Effect of cropping systems and arbuscular mycorrhizal fungi on soil microbial activity and root nodule nitrogenase

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Effect of cropping systems and arbuscular mycorrhizal

fungi on soil microbial activity and root nodule nitrogenase

Mohammad Javad Zarea



, Nasrin Karimi


, Ebrahim Mohammadi Goltapeh




Amir Ghalavand



Department of Agronomy and Plant Breeding, Faculty of Agriculture, Ilam University, P.O. Box 69315516, Ilam, Iran

bDepartment of Plant Pathology, Faculty of Agriculture, Tarbiat Modarres University, Tehran, Iran c

Department of Agronomy, Faculty of Agriculture, Tarbiat Modarres University, Tehran, Iran Received 9 March 2011; accepted 12 April 2011

Available online 18 May 2011

KEYWORDS Nitrogenase; Microbial activity; Soil enzymes; Mixed cropping; Glomus mosseae

Abstract Forage legumes are used to enhancement soil fertility of the agro ecosystem. Under-standing effect of them on agro ecosystem soil status during when these legumes growing and after that is essential. In one experiment the effects of inoculation with the arbuscular mycorrhizal fungi (AMF), Glomus mosseae, and mixed cropping systems (MCS) on forage biomass yield, nitrogen production, nitrogenase activity and after harvesting on soil microbial activity were studied at various mixed cropping ratios of berseem clover (Trifolium alexandrinum L., B) to Persian clover (Trifolium resupinatum L., P) (B:P = 1:0, 3:1, 1:1, and 1:3). In the second experiment, the effect of treatments on soil microbial activity were studied by soil collection after clover harvesting and 8-week soil incubations in the laboratory. MCS had positive effects on root and shoot dry weight. The effects of AMF on plant yield were positive. AMF affected the fraction root and the vertical root distribution. Plants colonized by AMF showed shorter roots than control plants. At cut 1, with the AMF colonization, the greatest nitrogenase activity (79.61 lmol C2H4g dwt1h1) of root

nod-ule was observed with B:P = 3:1. At cut 2, the Persian clover plants colonized by G. mosseae in the mixed crop (1:3) had a higher nitrogenase activity (77.38 lmol C2H4g dwt1h1). The greatest

* Corresponding authors. Tel.: +98 912 2219385; fax: +98 841 2227015.

E-mail addresses:mj.zarea@ilam.ac.ir(M.J. Zarea), emgoltapeh@ modares.ac.ir,emgoltapeh@yahoo.com(E.M. Goltapeh).

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Peer review under responsibility of King Saud University. doi:10.1016/j.jssas.2011.04.003

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nitrogen accumulation in the aboveground biomass, 23.5 mg g1forage dry matter, was obtained with mixed cropping (B:P = 1:1) in the presence of the AMF colonization. Microbial activity mea-sured as substrate-induced respiration and activities of dehydrogenase, alkaline phosphatase, and acid phosphatase enzymes responded positively to AMF colonization; with the greatest activities for B:P = 1:3.

ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Intercropping offers farmers the opportunity to exploit nat-ure’s principle of diversity on their farms. The intercropping system greatly contributes to crop production by its effective utilization of resources, as compared to the monoculture crop-ping system (Inal et al., 2007). Currently, this system is attract-ing increasattract-ing interestattract-ing in low-input crop production systems and is being extensively investigated (Inal et al., 2007). Inter-specific root interactions affect nutrient mobilization in the rhi-zosphere and contribute efficiently to nutrient acquisition by intercropping (Wasaki et al., 2003). It has been well docu-mented that an important N-transfer takes place in intercrop-ping systems of legumes with cereals (Martin et al., 1991). Sharma and Gupta (2002) showed that N-nutrition of pearl millet was greatly improved by intercropping with legumes. Inter-specific facilitation of P-uptake by intercropped species has also been reported (Inal et al., 2007). These effects on P-utilization are related to the release or activation of enzymes (e.g. like acid phosphates) and root exudation of carboxylates, phosphatases, and phytase under P deficiency which improve solubility and uptake of P in rhizosphere (Neumann and

Ro¨mheld, 1999; Inal et al., 2007). Plant roots release enzymes like phosphatases (Yun and Kaeppler, 2001), phytase (Li et al.,

1997), and carboxylates (Veneklaas et al., 2003) under P defi-ciency, allowing mobilization and utilization of P in soil under P deficiency stress.

The symbiotic relationship between AMF and the roots of higher plants contributes significantly to plant nutrition and growth (Auge´, 2001), and has been shown to increase the pro-ductivity of a variety of agronomic crops. These positive re-sponses in productivity to AM colonization have mainly been attributed to the enhanced uptake by AM of relatively immobile soil ions (Marschner and Dell, 1994; Liu et al.,

2000), but also involve the enhanced uptake and transport of far more mobile nitrogen (N) ions, particularly under drought conditions (Tobar et al., 1994).

The primary benefit of promoting biological nitrogen (N2)

fixation in grain and forage legumes is the enhancement of yield potentials without the use of exogenous N fertilizers. Rhizobium plays an important role as microbial endos-ymbionts in the supply of N to legumes growing in nutrient deficient soils (Azco´n et al., 1979). In most reported AMF have had a positive influence on biological nitrogen (N2)

fix-ation in grain and forage legumes (Azco´n et al., 1979; Barea

et al., 1987, 1989, 1992, 2002). In many experiments co-inoc-ulation with both symbionts resulted in higher plant biomass and better nitrogen and phosphorus acquisition, although these effects were also dependent on the specific symbiont (Azco´n et al., 1991). Tripartite symbiotic associations were more effective than AMF or Rhizobium inoculation alone in respect to the uptake of nitrogen and phosphorus by plants (Azco´n et al., 1991).

Soil enzymes are derived primarily from soil fungi, bacteria, plant roots, microbial cells, plant, and animal residues, etc. (Brown, 1973; Cao et al., 2003; Tarafdar and Marschner,

1994) and play a significant role in mediating biochemical transformations involving organic residue decomposition and nutrient cycling in soil (Martens et al., 1992; McLatchey and Reddy, 1998). Land management and utilization methods, crop species and cultivation systems, etc. can affect soil enzymatic activity. Thus, this parameter can be used as a sensitive index to reveal changes of soil quality due to land management, and to monitor soil microorganism activity re-lated to soil nutrient transformation. Because all biochemical transformations in soil are dependent on, or related to the presence of enzymes, studying soil enzyme activities provides insight into biochemical processes in soil and may also reflect the differences in microbial dynamics and population.

AM-induced changes in plant physiology affect the micro-bial populations, both quantitatively and qualitatively, in either the rhizosphere and/or the rhizoplane. Therefore, the rhizosphere of a mycorrhizal plant can have features that differ from those of a non-mycorrhizal plant (Barea et al., 2002;

Johansson et al., 2004). Soil microbial activity has been shown to depend on the presence of AM fungi and plant species (Wamberg et al., 2003; Lo´pez-Gutie´rrez et al., 2004; Baligar

et al., 2005; Marschner and Timonen, 2006). The AM symbi-otic status changes the chemical composition of root exudates, while the development of an AM soil mycelium, which can act as a carbon source for microbial communities, introduces physical modifications into the environment surrounding the roots (Barea et al., 2005).

Berseem clover (Trifolium alexandrinum L., B) and Persian clover (Trifolium resupinatum L., P) are annual leguminous forage or cover crop species, well adapted to the semiarid con-ditions of Mediterranean areas. They are high-yielding, nutri-tious, cool-season forage crops (Knight, 1985), grown in pure stands or in mixtures with annual grass species for overwinter grazing and for harvested forage in spring (Martiniello, 1999;

Stringi et al., 1987). They can be grazed or cut for fodder 2–5 times a year, from late autumn to late spring in average seasons (if sown in early March), up to six times when irrigated, and twice as a summer crop (Zarea et al., 2008).

We report here the results of an investigation effect of AMF, Glomus mosseae, colonization and MCS on forage bio-mass yield, nitrogen production and root nodules nitrogenase activity and then, after harvesting the clovers, on soil microbial activity collected at various mixed cropping ratios of berseem clover to Persian clover. We have chosen an intercropping sys-tem including two N-fixing (legume) crops instead of a syssys-tem based on a fixing (legume), as the model in designing agricul-tural systems, to break down current intensive cropping system, wheat––wheat, that is prevalent under semi-arid areas. For better nutrient management in semi-arid areas, an increased use of the biological potential is important. It is


shown that different plant species can influence the composi-tion and number of soil microorganisms due to variacomposi-tions in the quantity and quality of root exudates (Curl and Truelove,

1986; Bowen and Rovira, 1991). Bardgett et al. (1998) and Wardle et al. (1999) have reported that the abundance, activ-ity, and composition of soil decomposer communities may vary markedly with different plant species, or specific func-tional plant groups such as legumes. Experiments of mixed versus monocultured plant species have demonstrated that plant mixtures can produce greater plant biomass (Forrester

et al., 2004), and improve nutrient cycling and soil fertility (Montagnini, 2000). The increase in plant biomass in the mix-ture may have strong effects on soil microbial community. In most terrestrial ecosystems microbial biomass and growth have been shown to increase with increasing carbon input (Spehn et al., 2000).

The hypotheses of this research were that: (1) the agro eco-system will more affected by intercropping (Persian and ber-seem clover); (2) AMF will have different effects in each intercropping system; and (3) effect of intercropping after har-vesting may will have different effects on soil properties.

2. Materials and methods 2.1. First experiment 2.1.1. Site description

Field experiments were conducted at the research farm of the Seed and Plant Improvement Institute, Karaj (54500N,

55350W, and 1312 m above sea level), Iran, during the dry

sea-son. The experimental sites were located in a semiarid region. The rainfall is restricted to five and a half months a year, from November to February, with negligible rainfall during spring and no rainfall in summer (May–August). The average maxi-mum temperature from May to July is very high (26.5– 44C), with a mean of 27 C. In October, temperature reaches a maximum of 48C in August, and falls to a minimum of 12C. The soil at the site is classified as medium black, clayey, and shallow (15–20 cm in depth).Table 1shows the physical and chemical analysis of soil across the locations before the experiment.

2.1.2. Treatments

The experimental design was a split-plot design with three ran-domized complete blocks, with the main plot treatments with or without AMF (G. mosseae) inocula, in three replications. The subplot treatments included five cropping systems (CS): stand ratios of 1:0 (84:0 plants m2), 3:1 (63:21 plants m2), 1:1 (42:42 plants m2), and 1:3 (21:63 plants m2) 0:1 (0:84 plants m2) of berseem clover to Persian clover (B:P). The clover density was 84 plants m2. Plots with AM inocu-lum received 60 g m2of mycorrhizal inoculum, (an average of 115 spores g1soil and root fragments with 85% of colo-nized roots length), applied and incorporated below the soil surface (seed bed).

Berseem and Persian clovers were hand seeded at a depth of 1.0–1.5 cm by surface broadcasting and the seed was incorpo-rated by raking. The spring conditions were very dry, so the plots were irrigated before and after seeding. The plots were hand weeded. Our previous research (unpublished) examined the correlations between the seeding densities and plant stands in both field and greenhouse environments for 2 years. The field establishment rates for the Persian and berseem clover seeds were 40% and 70% of the seed sowing rate, respectively. The clover seeding rates were based on these data, but ulti-mately, the seedlings were thinned to 84 plants m2 in the appropriate ratios. The plots, 3 m· 6 m in size, were separated by heavy gauge polythene barriers buried to a depth of 0.5 m into the soil, which stood (initially) 10–15 cm above the ground. The sowing date was May 18. The necessary plant protection, irrigation, and other management practices were followed during crop growth. No pesticides were applied. The clovers were grown with irrigation, given at 10-day inter-vals, when water to a height of 65 mm was applied. No serious incidence of insects or diseases was observed.

The AMF inoculum of G. mosseae was purchased from the Agricultural and Biotechnology Research Institute, Iran, and was a pure culture of G. mosseae isolated from Karaj (Iran). It was selected because it was commercially available in Iran and had been reported to increase the growth of some clover species. The mycorrhizal fungus inocula consisted of spores and hyphal root fragments from a stock culture of G. mos-seae. Mycorrhizal inoculum was multiplied in an open pot cul-ture of maize and consisted of soil, spores, hyphae, and AM root fragments. The field of experiment was fallow for 2 years before the experiment beginning, and we also used a wet siev-ing technique to extract the spores at the initiate the experi-ment. We also used the most probable number (MPN) test to determine the number of propagules (mL1) in the original sample. Because the mycorrhizal spore propagules extracted (1–2 per kg) with the wet sieving technique and the MPN of the propagules (0.018 cm3) from the native soil were extre-mely low, no attempt was made to fumigate the soil. The mycorrhizal colonization rate (%) of both clover species was assayed before the first and second cuttings.

2.1.3. Measurements

Crops were harvested twice by sickle at ground level, once be-fore corn were sown and twice bebe-fore wheat were sown in this area. The first cut, regrowth harvesting (cut 2), and total aboveground biomass yields were recorded. At cut 2, the observations on root dry matter development were extended by taking 10 soil cores of 30 cm deep with a diameter of 5 cm to assess root dry matter. The soil cores were cut in three

Table 1 Soil physical and chemical properties across the locations before the experiment.

Soil property

Caution exchange capacity (CEC) 8.4–11.0

pH (CaCl2) 7.1–7.8 Organic matter 1.08–1.09%, Soil chemical N% 0.63 P (mg kg1) 12.2–13.2 K (kg ha1) 152–287 Mn (mg kg1) 68.6 Cu (mg kg1) 3.1 Zn (mg kg1) 1.2 Fe (mg kg1) 270


parts of 10 cm each and pooled layer wise. Soil was washed out until clean roots remained. The roots were dried for 24 h at 70C. Samples were cut from an inner plant area of 2 m2

at 5–7.5 cm above the soil level and the clover biomasses were separated by species. Shoot samples were oven dried at 70C until daily checks indicated no further reduction in weight. The dried samples were weighed and allowed to remain as green manure on each plot. The dried herbage was ground, passed through a 0.5 mm screen, and analyzed for N concen-tration (Baruah and Barthakur, 1997).

The mycorrhizal colonization assessment was performed with the method described byBrundrett et al. (1996). We sam-pled 100:0, 75:25, 50:50, 25:75, and 0:100 intersections per root cluster, for a total of 100 intersections per sample of B:P plants from each treatment. The samples were washed in distilled water, stained with trypan blue, and the mycorrhizal coloniza-tion levels determined using the gridline intersect method of Giovanetti and Mosse (1980).

2.1.4. Acetylene reduction assay (ARA)

Acetylene reduction assay (ARA) assessment was performed with the method described by Alef and Kleiner (1995). NA on roots nodules was studied by the acetylene reduction tech-nique at cuts 1 and 2. Briefly, 100:0, 75:25, 50:50, 75:25, and 0:100 number of root noodle of the B:P was placed in vessels stopped with Suba-Seals and placed in the incubation system. Thirty nodulated root samples of both clover species, each with 3–4 mature nodules, were used. Each nodulated root sam-ple was excised from lateral roots by carefully cutting with sharp scissors late in the afternoon, leaving a sufficient length of root to avoid damage to nodules. The nodulated root sam-ples were washed in distilled water, dried, and placed in three separate gas-tight vials each containing 10% acetylene, whilst a third nodulated root sample was placed in a vial containing no acetylene to account for root ethylene production. In addition, control samples were taken, containing only roots without nodules in order to detect any non-nodule related acetylene-reduction activity by the roots. Vials containing only air and acetylene were also used to ensure the absence of ethylene in the acetylene gas. Samples were assayed after 24 h for ethylene by injection into a Hewlett–Packard gas chro-matograph with a Poropak R column (shimadzu, GC-148B, Japan). Ethylene production per hour was finally related to the dry weight of the root nodules. These observations were replicated three times in three weekly samplings. Nodule dry weight was determined after oven-drying for 24 h at 70C. Moles of ethylene produced per gram of nodules per hour of incubation period was calculated as the measure of NA. 2.2. Second experiment

2.2.1. Treatments and soil sampling

After harvesting the clovers, effect of MCS and AMF inocula on soil enzymatic was assayed. To investigate the effects of first experiment treatments on soil biological activity, soil samples of all plots were obtained from the same 0–30 cm depth. Bulk densities were not significantly different (1.16 ± 0.035) among the different plots. Therefore, treatments consisted of split-plot design with three randomized complete blocks, with the main plot treatments with or without G. mosseae inocula. Samples of soil (5 kg) were kept separately in 750 ml plastic containers in the laboratory. Soil water content of treatments soil samples

was at 30% (dry weight basis). Soils were not air-dried and stored with this moisture content. Soils stored for about 8 weeks before being sieved through a 2 mm mesh, to remove organic debris and gravel, and to destroy large aggregates. For each soil, there were three replicate containers for the various MCS.

2.2.2. Soil analysis

Substrate-induced respiration (SIR) was determined by adding 5 ml of glucose solution (32 mg/ml) to 25 g subsamples of soil had been air-dried and placing them in 1 l jars for 6 h. The car-bon dioxide (CO2) respired during this period, was trapped in

20 ml 0.02 M NaOH, and subsequently measured by titration with HCl to a phenolphthalein endpoint after adding excess BaCl2(Anderson, 1982). The activity of the ALP and ACP

en-zymes were measured by the methods described byTabatabai

(1994). Briefly, a fresh soil sample of 1 g was placed in 50 ml test tube, to which one drop of toluene and 4 ml of modified universal buffer (pH 11 for ALP and pH 6.5 for ACP) was added. The samples were incubated with p-nitrophenyl phos-phatase at 37C for 60 min. After filtration, the yellow color intensity was measured by spectrophotometer at 420 nm. The enzyme activities were expressed as mg p-nitrophenol (PNP) released per gram soil within 1 h. The measurements were car-ried out in three replicates and the activity of enzymes was averaged for all three replicates. Dehydrogenase activity was determined according toGarcı´a et al. (1997). For this, 1 g of soil was exposed to 0.2 ml of 0.4% INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride) in distilled water for 20 h at 22C in darkness. The INTF (iodo-nitrotetrazoli-umformazan) formed was extracted with 10 ml of methanol by shaking vigorously for 1 min and filtering though a What-man No. 5 filter paper. INTF was measured spectrophotomet-rically at 490 nm.

2.3. Data analysis

The analysis of variance (ANOVA) for both experiments was based on the common mixed model for a split-plot randomized complete blocks design. All measured variables were assumed to be normally distributed and statistical analysis by ANOVA was performed usingSAS software (1990). The normal distri-bution of the data was determined using the Shapiro–Wilk W test. The significance of the differences between treatments was estimated using the LSD range test, and a main effect or inter-action was deemed significant at P 6 0.05. Linear regressions were carried out using Spearman correlation coefficient.

3. Results

3.1. Mycorrhiza colonization value

Clover plants were colonized after all treatments involving inoculation with AMF. The percentage mycorrhizal root colonization was significantly (P 6 0.01) greater in all the mycorrhizal treatments than in the non-inoculated controls. Non-inoculated treatments (control) had only 4–7% coloniza-tion from indigenous mycorrhizal fungus. However by harvest-ing-time, results showed that both clovers species colonized by G. mosseae had significantly better performances on growth parameter when compared to the control treatment colonized


by indigenous mycorrhizal fungus which may relate to the extremely lower of spore propagules indigenous mycorrhizal fungus. Inoculated treatments (F = 2366.8, P 6 0.0001) had higher mycorrhizal colonization. The MCS had no effect on the mycorrhizal colonization value. Mycorrhizal coloniza-tion values of 40.9%, 45.3%, 45.6%, 44.43%, and 42% were observed for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 intersections root of the BP plants (Fig. 1).

3.2. Shoot growth

MCS and AMF had significant positive effects on forage yield (Table 2). There were no direct interactive effects between MCS and AMF on plant yield (Table 2). At cuts 1 and 2, the berseem clover was roughly taller than the Persian clover, regardless of the stand ratio. The average rates of plant height were 15–54 cm (cut 1) and 50–54 cm (cut 2) for Persian clover and berseem clover, respectively. At cuts 1 and 2, the berseem clover and Persian clover monocultures had the highest yields, and the berseem clover monoculture had the highest yield at cut 1 (Table 2). The dry matter yields of Persian clover in mixed crops were lower than the berseem clover yields in monocultures in cut 1, but the total yields of the mixtures were higher than those of the berseem clover monoculture. The total mixed cropping forage yield was highest in MCS, at 3:1 kg ha1.

At cut 2, the Persian clover monoculture had a higher yield than the berseem monoculture (Table 2). Biomass production of Persian clover was the faster developing at cut 2, mainly due to its relatively lower temperature and higher root accumula-tion. For the species represented in this study, no clear relation between total dry matter accumulation and maximum height was observed. Persian clover, the species with the higher amount of accumulated dry matter at cut 2, was the shorter species. The berseem clover forage yield was also significantly higher in the monoculture crop (1:0) than in the mixed crops in the second cut (Table 2). The maximum dry matter yield for berseem clover under MCS was obtained for 3:1, followed by 1:1. Among the Persian clover intercrop yields, the dry mat-ter yield of the Persian clover increased with reductions in the berseem clover plants but was significantly lower than that of the monoculture crop (Table 2). The same statement is true for berseem clover since dry matter yield of berseem clover in-creases with reductions in Persian clover but is lower than of

the monoculture. At the second cut (regrowth), the B:P ratio of 1:3 had the highest total forage intercrop yield (Table 2). There was no difference between the ratios 1:3 and 1:1. The to-tal yields of the mixtures (cuts 1 plus 2) were obtained for 1:1 and 1:3, followed by 3:1 (Table 2).

3.3. Root growth

Total root dry weight, the fraction root and the vertical root distribution in the upper 30 cm of the soil are presented in

Tables 3 and 4. Total root dry weight of Persian clover was significantly higher than the total root dry weight of berseem clover (Table 3).

MCS had significant positive effects on root weight produc-tion (Table 3). At cuts 1 and 2, the Persian clover monocul-tures had the highest root weight than berseem clover monoculture (Table 3). At cuts 1 and 2, the dry matter roots of berseem clover in mixed crops were higher than those of that in monoculture (Table 3). At cut 1, the total mixed crop-ping dry matter roots was highest in MCS, at 1:3 g plant1. At cut 2, the maximum root dry weight was obtained for 0:1, 1:3, 1:1, and 3:1. The total root dry matters of the mixtures (cuts 1 plus 2) were affected by various ratios of mixtures (Table 3). The B:P ratio of 1:3 had the highest total root dry mass inter-crop yield (Table 3). AM fungi had no effect on root dry mass (Table 3). There were no direct interactive effects between MCS and AMF on root biomass production (Table 3). At cuts 1 and 2, the maximum ratio of shoot to root (S/R) was ob-tained for 1:0 and 1:3, respectively. The B:P ratio of 1:3 had the highest total S/R. The ratio of S/R tended to increase with inoculating clover with AM fungus (Table 3).

The differences in vertical root distribution were observed (Table 4). Persian clover had a higher fraction (0.8) of its root material in the top soil layer (0–10 cm) than berseem clover (0.5). Berseem clover in turn had a higher fraction root dry matter in the lower zone (Table 4). Result showed the differ-ences in vertical root distribution affected by MCS (Table 4). AMF limited vertical root distribution in the upper 10 cm of the soil (Table 4). Non-mycorrhizal plants had higher root dis-tribution in the downer 10 cm of the soil. There were no direct interactive effects between MCS and AMF on root distribu-tion (Table 4).

3.4. Nitrogenase activity

The main effects of AM and MCS on NA were statistically sig-nificant at both cuts 1 and 2 (Table 5). The NA of root nodule was increased by AM and MCS at B:P = 3:1 and B:P = 1:3 at first cut and second cut, respectively. There was no significant difference between the ratios 1:3 and 1:1 at cut 2 (Table 5). The AMF colonization was correlated with nitrogenase activity (Fig. 2). There was synergism between the two clovers on nitrogenase activity (Table 5). The nitrogenase activity in ber-seem clover at cut 1 is increased by the presence of a relatively small amount of Persian clover, while at cut 2, nitrogenase activity of Persian clover is increased by a small amount of ber-seem clover.

3.5. Shoot N concentration

The main effects of AMF and MCS treatments and their inter-actions on N concentration were statistically significant

0 10 20 30 40 50 60 B B(3:1) BP(1:1) BP(1:3) P B B(3:1) BP(1:1) BP(1:3) P e a e s s o m e s u m o l G + e a e s s o m e s u m o l G

-Mycorrhiza colonization rate (%)

Figure 1 Mycorrhizal colonization rate (%) of single or mixed cropping clovers (P and/or B) inoculated or not with mycorrhiza. Means ± S.E., P < 0.05.


(Fig. 3). Shoot N concentration (23.5 mg g1) after treatment with AMF at culture ratios of B:P = 1:1 was significantly higher than that after other treatments (Fig. 3). The clear rela-tionship between N concentration and nitrogenase activity is illustrated in Fig. 4. The nodules on roots systems that containing the higher NA activity was clearly much shoot N concentration than those of nodules on root systems with less-abundant NA activity.

3.6. Microbial activity

The main effects of AMF and MCS treatments on organic matter were statistically significant (Table 6). The percent

organic matter varied with the MCS and AMF inoculant. The highest percent organic matter (1.43 was found in the %) was found at BP 1:3 and was further increased at AMF inoculant plots (Table 6).

The microbial respiration (SIR), DHA, ALP, and ACP enzymes from the incubated soils of mycorrhizal and non-mycorrhizal plants with varies cropping systems are shown in Table 6. Results indicated main treatment effects of MCS and AM fungus, and their interaction on SIR, DHA, ALP, and ACP enzymes microbial metabolic (Table 6).

The soil obtained from B:P = 1:3 had a much greater SIR (Table 6). There was no significant difference between the ra-tios 1:3 and 1:1. AMF had significant positive effects on SIR

Table 2 Dry for age yield of single or mixed cropping clovers (P and/or B) colonized or not with mycorrhiza.

Treatmentsa Cut 1 forage root yield in monoculture

and mixture (kg ha1)

Cut 1 forage root yield in monoculture and mixture (kg ha1)

Cuts 1 and 2 yields (kg ha1) Berseem clover Persian clover Mixtures total yields Berseem clover Persian clover Mixtures total yields Mixtures total yields (cuts 1 plus 2)

+AM 989.92a 726.83a 1374.8a 2608.40a 3377.67a 4788.8a 6163.7a

AM 924.15b 662.37b 1269.2b 2450.50b 3230.00b 4544.4b 5813.6b

LSD (0.05)a 19.72 19.25 8.3 138.16 129.28 213.9 209.3

F 2123.8*** 324.4*** 1075.4*** 15.8** 19.5** 40.2*** 80.7***

B(1:0) 1545.65a – 1545.6b 4123.53a – 4123.5e 5669.1c

B(3:1) 1272.2b 372.9d 1645.1a 2778.20b 1535.67d 4313.8d 5959a

BP(1:1) 756.1c 659.31c 1417.4c 2100.87c 2788.00c 4888.8b 6306.3a

BP(1:3) 254.1d 791.40b 1047.2d 1115.20d 4173.00b 5288.2a 6335.4a

P(0:1) – 954.8a 954.8e – 4718.67a 4718.6c 5673.4c

LSD (0.05) 4.39 11.02 10.7 86.09 102.91 129.21 130.6

F 160,405*** 4732.5*** 7204.1*** 1015.6*** 1837.6*** 11,545*** 55.79***

Cropping systems vs. AMb Ns Ns Ns Ns Ns Ns Ns

Different letters (a–c) within a column indicate significant differences at the 0.05 level; NS: not significant. a Least significant differences between treatments at P = 0.05.

b interaction between treatment. **Fvalues significant at P < 0.01. ***Fvalues significant at P < 0.001.

Table 3 Root dry matter (g plant1) of single or mixed cropping clovers (P and/or B) colonized or not with mycorrhiza.

Treatments Cut 1 root dry matter (g plant1) Cut 1 root dry matter (g plant1) Cuts 1 and 2 root dry

matter (g plant1) Berseem clover Persian clover Mixtures total yields Berseem clover Persian clover Mixtures total yields Mixtures total yields +AM 2.9 8.3 9 3.5 7.8 8.7 17.8 AM 2.8 8.5 9.2 3.4 7.6 8.3 17.5 LSD (0.05)a 0.3 0.6 0.45 0.25 1.6 1.3 0.9 F 1ns 0.5ns 0.15ns 0.39ns 0.97ns 2.5ns 0.2 B (1:0) 6.2a – 6.2c 5.3a – 5c 11d B (3:1) 3.4b 5.1d 8.5b 3.6b 5.7b 5. 2b 13.6c BP (1:1) 2.1c 7.6bc 9.6b 2.7c 6.5b 9.3a 18.9b

BP (1:3) 1.9c 8.7ab 10.9a 2.2d 8.9a 9.2a 20.2a

P (0:1) – 9.9a 9.9ab 9.6a 9.6a 19.6ab

LSD (0.05) 0.35 1.3 1.1 0.4 0.8 0.6 1.4

F 85.94*** 8.1*** 54.8*** 90.2*** 49.6*** 66.2*** 93.2***

Cropping systems vs. AMb Ns Ns Ns Ns Ns Ns Ns

Different letters (a–d) within a column indicate significant differences at the 0.05 level; NS: not significant. **

Fvalues significant at P < 0.01.

a Least significant differences between treatments at P = 0.05. b Interaction between treatment.


(Table 6). There were no direct interactive effects between MCS and AMF on SIR (Table 6). There was higher ALP activity in mycorrhizal treatments regardless of MCS. How-ever, B:P = 1:3 increased the activity of ALP in both non-mycorrhizal and non-mycorrhizal treatments. The activity of this enzyme was not affected by an interaction of MCS and AMF. The main treatment effects of MCS and AMF and their interaction on the activity of ACP was statistically significant (Table 6). AM fungus increased the activity of ACP, whereas MCS at B:P = 3:1 increased it regardless of AM fungal treat-ments (Table 6). Results also showed that AMF effect on ALP activity is more striking than on ACP activity (Table 6). There was a beneficial effect of both mixture cropping at B:P = 1:1 and B:P = 1:3 and AMF inoculation on DHA (Table 6).

The activity of this enzyme was not affected by an interaction of MCS and AMF.

4. Discussion 4.1. Shoot growth

When plant species are intercropped, it is likely that yield advantages occur as a result of the complementary use of resources by the component crops (Evans, 1960; Grimes

et al., 1983; Anil et al., 1998). These results show that the pro-ductions of forage berseem clover and Persian clover monocul-tures were higher than that in the mixed crop culmonocul-tures. This was partly the result of the greater plant populations in the monocrops. Moreover, these crops were not subjected to in-ter-specific competition. At the first cut, the superiority of the forage yields of berseem clover over those of Persian clover was perhaps attributable to a higher number of tillers and branches (data not shown). Despite tiller formation, the higher forage yield of the berseem clover relative to that of the Persian clover was possibly because of the more rapid accumulation of dry matter in the berseem clover. Compared with berseem clo-ver, Persian clover generally has limited growth, especially when subjected to high temperatures. The higher temperatures during cut 1 reduced the growth rate of Persian clover com-pared with that of berseem clover, indicating that berseem clo-ver better tolerates high temperatures. Furthermore, during cut 1, photosynthesis assimilation in Persian clover compared to berseem clover more served in root (data not shown) that help Persian clover regrowth.

At cut 2, the crop experienced inter-specific competition. The yield of the berseem clover was affected by the Persian clo-ver. Although the Persian clover reduced the yield of the ber-seem clover, an overall benefit was observed when the yields of both crops were considered together (1:3). The aboveground dry matter of the Persian clover regrowth increased more dur-ing the growth season because of a higher number of tillers and branches compared with those at cut 1 (data not shown).

Table 5 Root nodules nitrogenase activity of single or mixed cropping clovers (P and/or B) colonized or not with mycorrhiza.

Treatments ARA

Cut 1 Cut 1 Average cut 1 plus cut 2

+AM 67.180a 66.3a 66.7

AM 55.84b 60.19b 58 LSD (0.05)a 1.5656 4.52 1.5 F 590.31*** 13.83*** 107.1*** B(1:0) 47.01c 46.9d 46.9b B(3:1) 79.61a 64.76b 72.1a BP(1:1) 71.05b 72.86a 71.9a BP(1:3) 71.58b 77.38a 74.4a P(0:1) 38.28d 54.35c 46.3b LSD (0.05) 1.32 5.51 2.8 F 1165.8*** 47.34*** 233.5*** Cropping systems vs. AMb Ns Ns Ns

Different letters (a–d) within a column indicate significant differences at the 0.05 level; NS: not significant. a Least significant differences between treatments at P = 0.05.


Interaction between treatment. **

Fvalues significant at P < 0.01. ***

Fvalues significant at P < 0.001.

Table 4 The vertical root distribution (fraction) over the upper 30 cm of the soil.

Treatments Root distribution

0–10 cm 10–20 cm 0 20–30 cm

+AM 0.79a 0.13b 0.07b

AM 0.74b 0.22a 0.08a

LSD (0.05)a 0.02 0.04 0.004 F 184.4*** 303.7*** 658.1*** B(1:0) 0.5b 0.28a 0.12a B(3:1) 0.8a 0.14bc 0.06c BP(1:1) 0.8a 0.14c 0.06c BP(1:3) 0.8a 0.15bc 0.07b P(0:1) 0.8a 0.16b 0.07b LSD (0.05) 0.01 0.02 0.002 F 663*** 103.3*** 1064.8*** Cropping systems vs. AMb Ns Ns Ns

Different letters (a–c) within a column indicate significant differ-ences at the 0.05 level; NS: not significant.


Least significant differences between treatments at P = 0.05. b

Interaction between treatment. **

Fvalues significant at P < 0.01. ***


The total intercropping yield (cuts 1 and 2) gives an accu-rate assessment of the greater biological efficiency of the inter-cropping system. The total interinter-cropping yield values indicate that berseem clover recorded a yield advantage at B:P ratios of 1:3 and 1:1 in the intercropping system, attributed to crop complementarities. This advantage is probably the result of different above- and below-ground growth habits and the mor-phological characteristics of the intercrop components, allow-ing their more efficient utilization of plant growth resources, i.e., water, nutrients, and radiant energy (Fukai and Trenbath,

1993). There has been no previous attempt to determine the yield of Persian/berseem clovers as a summer crop in this area, so we cannot compare this result with those of other research in the literature. However, in other areas, such as Michigan, Shrestha et al. (1998) reported a total annual forage yield of 5400 kg ha1when berseem clover was harvested in two cut-tings in a year following spring seeding, and in Montana, Westcott et al. (1995) reported forage yields from a two-cutting system of 7700 kg ha1. Clovers exhibit various yields in re-sponse to climate and cropping dates. However, in another

0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10

Mycorrhiza colonization rate (%)

0 2 4 6 8 10

Mycorrhiza colonization rate (%)

RAR r=-0.7 p= 0.001 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60

Mycorrhiza colonization rate (%)

RAR r=0.44 p=0.083 0 10 20 30 40 50 60 70 80 90 100 ARA r=0.7 p=0.003 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60

Mycorrhiza colonization rate(%)

RAR r=- 0.712 p= 0.002

Cut 1

Cut 2





Figure 2 Correlation between (a) plant involving inoculation with AMF and nitrogenase activity, (b) plant non-inoculated with AMF and nitrogenase activity at cuts 1 and 2, mycorrhiza colonization rate is square root transformed. Pearson correlation coefficient and its significance is given. 0 5 10 15 20 25 30 P BP(1:3) BP(1:1) BP(3:1) B P BP(1:3) BP(1:1) BP(3:1) B -Glomuse mosseae +Glomuse mosseae

N concentration (mg g


LSD=1.9 F = 994.26

Figure 3 Shoot N-concentration (mg g1) of single or mixed cropping clovers (P and/or B) inoculated or not with and mycorrhiza. Means ± S.E., P < 0.05.

0 5 10 15 20 25 30 0 20 40 60 80 100 RAR

Shoot N concentration (mg plant

-1) r=0.6 p=0.001

Figure 4 Correlation between shoot N concentration and nitrogenase activity. Pearson correlation coefficient and its signif-icance is given.


study, a ratio of Persian clover to berseem clover of 3:1 had a higher forage dry matter yield than those at other mixed crop-ping ratios (Zarea et al., 2008). However, in this study, there was no significant difference in the dry forage yields among the various mixed cropping ratios.

AMF fungi had a positive effect on the forage dry matter. The synergistic symbiosis between plants and AMF has been the subject of intensive research (Smith and Read, 1997; Clark

and Zeto, 2000; Hodge, 2000; Huat et al., 2002; Yu and Cheng, 2003; Zarea et al., 2010). This advantage is probably the result of the greater uptake of nutrients, especially N and P or higher shoot-to-root ratio. Negative effects of AM fungi on root growth have been previously observed (Marschner, 1995;

Marschner and Crowley, 1996; Khalil et al., 1994, 1999; Smith and Read, 1997). The higher shoot-to-root ratio (or lower root-to-shoot ratio) may also indicate the improved plant nutrition with AM fungus association (Khalil et al., 1999). 4.2. Nitrogenase activity

Mycorrhizal treatment led to clear increases in NA. Improve-ment N uptake by AMF must decreased nitrogenase enzyme. But as discussed byStreeter (1988), there is variation in this re-sponse depending on the host–Rhizobium spp. association. For example, with arrowleaf clover (T. vesiculosum Savi.), greater lotus (Lotus pedunculatus Cav.), and soybean [Glycine max (L.) Merr.], N fertilization increased herbage or grain yields and, in some cases, increased nodulation and N2 fixation

(Hojjati et al., 1978; Wedderburn, 1983; Abdel Wahab and

Abd-Alla, 1996). A high P demand of the N-fixation process can be supplied by AMF, and mycelial growth and mycorrhi-zal formation are enhanced by Rhizobium; a synergistic process that results in improved rates of P uptake and N-fixation (Azco´n et al., 1991; Xavier and Germida, 2003). Inoculation with AMF stimulates nodulation and nitrogen fixation in legume crops (Abbott and Robson, 1977).Powell

(1976) found that nitrogen fixation in several legume crops inoculated with favorable Rhizobium strains in phosphorus deficient soil depended on phosphorus provided by AMF.

4.3. Shoot N concentration

The better uptake of N in the mixed system is attributed to the low levels of competition for nutrients between berseem clover and Persian clover because the duration of and variations in their rooting and shooting habits differ, especially during cut 1. The competition for resources between berseem and Persian clovers is very low, probably because Persian clover has shal-lower roots and a shal-lower growth rate at cut 1 (data not shown). Although, many reports have indicated better nutrient uptake in intercropping systems, in such studies, one cropping partner is always a legume and the other a non-legume crop. There have been few reports of an improvement in N uptake achieved with a cereal/cereal intercropping system relative to the N uptake in sole cropping. There is a close relationship be-tween yield advantage and the nutrient uptake by the inter-cropped species (Morris and Garrity, 1993).

These results confirm that clover mixtures acquire large amounts of N from atmospheric fixation or the soil when in symbiosis with AMF. This is probably attributable to the more successful competition with weeds for N (data not shown) and the better use of the resources because the AMF symbiosis en-hances plant growth and increases the plants’ access to forms of N that are unavailable to non-mycorrhizal plants. This corresponds to the observations of Barea et al. (1987) and

Azco´n-Aguilar et al. (1993), who found greater N uptake in plants inoculated with AMF.

4.4. Microbial activity

Higher activities of enzymes in soil incubated for 8 weeks from various cropping systems may be related to generally higher or-ganic matter provided through root and external AMF myce-lium decomposition. This is a food source that creates an ideal soil environment for microbial populations and their asso-ciated activities; and some of the highest respiration and enzyme activities were noted in these plots. The higher organic matter levels in the BP 1:3 plots also support the beneficial effects of mixture cropping or AMF colonization. An explanation for

Table 6 Effects of cropping systems and mycorrhiza on OM, SIR, DHA, phosphatases enzyme activity.

Treatment OM% SIR (mg C kg1soil) Phosphatase (mg p-nitrophenol g1soil h1) DHA (lg INTF/g\h)

Alkaline phosphatase Acid phosphatase

+AM 1.5a 5.7a 437.6a 156.5 826.0a

AM 1.1b 4.8b 292.8b 125.0b 612.8b

LSD (0.05)a 0.3 0.2 139.0 23.0 53.5

F 163.39*** 114.82*** 499.40*** 24.24*** 114.82***

B(1:0) 1.2c 5.0c 326.6c 120.1c 641.2c

B(3:1) 1.2c 5.14bc 364.5b 129.4bc 676.2bc

BP(1:1) 1.3c 5.43a 379.8ab 141.4bc 747.9a

BP(1:3) 1.4a 5.62a 391.8a 164.1a 795.4a

P(0:1) 1.3ab 5.3ab 363.1b 148.8ab 736.2ab

LSD (0.05) 0.1 0.2 21.7 21.4 66.6

F 6.83*** 7.50*** 11.49*** 5.7*** 7.5***

Cropping systems vs. AMb Ns Ns Ns Ns Ns

Different letters (a–c) within a column indicate significant differences at the 0.05 level; NS: not significant. **Fvalues significant at P < 0.01.

a Least significant differences between treatments at P = 0.05. bInteraction between treatment.


these mixed cropping effects in enzyme activities may involve the different root growth parameters of beseem and Persian clover plants. Previous reports by other workers have suggested root growth differences can be positive regulators of soil micro-bial biomass (Lynch and Panting, 1980). Dry matter accumula-tions of Persian clover roots were linear with time. At cut 1, photosynthesis assimilation in Persian clover was greater in roots compared to berseem clover. Root dry matter accumula-tions were 4–6 times greater for Persian than berseem clover, especially at cut 2 (data not shown). This huge dry matter accu-mulation of Persian clover roots may increase organic matter levels and subsequently microbial activity. When both clover species were present, different qualitative exudation from roots may activate different groups of microbes from those activated when one clover species is present.

Soil enzymatic activity can reflect the strength of biochem-ical process in soil, and the rate of decomposition and transfor-mation of organic manure in soil was mainly controlled by soil microbes and enzyme activities (Yang et al., 2008). When soil microbes and enzymes activities were higher, organic manure decomposed and transformed more quickly, and then released more nutrients per unit time. The nutrients from the decompo-sition and transformation of organic manure can be main-tained in an available form in soil, and thus can supply plant growth and development needs (Yang et al., 2008). For exam-ple, phosphatase can hydrolyze some kind of organic com-pounds containing P into inorganic P, which can be absorbed and utilized by plants, thus soil enzyme activity was positively correlated with soil available nutrients. Soil enzymatic activities can indicate the function of soil ecosystem because they can reflect the intensity and direction of various biochemical processes occurred in soil, and also correlate clo-sely with soil fertility (Yang and Wang, 2002; He et al., 2002). The results of these investigations indicate that MCS and AMF stimulated soil microbial activities, depending on the MCS ratio. The combined effects of MCS and AMF were ben-eficial to plant growth, N uptake, NA of root nodules, and microbial activity. These values were affected by the ratio of berseem to Persian clover in the mixture and time (8-week soil incubations). In our previous report, the greatest NA of free-living rhizosphere and soil microbial biomass was observed with B:P = 3:1 at cut 2 (Zarea et al., 2009). Therefore, the present research has revealed another factor, time, that ap-pears to be significant in overall effects of AMF and MCS on soil biology, and to agriculture that uses the model in designing agricultural systems.


The author thanks Professor Rosario Azco´n, Departamento de Microbiologı´a del Suelo y Sistemas Simbio´ticos, for her comments on the earlier manuscript. Constructive comments on an earlier draft of this paper by two anonymous reviewers are gratefully acknowledged.


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