Annals of Microbiology, 57 (1)1-7 (2007)
Influence of arbuscular mycorrhizal fungi on microbes and enzymes of soils from different cultivated densities of red clover
Ming-Yuan WANG1, Ren-Xue XIA1*, Qiang-Sheng WU2, Ji-Hong LIU3, Li-Ming HU1
1Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, P.R. China; 2College of Horticulture and Gardening, Yangtze University, Jingzhou, 434023, P.R. China; 3National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan, 430070, P.R. China
Received 26 September 2006 / Accepted 26 January 2007
Abstract - Effects of arbuscular mycorrhizal fungus (AMF) Glomus mosseae on plant growth, soil microbial populations and enzymes activities of soils were studied in red clover (Trifolium pratense L.) grown in pots at different cultivated densities. Seeds of red clover were sown with 50 g inoculums of G. mosseae per pot. After a week, the plants were thinned to 20, 30, 40, 50 and 60 seedlings per pot. Three months after treatment, AMF inoculation significantly stimulated plant growth. Quantities of vesicles and
spores, arbuscules and hyphae were the highest when 30 and 50 seedlings were grown per pot, respectively. However, no root was infected in control plants. In all the soil sites, the numbers of fungi and bacteria were followed in the order: root >root surface >rhizospheric. It was indi-cated that arbuscular mycorrhizal fungus decreased the numbers of fungi and bacteria but improved growth of actinomycetes. Compared to control plants, AMF stimulated activities of phosphatase and urease but decreased invertase.
Key words:arbuscular mycorrhizal fungi, invertase, phosphatase, soil microbes, urease.
INTRODUCTION
Arbuscular mycorrhizal fungi (AMF) inhabit both plant roots and surrounding soil, where they can benefit their host plants in several ways, including more phosphorus nutrition (Toro et al., 1998), improved plant growth (Wu and Xia, 2004), increased drought tolerance (Ruíz-Lozano and Azcón, 1995; Wu et al., 2006), and root pathogens (Pozo et al., 1999) and improved soil environments (G aur and Adholeya, 2004).
Formation of arbuscular mycorrhizal (AM) symbiosis influences the composition of the microbial community in the rhizospheric soil (Schreiner et al., 1997) and its activity (Olsson et al., 1996), which is
ascribed to competition for energy-rich carbon compounds (Christensen and Jakobsen, 1993) or to indirect influence on quantity and quality of plant root exudates and soil structure (Johansson et al., 2004). Mycorrhizal fungi can enhance the number and activ-ity of beneficial soil organisms like nitrogen fixers and phos-phate solubilisers leading to improvement of plant growth (Linderman, 1992), and enhance in number of aerobic bac-teria in the rhizosphere (Krishnaraj and Sreenivasa, 1992). However, it was reported that there was no effect of AMF on the total number of bacteria (Waschkies et al., 1994), but those in the rhizosphere of mycorrhizal plants changed to more facultative anaerobic bacteria and less fluorescent pseudomonads (Meyer and Linderman, 1986). Song et al. (2004) reported that AMF had no effect on microbial popu-lations in rhizospheric soil, but increased the number of bac-teria, actinomycetes and azotobacters in the root surface and root itself.
The effects acted by the association between AMF and soil microbes. Several studies indicated that metabolic char-acteristics of intra- or extra-radical fungal structures were closely related to the mycorrhizospheric microbial interac-tions during AMF inoculation (Tisserant et al., 1993; Guillemin et al., 1995). It has been shown that some bac-teria were attached to hyphae and germinated spores, but there was different degree of attachment between different bacterial strains (Bianciotto et al., 1996; Artursson and Jansson, 2003). Walley and Germida (1996) reported that bacteria adhering to the AM
mycelium might live on hyphal exudates and/or use the mycelium as a vehicle for coloni-sation of the rhizosphere.
Although plant composition and diversity have effect on ecosystem (McG rady-Steed et al., 1997; Naeem and Li, 1997), little is known about how they influence microbes. At present, most of reports about the association between AMF and soil microorganisms were studied in vitro. Furthermore, information on quantities of bacteria, fungi and actino-mycetes in rhizospheric niche (rhizospheric, root surface, root) is still lacking.
In this paper, in order to study the effects of association between AMF and plant densities on soil microbes, we examined the distribution of soil microorganisms (bacteria, fungi and actinomycetes) at different soil sites in different cultivated densities with AMF and control treatments, and analysed a variety of enzymes activities in the rhizospheric niche of red clover (Trifolium pratense L.
) seedlings.
MATERIALS AND METHODS
Pla nt ma teria ls a nd pla nt culture. Seeds of red clover (Trifolium pratense L.) were marinated in tap water for 2 h, surface sterilised with 0.5% (w/v) KMnO4for 20 min, rinsed in distilled water and germinated on wet filter papers in Petri dishes at 26 °C overnight. The seeds were sown in white plastic pots with 14 cm (down dia.) x 18 cm (depth) x 20 cm (up dia.) containing 1.7 kg of autoclaved media com-posed of yellow soil/vermiculite/ sphagnum (5/2/1, v/v/v) at pH 5.9, which had 1.3% organic matter, 29.97 mg·kg-1 available phosphorus, 147.47 mg·kg-1alkalihydrolyzable nitrogen, and 140.89 mg·kg-1available potassium. Mycorrhiz a l inoculum. Mycorrhizal inoculums (Mycorrhizal Biotechnology Laboratory, Laiyang Agricultural College) consisted of spores, soil, hyphae and infected clover root fragments from a stock culture of Glomus mosseae.Nearly 50 g of mycorrhizal inoculums were placed 5 cm below seeds in each pot, which was placed in green-house, where the average day/night temperature was 25/15 °C with a relative humidity of 60%. The pots were watered with distilled water every two days.
The experiment consisted of two factors: (i) half of the pots were inoculated with AM fungus G. mosseae, and the others were added the equal substrates (as control); (ii) five levels of cultivated densities (20, 30, 40, 50, 60 seedlings per pot in respective treatment) with red clover. Each treat-ment was replicated six times.
Determina tions of pla nt bioma ss a nd mycorrhiza l colonisation. Plants were harvested after three months, and parts of plants were separated and dried at 72 °C for 48 h. A fraction of roots were carefully washed to remove soil, chopped into 1 cm long pieces and fixed by FAA solution (mixture of formalin/acetic acid/ethanol, 13/5/200, v/v/v) for 24 h. Afterwards, root samples were cleared with 10% KOH (w/v) solution and stained with 0.05% (v/v) trypan blue in lactophenol (Phillips and Hayman, 1970). The AM infected roots were observed under microscope and the infected per-centage was calculated by the following formula:
AM infected percentage (%) = 100 x root length infected / all root length observed
Qua ntities of hypha e a nd spores in the rhizospheric soil. Substrates in rhizosphere were collected, dried in room temperature, and then sieved through stainless sieve with 2 mm diameter. The hyphal length of G. mosseae was record-ed according to Bethlenfalvay and Ames (1987). Spores were isolated from soil using the wet sieving and sucrose gradient centrifugation techniques (G erdemann and Nicolson, 1963), and observed under microscopy.
Test of microbes in the rhizospheric soil. Rhizospheric soil, surface soil and roots were harvested according to Riley and Barber (1969, 1970). The population of microbes was determined by a plate dil
ution method (Johnson and Curl, 1972). Aliquots of soil (approximately 1 g) were homogenised with 9 ml sterilised distilled water, and shak-en for 30 min. Fungi were grown on PDA medium (Johnson and Curl, 1972); bacteria were grown on Beef extract Peptone agar (pH 7.2) containing peptone 10 g, beef extract 5 g, NaCl 5 g, and agar 15 g in 1 litre of distilled water; actinomycetes were enumerated using Sodium Caseinate agar (pH 6.7) containing sodium caseinate 0.2 g, K2HPO4 0.5 g, MgSO4 0.2 g, FeCl3 0.01g, and 15 g agar in 1 litre of distilled water. Plates were incubated at 25 °C for 5-7 days, except for the bacteria plates at 30 °C for 30 h.
Activities of enzymes in the rhizospheric soil. Activities of phosphatase were tested according to Nannipieri et al. (1980) with little minor modification. Two ml of 0.1 M maleate buffer (pH 6.5) and 0.5 ml of 0.115 M p-nitrophenyl phosphate disodium were added to 0.5 g soil, and then incubated at 37 °C for 90 min, cooled at 2 °C for 15 min before adding 0.5 ml of 0.5 M CaCl2and 2 ml of 0.5 M NaOH, then followed by centrifugation at 4000 x g for 5 min. Activities of phosphatase were determined with UV-2450 Spectrophotometer (SHIMADZU, Japan) at 398 nm. Phosphatase activity was expressed as µmol p-nitrophenol hydrolysed g-1dry soil h-1.
The urease activities were determined by a modified method proposed by Kandeler and Gerber (1988). Soil (5 g) was incubated in 1 ml methylbenzene for 15 min, after 10 ml urea (10%, w/v) and 20
ml citrate buffer (pH 6.7, 1 M) were added. After incubation for 24 h at 37 °C, the solution was filtered, and about 1.5 ml of filtrate was mixed with 10 ml distilled water, 2 ml sodium phenolate hydroxide, and 1.5 ml sodium hypochlorite. Urease activities were deter-mined 20 min later with UV-2450 Spectrophotometer at 578 nm. Urease activity was expressed as mg NH4-N released g-1dry soil in 24 h.
Activities of invertase were analysed following a stan-dard methodology reported by Zhao and Jiang (1986). Nearly 2.5 g air-dried soil was incubated in 3 ml methyl-benzene, 2.5 ml phosphate buffer (pH 5.5) and 7.5 ml sucrose solution (8%, w/v) for 12 h at 37 °C, followed by filtration. Approximately 0.25 ml filtrate was mixed with 1.5 ml 3,5-dinitrosalicylic acid, boiled for 5 min, then cooled with running tap water for 3 min. The activity of invertase was determined with UV-2450 Spectrophotometer at 508 nm. Invertase activity was expressed as µg glucose pro-duced g-1dry soil in 24 h.
Statistical analysis. The experimental data were statisti-cally analysed by variance (ANOVA) with SAS 8.1 software. Probabilities of significance were used to test the signifi-cance among treatments and interactions, and LSD (P < 0.05) was used to compare the means.
RESULTS AND DISCUSSION
AM colonisation
Roots of red clover inoculated with AMF were infected under different cultivated densities. No colonisation was found in control treatments. The percentage of AM colonisation decreased gradually from 75.0 to 26.1% with increase in the density of red clover. Both the vesicles and density of spores in 30 seedlings per pot were the highest, which were significantly higher from the other treatments. But the quantities of arbuscules and hyphae showed the maximum in pot with 50 seedlings (Table 1).
The result was in line with Chen et al.(2003), which showed that higher cultivated density limited the AM colonisation. It was due to the high density that restrained host plant growth, resulted in the decrease of AMF infection.
2M.-Y. Wang et al.
Plant growth
Dry weight of red clover was the highest in each treatment with 30 seedlings per pot. AMF significantly promoted the shoot dry weight compared with control treatments, and the maximum elevation was up to 42.6% (in 30 seedlings per pot) (Table 2).
Under different density of red clover, significant differ-ence of biomass of shoot and root between mycorrhizal and control plants was observed, which showed that the weight was always the highest in pots with 30 seedlings. The main problem was probably caused by nutrient competition. Excessive cultivated densities hampered ventilation and transparency, and limited photosynthesis and accumulation of organic matter. It was indicated that AMF have positive effects on the growth and the biomass of red clover. Similar results have been reported elsewhere (Al-Karaki, 1998; Fidelibus et al., 2001; Jeffries et al., 2003; Wu and Xia, 2004), they were possibly attributed to the improvement of nutrient absorption from the soil, such as N, P, K, Mg, Cu, Zn (Marschner and Dell, 1994; Clark and Zeto, 2000), or the increased roots length density (Bryla and Duniway, 1997). In addition, plant growth-promoting rhizobacteria or the interact with AMF may also increase plant growth by improving nutrition and disease suppressing (Broek and Vanderleyden, 1995; Artursson et al., 2006).Numbers of fungi, bacteria and actinomycetes Number of the fungi adhering to the rhizosphere, root sur-face and root was the largest at the density of 50 seedlings per pot, which was significantly higher from other treat-ments. The roots possessed more fungi than root surface, while rhizosphere had the least. And the total quantities of fungi were significantly lower in treatments inoculated with AMF compared to control from 15.1 to 27.0%, which implied that AMF restrained soil fungi, in agreement with Waschkies et al.(1994) (Table 3).
Root of red clover in 50 seedlings per pot had the largest number of bacteria (2.8 x 109), followed by root surface and rhizosphere (Table 4). When plant density rose from 20 to 60 seedlings per pot, the number of bacteria adhering to root surface, rhizosphere and the total numbers decreased, showing significant difference between AMF and control treatments. It also suggested that higher density of red clover interfered with the growth of bacteria within a small niche. On the other hand, the total numbers of bacteria fol-lowed the order of root > root surface > rhizosphere, which showed that the quantity decreased as the distance increas-es from the root. Artursson et al.(2005) also reported that AMF had impact on the composition of bacteria communi-ties. The effect on bacteria community was due to the myc-orrhizal establish that changed the exudates composition,
Ann. Microbiol., 57 (1), 1-7 (2007)3
TABLE 1 - The infected status of Trifolium pratense L. root in different treatments
Densities AMF status Root colonisation Vesicles Arbuscules Spores density Mycelial density (seedlings pot-1)status(%)(cm-1)(cm-1)(100 g-1 soil)(mg-1soil)
20AMF75.0a0.5c0.5b60c 3.37b NM0c0d0c0d0c
30AMF63.9a 2.3a 1.2a160a 3.45b NM0c0d0c0d0c
40AMF40.0b 1.1b 1.5a120a 4.18b NM0c0d0c0d0c
50AMF28.9b 1.2b 1.7a80bc14.67a NM0c0d0c0d0c
60AMF26.1b0.3c0.6b60c 5.00b NM0c0d0c0d0c
AMF: arbuscular mycorrhizal fungi; NM: no arbuscular mycorrhizal fungi. Values in each column followed by the different letter are sig-nificantly different (P = 0.05) according to LSD test.
TABLE 2 - Plant biomass of Trifolium pratense L. in different treatments
Densities AMF status Dry weight (g pot-1)
(seedlings pot-1)
Shoot Root
20AMF19.31cd 3.77ef
NM17.64d 3.10f
30AMF33.50a8.16a
NM23.50bc7.01ab
40AMF26.13b 6.52bc
NM19.46cd 5.45cd
50AMF25.85b 5.00de
NM19.18cd 3.87ef
60AMF25.38b 4.81de
NM18.71cd 3.78ef
AMF: arbuscular mycorrhizal fungi; NM: no arbuscular mycorrhizal fungi. Values in each col-
umn followed by the different letter are significantly different (P = 0.05) according to LSD test.
which was a nutrient source to the associated bacteria (Barea, 2000; Linderman, 2000). Fungi and bacteria were suppressed by AMF, which was due to the C resources com-petition (Baggi, 2000).
The actinomycetes in rhizosphere and root itself increased gradually until the peak at the density of 40 seedlings per pot, but in the total and root surface at den-sity of 30 ones (Table 5). The quantities in root surface were relatively higher to the ones adhering to both rhizos-phere and root itself. It seemed that the total numbers were significantly higher in mycorrhizal soils than in con-trol ones. The result was in accordance with Song et al. (2004), who reported that AMF have closely relation with actinomycetes. Diem (1997) reported the formation of mycorrhizae could affect the growth of Frankia, though there was no competition between AMF and Frankia in infectious site. Perhaps root exudates and cells fell off the host roots could provide adequate nutrient to those microbes (Ban, 2003).
Activities of enzymes
冶金专业Significant differences in the activities of phosphatase, invertase and urease are shown in Table 6. Phosphatase activities were the highest at 50 seedlings per pot, but there were no regulation with invertase and urease. It seems that AMF enhanced activities of phosphatase, as has been
4M.-Y. Wang et al.
TABLE 3 - The numbers of fungi at different soil sites in different treatments
AMF status Densities Fungi (x 106CFU)
(seedlings pot-1)
Rhizosphere Root surface Root Total
AMF2013.43b14.20d18.43d46.07d
30 5.53cd 5.70fg 6.27f17.50g
407.50c7.67ef7.93ef23.10f
5016.63a34.8b38.70b90.11b
60 2.94d e 3.63g 5.53f12.10h
NM2016.90b18.83c22.17c57.90c 307.60c de7.90ef8.47ef23.97f
408.83cd9.96e10.40e29.20e
5022.47b45.00a52.31a119.80a
60 4.05e 4.53g 6.67f15.23hg Significance
AMF******** Densities********
AMF x densities NS******
AMF: arbuscular mycorrhizal fungi; NM: no arbuscular mycorrhizal fungi. Values in each column followed by the different letter are sig-nificantly different (P = 0.05) according to LSD test. * P < 0.05; ** P < 0.01; NS: not significant.
TABLE 4 - The numbers of bacteria at different soil sites in different treatments
AMF status Densities Bacteria (x 108CFU)
(seedlings pot-1)
Rhizosphere Root surface Root Total
AMF2010.05a11.23a16.70bcd37.99a
30 5.93bcd10.70ab16.90bc33.53ab
40 5.40cd8.73bc17.47b31.60ab
50 6.80bc8.50cde20.27a35.57a
60 4.50de 5.73fg14.63de24.87d
NM2012.42ab14.40bcd20.50de47.32bc 307.60de13.87cde21.33de42.80cd
407.33DE11.57def23.77cde42.67d
508.80cde10.67ef28.00bcd47.47cd
60 5.90e7.93g17.01e30.82e Significance
AMF******** Densities********
AMF x densities NS NS NS NS
AMF: arbuscular mycorrhizal fungi; NM: no arbuscular mycorrhizal fungi. Values in each column followed by the different letter are significantly different (P = 0.05) according to LSD test. * P < 0.05; ** P < 0.01; NS: not significant.
reported by Dodd et al.(1987) and Wang (2006), but decreased invertase.
Changes, accumulation and decomposition of sub-stances in soils were a complicated process, in which soil enzymes play a very important role. Phosphatase is an important enzyme, which can transform organic phosphorus and hydrolyse them into inorganic phosphorus that can be absorbed by plants (Pascual et al., 2002).
Urease is a nickel-containing enzyme decomposing urea to carbon dioxide and ammonia that takes part in nitrogen metabolism of plants and microorganisms (Burne and Chen, 2000). The soil urease mainly come from soil microorgan-isms and plants, but no significant difference or relation between urease and soil microbes was found in this report, which may due to soil pH or root exudates in small soils zone. However, effects of interaction with AMF and red clover densities on urease were significant difference.
Invertase can decompose sucrose into fructose and glu-cose, which can be used as nutrient for plants and soil microorganisms. In this paper, as a result, competition between AMF and soil microbes for carbon resources derived from invertase led to growth impediment of microorganisms. Vázquez et al.(2000) reported that the increased enzyme activities found in rhizosphere of mycor-rhizal plants indicated an increase in C and nutrient leakage from roots.
Ann. Microbiol., 57 (1), 1-7 (2007)5
TABLE 5 - The numbers of actinomycetes at different soil sites in different treatments
AMF status Densities Actinomycetes (x 105CFU)
(seedlings pot-1)
Rhizosphere Root surface Root Total
AMF2011.37efg15.00e10.70d37.07e 3013.97de67.50b24.13c105.60b
4022.13b41.00d28.53ab91.67c
5011.13def14.10e 1.20e26.43ef
607.10g13.30e 1.27e21.62f
NM208.00cd11.00e7.17d26.14d 3010.67c56.87a18.33b85.85a
4019.00a31.33c24.77a75.10b
508.90cd11.67e0.80e21.36e
60 5.43fg10.73e0.93e17.10ef Significance
AMF******** Densities********
AMF x densities NS NS NS*
AMF: arbuscular mycorrhizal fungi; NM: no arbuscular mycorrhizal fungi. Values in each column followed by the different letter are sig-nificantly different (P = 0.05) according to LSD test. * P < 0.05; ** P < 0.01; NS: not significant.
TABLE 6 - The activities of soil phosphatase, invertase, urease in different treatments
AMF status Densities Phosphatase Invertase Urease
(seedlings pot-1)(μmol PNP g-1h-1)(μg glucose g-1)(mg NH4-N g-1)
AMF20 2.99cd17.13b 3.01c
30 3.34bcd 6.20ef 4.54a
40 3.44bc13.04c 2.15de
50 3.95a 5.04f 1.81e
60 3.67ab17.94b 4.80a
NM20 2.02f20.05a 1.87e
30 2.24f8.62d 2.38d
40 2.35ef16.37b 1.18f
50 2.85de7.23de0.81f
60 2.33f21.17a 3.57b
Significance
AMF******
Densities******
AMF x densities NS NS**
AMF: arbuscular mycorrhizal fungi; NM: no arbuscular mycorrhizal fungi. Values in each column fol-
lowed by the different letter are significantly different (P = 0.05) according to LSD test. * P < 0.05;
** P < 0.01; NS: not significant; PNP: p-nitrophenol.
Our findings provide that association between AMF and different plant cultivated densities have significant effect on the total quantities of soil fungi and actinomycetes, but no significant effect on the total bacteria. However, what hap-pened on earth between AMF, plant and soil microbes need much more study.
Acknowledgements
The authors are grateful to Run-Jin Liu for providing the AMF inoculums. We also wish to thank Xue-Mei Cai (University of Chicago Hospitals, USA) and Jing-Dong Ye (Howard Huges Medical Institute, USA) for supports. In addition, the authors acknowledged Fa-Yuan Wang (Department of Resources and Environmental Science, Agricultural College, Henan University of Science and Technology, China) for modification. This work was finan-cially supported by Ministry of Science and Technology, P.R. China (2004EP090019).
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