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Arbuscular mycorrhizal fungus enhances crop yield and P-uptake of maize (Zea mays L.): A field case study on a sandy loam soil as affected by long-term P-deficiency fertilization
Soil Biology & Biochemistry 41 (2009) 2460–2465
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Arbuscular mycorrhizal fungus enhances crop yield and P-uptake of maize (Zea mays L.): A field case study on a sandy loam soil as affected by long-term P-deficiency fertilization Junli Hu, Xiangui Lin*, Junhua Wang, Jue Dai, Xiangchao Cui, Ruirui Chen, Jiabao Zhang State Key Laboratory of Soil and Sustainable Agriculture, Joint Open Laboratory of Soil and the Environment, Hongkong Baptist University & Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road 71, Nanjing 210008, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2009 Received in revised form 28 August 2009 Accepted 1 September 2009 Available online 15 September 2009
The P efficiency, crop yield, and response of maize to arbuscular mycorrhizal fungus (AMF) Glomus caledonium were tested in an experimental field with long-term (18-year) fertilizer management. The experiment included five fertilizer treatments: organic amendment (OA), half organic amendment plus half mineral fertilizer (1/2 OM), mineral fertilizer NPK, mineral fertilizer NK, and the control (without fertilization). AMF inoculation responsiveness (MIRs) of plant growth and P-uptake of maize were estimated by comparing plants grown in unsterilized soil inoculated with G. caledonium and in untreated soil containing indigenous AMF. Soil total P, available P, microbial biomass P, alkaline phosphatase activity, plant biomass, crop yield and total P-uptake of maize were all significantly increased (P < 0.05) by the application of OA, 1/2 OM, and NPK, but not by the application of NK. Specifically, the individual crop yield of maize approached zero in the NK-fertilized soils, as well as in the control soils. All maize plants were colonized by indigenous AMF, and the root colonization at harvest time was not significantly influenced by fertilization. G. caledonium inoculation increased mycorrhizal colonization significantly (P < 0.05) only with the NK treatment, and produced low but demiurgic crop yield in the control and NK-fertilized soils. Compared to the inoculation in balanced-fertilized soils, G. caledonium inoculation in either the NK-fertilized soils or the control soils had significantly greater (P < 0.05) impacts on soil alkaline phosphatase activity, stem length, plant biomass, and total P-uptake of maize, indicating that AMF inoculation was likely more efficient in extremely P-limited soils. These results also showed that balanced mineral fertilizers and organic amendments did not differ significantly in their effects on MIRs in these soils. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: AMF inoculation responsiveness Glomus caledonium Mineral fertilizer Organic amendment P efficiency Soil alkaline phosphatase Soil microbial biomass P
1. Introduction Organic and inorganic fertilizers are used primarily to increase nutrient availability to plants (Chu et al., 2007). Traditional organic fertilizers are by-products of the food and agricultural industries, which are carbon-based and decomposed by soil microorganisms to release nutrients gradually in time with plant growth. In China, there are concerns about soil fertility degradation by replacement of organic fertilizers with inorganic fertilizers. Thus, a number of longterm experiments were set up in agricultural regions at the end of the 1980s to monitor changes in soil fertility with application of either inorganic fertilizers or organic amendments or a mixed application of both (Cai and Qin, 2006). Results obtained from long-term experiments have already substantially improved our knowledge of
* Corresponding author. Tel.: þ86 25 86881589; fax: þ86 25 86881000. E-mail addresses: [email protected] (J. Hu), [email protected] (X. Lin). 0038-0717/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2009.09.002
changes in soil productivity with various fertilization practices. There are many reports showing that soil fertility has declined with continuous application of inorganic fertilizers; therefore, intensive cropping with no return of crop residues and other organic inputs is assumed to be a non-sustainable practice (Singh et al., 2004). On the other hand, there are also numerous reports showing that balanced application of inorganic fertilizers maintains soil productivity and increases crop yields (Rasmussen and Collins, 1991; Cavazzna and Volk, 1996; Glendining et al., 1996; Buyanovsky and Wagner, 1998; Kanchikerimath and Singh, 2001; Shen et al., 2004; Cai and Qin, 2006). Thus, balanced fertilization of major plant nutrient elements could be beneficial for the growth of plant aboveground parts and roots (Chu et al., 2007). However, farmers are often forced to make decisions about their fertilizer strategy that reflect economic rather than agronomic pressure. As a result, unbalanced fertilization is unfortunately widespread (Chu et al., 2007). Phosphorus (P) is one of the major essential macronutrients limiting plant growth owing to its low bioavailability in soils
J. Hu et al. / Soil Biology & Biochemistry 41 (2009) 2460–2465
(Feng et al., 2004). For instance, Huang-Huai-Hai Plain, one of the most important agricultural regions in China (Yang and Janssen, 1997), is located in low reaches of the Yellow, Huai, and Hai rivers within an area of 350,000 km2 (Cai and Qin, 2006). However, the soil was extremely deficient in P – thus, crops growing in the farmland that had received no P for 18 years were clearly P limited and did not respond to nitrogen (N) and potassium (K) fertilization (Hu et al., 2009), and both wheat (Triticum aestivum L.) and maize (Zea mays L.) yields were always significantly lower in P-deficient soils than in P-fertilized soils (Cai and Qin, 2006). Despite large research efforts devoted to increasing the P availability to plants, several processes of the P cycle in soils remain obscure (Song et al., 2002). Microorganisms able to solubilize and mineralize P pools in soils are considered to be vital (Rodrı´guez and Fraga, 1999), since they can contribute to the efficiency of plants at acquiring P from the soil (Oliveira et al., 2009). Among other soil microorganisms, arbuscular mycorrhizal fungi (AMF), as ubiquitous mutualists found in both natural and agricultural ecosystems, provide a direct link between soil and roots, and are renowned for their ability to increase plant mineral nutrients, notably P (Leyval et al., 1997; Gaur and Adholeya, 2004; Bush, 2008). Consequently, although the dry matter production of plants is independent of the presence of AMF diasporas, another plant-production related parameter, estimated as the plant uptake of P, is dependent on the presence of AMF diasporas (Mårtensson and Carlgren, 1994). AMF may not always play a vital role in the nutrition and growth of plants in many agricultural ecosystems, especially high-input agriculture (Ryan and Graham, 2002). High available soil P may be detrimental to mycorrhizal colonization and may limit their benefits in agroecosystems because of the large application of readily soluble fertilizers. Obviously, agricultural practices such as the use of fertilizers may have severe impacts on AMF diasporas and activities (Douds and Millner, 1999; Hu et al., 2009). As a result, investigating long-term effects of fertilization on AMF inoculation efficiency may help to ensure an opportunity for the utilization of AMF in agroecosystems. We hypothesized that AMF inoculation could alter the P efficiency of agroecosystems and play an important role in enhancing maize growing in soils under P-deficiency fertilization relative to balanced fertilization. The objective of our study was to investigate AMF inoculation impacts on plant biomass, crop yield, and P-uptake of maize, as well as mycorrhizal colonization, soil microbial biomass P, and soil alkaline phosphatase activity, in an experimental field with long-term (18-year) fertilizer management. This work may contribute to our understanding of the impacts of fertilization on soil microorganisms and soil health, and to developing AMF application schemes based on specific functions in arable soils. 2. Materials and methods 2.1. Experimental field description The long-term field fertilizer experiment was established in September 1989, in the Fengqiu Agro-Ecological Experimental Station (35 000 N, 114 240 E) of the Chinese Academy of Sciences, Henan Province, China. The soil was derived from alluvial sediments of the Yellow River and is classified as an aquic inceptisol (a calcareous, fluvo-aquic sandy loam). The surface soil contained 4.5 g kg1 of organic C, 0.45 g kg1 of total N and 9.51 mg kg1 of available N, 0.50 g kg1 of total P and 1.93 mg kg1 of available P, 18.6 g kg1 of total K and 78.8 mg kg1 of available K, and had a pH (H2O) of 8.6 at the beginning of the fertilizer experiment. Five treatments (four replicates of each) were established in completely randomized blocks in 20 plots (9.5 5 m) under a rotation of winter wheat and summer maize: organic amendment (OA), half organic amendment plus half mineral fertilizer (1/2 OM), mineral NPK fertilizer (NPK), mineral NK fertilizer (NK), and the control (without fertilization). For NPK
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treatment, N, P, and K were applied in the form of urea (300 kg N ha1 per year), super phosphate (60 kg P ha1 per year), and potassium sulfate (250 kg K ha1 per year), respectively, while no P was applied for the NK treatment. The OA and 1/2 OM treatments were designed to give the same application rates of N, P, and K as those given in the NPK treatment. The organic amendment was a composted mixture of wheat straw, oil cake, and cotton cake in a ratio of 100:40:45. All P, K, and organic amendments were applied as basal fertilizers, whereas urea was added in two applications as both basal and supplementary fertilizers. Basal fertilizers were evenly broadcast onto the soil surface and immediately incorporated into the plowed layer by tillage before sowing of maize in June and wheat in October. Supplementary fertilizer urea was also applied to the soil surface and brought into the plowed layer with irrigation water. Detailed information on the experimental design and field management has been described by Meng et al. (2005). Each plot received the same fertilizer management every year from 1989 to 2008. 2.2. AMF inoculation and maize harvest A micro-plot method was used to determine AMF inoculation efficiency in each replicate plot of each treatment in 2008. On June 6, maize was directly sown by hand. The distances between rows and hills were 70 cm and 30 cm, respectively. In each plot, two square polyvinyl chloride (PVC) plastic boxes (30 30 32.5 cm) were inserted ca. 30 cm below the surface of the soil (2.5 cm was aboveground). Subsequently, the soil of one box was inoculated with 250 g of air-dried and sieved (2 mm) AM inocula (þM), while the other was inoculated with an equivalent amount of nonmycorrhizal inoculum (M). The tested AM inocula contained only one AM strain, Glomus caledonium (Nicol. & Gerd.) Trappe & Gerdemann 90036, which was also isolated from a fluvo-aquic soil in Hennan Province, China (Liao et al., 2003). As a mixture of rhizospheric soil containing spores, hyphae, and mycorrhizal root fragments, AM inocula were propagated on white clover (Trifolium repens L.) grown in a sandy soil in pots for two successive propagation cycles (4 months each). The nonmycorrhizal inoculum was prepared under the same conditions. Four maize seeds were sown into each box. On July 7, the maize seedlings in each plot were thinned to ca. 48,000 ha1 (Ding et al., 2007) and one seedling was left in the center of each box. On September 20, the stem length of each maize plant was measured, then the mature maize was harvested by hand, and soil samples were collected at a depth of 0–15 cm. Most maize roots in each box were also collected after harvest, while tiny maize roots left in the soil samples were removed for mycorrhizal colonization assessment. Fresh soil samples were sieved (<2 mm), removing plant materials and stones, for the analysis of alkaline phosphatase activity and microbial biomass P, while dried and ground soil samples were used for the analysis of pH, total P, and available P. 2.3. Mycorrhizal colonization and plant analysis Mycorrhizal colonization in fresh roots collected from soil samples was assessed with light microscopy by the grid-line intersect method (Giovannetti and Mosse, 1980) after clearing with 10% KOH and staining with acid fuchsin (Phillips and Hayman, 1970). Maize plants were divided into grains, shoots, and roots, and weighed after oven drying at 70 C for 48 h. All roots were thoroughly rinsed with tap water before drying, whereas tiny roots used for assessing mycorrhizal colonization were ignored in evaluating biomass. Subsamples of dried and ground grains, shoots, and roots were taken for immediate nitric-perchloric acid digestion in HNO3 (70%):HClO4 (70%) mixture (6:1 v/v) (Zhu et al., 2003), followed by molybdenum-ascorbic acid colorimetry (Hanson, 1950), to measure tissue P concentration.
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J. Hu et al. / Soil Biology & Biochemistry 41 (2009) 2460–2465
2.4. Soil chemical and biochemical property analysis
3.2. Soil microbial biomass P and alkaline phosphatase activity
Soil pH was determined with a glass electrode using a soil-towater ratio of 1:2.5. Soil total P was determined by HF-HClO4 digestion (Jackson, 1958) and molybdenum-blue colorimetry. Soil available P was determined by extraction with sodium bicarbonate followed by the molybdenum-blue method (Olsen et al., 1954). Soil microbial biomass P was determined by a chloroform fumigation– extraction method (Hedley and Stewart, 1982). Soil alkaline phosphatase activity was determined using 2 g (wet weight) aliquots of the soil according to the method of Tabatabai (1982), and is given in units of mg p-nitrophenol produced g1 soil 24 h1. All these results were expressed on an oven-dried soil weight basis by correcting for water content in the soil (105 C, 24 h).
Soil microbial biomass P and alkaline phosphatase activity differed significantly among fertilization regimes, in the order: Control, NK < NPK < 1/2 OM < OA (Fig. 1). These findings were consistent with soil total P and available P content, which were lower in P-deficiency treatments than in balanced-fertilization treatments (Table 1). Nevertheless, G. caledonium inoculation greatly increased soil microbial biomass P and alkaline phosphatase activity (P < 0.05) in Control, NK, and NPK treatments, while having no significant effect in OA and 1/2 OM treatments.
2.5. Statistical analysis AMF inoculation responsiveness (MIR) is expressed as percentage increase in stem length, plant biomass, total P-uptake of maize, soil available P, microbial biomass P, and alkaline phosphatase activity, and was calculated using the following equation:
MIR ¼ ½DðþMÞ DðMÞ =DðMÞ 100% where D(þM) and D(M) are data (from either plant or soil) with and without AMF inoculation respectively. The means and standard deviations of four replicates were calculated. An analysis of variance was carried out using the OneANOVA procedure with SPSS software while the comparison of mean effects was based on least significant difference multiple-comparison tests. Differences were considered significant at P < 0.05. 3. Results 3.1. Soil pH, total P, and available P contents Soil pH declined significantly (P < 0.05) under all fertilizer treatments, in the following order: Control > NK > NPK, 1/2 OM, OA (Table 1). Since the soil was characterized by low P content, fertilization except for the P-deficiency treatment (NK) significantly increased (P < 0.05) soil total P and available P content. Specifically, soil total P and available P under the OA, 1/2 OM, and NPK treatments were 0.57 and 9.80, 0.68 and 9.16, and 0.80 and 6.33 times greater, respectively, than those of the control. However, G. caledonium inoculation decreased soil pH significantly (P < 0.05) in all treatments, and increased soil available P content significantly (P < 0.05) in OA and 1/2 OM treatments. On the other hand, soil total P content tended to decrease with the inoculation of G. caledonium in most fertilizer treatments, especially with balanced fertilization.
3.3. Stem length, shoot biomass, root biomass, and crop yield Stem length, shoot biomass, root biomass, and crop yield of maize were all greatly increased (P < 0.05) by the application of OA or NPK, as well as 1/2 OM, but not by the NK treatment (Fig. 2). Specifically, the individual crop yield of maize approached zero in the NK-fertilized soils, as well as in the control soils. However, G. caledonium inoculation in the control and NK-fertilized soils resulted in significant increases (P < 0.05) in stem length and root biomass. In addition, G. caledonium inoculation produced low but demiurgic crop yield in these two long-term P-deficiency-fertilized soils. 3.4. Mycorrhizal colonization and individual P-uptake Without AMF inoculation, all maize plants were colonized by indigenous AMF, and the root colonization at harvest time was not significantly influenced by fertilizer management, while individual P-uptake by maize plant under the 1/2 OM, OA, and NPK treatments was 29, 29, and 20 times greater, respectively, than that of the control (Fig. 3). These were consistent with soil P status, particularly available P contents (Table 1). However, G. caledonium inoculation significantly increased (P < 0.05) mycorrhizal colonization only in the case of the NK treatment. Thus, the mycorrhizal colonization of maize inoculated with G. caledonium was significantly higher (P < 0.05) with NK treatment than with NPK, 1/2 OM, and OA treatments. However, individual P-uptake by maize plant was not significantly affected by G. caledonium inoculation with all fertilizer treatments. 3.5. Mycorrhizal inoculation responsiveness By way of comparison, G. caledonium inoculation had the greatest impacts in the control soils, and had greater impacts in the NK-fertilized soils than in balanced-fertilized soils. Specifically, G. caledonium inoculation in the control and NK-fertilized soils resulted in significantly higher (P < 0.05) impacts on stem length, shoot biomass, P-uptake of maize and soil alkaline phosphatase
Table 1 Soil pH, total P, and available P with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. Treatment
pH (H2O)
Total P (g kg1)
Available P (mg kg1)
M
Control NK NPK 1/2O M OA
9.1 9.0 8.8 8.8 8.7
(0.0)A (0.1)B (0.1)CD (0.1)CD (0.1)DE
0.49 0.48 0.88 0.83 0.77
(0.05)C (0.01)C (0.07)A (0.10)AB (0.03)AB
1.1 1.0 8.2 11.4 12.1
(0.6)D (0.8)D (2.1)BC (4.2)B (2.5)B
þM
Control NK NPK 1/2O M OA
8.9 8.8 8.6 8.5 8.4
(0.1)BC (0.1)CD (0.1)EF (0.2)FG (0.1)G
0.43 0.47 0.77 0.74 0.74
(0.05)C (0.10)C (0.08)AB (0.11)B (0.07)B
1.8 1.7 11.1 20.9 21.2
(0.6)CD (1.1)CD (2.5)B (10.1)A (5.1)A
OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization; þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Standard deviations are given in parentheses. Values within the same column not followed by the same letter differ significantly (P < 0.05).
J. Hu et al. / Soil Biology & Biochemistry 41 (2009) 2460–2465
b
15 -M
12
+M
ab b
b 9
a
ab
c
c
c
6
d
d
3
Soil ALP activity (mg g-1 24h-1)
Soil microbial biomass P (mg kg-1)
a
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0.75 -M
+M
a
0.60
a ab b
b
0.45
c
c
c
0.30 d
d
0.15 0.00
0 CK
NK
NPK
1/2OM
OA
Control
NK
NPK
1/2OM
OA
Fig. 1. Soil microbial biomass P (a) and alkaline phosphatase (ALP) activity (b) with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization. þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Vertical T bars indicate standard deviations. Bars not topped by the same letter indicate a significant difference in values (P < 0.05).
activity, than in balanced-fertilized soils (Table 2). Incidentally, changes in stem length, shoot biomass, crop yield and total P-uptake of maize in response to G. caledonium inoculation were all insignificant in balanced-fertilized soils, including NPK, 1/2 OM, and OA. 4. Discussion 4.1. Changes in soil quality and plant growth in response to long-term fertilization In China, an estimated 43–48% of soils have low levels (<10 mg P kg1) of available P (Tang et al., 2008). As a result, P is a major constraint to crop production on either red soils or calcareous soils (Takeda et al., 2009; Shen et al., 2004), and P
-M
+M
240 Stem length (cm)
b
300
180
c bc
bc
bc
ab a
d
d e
e 120 60
Individual shoot biomass (g)
a
fertilizer is the most effective way to improve the P supply for crop production (Johnston, 1994; Lo¨bermann et al., 2007) and to increase crop yields (Saı¨dou et al., 2003; Li et al., 2005). Rational fertilization, particularly for P, is one of the most important measures to improve crop yield and at the same time protect the environment (Tang et al., 2008). In our present experiment, soil P supply capacity increased by 18-year application of OA, NPK or 1/2 OM (Table 1); hence both plant biomass and crop yield were greatly enhanced by balanced fertilization (Fig. 2). However, soil total P and available P content in these balanced-fertilization treatments were 0.53–0.55 g kg1 and 3.2–6.2 mg kg1 in 1994 (Qin et al., 1998), and were 0.60–0.66 g kg1 and 4.5–7.4 mg kg1 in 2002 (data not published). Obviously, all these levels were slightly higher than the original contents in 1989 and significantly lower than the present
0 NK
NPK
1/2OM
+M
120
ab b ab
ab
ab
90 60
c c
30
OA
c
c
20 -M
+M ab
16
a a
ab b
12
ab
8 c
d
NK
c
4
NPK
1/2OM
200 -M
+M
160 ab
b
Control
Individual crop yield (g)
Individual root biomass (g)
a -M
0
Control
c
150
a a
a
OA
a a
a
120 80 b
b
40 b
b
0
0 Control
NK
NPK
1/2OM
OA
Control
NK
NPK
1/2OM
OA
Fig. 2. Stem length (a), shoot biomass (b), root biomass (c), and crop yield (d) of maize with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization. þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Vertical T bars indicate standard deviations. Bars not topped by the same letter indicate a significant difference in values (P < 0.05).
J. Hu et al. / Soil Biology & Biochemistry 41 (2009) 2460–2465
Mycorrhizal colonization (%)
a 75
-M
a
+M
60 45 30
ab
b
b
b
b b
b
b b
15
b 600 Individual P-uptake (mg)
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-M
ab ab b
360
b
240 120 c
0
ab a
+M
480
c c
c
0
Control
NK
NPK
1/2OM
Control
OA
NK
NPK
1/2OM
OA
Fig. 3. Mycorrhizal colonization (a) and total P-uptake (b) of maize with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization. þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Vertical T bars indicate standard deviations. Bars not topped by the same letter indicate a significant difference in values (P < 0.05).
contents, suggesting that nutrient accumulation in the soils may change slowly over time. On the contrary, P-deficiency fertilization did not benefit soil P. In 1994, soil total P and available P content in control and the NK treatment were 0.48–0.49 g kg1 and 1.2–1.3 mg kg1, respectively (Qin et al., 1998). However, the soil available P content was significantly lower than the primal content in 1989 and slightly higher than the present content, suggesting that soil fertility degradation is very quick under P-deficiency fertilization treatment. As a result, crops growing in soil that had received no P for 18 years were clearly P limited (Hu et al., 2009), and the crop yields in plastic box studies approached zero in this experiment. Similarly, soil microbial biomass P and alkaline phosphatase activity did not respond to N and K fertilization (Fig. 1). Since soil microorganisms are critical to the maintenance of soil function because of their contributions to the biogeochemical cycling of many elements, understanding soil microbial ecology is increasingly recognized as important for the restoration and sustainability of ecosystems (Steenwerth et al., 2002; Potthoff et al., 2006). Chu et al. (2007) studied soil microbial properties in the same field plots, and observed that balanced fertilization resulted in higher soil microbial biomass C and dehydrogenase activity than P-deficiency fertilization, findings which were consistent with crop yield as reported by Cai and Qin (2006). These results demonstrated the benefit of balanced fertilization, as well as the role of P, in maintaining soil P status, promoting biomass and activity of microorganisms, as well as crop yield and P-uptake. 4.2. Changes in plant growth and P-uptake in response to mycorrhizal inoculation With the current tendency for a reduced use of agrochemicals, research is currently aimed at crop yield improvement and at yield sustainability; thus, microbial-based approaches have been proposed to improve crop yield (Covacevich et al., 2007). For instance, improving plant uptake of P from soil is an obvious alternative to the management of low P soils and the enhancement of use of
P fertilizers (Zhu et al., 2003). Plant traits that can influence P-uptake efficiency include rhizosphere acidification, root exudation of organic anions, root morphology, uptake kinetics and mycorrhizal association (Smith et al., 1999). However, a mutualistic association between plants and AMF is widespread and of particular importance in improving plant P-uptake efficiency (Smith and Read, 1997; Bush, 2008). Since the beneficial effects of AMF are due mainly to the ability of hyphae to acquire P well beyond the limits of the rhizosphere depletion zone (Li et al., 1991), mycorrhizal roots acquire P more efficiently than nonmycorrhizal roots, especially at low soil fertility levels (Bush, 2008). It has been widely reported that responsiveness of host plants to (or dependency on) mycorrhizal colonization, in terms of improved growth and/or P-uptake, varies among crop species and is sensitive to soil P availability (Smith and Read, 1997; Zhu et al., 2003). Our previous findings also indicated that mycorrhizal plants are more dependent on AMF in P-poor soils than in P-fertilized soils (Hu et al., 2009). Mechanisms causing increased P-uptake upon AMF inoculation are not fully understood, but seem to be due to rhizosphere acidification, alkaline phosphatase activity elevation, and available P augmentation in this field experiment (Table 1; Fig. 2). Since P additions increase the amounts of easily soluble P in the soils, P fertilizers may adversely affect indigenous and/or exotic AMF diasporas and their activities (Mårtensson and Carlgren, 1994; Hu et al., 2009). Consequently, AMF inoculation did not accelerate mycorrhizal colonization significantly in P-fertilized soils in our present study (Fig. 3). Our results emphasized the role of AMF inoculation in enhancing soil microbial biomass P and alkaline phosphatase activity, promoting plant biomass and crop yield, as well as total P-uptake of maize in long-term P-deficiency-fertilized soils. Nevertheless, evaluation of the importance of inoculated AMF for plant growth needs to take into account the molecular identification of the colonized AMF in root systems since mycorrhizal colonization may not reflect the mutualistic efficiency of an inoculated AMF in terms of enhancement of host growth and P-uptake, especially in unsterilized soils containing indigenous AMF.
Table 2 Mycorrhizal inoculation responsiveness (MIR) of maize and soil in the field as affected by long-term (18-year) fertilizer management (%). Treatment
Stem length
Shoot biomass
Root biomass
P-uptake
Available P
Microbial P
ALP activity
Control NK NPK 1/2O M OA
29 17 4 1 5
142 87 8 9 3
208 134 11 3 5
317 229 20 9 32
83 81 63 79 74
100 89 40 21 18
68 70 21 13 3
(6)A (2)B (7)C (8)C (2)C
(65)A (19)B (27)C (27)C (10)C
(177)A (22)AB (52)B (31)B (36)B
(111)A (115)A (55)B (15)B (64)B
(61)A (39)A (72)A (20)A (15)A
(27)A (55)A (29)AB (32)B (14)B
(28)A (19)A (14)B (8)B (14)B
OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization; ALP, alkaline phosphatase. Standard deviations are given in parentheses. Values within the same column not followed by the same letter differ significantly (P < 0.05).
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5. Conclusion P-fertilization greatly increased the P supply of soils, therefore, both plant biomass and crop yield of maize were greatly enhanced by the application of OA or balanced mineral fertilizer. In contrast, maize grown in soil that had received no P for 18 years were clearly P limited, and the individual crop yield approached zero in this experiment. However, soil microbial biomass P and alkaline phosphatase activity did not respond to N and K fertilization, either. Compared to the inoculation in balanced-fertilized soils, G. caledonium inoculation in NK and the control soils resulted in higher responsiveness of soil microbial biomass P, soil alkaline phosphatase activity, plant biomass, crop yield and P-uptake of maize, suggesting that AMF inoculation is likely more efficient in long-term P-deficiency-fertilized soils. Acknowledgements This work was supported by the Knowledge Innovation Program of Chinese Academy of Sciences (Kzcx2-yw-408, ISSASIP0703, Kscx1-yw-09-05) and the National Basic Research Program of China (2005CB121108). We are grateful to Shengwu Qin, Linyun Zhou, Jinfang Wang, and Jian Liu, of the Institute of Soil Science, Chinese Academy of Sciences (ISSCAS), for their excellent field management and kind support with field experiments. We would like to thank Dr. Linda Cameron and Dr. Haiyan Chu of Queen’s University, for their great assistance in manuscript revision. We also wish to acknowledge Dr. Rui Yin and Dr. Huayong Zhang of the ISSCAS, as well as two anonymous reviewers, whose comments and suggestions greatly improved the quality of this manuscript. References Bush, J.K., 2008. The potential role of mycorrhizae in the growth and establishment of Juniperus seedlings. In: Van Auken, O.W. (Ed.), Western North American Juniperus Communities. Springer, New York, pp. 111–130. Buyanovsky, G.A., Wagner, G.H., 1998. Changing role of cultivated land in the global carbon cycle. Biology and Fertility of Soils 27, 242–245. Cai, Z.C., Qin, S.W., 2006. Dynamics of crop yields and soil organic carbon in a longterm fertilization experiment in the Huang-Huai-Hai Plain of China. Geoderma 136, 708–715. Cavazzna, J., Volk, T., 1996. Assessing long-term impacts of increasing crop productivity on atmospheric CO2. Energy Policy 24, 403–411. Chu, H., Fujii, T., Morimoto, S., Lin, X., Yagi, K., Hu, J., Zhang, J., 2007. Soil microbial biomass, dehydrogenase activity, bacterial community structure in response to long-term fertilizer management. Soil Biology & Biochemistry 39, 2971–2976. Covacevich, F., Echeverrı´a, H.E., Aguirrezabal, L.A.N., 2007. Soil available phosphorus status determines indigenous mycorrhizal colonization of field and glasshousegrown spring wheat from Argentina. Applied Soil Ecology 35, 1–9. Ding, W., Meng, L., Yin, L., Cai, Z., Zheng, X., 2007. CO2 emission in an intensively cultivated loam as affected by long-term application of organic manure and nitrogen fertilizer. Soil Biology & Biochemistry 39, 669–679. Douds, D.D., Millner, P.D., 1999. Biodiversity of arbuscular mycorrhizal fungi in agroecosystems. Agriculture, Ecosystem & Environment 74, 77–93. Feng, K., Lu, H.M., Sheng, H.J., Wang, X.L., Mao, J., 2004. Effect of organic ligands on biological availability of inorganic phosphorus in soils. Pedosphere 14, 85–92. Gaur, A., Adholeya, A., 2004. Prospects of arbuscular mycorrhizal fungi in phytoremediation of heavy metal contaminated soils. Current Science 86, 528–534. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular–arbuscular mycorrhizal infection in roots. New Phytologist 84, 489–500. Glendining, M.J., Powlson, D.S., Poulton, P.R., Bradbury, N.J., Palazzo, D., Li, X., 1996. The effects of long-term applications of inorganic nitrogen fertilizer on soil nitrogen in the Broadbalk Wheat Experiment. The Journal of Agricultural Science 127, 347–363. Hanson, W.C., 1950. The photometric determination of phosphorus in fertilisers using the phosphovanado–molybdate complex. Journal of the Science of Food and Agriculture 1, 172–173. Hedley, M.J., Stewart, J.W.B., 1982. Method to measure microbial phosphate in soil. Soil Biology & Biochemistry 14, 337–385. Hu, J., Lin, X., Wang, J., Chu, H., Yin, R., Zhang, J., 2009. Population size and specific potential of P-mineralizing and -solubilizing bacteria under long-term P-deficiency fertilization in a sandy loam soil. Pedobiologia. doi:10.1016/j.pedobi.2009.02.002. Jackson, M.L., 1958. Soil Chemical Analysis. Prentice-Hall, Inc., Englewood Cliffs, NJ, pp. 111–133.
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Arbuscular mycorrhizal fungus enhances crop yield and P-uptake of maize (Zea mays L.): A field case study on a sandy loam soil as affected by long-term P-deficiency fertilization Junli Hu, Xiangui Lin*, Junhua Wang, Jue Dai, Xiangchao Cui, Ruirui Chen, Jiabao Zhang State Key Laboratory of Soil and Sustainable Agriculture, Joint Open Laboratory of Soil and the Environment, Hongkong Baptist University & Institute of Soil Science, Chinese Academy of Sciences, East Beijing Road 71, Nanjing 210008, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2009 Received in revised form 28 August 2009 Accepted 1 September 2009 Available online 15 September 2009
The P efficiency, crop yield, and response of maize to arbuscular mycorrhizal fungus (AMF) Glomus caledonium were tested in an experimental field with long-term (18-year) fertilizer management. The experiment included five fertilizer treatments: organic amendment (OA), half organic amendment plus half mineral fertilizer (1/2 OM), mineral fertilizer NPK, mineral fertilizer NK, and the control (without fertilization). AMF inoculation responsiveness (MIRs) of plant growth and P-uptake of maize were estimated by comparing plants grown in unsterilized soil inoculated with G. caledonium and in untreated soil containing indigenous AMF. Soil total P, available P, microbial biomass P, alkaline phosphatase activity, plant biomass, crop yield and total P-uptake of maize were all significantly increased (P < 0.05) by the application of OA, 1/2 OM, and NPK, but not by the application of NK. Specifically, the individual crop yield of maize approached zero in the NK-fertilized soils, as well as in the control soils. All maize plants were colonized by indigenous AMF, and the root colonization at harvest time was not significantly influenced by fertilization. G. caledonium inoculation increased mycorrhizal colonization significantly (P < 0.05) only with the NK treatment, and produced low but demiurgic crop yield in the control and NK-fertilized soils. Compared to the inoculation in balanced-fertilized soils, G. caledonium inoculation in either the NK-fertilized soils or the control soils had significantly greater (P < 0.05) impacts on soil alkaline phosphatase activity, stem length, plant biomass, and total P-uptake of maize, indicating that AMF inoculation was likely more efficient in extremely P-limited soils. These results also showed that balanced mineral fertilizers and organic amendments did not differ significantly in their effects on MIRs in these soils. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: AMF inoculation responsiveness Glomus caledonium Mineral fertilizer Organic amendment P efficiency Soil alkaline phosphatase Soil microbial biomass P
1. Introduction Organic and inorganic fertilizers are used primarily to increase nutrient availability to plants (Chu et al., 2007). Traditional organic fertilizers are by-products of the food and agricultural industries, which are carbon-based and decomposed by soil microorganisms to release nutrients gradually in time with plant growth. In China, there are concerns about soil fertility degradation by replacement of organic fertilizers with inorganic fertilizers. Thus, a number of longterm experiments were set up in agricultural regions at the end of the 1980s to monitor changes in soil fertility with application of either inorganic fertilizers or organic amendments or a mixed application of both (Cai and Qin, 2006). Results obtained from long-term experiments have already substantially improved our knowledge of
* Corresponding author. Tel.: þ86 25 86881589; fax: þ86 25 86881000. E-mail addresses: [email protected] (J. Hu), [email protected] (X. Lin). 0038-0717/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2009.09.002
changes in soil productivity with various fertilization practices. There are many reports showing that soil fertility has declined with continuous application of inorganic fertilizers; therefore, intensive cropping with no return of crop residues and other organic inputs is assumed to be a non-sustainable practice (Singh et al., 2004). On the other hand, there are also numerous reports showing that balanced application of inorganic fertilizers maintains soil productivity and increases crop yields (Rasmussen and Collins, 1991; Cavazzna and Volk, 1996; Glendining et al., 1996; Buyanovsky and Wagner, 1998; Kanchikerimath and Singh, 2001; Shen et al., 2004; Cai and Qin, 2006). Thus, balanced fertilization of major plant nutrient elements could be beneficial for the growth of plant aboveground parts and roots (Chu et al., 2007). However, farmers are often forced to make decisions about their fertilizer strategy that reflect economic rather than agronomic pressure. As a result, unbalanced fertilization is unfortunately widespread (Chu et al., 2007). Phosphorus (P) is one of the major essential macronutrients limiting plant growth owing to its low bioavailability in soils
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(Feng et al., 2004). For instance, Huang-Huai-Hai Plain, one of the most important agricultural regions in China (Yang and Janssen, 1997), is located in low reaches of the Yellow, Huai, and Hai rivers within an area of 350,000 km2 (Cai and Qin, 2006). However, the soil was extremely deficient in P – thus, crops growing in the farmland that had received no P for 18 years were clearly P limited and did not respond to nitrogen (N) and potassium (K) fertilization (Hu et al., 2009), and both wheat (Triticum aestivum L.) and maize (Zea mays L.) yields were always significantly lower in P-deficient soils than in P-fertilized soils (Cai and Qin, 2006). Despite large research efforts devoted to increasing the P availability to plants, several processes of the P cycle in soils remain obscure (Song et al., 2002). Microorganisms able to solubilize and mineralize P pools in soils are considered to be vital (Rodrı´guez and Fraga, 1999), since they can contribute to the efficiency of plants at acquiring P from the soil (Oliveira et al., 2009). Among other soil microorganisms, arbuscular mycorrhizal fungi (AMF), as ubiquitous mutualists found in both natural and agricultural ecosystems, provide a direct link between soil and roots, and are renowned for their ability to increase plant mineral nutrients, notably P (Leyval et al., 1997; Gaur and Adholeya, 2004; Bush, 2008). Consequently, although the dry matter production of plants is independent of the presence of AMF diasporas, another plant-production related parameter, estimated as the plant uptake of P, is dependent on the presence of AMF diasporas (Mårtensson and Carlgren, 1994). AMF may not always play a vital role in the nutrition and growth of plants in many agricultural ecosystems, especially high-input agriculture (Ryan and Graham, 2002). High available soil P may be detrimental to mycorrhizal colonization and may limit their benefits in agroecosystems because of the large application of readily soluble fertilizers. Obviously, agricultural practices such as the use of fertilizers may have severe impacts on AMF diasporas and activities (Douds and Millner, 1999; Hu et al., 2009). As a result, investigating long-term effects of fertilization on AMF inoculation efficiency may help to ensure an opportunity for the utilization of AMF in agroecosystems. We hypothesized that AMF inoculation could alter the P efficiency of agroecosystems and play an important role in enhancing maize growing in soils under P-deficiency fertilization relative to balanced fertilization. The objective of our study was to investigate AMF inoculation impacts on plant biomass, crop yield, and P-uptake of maize, as well as mycorrhizal colonization, soil microbial biomass P, and soil alkaline phosphatase activity, in an experimental field with long-term (18-year) fertilizer management. This work may contribute to our understanding of the impacts of fertilization on soil microorganisms and soil health, and to developing AMF application schemes based on specific functions in arable soils. 2. Materials and methods 2.1. Experimental field description The long-term field fertilizer experiment was established in September 1989, in the Fengqiu Agro-Ecological Experimental Station (35 000 N, 114 240 E) of the Chinese Academy of Sciences, Henan Province, China. The soil was derived from alluvial sediments of the Yellow River and is classified as an aquic inceptisol (a calcareous, fluvo-aquic sandy loam). The surface soil contained 4.5 g kg1 of organic C, 0.45 g kg1 of total N and 9.51 mg kg1 of available N, 0.50 g kg1 of total P and 1.93 mg kg1 of available P, 18.6 g kg1 of total K and 78.8 mg kg1 of available K, and had a pH (H2O) of 8.6 at the beginning of the fertilizer experiment. Five treatments (four replicates of each) were established in completely randomized blocks in 20 plots (9.5 5 m) under a rotation of winter wheat and summer maize: organic amendment (OA), half organic amendment plus half mineral fertilizer (1/2 OM), mineral NPK fertilizer (NPK), mineral NK fertilizer (NK), and the control (without fertilization). For NPK
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treatment, N, P, and K were applied in the form of urea (300 kg N ha1 per year), super phosphate (60 kg P ha1 per year), and potassium sulfate (250 kg K ha1 per year), respectively, while no P was applied for the NK treatment. The OA and 1/2 OM treatments were designed to give the same application rates of N, P, and K as those given in the NPK treatment. The organic amendment was a composted mixture of wheat straw, oil cake, and cotton cake in a ratio of 100:40:45. All P, K, and organic amendments were applied as basal fertilizers, whereas urea was added in two applications as both basal and supplementary fertilizers. Basal fertilizers were evenly broadcast onto the soil surface and immediately incorporated into the plowed layer by tillage before sowing of maize in June and wheat in October. Supplementary fertilizer urea was also applied to the soil surface and brought into the plowed layer with irrigation water. Detailed information on the experimental design and field management has been described by Meng et al. (2005). Each plot received the same fertilizer management every year from 1989 to 2008. 2.2. AMF inoculation and maize harvest A micro-plot method was used to determine AMF inoculation efficiency in each replicate plot of each treatment in 2008. On June 6, maize was directly sown by hand. The distances between rows and hills were 70 cm and 30 cm, respectively. In each plot, two square polyvinyl chloride (PVC) plastic boxes (30 30 32.5 cm) were inserted ca. 30 cm below the surface of the soil (2.5 cm was aboveground). Subsequently, the soil of one box was inoculated with 250 g of air-dried and sieved (2 mm) AM inocula (þM), while the other was inoculated with an equivalent amount of nonmycorrhizal inoculum (M). The tested AM inocula contained only one AM strain, Glomus caledonium (Nicol. & Gerd.) Trappe & Gerdemann 90036, which was also isolated from a fluvo-aquic soil in Hennan Province, China (Liao et al., 2003). As a mixture of rhizospheric soil containing spores, hyphae, and mycorrhizal root fragments, AM inocula were propagated on white clover (Trifolium repens L.) grown in a sandy soil in pots for two successive propagation cycles (4 months each). The nonmycorrhizal inoculum was prepared under the same conditions. Four maize seeds were sown into each box. On July 7, the maize seedlings in each plot were thinned to ca. 48,000 ha1 (Ding et al., 2007) and one seedling was left in the center of each box. On September 20, the stem length of each maize plant was measured, then the mature maize was harvested by hand, and soil samples were collected at a depth of 0–15 cm. Most maize roots in each box were also collected after harvest, while tiny maize roots left in the soil samples were removed for mycorrhizal colonization assessment. Fresh soil samples were sieved (<2 mm), removing plant materials and stones, for the analysis of alkaline phosphatase activity and microbial biomass P, while dried and ground soil samples were used for the analysis of pH, total P, and available P. 2.3. Mycorrhizal colonization and plant analysis Mycorrhizal colonization in fresh roots collected from soil samples was assessed with light microscopy by the grid-line intersect method (Giovannetti and Mosse, 1980) after clearing with 10% KOH and staining with acid fuchsin (Phillips and Hayman, 1970). Maize plants were divided into grains, shoots, and roots, and weighed after oven drying at 70 C for 48 h. All roots were thoroughly rinsed with tap water before drying, whereas tiny roots used for assessing mycorrhizal colonization were ignored in evaluating biomass. Subsamples of dried and ground grains, shoots, and roots were taken for immediate nitric-perchloric acid digestion in HNO3 (70%):HClO4 (70%) mixture (6:1 v/v) (Zhu et al., 2003), followed by molybdenum-ascorbic acid colorimetry (Hanson, 1950), to measure tissue P concentration.
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2.4. Soil chemical and biochemical property analysis
3.2. Soil microbial biomass P and alkaline phosphatase activity
Soil pH was determined with a glass electrode using a soil-towater ratio of 1:2.5. Soil total P was determined by HF-HClO4 digestion (Jackson, 1958) and molybdenum-blue colorimetry. Soil available P was determined by extraction with sodium bicarbonate followed by the molybdenum-blue method (Olsen et al., 1954). Soil microbial biomass P was determined by a chloroform fumigation– extraction method (Hedley and Stewart, 1982). Soil alkaline phosphatase activity was determined using 2 g (wet weight) aliquots of the soil according to the method of Tabatabai (1982), and is given in units of mg p-nitrophenol produced g1 soil 24 h1. All these results were expressed on an oven-dried soil weight basis by correcting for water content in the soil (105 C, 24 h).
Soil microbial biomass P and alkaline phosphatase activity differed significantly among fertilization regimes, in the order: Control, NK < NPK < 1/2 OM < OA (Fig. 1). These findings were consistent with soil total P and available P content, which were lower in P-deficiency treatments than in balanced-fertilization treatments (Table 1). Nevertheless, G. caledonium inoculation greatly increased soil microbial biomass P and alkaline phosphatase activity (P < 0.05) in Control, NK, and NPK treatments, while having no significant effect in OA and 1/2 OM treatments.
2.5. Statistical analysis AMF inoculation responsiveness (MIR) is expressed as percentage increase in stem length, plant biomass, total P-uptake of maize, soil available P, microbial biomass P, and alkaline phosphatase activity, and was calculated using the following equation:
MIR ¼ ½DðþMÞ DðMÞ =DðMÞ 100% where D(þM) and D(M) are data (from either plant or soil) with and without AMF inoculation respectively. The means and standard deviations of four replicates were calculated. An analysis of variance was carried out using the OneANOVA procedure with SPSS software while the comparison of mean effects was based on least significant difference multiple-comparison tests. Differences were considered significant at P < 0.05. 3. Results 3.1. Soil pH, total P, and available P contents Soil pH declined significantly (P < 0.05) under all fertilizer treatments, in the following order: Control > NK > NPK, 1/2 OM, OA (Table 1). Since the soil was characterized by low P content, fertilization except for the P-deficiency treatment (NK) significantly increased (P < 0.05) soil total P and available P content. Specifically, soil total P and available P under the OA, 1/2 OM, and NPK treatments were 0.57 and 9.80, 0.68 and 9.16, and 0.80 and 6.33 times greater, respectively, than those of the control. However, G. caledonium inoculation decreased soil pH significantly (P < 0.05) in all treatments, and increased soil available P content significantly (P < 0.05) in OA and 1/2 OM treatments. On the other hand, soil total P content tended to decrease with the inoculation of G. caledonium in most fertilizer treatments, especially with balanced fertilization.
3.3. Stem length, shoot biomass, root biomass, and crop yield Stem length, shoot biomass, root biomass, and crop yield of maize were all greatly increased (P < 0.05) by the application of OA or NPK, as well as 1/2 OM, but not by the NK treatment (Fig. 2). Specifically, the individual crop yield of maize approached zero in the NK-fertilized soils, as well as in the control soils. However, G. caledonium inoculation in the control and NK-fertilized soils resulted in significant increases (P < 0.05) in stem length and root biomass. In addition, G. caledonium inoculation produced low but demiurgic crop yield in these two long-term P-deficiency-fertilized soils. 3.4. Mycorrhizal colonization and individual P-uptake Without AMF inoculation, all maize plants were colonized by indigenous AMF, and the root colonization at harvest time was not significantly influenced by fertilizer management, while individual P-uptake by maize plant under the 1/2 OM, OA, and NPK treatments was 29, 29, and 20 times greater, respectively, than that of the control (Fig. 3). These were consistent with soil P status, particularly available P contents (Table 1). However, G. caledonium inoculation significantly increased (P < 0.05) mycorrhizal colonization only in the case of the NK treatment. Thus, the mycorrhizal colonization of maize inoculated with G. caledonium was significantly higher (P < 0.05) with NK treatment than with NPK, 1/2 OM, and OA treatments. However, individual P-uptake by maize plant was not significantly affected by G. caledonium inoculation with all fertilizer treatments. 3.5. Mycorrhizal inoculation responsiveness By way of comparison, G. caledonium inoculation had the greatest impacts in the control soils, and had greater impacts in the NK-fertilized soils than in balanced-fertilized soils. Specifically, G. caledonium inoculation in the control and NK-fertilized soils resulted in significantly higher (P < 0.05) impacts on stem length, shoot biomass, P-uptake of maize and soil alkaline phosphatase
Table 1 Soil pH, total P, and available P with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. Treatment
pH (H2O)
Total P (g kg1)
Available P (mg kg1)
M
Control NK NPK 1/2O M OA
9.1 9.0 8.8 8.8 8.7
(0.0)A (0.1)B (0.1)CD (0.1)CD (0.1)DE
0.49 0.48 0.88 0.83 0.77
(0.05)C (0.01)C (0.07)A (0.10)AB (0.03)AB
1.1 1.0 8.2 11.4 12.1
(0.6)D (0.8)D (2.1)BC (4.2)B (2.5)B
þM
Control NK NPK 1/2O M OA
8.9 8.8 8.6 8.5 8.4
(0.1)BC (0.1)CD (0.1)EF (0.2)FG (0.1)G
0.43 0.47 0.77 0.74 0.74
(0.05)C (0.10)C (0.08)AB (0.11)B (0.07)B
1.8 1.7 11.1 20.9 21.2
(0.6)CD (1.1)CD (2.5)B (10.1)A (5.1)A
OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization; þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Standard deviations are given in parentheses. Values within the same column not followed by the same letter differ significantly (P < 0.05).
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b
15 -M
12
+M
ab b
b 9
a
ab
c
c
c
6
d
d
3
Soil ALP activity (mg g-1 24h-1)
Soil microbial biomass P (mg kg-1)
a
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0.75 -M
+M
a
0.60
a ab b
b
0.45
c
c
c
0.30 d
d
0.15 0.00
0 CK
NK
NPK
1/2OM
OA
Control
NK
NPK
1/2OM
OA
Fig. 1. Soil microbial biomass P (a) and alkaline phosphatase (ALP) activity (b) with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization. þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Vertical T bars indicate standard deviations. Bars not topped by the same letter indicate a significant difference in values (P < 0.05).
activity, than in balanced-fertilized soils (Table 2). Incidentally, changes in stem length, shoot biomass, crop yield and total P-uptake of maize in response to G. caledonium inoculation were all insignificant in balanced-fertilized soils, including NPK, 1/2 OM, and OA. 4. Discussion 4.1. Changes in soil quality and plant growth in response to long-term fertilization In China, an estimated 43–48% of soils have low levels (<10 mg P kg1) of available P (Tang et al., 2008). As a result, P is a major constraint to crop production on either red soils or calcareous soils (Takeda et al., 2009; Shen et al., 2004), and P
-M
+M
240 Stem length (cm)
b
300
180
c bc
bc
bc
ab a
d
d e
e 120 60
Individual shoot biomass (g)
a
fertilizer is the most effective way to improve the P supply for crop production (Johnston, 1994; Lo¨bermann et al., 2007) and to increase crop yields (Saı¨dou et al., 2003; Li et al., 2005). Rational fertilization, particularly for P, is one of the most important measures to improve crop yield and at the same time protect the environment (Tang et al., 2008). In our present experiment, soil P supply capacity increased by 18-year application of OA, NPK or 1/2 OM (Table 1); hence both plant biomass and crop yield were greatly enhanced by balanced fertilization (Fig. 2). However, soil total P and available P content in these balanced-fertilization treatments were 0.53–0.55 g kg1 and 3.2–6.2 mg kg1 in 1994 (Qin et al., 1998), and were 0.60–0.66 g kg1 and 4.5–7.4 mg kg1 in 2002 (data not published). Obviously, all these levels were slightly higher than the original contents in 1989 and significantly lower than the present
0 NK
NPK
1/2OM
+M
120
ab b ab
ab
ab
90 60
c c
30
OA
c
c
20 -M
+M ab
16
a a
ab b
12
ab
8 c
d
NK
c
4
NPK
1/2OM
200 -M
+M
160 ab
b
Control
Individual crop yield (g)
Individual root biomass (g)
a -M
0
Control
c
150
a a
a
OA
a a
a
120 80 b
b
40 b
b
0
0 Control
NK
NPK
1/2OM
OA
Control
NK
NPK
1/2OM
OA
Fig. 2. Stem length (a), shoot biomass (b), root biomass (c), and crop yield (d) of maize with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization. þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Vertical T bars indicate standard deviations. Bars not topped by the same letter indicate a significant difference in values (P < 0.05).
J. Hu et al. / Soil Biology & Biochemistry 41 (2009) 2460–2465
Mycorrhizal colonization (%)
a 75
-M
a
+M
60 45 30
ab
b
b
b
b b
b
b b
15
b 600 Individual P-uptake (mg)
2464
-M
ab ab b
360
b
240 120 c
0
ab a
+M
480
c c
c
0
Control
NK
NPK
1/2OM
Control
OA
NK
NPK
1/2OM
OA
Fig. 3. Mycorrhizal colonization (a) and total P-uptake (b) of maize with or without Glomus caledonium inoculation in the field as affected by long-term (18-year) fertilizer management. OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization. þM, Glomus caledonium inoculation; M, nonmycorrhizal inoculum. Vertical T bars indicate standard deviations. Bars not topped by the same letter indicate a significant difference in values (P < 0.05).
contents, suggesting that nutrient accumulation in the soils may change slowly over time. On the contrary, P-deficiency fertilization did not benefit soil P. In 1994, soil total P and available P content in control and the NK treatment were 0.48–0.49 g kg1 and 1.2–1.3 mg kg1, respectively (Qin et al., 1998). However, the soil available P content was significantly lower than the primal content in 1989 and slightly higher than the present content, suggesting that soil fertility degradation is very quick under P-deficiency fertilization treatment. As a result, crops growing in soil that had received no P for 18 years were clearly P limited (Hu et al., 2009), and the crop yields in plastic box studies approached zero in this experiment. Similarly, soil microbial biomass P and alkaline phosphatase activity did not respond to N and K fertilization (Fig. 1). Since soil microorganisms are critical to the maintenance of soil function because of their contributions to the biogeochemical cycling of many elements, understanding soil microbial ecology is increasingly recognized as important for the restoration and sustainability of ecosystems (Steenwerth et al., 2002; Potthoff et al., 2006). Chu et al. (2007) studied soil microbial properties in the same field plots, and observed that balanced fertilization resulted in higher soil microbial biomass C and dehydrogenase activity than P-deficiency fertilization, findings which were consistent with crop yield as reported by Cai and Qin (2006). These results demonstrated the benefit of balanced fertilization, as well as the role of P, in maintaining soil P status, promoting biomass and activity of microorganisms, as well as crop yield and P-uptake. 4.2. Changes in plant growth and P-uptake in response to mycorrhizal inoculation With the current tendency for a reduced use of agrochemicals, research is currently aimed at crop yield improvement and at yield sustainability; thus, microbial-based approaches have been proposed to improve crop yield (Covacevich et al., 2007). For instance, improving plant uptake of P from soil is an obvious alternative to the management of low P soils and the enhancement of use of
P fertilizers (Zhu et al., 2003). Plant traits that can influence P-uptake efficiency include rhizosphere acidification, root exudation of organic anions, root morphology, uptake kinetics and mycorrhizal association (Smith et al., 1999). However, a mutualistic association between plants and AMF is widespread and of particular importance in improving plant P-uptake efficiency (Smith and Read, 1997; Bush, 2008). Since the beneficial effects of AMF are due mainly to the ability of hyphae to acquire P well beyond the limits of the rhizosphere depletion zone (Li et al., 1991), mycorrhizal roots acquire P more efficiently than nonmycorrhizal roots, especially at low soil fertility levels (Bush, 2008). It has been widely reported that responsiveness of host plants to (or dependency on) mycorrhizal colonization, in terms of improved growth and/or P-uptake, varies among crop species and is sensitive to soil P availability (Smith and Read, 1997; Zhu et al., 2003). Our previous findings also indicated that mycorrhizal plants are more dependent on AMF in P-poor soils than in P-fertilized soils (Hu et al., 2009). Mechanisms causing increased P-uptake upon AMF inoculation are not fully understood, but seem to be due to rhizosphere acidification, alkaline phosphatase activity elevation, and available P augmentation in this field experiment (Table 1; Fig. 2). Since P additions increase the amounts of easily soluble P in the soils, P fertilizers may adversely affect indigenous and/or exotic AMF diasporas and their activities (Mårtensson and Carlgren, 1994; Hu et al., 2009). Consequently, AMF inoculation did not accelerate mycorrhizal colonization significantly in P-fertilized soils in our present study (Fig. 3). Our results emphasized the role of AMF inoculation in enhancing soil microbial biomass P and alkaline phosphatase activity, promoting plant biomass and crop yield, as well as total P-uptake of maize in long-term P-deficiency-fertilized soils. Nevertheless, evaluation of the importance of inoculated AMF for plant growth needs to take into account the molecular identification of the colonized AMF in root systems since mycorrhizal colonization may not reflect the mutualistic efficiency of an inoculated AMF in terms of enhancement of host growth and P-uptake, especially in unsterilized soils containing indigenous AMF.
Table 2 Mycorrhizal inoculation responsiveness (MIR) of maize and soil in the field as affected by long-term (18-year) fertilizer management (%). Treatment
Stem length
Shoot biomass
Root biomass
P-uptake
Available P
Microbial P
ALP activity
Control NK NPK 1/2O M OA
29 17 4 1 5
142 87 8 9 3
208 134 11 3 5
317 229 20 9 32
83 81 63 79 74
100 89 40 21 18
68 70 21 13 3
(6)A (2)B (7)C (8)C (2)C
(65)A (19)B (27)C (27)C (10)C
(177)A (22)AB (52)B (31)B (36)B
(111)A (115)A (55)B (15)B (64)B
(61)A (39)A (72)A (20)A (15)A
(27)A (55)A (29)AB (32)B (14)B
(28)A (19)A (14)B (8)B (14)B
OA, organic amendment; 1/2 OM, half organic amendment plus half mineral fertilizer; NPK, mineral NPK fertilizer; NK, mineral NK fertilizer; control, without fertilization; ALP, alkaline phosphatase. Standard deviations are given in parentheses. Values within the same column not followed by the same letter differ significantly (P < 0.05).
J. Hu et al. / Soil Biology & Biochemistry 41 (2009) 2460–2465
5. Conclusion P-fertilization greatly increased the P supply of soils, therefore, both plant biomass and crop yield of maize were greatly enhanced by the application of OA or balanced mineral fertilizer. In contrast, maize grown in soil that had received no P for 18 years were clearly P limited, and the individual crop yield approached zero in this experiment. However, soil microbial biomass P and alkaline phosphatase activity did not respond to N and K fertilization, either. Compared to the inoculation in balanced-fertilized soils, G. caledonium inoculation in NK and the control soils resulted in higher responsiveness of soil microbial biomass P, soil alkaline phosphatase activity, plant biomass, crop yield and P-uptake of maize, suggesting that AMF inoculation is likely more efficient in long-term P-deficiency-fertilized soils. Acknowledgements This work was supported by the Knowledge Innovation Program of Chinese Academy of Sciences (Kzcx2-yw-408, ISSASIP0703, Kscx1-yw-09-05) and the National Basic Research Program of China (2005CB121108). We are grateful to Shengwu Qin, Linyun Zhou, Jinfang Wang, and Jian Liu, of the Institute of Soil Science, Chinese Academy of Sciences (ISSCAS), for their excellent field management and kind support with field experiments. We would like to thank Dr. Linda Cameron and Dr. Haiyan Chu of Queen’s University, for their great assistance in manuscript revision. We also wish to acknowledge Dr. Rui Yin and Dr. Huayong Zhang of the ISSCAS, as well as two anonymous reviewers, whose comments and suggestions greatly improved the quality of this manuscript. References Bush, J.K., 2008. The potential role of mycorrhizae in the growth and establishment of Juniperus seedlings. In: Van Auken, O.W. (Ed.), Western North American Juniperus Communities. Springer, New York, pp. 111–130. Buyanovsky, G.A., Wagner, G.H., 1998. Changing role of cultivated land in the global carbon cycle. Biology and Fertility of Soils 27, 242–245. Cai, Z.C., Qin, S.W., 2006. Dynamics of crop yields and soil organic carbon in a longterm fertilization experiment in the Huang-Huai-Hai Plain of China. Geoderma 136, 708–715. Cavazzna, J., Volk, T., 1996. Assessing long-term impacts of increasing crop productivity on atmospheric CO2. Energy Policy 24, 403–411. Chu, H., Fujii, T., Morimoto, S., Lin, X., Yagi, K., Hu, J., Zhang, J., 2007. Soil microbial biomass, dehydrogenase activity, bacterial community structure in response to long-term fertilizer management. Soil Biology & Biochemistry 39, 2971–2976. Covacevich, F., Echeverrı´a, H.E., Aguirrezabal, L.A.N., 2007. Soil available phosphorus status determines indigenous mycorrhizal colonization of field and glasshousegrown spring wheat from Argentina. Applied Soil Ecology 35, 1–9. Ding, W., Meng, L., Yin, L., Cai, Z., Zheng, X., 2007. CO2 emission in an intensively cultivated loam as affected by long-term application of organic manure and nitrogen fertilizer. Soil Biology & Biochemistry 39, 669–679. Douds, D.D., Millner, P.D., 1999. Biodiversity of arbuscular mycorrhizal fungi in agroecosystems. Agriculture, Ecosystem & Environment 74, 77–93. Feng, K., Lu, H.M., Sheng, H.J., Wang, X.L., Mao, J., 2004. Effect of organic ligands on biological availability of inorganic phosphorus in soils. Pedosphere 14, 85–92. Gaur, A., Adholeya, A., 2004. Prospects of arbuscular mycorrhizal fungi in phytoremediation of heavy metal contaminated soils. Current Science 86, 528–534. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular–arbuscular mycorrhizal infection in roots. New Phytologist 84, 489–500. Glendining, M.J., Powlson, D.S., Poulton, P.R., Bradbury, N.J., Palazzo, D., Li, X., 1996. The effects of long-term applications of inorganic nitrogen fertilizer on soil nitrogen in the Broadbalk Wheat Experiment. The Journal of Agricultural Science 127, 347–363. Hanson, W.C., 1950. The photometric determination of phosphorus in fertilisers using the phosphovanado–molybdate complex. Journal of the Science of Food and Agriculture 1, 172–173. Hedley, M.J., Stewart, J.W.B., 1982. Method to measure microbial phosphate in soil. Soil Biology & Biochemistry 14, 337–385. Hu, J., Lin, X., Wang, J., Chu, H., Yin, R., Zhang, J., 2009. Population size and specific potential of P-mineralizing and -solubilizing bacteria under long-term P-deficiency fertilization in a sandy loam soil. Pedobiologia. doi:10.1016/j.pedobi.2009.02.002. Jackson, M.L., 1958. Soil Chemical Analysis. Prentice-Hall, Inc., Englewood Cliffs, NJ, pp. 111–133.
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