social pharmacy d-pharm 1st year by Pragati K. Mahajan
2006 Caravaca et al liquid organic amendments and retama
1. Biol Fertil Soils (2006) 43: 30–38
DOI 10.1007/s00374-005-0058-1
ORIGINA L PA PER
F. Caravaca . G. Tortosa . L. Carrasco .
J. Cegarra . A. Roldán
Interaction between AM fungi and a liquid organic amendment
with respect to enhancement of the performance
of the leguminous shrub Retama sphaerocarpa
Received: 6 June 2005 / Revised: 14 October 2005 / Accepted: 18 October 2005 / Published online: 14 December 2005
# Springer-Verlag 2005
Abstract This study examined the interactions between Keywords Aggregate stability . Arbuscular mycorrhizal
the 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 activities
mus mosseae, and the addition of a liquid organic amend-
ment at different rates (0, 50, 100 or 300 mg C of liquid
amendment per kilogram soil) obtained by alkaline extrac- Introduction
tion of composted dry olive residue with respect to their
effects 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 and
treatments 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 and
G. mosseae increased plant growth to a greater extent than other nutrients (Moro et al. 1997). A leguminous species
the addition of the amendment (about 35% greater than common in semi-arid environments of south-east Spain is
plants grown in the amended soil and about 79% greater Retama sphaerocarpa (L.) Boissier, which has a deep root
than control plants) and both treatments produced similar system, whose functionality is maintained at depths of
increases in soil aggregate stability (about 31% higher than more than 25 m (Haase et al. 1996) that allows the plant
control soil). The organic amendment produced a very sig- access to deep water sources; in addition, this shrub is
nificant 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 conspicuous
which were proportional to the amendment rate. Only the understorey vegetation of annual and perennial species
combined treatment involving the addition of a medium constitute “fertility islands” (Moro et al. 1997), which are
dose 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 the
produced an additive effect on the plant growth with respect benefits of AM inoculation of these shrubs in semi-arid
to the treatments applied individually (about 77% greater soils (Caravaca et al. 2003a). The selection of efficient AM
than 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 organic
Department 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 its
P.O. Box 164, Campus de Espinardo, high C and mineral nutrient content (Nogales et al. 1999).
30100 Murcia, Spain
e-mail: fcb@cebas.csic.es However, these residues contain phenolic compounds that
Tel.: +34-968-396337 may have a negative effect on microbial activity, inhibit the
Fax: +34-968-396213 establishment of AM symbioses and decrease the plant
2. 31
Table 1 Chemical, biochemical, microbiological and physical Materials and methods
characteristics of the soil
pH (H2O) 8.5±0.0a Materials
EC (1:5, μs cm−1) 225±2
Texture 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 sediments
Total carbohydrates (μg g−1) 552±20 (Table 1). It was collected from Los Cuadros in the
Water-soluble C (μg g−1) 100±1 province of Murcia, south-east Spain (coordinates 1°05'W
Water-soluble carbohydrates (μg g−1) 8±0 and 38°10'N). The climate is semi-arid Mediterranean with
Total N (g kg−1) 0.95±0.02 an average annual rainfall of 300 mm and a mean annual
Available P (μg g−1) 7±0 temperature of 19.2°C; the potential evapotranspiration
Extractable 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 extracted
Dehydrogenase (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. The
Protease-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 at
Phosphatase (μmol PNP g−1 h−1) 0.28±0.02
25°C and 12 h at 70°). The suspension was centrifuged at
β-Glucosidase (μmol PNP g−1 h−1) 0.46±0.01
14,644×g for 20 min. After centrifugation the particulate
Aggregate 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, frequently
growth (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 the
type of fungi and the time of inoculation (Martín et al. Mycorrhizal inoculation of seedlings
2002). DOR composting could minimise its antimicrobial
effect, but the composted DOR may contain phytotoxic The mycorrhizal fungi used in the experiment, Glomus
compounds (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/or
through the improvement in soil structure, cation exchange
capacity, microbiological activity and for complexing cer- Table 2 Chemical characteristics of the KOH-soluble extract of dry
tain soil ions. However, there are no data on the effects of olive residue
this organic fraction extracted from DOR on colonization of Ash (%) 30.4
roots by AM fungi or on soil properties in revegetation pH 9.83
programmes. Our hypothesis was that the combined actions EC (mS cm−1) 9.78
of this liquid organic amendment and mycorrhiza on the Organic matter (%) 69.6
growth of Mediterranean shrub species could be higher than Extractable C (g l−1) 8.6
the 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) 6.5
actions between the inoculation with three AM fungi and Water-soluble carbohydrates (g l−1) 1.03
the addition of a liquid organic amendment obtained by Phenols (g l−1) 1.58
alkaline extraction of composted DOR, with respect to their Total N (mg l−1) 320
effects on the growth of R. sphaerocarpa seedlings in a Na (mg l−1) 186
degraded semi-arid Mediterranean soil, and (2) to establish K (mg l−1) 1,776
the optimum levels of amendment addition, thereby avoid- P (mg l−1) 62
ing possible negative effects on plant growth, AM coloni- Ca (mg l−1) 136
zation and soil properties considered to be indicators of soil Mg (mg l−1) 28.9
quality such as labile C fractions (microbial biomass C and
Fe (mg l−1) 3.4
water-soluble C), enzyme activities (dehydrogenase, ure-
Cu (mg l−1) 0.2
ase, protease-BAA, and β-glucosidase) and soil aggregate
Mn (mg l−1) 0.3
stability.
Zn (mg l−1) 1.2
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
8.47ab
zosphere 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
3
germinated, seedlings were transplanted into the growth
substrate consisting of peat and cocopeat (1:1, v/v). The
254cde
8.43ab
corresponding AM inoculum was applied at a rate of 5%
(v/v). The same amount of an autoclaved mixture of the
2
inocula was added to control plants, supplemented with
a filtrate (Whatman No. 1 paper) of the pot culture of all
8.40ab
283e
three AM fungi to provide the microbial populations
1
accompanying the mycorrhizal fungi. Inoculated and non-
G. mosseae
inoculated seedlings were grown for 8 months under nurs-
209bcd 270de
ery conditions without any chemical fertiliser treatment.
8.39a
0
18.5fg 17.0cdefg 19.2g
Experimental design and layout
8.34a
3
The experiment was a mesocosm assay, conducted as a
completely randomized factorial design with two factors.
200bc 225cde 249cde
347bcde 357de 290ab
The first factor had four levels: without and with the
8.47ab 8.46ab 8.34a
addition of low, medium or high doses of the liquid
2
amendment to the soil. The second factor had four levels:
non-inoculation and inoculation of R. sphaerocarpa plants
G. deserticola
with one of three AM fungi (G. intraradices, G. deserticola
1
or G. mosseae) in the nursery. Four replicates per treatment
13.7bcd 14.6bcde 15.0bcdef 11.0a
were 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
0
miculite at a ratio of 3:1 (v/v), were placed in 3.6-l pots. In
March 2003, R. sphaerocarpa seedlings (inoculated and
240cde
8.48ab
non-inoculated) were transplanted to the pots (one per pot).
303abcd 315abcde 320abcde 374e
Two weeks after planting, the amendment was applied to
3
the 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
8.44ab
300 mg C liquid amendment kg−1 soil. The experiment was
213bcd 228cde 184a
carried out in the nursery of the University of Murcia (Murcia,
2
Spain) without the addition of chemical fertiliser. The plants
were well watered and kept outdoors under ambient irradi-
G. intraradices
8.31a
ance, temperature and air humidity. One year after planting,
1
the plants were harvested and rhizosphere soil samples of the
pots were taken. Rhizosphere soil samples, air-dried to 20%
13.5bc 13.1b 14.8bcde 10.8a
8.34a
moisture content and sieved to less than 2 mm, were divided
0
into two subsamples. One subsample was stored at 2°C for
microbiological analysis, and another subsample was allowed
to dry at room temperature for physical–chemical analysis.
8.58b 8.60b
310abcd 287ab 294abc 278a
208bcd 188ab 174a
3
Plant analyses
2
Basal stem diameters and heights of plants were measured
8.58b
with callipers and rulers. Fresh and dry (105°C, 5 h) masses
No mycorrhiza
of shoots and roots were recorded. Plant tissues were
1
ground before chemical analysis. Plant P was determined
DOR Dry olive residue
10.6a
colorimetrically according to Murphy and Riley (1962)
8.56b
Water-soluble 183a
after digestion in nitric–perchloric acid (5:3) for 6 h at
a
0
210°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 a
thermic conductivity detector. Plant K was estimated by
flame photometry (Schollemberger and Simon 1954).
a
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 of
Hayman 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 the
Soil 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 analysis
water-soluble C was determined with an automatic carbon
analyser 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 their
extraction method (Vance et al. 1987). Ten grams of soil at interaction effects on measured variables were tested by a
60% of field capacity was fumigated in a 125-ml Erlenmeyer two-way analysis of variance, and comparisons among
flask with purified CHCl3 for 24 h. After removal of means were made using the least significant difference
residual CHCl3, 40 ml of 0.5 M K2SO4 solution was added (LSD) test calculated at p<0.05. Statistical procedures were
and the sample was shaken for 1 h before filtration of the carried out with the software package SPSS 10.0 for
mixture. 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 and
non-fumigated samples divided by the calibration factor Results
KEC=0.38.
Dehydrogenase activity was determined according to Soil parameters
Trevors et al. (1982). For this, 1 g of soil at 60% of field
capacity was exposed to 0.2 ml of 0.4% 2-p-iodophenyl-3-p- Only the mycorrhizal treatments with G. intraradices and
nitrophenyl-5-phenyltetrazolium chloride (INT) in distilled G. mosseae significantly decreased soil pH (Tables 3 and
water 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 conductivity
through 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 buffer
at pH 7; 1 M urea (Tabatabai and Bremner 1972) and Table 4 Two-factor ANOVA (mycorrhizal inoculation treatments
0.03 M BAA (Ladd and Butler 1972) were used as and amendment addition) for all parameters studied in the
substrates, respectively. Two millilitres of buffer and 0.5 ml rhizosphere soil of R. sphaerocarpa seedlings 1 year after planting
of 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
þ
90 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 0.814
glucopyranoside (PNG, 0.05 M) as substrate. This assay is Electrical 0.669 0.079 0.169
based on the release and detection of p-nitrophenol (PNP). conductivity
Two millilitres of 0.1 M maleate buffer (pH 6.5) and 0.5 ml Water-soluble C 0.615 0.003 0.987
of substrate were added to 0.5 g of sample and incubated at Aggregate stability <0.001 0.014 0.362
37°C for 90 min. The reaction was stopped with tris(hydro Microbial biomass C <0.001 0.587 0.806
xymethyl)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 0.001
trophotometry 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 0.384
the method described by Lax et al. (1994). Sieved (0.2– Shoot 0.037 0.001 0.211
4 mm) soil (4 g) was placed on a small, 0.250-mm sieve Root 0.604 0.353 0.163
and wetted by spray. After 15 min the soil was subjected to
N foliar 0.006 <0.001 0.018
an artificial rainfall of 150 ml with energy of 270 J m−2.
P foliar 0.002 <0.001 0.003
The remaining soil on the sieve was placed in a previously
K foliar 0.319 0.001 0.028
weighed capsule (T), dried at 105°C and weighed (P1).
Colonization 0.861 <0.001 0.547
Then the soil was soaked in distilled water and, after 2 h,
5. 34
Table 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