Plant Soil (2012) 357:215–226 DOI 10.1007/s11104-012-1155-1
REGULAR ARTICLE
Organic amendments and land management affect bacterial community composition, diversity and biomass in avocado crop soils Nuria Bonilla & Francisco M. Cazorla & Maira Martínez-Alonso & José M. Hermoso & Jorge J. González-Fernández & Núria Gaju & Blanca B. Landa & Antonio de Vicente
Received: 21 September 2011 / Accepted: 30 January 2012 / Published online: 23 February 2012 # Springer Science+Business Media B.V. 2012
Abstract Background and aims The avocado-producing area of southern Spain includes conventional orchards and organic orchards that use different organic amendments. To gain insight into the effects of these amendments, physicochemical properties and microbial communities of the soil were analysed in a representative set of commercial and experimental orchards. Methods The population size of several groups of culturable microorganisms was determined by plating on different selective media. Bacterial community structure was studied by denaturing gradient gel electrophoresis (DGGE) Results Commercial composts showed the largest effects, especially the animal compost, enhancing the
population sizes of some microbial groups and affecting bacterial community structure in superficial and deep soil layers. Moreover, animal and vegetal compost, manure and blood meal addition are related to high bacterial diversity in the superficial soil layer. Conclusions All of the organic amendments used in this study affect soil properties in one or more of the characteristics that were analysed. Culturable microbial population data revealed the most evident effects of some of the organic treatments. However, molecular analysis of soil bacterial communities by DGGE allowed the detection of the influence of all of the analysed amendments on bacterial community composition. This effect was stronger in the superficial layer of the avocado soil.
Responsible Editor: Harsh P. Bais. Electronic supplementary material The online version of this article (doi:10.1007/s11104-012-1155-1) contains supplementary material, which is available to authorized users. N. Bonilla : F. M. Cazorla (*) : A. de Vicente Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC). Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain e-mail:
[email protected]
J. M. Hermoso : J. J. González-Fernández IHSM-UMA-CSIC, Departamento de Fruticultura, EE “La Mayora”, 29750 Algarrobo costa, Spain
M. Martínez-Alonso : N. Gaju Departamento de Genética y Microbiología, Facultad de Biociencias, Universidad Autónoma de Barcelona, 08193 Bellaterra, Spain
B. B. Landa Departamento de Protección de cultivos, Instituto de Agricultura Sostenible (IAS-CSIC) Finca Alameda del Obispo, 4084-14080 Córdoba, Spain
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Keywords Organic crop . Community structure . Microbial diversity . Manure . Compost . Almond shells . DGGE
Introduction The avocado, Persea americana Miller, is a significant fruit crop in tropical and subtropical regions (Pérez-Jiménez 2008). Persea americana is species of tree that apparently originated in Central America. The main avocado-producing and exporting countries are located in the Americas. In Europe, avocado production is restricted to the south of Spain and Portugal, and Spain is one of the main exporter countries to the European Union, the most important international avocado market (Galán and Farré 2005). For several years, organic avocado production methods have spread throughout southern Spain due to the amenability of this crop to organic management. In these orchards, organic amendment or mulch additions are among the most popular actions performed by farmers. Soil organic matter is fundamental to the long-term sustainability of agroecosystems and plays a critical role in global biochemical cycles (Fonte et al. 2009). Moreover, as a rainforest native, the avocado is accustomed to growing in soil with high organic matter content (Galán and Farré 2005). Several studies have reported the effects of land management techniques and organic amendment addition on crop soil quality and the consequent impact on plant health and crop yield (Maeder et al. 2002; Bailey and Lazarovits 2003; Roy et al. 2010; Yan and Gong 2010). The quantity and quality of organic matter input affect both physicochemical properties of the soil and biotic factors related to the soil microbiota, such as microbial biomass, microbial diversity and community structure (Sun et al. 2004; Saison et al. 2006; CejaNavarro et al. 2010; Wallis et al. 2010). However, most reports on the effect of organic soil amendments are based on short-term experiments in experimental field plots and miss many of the slow changes that happen in the soil. Long-term experiments are vital to agricultural and environmental research (Powlson et al. 2011). In the past, microbial diversity was analysed by direct plate counts and, later, by physiological methods, such as community-level physiological profiling
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(CLPP). Culture-based methods are limited to the detection of only 0.1 to 10% of total microbial populations in soil (Wu et al. 2007; van Elsas et al. 2007). The development of new molecular techniques that are based on the characterisation of soil-extracted DNA offers great potential for expanding the microbial groups that were analysed to include the vast portion of non-culturable microorganisms (van Elsas et al. 2007). Most of these techniques rely on PCR amplification of the conserved and variable regions of the microbial genome, commonly 16S ribosomal DNA (rDNA) for bacteria and internal transcribed sequences (ITS) for fungi. Fingerprint methods, such as denaturing gradient gel electrophoresis (DGGE), separate the PCR product fragments, generating a different fingerprint for each microbial community. The fingerprint methods are very useful for comparing a large number of samples or treatments and, among these techniques, DGGE is one of the most well-established molecular tools in microbial ecology (Marzorati et al. 2008; Dini-Andreote et al. 2010). At present, massive sequencing methods are being incorporating into soil microbiology, allowing for the analysis of the majority of components of microbial soil communities (Will et al. 2010). However, they are not yet suitable and affordable for initial studies in which a large number of samples must be analysed (Kirk et al. 2004). Because no single method can fully detail a microbial soil community, a polyphasic approach, which combines different methods, offers the opportunity to correlate information, overcoming the disadvantages of any one technique (van Elsas et al. 2007). At present, conventional and organic commercial orchards coexist in the avocado-producing area of southern Spain where the organic farming practice of organic amendment or mulch application is common. Furthermore, several experimental orchards that test organic and conventional agricultural methods, including the addition of different organic amendments, were established in this area 15 years ago (JM Farré, personal communication). The aim of the present study was to evaluate for the first time the long-term impact on physicochemical soil properties and soil microbial communities of different organic amendments to avocado crop. These communities were analysed using a polyphasic approach, including both cultivation-based and cultivation-independent molecular methods.
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organic amendments) due to the orchards’ proximities and initial soil characteristics. Most relevant characteristics of the orchards and experimental plots are shown in Table 1.
Material and methods Orchard selection Soil samples were obtained from five orchards of cv. Hass avocado trees grafted onto cv. Topa-Topa seedling rootstocks, which were representative of those present in the Axarquía region (Málaga, Spain), the most important area for avocado production in Spain. Orchards were selected by considering their farming systems, their agronomic management policies and their use of organic amendments. The five sampled orchards included both commercial (‘Tío Palomo’ and ‘Sarmiento’) and experimental (‘Barranco’, ‘La Alegría’ and ‘La Mayora’) plots. Each of the three experimental orchards included trees that were treated with organic amendments (MY, AL, BRA, BRV—see Table 1 for the sample codes) and those that were not (MYC, ALC, BRC) in the same plot. Unamended trees were used as controls. As for commercial orchards, ‘Sarmiento’ (conventional management without organic amendment) may be considered a control for ‘Tío Palomo’ (organic management with
Soil sampling Three soil sampling campaigns were performed for this study and occurred in February 2007, December 2007 and May 2008. The sampling methodology for microbial analysis was established in a preliminary experiment in which the minimum number of trees per plot, the number of sampling points per tree and the size of the soil sample were optimised (Bonilla 2009). In the final, optimised sampling method, for each orchard or experimental plot, three different trees were chosen, and the samples were taken from four equidistant sites 1 m from the trunk base. Soil samples were taken with a corer with a 3 cm diameter to obtain two types of samples: superficial (0 to 5 cm deep) and deep (10 to 17 cm deep). The twelve samples from a given depth, either superficial or deep, at each orchard or experimental plot were pooled to provide a single
Table 1 Sample codes and characteristics of the avocado soils that were studied Orchard
Code Farming system
Net annual production (kg/tree)
Agrochemical use
Organic amendmentsa
2006/07 2007/08 Fertilisers Herbicides La Mayora
MY
Organic
32.1
MYC Conventional 21.6 La Alegría
Barranco
Sarmiento
112.6 89.1
No
No
Uncut pruning waste. Massive addition of almond shells in 2001
Yes
No
No organic amendments were added
AL
Organic
ND
ND
No
No
Uncut pruning waste. Massive addition of almond shells in 2002 (90 Tm/Ha)
ALC
Conventional ND
ND
Yes
No
No organic amendments were added
BRA Organic
3.5
61.8
No
No
Milled pruning waste. Annual addition of animal compost since 1997: 10 Tm/Ha of Fertiplus (Ferm O Feed, Schijndel, The Netherlands)
BRV
Organic
9
57.3
No
No
Milled pruning waste. Annual addition of vegetal compost since 1997: 10 Tm/Ha of solid compost (Fertiormont, Antequera, Spain)
BRC
Conventional
5.3
32.9
Yes
No
No organic amendments were added
SAR
Conventional 34
66.1
Yes
Yes
Milled pruning waste
Organic
30.4
No
No
Uncut pruning waste. Annual addition of manure (40 kg/tree) and blood meal (2 kg/tree) since 1996
Tío Palomo TP
36.7
ND no data a
In all of the avocado orchards, the dead fallen leaves were left on the soil following the traditional agriculture practice. The blanket of leaves acts as natural mulch. Decomposition can affect soil physicochemical properties
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composite soil sample. All soil samples were placed in cold storage and transported to the laboratory. Moist field soils were passed through a 2 mm sieve and freshly used for culturable microbial population analysis. Three subsamples of the soil were stored at −80°C for subsequent DNA extraction. For physicochemical analysis, the same three selected trees were sampled for each orchard or treatment in December 2007. Two points, one to the north and the other to the south of the trunk, were sampled to obtain deep and superficial soil samples of 1 kg each. Each soil sample was air dried and finely milled before analysis. Physicochemical analysis Detailed chemical analysis of the different soil samples was performed in duplicate in two different laboratories using conventional methodology (Laboratorio Caisur S.L., Granada, Spain and CEBAS-CSIC, Murcia, Spain). Three or four subsamples from each soil sample were analysed. The parameters considered for analysis were N, total C, organic C, total P, S, chlorides, electric conductivity (EC) and pH. These were further analysed by principal component analyses (PCA) using the demo version of the Multivariate Statistical Package (MVSP) v3.12e (Kovach Computing Service, Anglesey, UK). Significant differences among orchards were analysed by one-way analysis of variance (ANOVA) using the sample scores in the first PCA axis and, in case this was necessary, in the second PCA axis, and followed by Fisher’s protected least significant difference (LSD) test (P00.05) using SPSS software (SPSS Inc., Chicago, IL., USA) Culturable microbial populations Samples of 10 g of soil were suspended in 90 ml of sterile saline solution (0.85% NaCl) with 5 g of sterile gravel and mixed at 250 rpm for 30 min on an orbital shaker. Ten-fold serial dilutions were plated on different selective media. The microbial groups analysed and the selective media used for each one of them (in brackets) were as follows (Larkin and Honeycutt 2006): heterotrophic bacteria (Luria Bertoni [LB] agar with 100 mg of cycloheximide per litre), pseudomonads (King’s B [KB] agar with 75 mg of penicillin G, 45 mg of novobiocin and 100 mg of cycloheximide per litre), sporulating bacteria (LB agar with 100 mg
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of cycloheximide per litre), actinomycetes (water agar with 5 mg of polymyxin B, 1 mg of penicillin G and 100 mg of cycloheximide per litre) and fungi (potato dextrose agar [PDA] with 50 mg of chlortetracycline and 1 ml of tergitol NP-10 per litre). For isolation of sporulating bacteria, the dilutions were pre-treated at 80°C for 10 min before plating. Plates were incubated at 23°C for 48 h for bacteria and for 10 days for actinomycetes and fungi before estimating colony numbers. Microbial plate count data were log transformed before data analysis, and global comparisons were performed using InfoQuest FP 5.10 software (BioRad Laboratories, Richmond, CA, USA) by comparing the population size of the different microbial groups in deep and superficial layers of the soil for the three sampling times. Similarity among soils was calculated based on Pearson correlation index and the unweighted pair group method with arithmetic mean (UPGMA). To detect the specific effect of the organic treatments on the culturable microbial population, each organic-amended soil was compared to the respective unamended control soil. Value distribution of log transformed population data of each soil type, in all the three time samplings, were tested for normality using the Kolmogorov-Smirnov test (P00.05). Superficial and deep soil samples were analysed separately. Differences between amended and control soils were analyzed for statistical significance by the parametric Student’s ttest (P00.05) or by nonparametric Mann–Whitney U test (P00.05), depending of the fit ability of the value distribution to a normal law, using SPSS software. Soil DNA extraction Soil DNA extraction was performed using the FastDNA SPIN Kit for Soil (Qbiogene, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol, using 0.4 g of soil. Three replicate DNA extractions were performed from each composite soil sample. The quantity and quality of the extracted DNA was checked by agarose gel electrophoresis and by spectrophotometer measurement at wavelength 260 nm and 280 nm. All DNA samples were stored at −20°C for further analyses. PCR-DGGE analysis The DGGE analysis was performed only on samples from December 2007. This time of sampling was
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chosen as the most representative based on the data of culturable microorganisms and availability of chemical analysis data. The three replicate DNA extractions were analysed separately by PCRDGGE. PCR amplification of the variable region of the bacterial 16S rDNA was performed with the universal bacterial primers 341F-GC and 907R as described by Muyzer et al. (2004). The PCR mixture and conditions were the same as those described in the original study. PCR products were analysed for size and quantity by agarose gel electrophoresis and ethidium bromide staining. DGGE analyses were conducted using a D-Code Universal Detection System (Bio-Rad Laboratories). One microgram of PCR product was loaded into a well of a 6% polyacrylamide gel (acrylamide:bis-acrylamide 37.5:1) containing a gradient of 30% to 70% denaturants (100% denaturant concentration was defined as 7 M urea and 40% v/v deionised formamide). Electrophoresis was performed in 1x Tris-acetate-EDTA (TAE) buffer at 60°C with a constant voltage of 75 V for 14 h. The gels were stained with ethidium bromide (0.5 μg/ml), destained in distilled water and photographed under UV illumination using a Gel Doc XR+ imaging system (Bio-Rad Laboratories). DGGE images were analysed using the InfoQuest FP 5.10 software (Bio-Rad Laboratories). Similarities of the DGGE profiles were calculated based on the Dice coefficient, and dendrograms were obtained using the UPGMA clustering algorithm. A band position tolerance from 0.5% to 2.5%, increasing towards the end of the fingerprint, was used for all gels. Band patterns were normalised using the marker lanes as a reference, allowing comparisons among samples loaded on different DGGE gels. Anyway, samples from amended and unamended soil from the same orchard (and also samples from the commercial orchards, TP and SAR) were always loaded in the same gel, allowing the direct comparison of the fingerprints. The cophenetic correlation coefficients were calculated to assess the robustness of the assigned clusters. The number of DGGE bands in each fingerprint, which was determined using the InfoQuest software, was used as an estimate of the apparent bacterial richness. The richness of amended soils was compared with their respective unamended control soil and tested for significance by Mann–Whitney U test (P00.05) using SPSS software.
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Results Physicochemical soil properties Physicochemical analyses revealed similar textures for all soil samples, which were determined to be sandyloam soils, containing between 12% and 24% clay and a mainly neutral pH, ranging between 6.5 and 7.7. An extract of the most relevant soil properties is shown as supplementary material in Table S1. The PCA of physicochemical data (Fig. 1) showed high homogeneity among deep soils, whereas superficial soils showed larger differences. Among the superficial soil samples, the largest differences were detected in the soil treated with animal compost (BRA) because of the high levels of total P and chlorides and in the soil amended with almond shells in the orchard “La Alegría” (AL) due to the high values of total N, total C, and organic C. The soil of the organic orchard “Tío Palomo” (TP), which was annually amended with manure and blood meal, was particularly different, showing the highest levels of total N, P, S and chlorides and also high values of total and organic C. Culturable microbial populations Culturable microbial populations showed significant (P
