
Phylogenetic analysis revealed that the isolates were grouped in the classes Actinobacteria, Bacilli, Gammaproteobacteria, Betaproteobacteria and Flavobacteriia. The isolates belonging to Betaproteobacteria (40 %) were the most abundant, followed by Bacilli (25 %), Actinobacteria and Gammaproteobacteria (15 %) and Flavobacteriia (5 %). The isolates were identified as Arthrobacter livingstonensis, Kocuria flava, Leifsonia lichenia, Bacillus vallismortis, Bacillus pumilus, Staphylococcus warneri, Burkholderia phenazinium, Burkholderia sp., Ralstonia pickettii, Rhodanobacter spathiphylli, Rhodanobacter sp., Pseudomonas thivervalensis and Chryseobacterium gallinarum (Table 1; Fig. 1).
Bacillus species colonies were generally opaque while R. spathiphylli, S. warneri, C. gallinarum, L. lichenia and K. flava were yellow. B. phenazinium was slightly beige, A. livingstonensis was off-white and R. pickettii and P. thivervalensis were white.
In previous studies, some bacterial species such as Bacillus species (Huang et al. 2012), and Legionella longbeachae (Koide et al. 2001), have been isolated and identified from various potting media or their ingredients such as compost, vermicompost and peat. Three bacterial species found in this study, including B. pumilus (Reddy 2014), B. vallismortis (Zhao et al. 2010) and R. spathiphylli (De Clercq et al. 2006) have been used as biological control agents. Moreover, P. thivervalensis (Achouak et al. 2000) is pathogenic to plants, and S. warneri (Kloos 1980) and R. pickettii (Stelzmueller et al. 2006) have been reported to cause diseases in humans. The other species, including L. lichenia, C. gallinarum, A. livingstonensis, B. phenazinium and K. flava have been previously reported from lichen (An et al. 2009), chicken (Kämpfer et al. 2014), moss-covered soil (Ganzert et al. 2011), soil (Bell and Turner 1973) and air (Zhou et al. 2008), respectively.
Differences in bacterial species composition and densities among different products could be related to differences in raw material from which the substrates were produced as well as differences in processing between different companies. For example, substrate #2, from which four bacterial species were recovered, was produced from different material (white and frozen black peat) (Table 1). The origin of bacteria in potting media products could be from the original plant waste products from which the potting media were produced (Messiha et al. 2009) or they could have been introduced during the processing or packaging of potting media (Al-Sadi et al. 2011). However, future studies might be required to investigate the source of contamination of these commercial products of potting media.
Our study reveals the association of different types of bacterial species with potting media, with some being potential pathogens of plants and humans, while others are potential biocontrol species or saprophytic species. To our knowledge, this is the first report of culturable A. livingstonensis, K. flava, L. lichenia, B. vallismortis, B. pumilus, S. warneri, B. phenazinium, Burkholderia sp., R. pickettii, R. spathiphylli, Rhodanobacter sp., P. thivervalensis and C. gallinarum from potting media.
Collection of samples and isolation of bacteria
In this study, 13 samples of potting media from different European countries were obtained from Sultan Qaboos Sea Port (Table 1). Each sample represents a different company. Bacteria were isolated using serial dilution technique on nutrient agar media (NA, OXOID, England, UK). In this technique, a sample suspension was prepared by adding 1 g potting medium sample to 10 ml sterile distilled water and mixed well for 15 min and vortexed. Each suspension was serially diluted 10−1–10−4. Then 0.2 ml was pipetted from the 4th dilution onto NA media, spread with a sterile glass spreader and incubated at 28 °C for bacterial observation. Bacterial colonies which appeared different in morphology or color from each other were transferred to new NA media. Eventually, the obtained isolates were maintained on NA slant agar at 4 °C as stock culture.
DNA extraction and PCR
For DNA extraction, single bacterial colonies were transferred to nutrient broth in 1.5 ml broth and incubated in a shaker (120 rpm) at 28 °C for 48 h. Total genomic DNA was extracted from all bacterial isolates using GenElute Bacterial Genomic DNA Kit (Sigma Aldrich, Germany) according to the manufacturer’s protocol. For PCR amplification, the 16S rRNA gene fragment was amplified using the universal primers 518F (5′-CCAGCAGCCGCGGTAATACG-3′) and 800R (5′-TACCAGGGTATCTAATCC-3′) using PuRe Taq Ready-To-Go PCR beads (GE Healthcare UK Limited, UK). Thermocycling was carried out with the following conditions: heating at 95 °C (2 min); then 40 cycles of 95 °C (40 s), 60 °C (1 min) and 72 °C (1 min); and a final extension step at 72 °C (10 min). The PCR products were sequenced using a commercial sequencing service provider (Macrogen Inc., Seoul, Korea).
Sequences of each isolate were refined using BioEdit sequence Alignment Editor (Hall 1999), in which the sequences obtained from reverse primers were transformed to the reverse complement orientation and aligned with the sequences obtained from forward primers to obtain consensus sequences. To analyze the relationships of the isolates to known bacterial species, the 20 sequences from this study, 53 sequences of type strains which had the closest relationship to the isolates and Aquifex pyrophilus (type strain Kol5a) as an outgroup were initially aligned using the Clustal W Multiple alignment (Thompson et al. 1994), checked visually and improved manually where necessary. Phylogenetic analysis of the 16S rRNA gene using the parsimony optimality criterion was performed in PAUP* 4.0b10 (Swofford 1998). Gaps were treated as missing data. Maximum parsimony (MP) analysis was conducted by heuristic searches consisting of 1000 stepwise random addition replicates and branch swapping by the tree-bisection-reconnection algorithm. For each MP analysis, 1000 bootstrap replicates using a heuristic search with simple sequence addition was performed to assess statistical support for branch stability.
AMA designed the experiments. HAA, SSA and IHA conducted the experiments. AMA, AN and HAA analyzed data and wrote the manuscript. All authors read and approved the final manuscript.
Authors would like to acknowledge Quarantine Officers (MAFW, Oman) for their help in supplying samples of potting media products. Thanks also to Sultan Qaboos University for financial support of the study through SR/AGR/CROP/13/01 and to VALE Oman for their support through EG/AGR/CROP/12/02.
The authors declare that they have no competing interests.
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- Achouak W, Sutra L, Heulin T, Meyer JM, Fromin N, Degraeve S, Christen R, Gardan L (2000) Pseudomonas brassicacearum sp. nov. and Pseudomonas thivervalensis sp. nov., two root-associated bacteria isolated from Brassica napus and Arabidopsis thaliana. Int J Syst Evol Microbiol 50(1):9–18View ArticleGoogle Scholar
- Al-Mazroui SS, Al-Sadi AM (2015) Highly variable fungal diversity and the occurrence of potentially plant pathogenic fungi in potting media, organic fertilizers and composts originating from 14 countries. J Plant Pathol 97:529–534Google Scholar
- Al-Sa’di AM, Drenth A, Deadman ML, Al-Said FA, Khan I, Aitken EAB (2008) Potential sources of Pythium inoculum into greenhouse soils with no previous history of cultivation. J Phytopathol 156:502–505View ArticleGoogle Scholar
- Al-Sadi AM, Al-Said FA, Al-Jabri AH, Al-Mahmooli IH, Al-Hinai AH, de Cock AWAM (2011) Occurrence and characterization of fungi and oomycetes transmitted via potting mixtures and organic manures. Crop Prot 30:38–44View ArticleGoogle Scholar
- Al-Sadi AM, Al-Mazroui SS, Phillips A (2015) Evaluation of culture-based techniques and 454 pyrosequencing for the analysis of fungal diversity in potting media and organic fertilizers. J Appl Microbiol 119:500–509View ArticleGoogle Scholar
- An SY, Xiao T, Yokota A (2009) Leifsonia lichenia sp. nov., isolated from lichen in Japan. J Gen Appl Microbiol 55(5):339–343. doi:10.2323/jgam.55.339 View ArticleGoogle Scholar
- Bell SC, Turner JM (1973) Iodinin biosynthesis by a pseudomonad. Biochem Soc Trans 1(3):751–753View ArticleGoogle Scholar
- De Clercq D, Van TrappenS, Cleenwerck I, Ceustermans A, Swings J, Coosemans J, Ryckeboer J (2006) Rhodanobacter spathiphylli sp. nov., a gammaproteobacterium isolated from the roots of Spathiphyllum plants grown in a compost-amended potting mix. Int J Syst Evol Microbiol 56(8):1755–1759. doi:10.1099/ijs.0.64131-0 View ArticleGoogle Scholar
- Ganzert L, Bajerski F, Mangelsdorf K, Lipski A, Wagner D (2011) Arthrobacter livingstonensis sp. nov. and Arthrobacter cryotolerans sp. nov., salt-tolerant and psychrotolerant species from antarctic soil. Int J Syst Evol Microbiol 61(4):979–984. doi:10.1099/ijs.0.021022-0 View ArticleGoogle Scholar
- Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
- Huang TP, Tzeng DDS, Wong ACL, Chen CH, Lu KM, Lee YH, Huang WD, Hwang BF, Tzeng KC (2012) DNA polymorphisms and biocontrol of Bacillus antagonistic to citrus bacterial canker with indication of the interference of phyllosphere biofilms. PLoS One. doi:10.1371/journal.pone.0042124 Google Scholar
- Jagielski T, Van Ingen J, Rastogi N, Dziadek J, Mazur PK, Bielecki J (2014) Current methods in the molecular typing of Mycobacterium tuberculosis and other mycobacteria. BioMed Res Int 2014:645802. doi:10.1155/2014/645802 Google Scholar
- Kämpfer P, Poppel MT, Wilharm G, Busse HJ, McInroy JA, Glaeser SP (2014) Chryseobacterium gallinarum sp. nov., isolated from a chicken, and Chryseobacterium contaminans sp. nov., isolated as a contaminant from a rhizosphere sample. Int J Syst Evol Microbiol 64(part 4):1419–1427. doi:10.1099/ijs.0.058933-0 View ArticleGoogle Scholar
- Kloos WE (1980) Natural populations of the genus Staphylococcus. Annu Rev Microbiol 34:559–592View ArticleGoogle Scholar
- Koide M, Arakaki N, Saito A (2001) Distribution of Legionella longbeachae and other legionellae in Japanese potting soils. J Infect Chemother 7(4):224–227. doi:10.1007/s101560170017 View ArticleGoogle Scholar
- Lindsay DSJ, Brown AW, Brown DJ, Pravinkumar SJ, Anderson E, Edwards GFS (2012) Legionella longbeachae serogroup 1 infections linked to potting compost. J Med Microbiol 61(2):218–222. doi:10.1099/jmm.0.035857-0 View ArticleGoogle Scholar
- Messiha NAS, van Bruggen AHC, Franz E, Janse JD, Schoeman-Weerdesteijn ME, Termorshuizen AJ, van Diepeningen AD (2009) Effects of soil type, management type and soil amendments on the survival of the potato brown rot bacterium Ralstonia solanacearum. Appl Soil Ecol 43(2–3):206–215. doi:10.1016/j.apsoil.2009.07.008 View ArticleGoogle Scholar
- Querido JFB, Agirre J, Marti GA, Guérin DMA, Silva MS (2013) Molecular techniques for dicistrovirus detection without RNA extraction or purification. BioMed Res Int 2013:218593. doi:10.1155/2013/218593 View ArticleGoogle Scholar
- Reddy PP (2014) Plant growth promoting rhizobacteria for horticultural crop protection. doi:10.1007/978-81-322-1973-6_1
- Stelzmueller I, Biebl M, Wiesmayr S, Eller M, Hoeller E, Fille M, Weiss G, Lass-Floerl C, Bonatti H (2006) Ralstonia pickettii—innocent by bystander or a potential threat? Clin Microbiol Infect 12(2):99–101. doi:10.1111/j.1469-0691.2005.01309.x View ArticleGoogle Scholar
- Swofford DL (1998) PAUP* phylogenetic analysis using parsimony (*and other methods), version 4. Sinauer Associates, SunderlandGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680View ArticleGoogle Scholar
- Whiley H, Taylor M, Bentham R (2011) Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH). J Microbiol Methods 86:304–309View ArticleGoogle Scholar
- Zhao Z, Wang Q, Wang K, Brian K, Liu C, Gu Y (2010) Study of the antifungal activity of Bacillus vallismortis ZZ185 in vitro and identification of its antifungal components. Bioresour Technol 101(1):292–297. doi:10.1016/j.biortech.2009.07.071 View ArticleGoogle Scholar
- Zhou G, Luo X, Tang Y, Zhang L, Yang Q, Qiu Y, Fang CX (2008) Kocuria flava sp. nov. and Kocuria turfanensis sp. nov., airborne actinobacteria isolated from Xinjiang, China. Int J Syst Evol Microbiol 58(6):1304–1307. doi:10.1099/ijs.0.65323-0 View ArticleGoogle Scholar