2006 Caravaca et al liquid organic amendments and retama

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2006 Caravaca et al liquid organic amendments and retama

  1. 1. Biol Fertil Soils (2006) 43: 30–38DOI 10.1007/s00374-005-0058-1 ORIGINA L PA PERF. Caravaca . G. Tortosa . L. Carrasco .J. Cegarra . A. RoldánInteraction between AM fungi and a liquid organic amendmentwith respect to enhancement of the performanceof the leguminous shrub Retama sphaerocarpaReceived: 6 June 2005 / Revised: 14 October 2005 / Accepted: 18 October 2005 / Published online: 14 December 2005# Springer-Verlag 2005Abstract This study examined the interactions between Keywords Aggregate stability . Arbuscular mycorrhizalthe inoculation with three arbuscular mycorrhizal fungi, fungi . Dry olive residue . Microbial biomass C .namely, Glomus intraradices, Glomus deserticola and Glo- Semi-arid environments . Soil enzyme activitiesmus mosseae, and the addition of a liquid organic amend-ment at different rates (0, 50, 100 or 300 mg C of liquidamendment per kilogram soil) obtained by alkaline extrac- Introductiontion of composted dry olive residue with respect to theireffects on growth of Retama sphaerocarpa seedlings and Legumes are among the most effective species in reveg-on some microbiological and physical properties of soil. etation programmes because of their ability to form sym-One year after planting, both mycorrhizal inoculation biotic associations with both nitrogen-fixing bacteria andtreatments and the addition of amendment had increased arbuscular mycorrhizal (AM) fungi. They have been con-plant growth and dehydrogenase, urease and benzoyl sidered useful for revegetation of dry Mediterranean hab-argininamide hydrolysing activities. The inoculation with itats that have low availability of nitrogen, phosphorus andG. mosseae increased plant growth to a greater extent than other nutrients (Moro et al. 1997). A leguminous speciesthe addition of the amendment (about 35% greater than common in semi-arid environments of south-east Spain isplants grown in the amended soil and about 79% greater Retama sphaerocarpa (L.) Boissier, which has a deep rootthan control plants) and both treatments produced similar system, whose functionality is maintained at depths ofincreases in soil aggregate stability (about 31% higher than more than 25 m (Haase et al. 1996) that allows the plantcontrol soil). The organic amendment produced a very sig- access to deep water sources; in addition, this shrub isnificant decrease in the levels of microbial biomass C and a capable of resisting the frequent droughts of arid and semi-strong increase in soil dehydrogenase and urease activities, arid zones. Also, R. sphaerocarpa and its conspicuouswhich were proportional to the amendment rate. Only the understorey vegetation of annual and perennial speciescombined treatment involving the addition of a medium constitute “fertility islands” (Moro et al. 1997), which aredose of amendment (100 mg C kg−1 soil) and the mycor- points of high biological activity scattered in a heteroge-rhizal inoculation with G. intraradices or G. deserticola neous landscape. Several studies have demonstrated theproduced an additive effect on the plant growth with respect benefits of AM inoculation of these shrubs in semi-aridto the treatments applied individually (about 77% greater soils (Caravaca et al. 2003a). The selection of efficient AMthan plants grown in the amended soil and about 63% fungi is a major topic in inoculation programmes, espe-greater than inoculated plants). cially in disturbed soils where the indigenous inoculum levels of AM fungi are considered as being low, which may limit the successful establishment of plants (Palenzuela et al. 2002; Azcón-Aguilar et al. 2003). The addition of organic materials can increase fertility and improve the physical and biological properties of de-F. Caravaca (*) . G. Tortosa .L. Carrasco . J. Cegarra . A. Roldán graded soils (Roldán et al. 1996). Among the organicDepartment of Soil and Water Conservation, amendments, the agronomic use of dry olive residue (DOR)CSIC-Centro de Edafología y Biología Aplicada del Segura, or “alperujo” has increased steadily in recent years due to itsP.O. Box 164, Campus de Espinardo, high C and mineral nutrient content (Nogales et al. 1999).30100 Murcia, Spaine-mail: fcb@cebas.csic.es However, these residues contain phenolic compounds thatTel.: +34-968-396337 may have a negative effect on microbial activity, inhibit theFax: +34-968-396213 establishment of AM symbioses and decrease the plant
  2. 2. 31Table 1 Chemical, biochemical, microbiological and physical Materials and methodscharacteristics of the soilpH (H2O) 8.5±0.0a MaterialsEC (1:5, μs cm−1) 225±2Texture Loam The soil was a Typic Haplocalcid (Soil Survey Staff 1999)Total organic C (g kg−1) 10.3±0.3 with a texture loam developed from Quaternary sedimentsTotal carbohydrates (μg g−1) 552±20 (Table 1). It was collected from Los Cuadros in theWater-soluble C (μg g−1) 100±1 province of Murcia, south-east Spain (coordinates 1°05WWater-soluble carbohydrates (μg g−1) 8±0 and 38°10N). The climate is semi-arid Mediterranean withTotal N (g kg−1) 0.95±0.02 an average annual rainfall of 300 mm and a mean annualAvailable P (μg g−1) 7±0 temperature of 19.2°C; the potential evapotranspirationExtractable K (μg g−1) 222±4 reaches 1,000 mm year−1.Microbial biomass C (μg g−1) 396±11 The amendment used was the organic fraction extractedDehydrogenase (μg INTF g−1) 51±1 with KOH from a composted DOR (Alburquerque et al.Urease (μmol NH3 g−1 h−1) 0.31±0.03 2005) collected from an olive mill in Granada, Spain. TheProtease-BAA (μmol NH3 g−1 h−1) 0.60±0.04 extract was obtained by mechanically shaking the com- posted DOR with 0.1 M KOH (1:20, w/v) for 24 h (12 h atPhosphatase (μmol PNP g−1 h−1) 0.28±0.02 25°C and 12 h at 70°). The suspension was centrifuged atβ-Glucosidase (μmol PNP g−1 h−1) 0.46±0.01 14,644×g for 20 min. After centrifugation the particulateAggregate stability (%) 11.5±0.4 matter was eliminated of supernatant. The analytical char-a Mean±standard error (N=6) acteristics of the amendment are shown in Table 2. The plant used for the reafforestation experiment was R. sphaerocarpa, a low-growing shrub reaching a height of 1.3–2.5 m and widely distributed in the Mediterranean area. It is also well adapted to drought and, therefore, frequentlygrowth (Martín et al. 2002; Linares et al. 2003). Studies used in the reafforestation of semi-arid disturbed lands.have shown that the effect of phenolic compounds con-tained in DOR on AM root colonization can vary with thetype of fungi and the time of inoculation (Martín et al. Mycorrhizal inoculation of seedlings2002). DOR composting could minimise its antimicrobialeffect, but the composted DOR may contain phytotoxic The mycorrhizal fungi used in the experiment, Glomuscompounds (Linares et al. 2003). On the other hand, the intraradices Schenck & Smith, Glomus deserticola (Trappe,organic fraction extracted with alkaline solutions from Bloss & Menge) and Glomus mosseae (Nicol & Gerd.) Gerd.organic materials has been used for improving plant growth & Trappe, were obtained from the collection of the exper-and nutrient uptake (Ayuso et al. 1996). This fraction may imental field station of Zaidín, Granada.have a positive effect on plant growth directly and/orthrough the improvement in soil structure, cation exchangecapacity, microbiological activity and for complexing cer- Table 2 Chemical characteristics of the KOH-soluble extract of drytain soil ions. However, there are no data on the effects of olive residuethis organic fraction extracted from DOR on colonization of Ash (%) 30.4roots by AM fungi or on soil properties in revegetation pH 9.83programmes. Our hypothesis was that the combined actions EC (mS cm−1) 9.78of this liquid organic amendment and mycorrhiza on the Organic matter (%) 69.6growth of Mediterranean shrub species could be higher than Extractable C (g l−1) 8.6the sum of individual effects. Fulvic acid C (g l−1) 2.1 The objectives of this study were (1) to assess the inter- Humic acid C (g l−1) 6.5actions between the inoculation with three AM fungi and Water-soluble carbohydrates (g l−1) 1.03the addition of a liquid organic amendment obtained by Phenols (g l−1) 1.58alkaline extraction of composted DOR, with respect to their Total N (mg l−1) 320effects on the growth of R. sphaerocarpa seedlings in a Na (mg l−1) 186degraded semi-arid Mediterranean soil, and (2) to establish K (mg l−1) 1,776the optimum levels of amendment addition, thereby avoid- P (mg l−1) 62ing possible negative effects on plant growth, AM coloni- Ca (mg l−1) 136zation and soil properties considered to be indicators of soil Mg (mg l−1) 28.9quality such as labile C fractions (microbial biomass C and Fe (mg l−1) 3.4water-soluble C), enzyme activities (dehydrogenase, ure- Cu (mg l−1) 0.2ase, protease-BAA, and β-glucosidase) and soil aggregate Mn (mg l−1) 0.3stability. Zn (mg l−1) 1.2
  3. 3. 32 14.0bcd 17.2defg 16.9cdefg 17.7efg The AM fungal inoculum consisted of a mixture of rhi- 310abcd 300abcd 324abcde 322abcde 354cde 248cde 8.47abzosphere soil from trap cultures (Sorghum sp.) containing Table 3 Soil physical–chemical properties of R. sphaerocarpa after 1 year of growth in response to mycorrhizal inoculation treatments and KOH extract of DOR addition (n=4)spores, hyphae and mycorrhizal root fragments. Once 3germinated, seedlings were transplanted into the growthsubstrate consisting of peat and cocopeat (1:1, v/v). The 254cde 8.43abcorresponding AM inoculum was applied at a rate of 5%(v/v). The same amount of an autoclaved mixture of the 2inocula was added to control plants, supplemented witha filtrate (Whatman No. 1 paper) of the pot culture of all 8.40ab 283ethree AM fungi to provide the microbial populations 1accompanying the mycorrhizal fungi. Inoculated and non- G. mosseaeinoculated seedlings were grown for 8 months under nurs- 209bcd 270deery conditions without any chemical fertiliser treatment. 8.39a 0 18.5fg 17.0cdefg 19.2gExperimental design and layout 8.34a 3The experiment was a mesocosm assay, conducted as acompletely randomized factorial design with two factors. 200bc 225cde 249cde 347bcde 357de 290abThe first factor had four levels: without and with the 8.47ab 8.46ab 8.34aaddition of low, medium or high doses of the liquid 2amendment to the soil. The second factor had four levels:non-inoculation and inoculation of R. sphaerocarpa plants G. deserticolawith one of three AM fungi (G. intraradices, G. deserticola 1or G. mosseae) in the nursery. Four replicates per treatment 13.7bcd 14.6bcde 15.0bcdef 11.0awere carried out, making a total of 64 pots. Two kilograms of substrate, consisting of soil and ver- 0, 1, 2 or 3 indicates 0, 5.8, 11.7 or 35.1 ml amendment kg−1 soil/vermiculite mixture, respectively 0miculite at a ratio of 3:1 (v/v), were placed in 3.6-l pots. InMarch 2003, R. sphaerocarpa seedlings (inoculated and 240cde 8.48abnon-inoculated) were transplanted to the pots (one per pot). 303abcd 315abcde 320abcde 374eTwo weeks after planting, the amendment was applied to 3the pots at a rate of 0, 5.8, 11.7 or 35.1 ml kg−1 soil/vermiculite Values in rows followed by the same letter are not significantly different (LSD, p<0.05)mixture, which corresponds to the addition of 0, 50, 100 or 8.44ab300 mg C liquid amendment kg−1 soil. The experiment was 213bcd 228cde 184acarried out in the nursery of the University of Murcia (Murcia, 2Spain) without the addition of chemical fertiliser. The plantswere well watered and kept outdoors under ambient irradi- G. intraradices 8.31aance, temperature and air humidity. One year after planting, 1the plants were harvested and rhizosphere soil samples of thepots were taken. Rhizosphere soil samples, air-dried to 20% 13.5bc 13.1b 14.8bcde 10.8a 8.34amoisture content and sieved to less than 2 mm, were divided 0into two subsamples. One subsample was stored at 2°C formicrobiological analysis, and another subsample was allowedto dry at room temperature for physical–chemical analysis. 8.58b 8.60b 310abcd 287ab 294abc 278a 208bcd 188ab 174a 3Plant analyses 2Basal stem diameters and heights of plants were measured 8.58bwith callipers and rulers. Fresh and dry (105°C, 5 h) masses No mycorrhizaof shoots and roots were recorded. Plant tissues were 1ground before chemical analysis. Plant P was determined DOR Dry olive residue 10.6acolorimetrically according to Murphy and Riley (1962) 8.56b Water-soluble 183aafter digestion in nitric–perchloric acid (5:3) for 6 h at a 0210°C. Plant N was determined by combustion at 1,020°C stability (%) C (μg g−1)in a carbon and nitrogen analyser and reduction, separation Aggregate pH (H2O) μs cm−1) EC (1:5,of N2 in a chromatographic column and measurement in athermic conductivity detector. Plant K was estimated byflame photometry (Schollemberger and Simon 1954). a
  4. 4. 33 The percentage of root length colonized by AM fungi was passed through the same 0.250-mm sieve with the assis-calculated by the gridline intersect method (Giovannetti and tance of a small stick to break the remaining aggregates.Mosse 1980) after staining with trypan blue (Phillips and The residue remaining on the sieve, which was made up ofHayman 1970). plant debris and sand particles, was dried at 105°C and weighed (P2). The percentage of stable aggregates with regard to the total aggregates was calculated by theSoil analyses following relationship (P1−P2)×100/(4−P2+T).Soil pH and electrical conductivity were measured in a 1:5(w/v) aqueous solution. In soil aqueous extracts (1:5), Statistical analysiswater-soluble C was determined with an automatic carbonanalyser for liquid samples (Shimadzu TOC-5050A). Data were log-transformed to achieve for normality. Microbial biomass C was determined by the fumigation– Amendment addition, mycorrhizal inoculation and theirextraction method (Vance et al. 1987). Ten grams of soil at interaction effects on measured variables were tested by a60% of field capacity was fumigated in a 125-ml Erlenmeyer two-way analysis of variance, and comparisons amongflask with purified CHCl3 for 24 h. After removal of means were made using the least significant differenceresidual CHCl3, 40 ml of 0.5 M K2SO4 solution was added (LSD) test calculated at p<0.05. Statistical procedures wereand the sample was shaken for 1 h before filtration of the carried out with the software package SPSS 10.0 formixture. The K2SO4-extracted C was measured as indi- Windows.cated for water-soluble C. Microbial biomass C was cal-culated as the difference between the C of fumigated andnon-fumigated samples divided by the calibration factor ResultsKEC=0.38. Dehydrogenase activity was determined according to Soil parametersTrevors et al. (1982). For this, 1 g of soil at 60% of fieldcapacity was exposed to 0.2 ml of 0.4% 2-p-iodophenyl-3-p- Only the mycorrhizal treatments with G. intraradices andnitrophenyl-5-phenyltetrazolium chloride (INT) in distilled G. mosseae significantly decreased soil pH (Tables 3 andwater for 20 h at 22°C in the dark. The iodonitrotetrazolium 4). However, neither amendment addition nor the interac-formazan (INTF) formed was extracted with 10 ml of tion of amendment × mycorrhizal inoculation had any sig-methanol by shaking vigorously for 1 min and filtering nificant effect on soil pH and electrical conductivitythrough Whatman No. 5 filter paper. INTF was measured (Table 4).spectrophotometrically at 490 nm. Urease and N-α-benzoyl-L-argininamide (BAA) hydro-lysing activities were determined in 0.1 M phosphate bufferat pH 7; 1 M urea (Tabatabai and Bremner 1972) and Table 4 Two-factor ANOVA (mycorrhizal inoculation treatments0.03 M BAA (Ladd and Butler 1972) were used as and amendment addition) for all parameters studied in thesubstrates, respectively. Two millilitres of buffer and 0.5 ml rhizosphere soil of R. sphaerocarpa seedlings 1 year after plantingof substrate were added to 0.5 g of sample, which was (P significance values)incubated at 30°C (for urease) or 39°C (for protease) for Amendment Mycorrhiza Interaction þ90 min. Both activities were determined as the NH4 re- (A) (M) (A×M)leased in the hydrolysis reaction. β-Glucosidase was determined using p-nitrophenyl-β-D- pH 0.700 <0.001 0.814glucopyranoside (PNG, 0.05 M) as substrate. This assay is Electrical 0.669 0.079 0.169based on the release and detection of p-nitrophenol (PNP). conductivityTwo millilitres of 0.1 M maleate buffer (pH 6.5) and 0.5 ml Water-soluble C 0.615 0.003 0.987of substrate were added to 0.5 g of sample and incubated at Aggregate stability <0.001 0.014 0.36237°C for 90 min. The reaction was stopped with tris(hydro Microbial biomass C <0.001 0.587 0.806xymethyl)aminomethane (THAM) according to Tabatabai Dehydrogenase 0.002 0.026 0.685(1982). The amount of PNP was determined by spec- Urease <0.001 <0.001 0.001trophotometry at 398 nm (Tabatabai and Bremner 1969). Protease 0.643 <0.001 0.807 The percentage of stable aggregates was determined by β-Glucosidase 0.818 0.293 0.384the method described by Lax et al. (1994). Sieved (0.2– Shoot 0.037 0.001 0.2114 mm) soil (4 g) was placed on a small, 0.250-mm sieve Root 0.604 0.353 0.163and wetted by spray. After 15 min the soil was subjected to N foliar 0.006 <0.001 0.018an artificial rainfall of 150 ml with energy of 270 J m−2. P foliar 0.002 <0.001 0.003The remaining soil on the sieve was placed in a previously K foliar 0.319 0.001 0.028weighed capsule (T), dried at 105°C and weighed (P1). Colonization 0.861 <0.001 0.547Then the soil was soaked in distilled water and, after 2 h,
  5. 5. 34Table 5 Soil microbial biomass C and enzyme activities of R. sphaerocarpa after 1 year of growth in response to mycorrhizal inoculation treatments and KOH extract of DOR addition (n=4) Water-soluble C concentrations were increased only 0.27abc 1.15ef 0.61d with the mycorrhizal inoculation treatments (Tables 3 and 53.4f 42ab 3 4), the greatest increases being observed in the inoculation treatment with G. mosseae (on average, about 40% greater 42.4de 1.15ef 0.69d 0.32c 46ab than in the soil of non-inoculated plants). Both amendment addition and inoculation with G. mos 2 seae significantly increased soil aggregate stability, reach- 0.29abc 30.2ab 0.19ab ing similar values at the end of the growth period (Tables 3 1.50g 73bc and 4). In the amended soils, the percentage of stable 1 G. mosseae aggregates did not vary with the dose of the amendment 0.28abc added. The highest aggregate stability was found in the 34.8bc 0.19ab 1.29fg 301e amended soil of plants inoculated with G. deserticola or G. 0 mosseae. The addition of the amendment decreased soil microbial 0.26abc 43.8de 0.56cd 0.57b biomass C for both non-inoculated and inoculated plants, 81c particularly for the plants inoculated with G. mosseae or 3 G. intraradices (Table 5). In general, microbial biomass C 46.0de 0.69bc decreased as the dose of amendment increased. Microbial 0.83e 0.31c 180d biomass C was not affected by the mycorrhizal inocula- 2 tion treatments (Table 4). With the exception of G. desert 0.27abc icola, the mycorrhizal inoculation alone produced sig- 31.3ab 0.75bc 0.24b 149d G. deserticola nificantly higher dehydrogenase and benzoyl arginina- 1 mide hydrolysing activities than the control soil (Table 5). In general, neither amendment addition nor mycorrhizal 0.25abc 29.8ab 0.14ab 0.46a inoculation modified β-glucosidase activity. The medium 288e and high dose of amendment significantly increased dehy- 0 drogenase and urease activities. Only urease activity 0.84cd 0.22ab 0, 1, 2 or 3 indicates 0, 5.8, 11.7 or 35.1 ml amendment kg−1 soil/vermiculite mixture, respectively 0.66d 47.5e increased proportionally with the increase in the amend- 35a ment dose. The interaction amendment × mycorrhizal 3 inoculation had no significant effect on the biochemical 40.9cd 0.56cd 0.68bc 0.29bc properties (Table 4). Values in rows followed by the same letter are not significantly different (LSD, p<0.05) 86c 2 30.0ab 0.16ab 0.96de 0.21a 167d G. intraradices 1 Table 6 Growth parameters, foliar nutrient content and root 0.25abc 34.8bc 0.14ab 0.95de infection of R. sphaerocarpa in response to mycorrhizal inoculation 296e treatments and KOH extract of DOR addition previous to planting (n=4) 0 0.25abc No G. G. G. 40.7cd 0.56cd 0.53ab mycorrhiza intraradices deserticola mosseae 23a 3 Shoot 0.5a 0.7a 0.7a 0.5a 40.6cd 0.48ab 0.23ab 0.43c 70bc (g dry wt. plant−1) 2 Root 0.6ab 0.8b 0.7ab 0.5a 0.24abc 0.19ab 0.54ab (g dry wt. 26.0a 177d No mycorrhiza plant−1) 1 Nitrogen 8a 13b 10ab 9a 0.26abc (mg plant−1) 26.1a 0.05a 0.44a 281e Phosphorus 0.6a 0.8ab 1.2b 1.0ab 0a (mg plant−1) DOR Dry olive residue (μmol PNP g−1 h−1) (μmol NH3 g−1 h−1) Potassium 7a 9a 9a 8a (μg INTF g−1 soil) (mg plant−1) Microbial biomass Dehydrogenase β-Glucosidase Colonization (%) 0.4a 61.9b 67.7b 51.8b Protease-BAA NH3 g−1 h−1) Urease (μmol C (μg g−1) Values in rows followed by the same letter are not significantly different (LSD, p<0.05) a 0, 1, 2 or 3 indicates 0, 5.8, 11.7 or 35.1 ml amendment kg−1 soil/ vermiculite mixture, respectively a

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