Scientia Horticulturae 129 (2011) 294–298
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Differences of hyphal and soil phosphatase activities in drought-stressed mycorrhizal trifoliate orange (Poncirus trifoliata) seedlings Qiang-Sheng Wu a,b,∗ , Ying-Ning Zou a , Xin-Hua He b,c,d a
College of Horticulture and Gardening, Yangtze University, 88 Jingmi Road, Jingzhou City, Hubei 434025, PR China Ministry of Agriculture Key Laboratory of Crop Nutrition and Fertilization, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China c Centre for Ecosystem Management, School of Natural Resources, Edith Cowan University, Joondalup, WA 6027, Australia d State Centre of Excellence for Ecohydrology and School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia b
a r t i c l e
i n f o
Article history: Received 29 January 2011 Received in revised form 25 March 2011 Accepted 30 March 2011 Keywords: Arbuscular mycorhizal fungi Drought stress Phosphatase Phosphorus Poncirus trifoliata Succinate dehydrogenase
a b s t r a c t Differences of hyphal and soil phosphatase activities between mycorrhizal and non-mycorrhizal plants were less studied under drought-stressed (DS) conditions. In a pot experiment, fungal alkaline phosphatase (FALP), and succinate dehydrogenase (FSDH), soil phosphatase activity, both soil and plant P contents were compared in 6.5-month-old trifoliate orange (Poncirus trifoliata) seedlings under 80 days of DS with or without inoculations by arbuscular mycorrhizal fungi (AMF, Glomus diaphanum, Glomus mosseae or Glomus versiforme). Plant growth and biomass production under DS were significantly higher in mycorrhizal than in non-mycorrhizal seedlings. Both the FALP and the FSDH activities under DS were significantly reduced in these three Glomus inoculated seedlings. In general, similar soil neutral and alkaline phosphatase activities, but significantly higher soil acid and total phosphatase activities, were exhibited in mycorrhizal than in non-mycorrhizal seedlings under both the well-watered (WW) and the DS. Both leaf and root P contents were significantly higher in the AM colonized seedlings, but soil available P contents were lower in the growth media with AM seedlings. Our results showed that higher hyphal enzymes’ activities, soil acid and total phosphatase activities, and plant P contents in AM colonized seedlings, particularly in Glomus mosseae-colonized seedlings and/or under DS, would result in a better growth of the host plants, which might be the basis for enhancing drought tolerance in plants. © 2011 Elsevier B.V. All rights reserved.
1. Introduction More than 50% of plant yields may be reduced by drought (Zlatev and Yordanov, 2004). For instance, citrus (Citrus L.), one of the most important commercial fruit trees in the south and southwest of China, is not able to grow or fruit well without frequent and timely irrigation (Bhusal et al., 2002). Arbuscular mycorrhizas (AMs), symbiotic associations between ∼80% of higher plants and soil arbuscular mycorrhizal fungi (AMF), can provide soil nutrients, particularly phosphorus (P), to host plants in exchange ∼20% of photosynthetic fixed carbon for the functioning of AMF (Parniske, 2008; Smith and Read, 2008). Meanwhile, there is increasing evi-
Abbreviations: AM, arbuscular mycorrhiza; AMF, arbuscular mycorrhizal fungi; DS, drought-stressed; FALP, fungal alkaline phosphatase; FSDH, fungal succinate dehydrogenase; TB, total hyphae; WW, well-watered. ∗ Corresponding author at: College of Horticulture and Gardening, Yangtze University, 88 Jingmi Road, Jingzhou City, Hubei 434025, PR China. Tel.: +86 716 8066262; fax: +86 716 8066262. E-mail address:
[email protected] (Q.-S. Wu). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.03.051
dence that AMs improve water relations of citrus plants under drought-stressed (DS) conditions (Levy and Krikun, 1980; Graham and Syvertsen, 1984; Augé, 2004; Wu et al., 2009). The potential mechanisms included greater osmotic adjustment and nutrient uptake (especially P), better soil structures, and improved reactive oxygen metabolism in mycorrhizal citrus seedlings (Augé, 2004; Wu et al., 2007, 2008; Wu and Zou, 2009). Studies have also found that mycorrhizal fungal hyphae participate in uptake and transport of water to host plants (Hardie, 1985; Augé, 2004; Allen, 2006, 2007). Mycorrhizal hyphae are generally divided into active and functional (Tang and Chen, 1999), and the activity of fungal alkaline phosphatase (FALP, active hyphae) or of fungal succinate dehydrogenase (FSDH, functional hyphae) has been suggested to be a potential efficiency marker in mycorrhizal symbiosis (Smith and Gianinazzi-Pearson, 1990; Guillemin et al., 1995; Joner and Johansen, 2000; Joner et al., 2000). However, response of mycorrhizal hyphae to drought stress is less studied. On the other hand, P becomes less mobile in arid soils, and an enhanced P acquisition by AMs would hence become more important in improving water relations of host plants (Hardie, 1985; Smith and Gianinazzi-Pearson, 1988; Augé, 2004; Allen, 2006,
Q.-S. Wu et al. / Scientia Horticulturae 129 (2011) 294–298
2007). Arbuscular mycorrhizal fungi also increase the activity of soil enzymes, including dehydrogenase, phosphatase and urease (Wang et al., 2006; Huang et al., 2009). For instance, soil phosphatase activity was increased with the increase of AM colonization (Chethan Kumar et al., 2008), leading more inorganic P available to the host plants since the release of inorganic P from organically bound P is mediated by soil phosphates (Wang et al., 2006). However, effects of AMF on soil phosphatase activity and P contents in citrus rhizosphere are poorly known particularly under DS conditions. We hypothesize that an enhanced plant P nutrition through the increase of soil phosphatase activity mediated by AMF may contribute drought tolerance to host plants. Using a highly droughtsensitive citrus rootstock [Poncirus trifoliata (L.) Raf.], the objectives of this research were to study: (1) possible effects of AM fungal species and drought stress on fungal hyphae, hyphal and soil phosphatase activities, plant and soil P content, and (2) whether an enhanced P nutrition through soil phosphatase and hyphal activities mediated by AM associations contributes drought tolerance to host plants. 2. Materials and methods 2.1. Experimental design and plant growth This experiment was a bifactorial randomized design consisting of four AMF inoculations (Glomus diaphanum, Glomus mosseae, Glomus versiforme, and non-AMF control) and two water (WW and DS) treatments. Each treatment had six replicates with a total of 48 pots. Seeds of trifoliate orange were sterilized with 70% alcohol for 15 min and germinated in wet filter paper under darkness at 28 ◦ C. Six 7-day-old seedlings were transplanted into a plastic pot (15 cm × 20 cm × 18 cm) containing 3.4 kg autoclaved soils (Xanthiudic ferralsols, US System, pH 5.4, 18.5 mg kg−1 available P and 8.7 organic C mg kg−1 soil) and thinned to three seedlings after 1 month transplanting. Before transplanting, fungal inocula of G. diaphanum Morton & Walker, G. mosseae (Nicol. & Gerd.) Gerdemann & Trappe and G. versiforme (Karsten) Berch (supplied by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China), were separately placed at 5 cm depth below the growth media (∼1400 spores per pot). The non-AMF control pots were received the same amount of ovendried (100 ◦ C) Glomus inocula. After 120 days of transplanting, two water treatments of well-watered (WW, 100% field water-holding capacity at −0.10 MPa) and drought-stressed (DS, 73% field waterholding capacity at −0.44 MPa) were performed for further 80 days by daily weighting and then distilled water supplement. Soil water potential was determined using a Psypro Dew Point Water Potential System (Wescor Inc., Logan, Utah, USA). The exerted level of drought stress was only for a comparative long-term period of 80 days, because trifoliate orange is a drought-sensitive plant with less root hair in its shallow root system. The seedlings were grown in a plastic greenhouse without light and temperature control till harvest for a total of 200 days between March and September. 2.2. Analyses of plant growth, plant and soil P contents and enzyme activity Plant height, stem diameter and leaf number were recorded before harvest when seedlings were 200 days old. Fresh leaves, stems and roots were then harvested and oven-dried at 75 ◦ C for 48 h. Fresh rhizospheric soils were collected from the root systems, air-dried and ground through a 4 mm sieve. A portion of fresh roots were cut into 1 cm segments and then divided into three parts for the analysis of active hyphae
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(FALP), functional hyphae (FSDH) and total hyphae (trypan bluestained infection, TB), respectively. Histochemical evaluation of FALP and FSDH was followed by Tisserant et al. (1993). Briefly, roots were soaked for 2 h at 25 ◦ C in solutions containing 0.05 mM Tric/citric acid (pH 9.2), 50 mg mL−1 sorbitol, 15 units mL−1 cellulase, and 15 units mL−1 pectinase. The distilled water rinsed roots were incubated in solutions [0.05 M Tris/citric acid (pH 9.2), 0.5 mg mL−1 MgCl2 anhydrous, 1 mg mL−1 ␣-naphthyl acid phosphate, 1 mg mL−1 fast blue RR salt, and 0.8 mg mL−1 MnCl2 tetrahydrate] for FALP or [0.2 M Tris/HCl (pH 7.4), 5 mM MgCl2 , 4 mg mL−1 nitro-blue tetrazonium, and 2.5 M Na-succinate] for FSDH overnight at 25 ◦ C and then cleared for 5 min with 1% or 3% active chlorine of NaClO for FALP or FSDH, respectively. Histochemical estimation of total hyphae was performed after clearing and staining with trypan blue (Phillips and Hayman, 1970). All fungal hyphae were observed in a microscope under 10× magnification and quantified by the method of Trouvelot et al. (1986). Analysis of plant P content was followed by Chapman and Pratt (1961), soil available P by Olsen et al. (1954) and phosphatase activity by Zhao and Jiang (1986). Soil acid, neutral or alkaline phosphatase was, respectively, extracted by the sodium acetate buffer (pH 5.0), citric acid–disodium hydrogen phosphate buffer (pH 7.0) or borate buffer (pH 10.0) and their activity was then analysed (Zhao and Jiang, 1986). The total phosphatase was the sum of acid, neutral and alkaline phosphatase activities. 2.3. Statistical analysis Data (means ± SE, n = 6) were subjected to a two-way ANOVA. Differences in means among treatments and interactions of DS and AMF treatments were compared by the LSD at 5% or 1% level with the SAS 8.1 software (SAS Institute Inc., Cary, NC, USA). 3. Results Plant height, stem diameter, leaf numbers and biomass production were significantly higher in the AMF colonized than in the non-AMF colonized seedlings under the WW or DS treatment, except for the total biomass production in the G. diaphanum inoculated seedlings under WW (Table 1). Between the WW and DS treatments, a total of 80 days drought stress generally inhibited plant growth of the AM colonized seedlings (Table 1). Among the three AMF inoculations, G. mosseae had the highest impact on plant growth regardless of soil water status. Root AM colonization was observed in all AM colonized seedlings including these endured 80 days of drought stress, but not in the non-AM controls (Table 2). The active hyphae (FALP), functional hyphae (FSDH) and total hyphae (TB) were generally significantly higher under the WW than under the DS treatment (Table 2). Among the three AMF inoculations, FALP, FSDH or TB was, respectively, highest in G. mosseae, higher in G. versiforme and least in G. diaphanum colonized seedlings. As a general rule, mycorrhizal seedlings had higher P content in either leaves (Fig. 1A) or roots (Fig. 1B) but less soil available P content (Fig. 2) under both WW and DS. Meanwhile, soil available P and root P contents were generally lower, but leaf P contents were generally higher under the WW than under the DS treatment. Among the three AMF inoculations, G. mosseae colonized seedlings had the lowest soil available P but the highest P contents in both leaves and roots under both WW and DS. Mycorrhizal seedlings under WW had significantly higher soil acid and total phosphatase activities, similar alkaline phosphatase activity, but varied neutral phosphatase activity, compared to the non-AMF inoculated seedlings under both WW and DS (Table 3). Among the three AMF inoculations, no differences in soil acid, neu-
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Table 1 Growth characteristics of AM or non-AM Poncirus trifoliata seedlings under well-watered (WW) and drought-stressed (DS) conditions. Water status
AMF
Plant height (cm)
Stem diameter (cm)
Dry weights (g plant−1 )
Leaf number per plant
Leaf WW
DS
Significance DS AMF DS × AMF
G. diaphanum G. mosseae G. versiforme Non-AMF G. diaphanum G. mosseae G. versiforme Non-AMF
20.2 40.6 33.7 14.7 18.4 29.9 19.0 13.5
± ± ± ± ± ± ± ±
1.5c 2.3a 3.4b 1.6de 4.4cd 0.7b 2.6c 1.2e
0.23 0.35 0.30 0.21 0.23 0.32 0.21 0.19
± ± ± ± ± ± ± ±
0.02c 0.03a 0.01b 0.01cd 0.04c 0.02b 0.01cd 0.00d
25.0 37.9 33.1 21.6 23.3 31.0 21.9 18.8
± ± ± ± ± ± ± ±
1.9c 2.2a 1.8b 2.5cd 3.9c 2.0b 3.1cd 1.6d
0.22 0.60 0.43 0.20 0.20 0.40 0.20 0.15
Stem ± ± ± ± ± ± ± ±
0.10c 0.03a 0.09b 0.01c 0.07c 0.03b 0.03c 0.02c
0.30 0.91 0.62 0.24 0.30 0.52 0.26 0.20
± ± ± ± ± ± ± ±
Root 0.02c 0.04a 0.13b 0.03c 0.12c 0.07b 0.11c 0.04c
0.31 0.69 0.48 0.28 0.30 0.45 0.29 0.25
Total ± ± ± ± ± ± ± ±
0.02c ± 0.03a 0.05b 0.04cd 0.03c 0.02b 0.02cd 0.03d
0.82 2.20 1.53 0.72 0.80 1.37 0.75 0.59
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
± ± ± ± ± ± ± ±
0.12c 0.09a 0.10b 0.07cd 0.08c 0.08b 0.10c 0.07d
Data (means ± SE, n = 6) followed by the same letter within a column indicate no significant difference among treatments at P < 0.05. ** P < 0.01. Table 2 Active hyphae (FALP), functional hyphae (FSDH) and total hyphae (TB) in AM Poncirus trifoliata seedlings under well-watered (WW) and drought-stressed (DS) conditions. Water status
AMF
FALP (%)
WW
G. diaphanum G. mosseae G. versiforme Non-AMF G. diaphanum G. mosseae G. versiforme Non-AMF
15.4 31.4 17.0 0 8.5 20.6 11.4 0
DS
Significance DS AMF DS × AMF
± ± ± ± ± ± ± ±
FSDH (%)
2.9cd 2.7a 2.3bc 0 1.6e 2.4b 1.8de 0
19.7 43.2 34.4 0 10.3 21.3 20.4 0
± ± ± ± ± ± ± ±
TB (%)
2.3c 3.5a 2.7b 0 1.0d 1.5c 3.1c 0
23.1 67.2 46.9 0 13.6 24.8 22.2 0
**
**
**
**
**
**
**
**
**
± ± ± ± ± ± ± ±
0.5c 11.4a 8.2b 0 2.4c 2.3c 7.8c 0
Data (means ± SE, n = 6) followed by the same letter within a column indicate no significant difference among treatments at P < 0.05. ** P < 0.01.
tral or alkaline phosphatase activity were observed under both WW and DS, except lower soil neutral phosphatase activity under the G. diaphanum inoculated seedlings. On the other hand, acid phosphatase accounted for 60–72% of the total soil phosphatase. 4. Discussion Our results showed that the mycorrhizal functional hyphae (FSDH) were generally higher than the active hyphae (FALP) under both WW and DS, and the active, functional and total hyphae were generally significantly decreased by DS. These hyphae had close relationships not only with nutrient accumulation, growth and metabolic activities of host plants (Tisserant et al., 1993; Abdel-
Fattah, 2001), but also with transfer of soil water to roots under DS (Khalvati et al., 2005). Mycorrhizal fungal hyphae include both live and dead hyphae, and the former participates in water transport (Allen, 2009). Functional hyphae are regarded as an index of hyphal vitality (Vivas et al., 2003), and active hyphae play an important role in biomass accumulation of host plants under DS (Tang and Chen, 1999). Therefore, the maintenance of AM hyphal activities under DS would help host plants to sustain greater nutritional (especially P) uptake and water transport. As a result, hyphal activities of AMF might be key factors to enhance drought tolerance in mycorrhizal plants. Soil P is usually in the form of orthophosphate that may be directly absorbed at the soil–root interface through root epidermis and
Table 3 Phosphatase activity in soils collected beneath non-AM and AM Poncirus trifoliata seedlings under well-watered (WW) or drought-stressed (DS) condition. Water status
AMF
WW
G. diaphanum G. mosseae G. versiforme Non-AMF G. diaphanum G. mosseae G. versiforme Non-AMF
Phosphatase activity (mg hydroxybenzene g−1 DW soil) Acid
DS
Significance DS AMF DS × AMF
1.65 1.70 1.63 1.14 1.96 2.09 1.89 1.58
Neutral ± ± ± ± ± ± ± ±
0.06cd 0.16bcd 0.13d 0.13e 0.10ab 0.23a 0.17abc 0.13d
0.26 0.51 0.52 0.34 0.36 0.34 0.46 0.46
**
*
**
NS
**
*
± ± ± ± ± ± ± ±
0.03d 0.11ab 0.07a 0.04cd 0.12bcd 0.12cd 0.05abc 0.14abc
Alkaline 0.41 0.48 0.43 0.41 0.43 0.57 0.42 0.42
± ± ± ± ± ± ± ±
0.09a 0.05a 0.04a 0.11a 0.05a 0.27a 0.01a 0.07a
NS NS NS
Data (means ± SE, n = 6) followed by the same letter within a column indicate no significant difference among treatments at P < 0.05. NS – not significant. * P < 0.05. ** P < 0.01.
Total 2.33 2.69 2.58 1.89 2.74 3.00 2.77 2.46 ** ** **
± ± ± ± ± ± ± ±
0.00d 0.08bc 0.12bcd 0.19e 0.23abc 0.26a 0.17ab 0.13cd
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Phosphorus has been considered as one of the major plant growth limiting factors, and its restricted mobility in soils constructs a depletion zone around roots (Brundrett and Abbott, 2002). The present study indicated that soil available P was indeed lower in both the non-AMF and the AMF colonized trifoliate seedlings (8–13 mg kg−1 ) than in the growth media (18.5 mg kg−1 ). Compared with the non-AMF inoculated seedlings, the mycorrhizal ones exhibited higher P contents in both leaves and roots under the WW or DS, which was in agreement with observations in onion (Allium cepa) (Nelsen and Safir, 1982), Sclerocarya birrea (Muok and Ishii, 2006), and coriander (Coriandrum sativum) (Farahani et al., 2008). Phosphorus efflux from arbuscles was related to FALP (active hyphae) (Kojima and Saito, 2004), and efficiency of mycorrhizal symbiosis was related to FSDH (functional hyphae) (Abdel-Fattah, 2001). When extraradical mycelia of AMF reached soluble P beyond the root P depletion zone, P could be taken up and then transported to aboveground (Karandoshov and Bucher, 2005). Moreover, plant roots with high acid phosphatase activity obviously had high potential to utilize soil organic P (Khade et al., 2010; Tawaraya et al., 2006). As a result, a better plant growth under DS could thus be enhanced by an improved P nutrition through hyphal and soil phosphatase activities mediated by AM associations (Koide, 1993; Smith and Read, 2008), though P nutrition, water absorption and plant growth were similar in 5-month-old G. intraradices colonized sour orange and Carrizo citrange under DS (Graham et al., 1987). Therefore, further studies under similar P level and plant growth are required to evaluate water relations between AMF and non-AMF colonizations. Fig. 1. Effects of mycorrhizal colonization on leaf (A) and root (B) P contents of Poncirus trifoliata seedlings under well-watered (WW) and drought-stressed (DS) conditions. Data (means ± SE, n = 6) followed by the same letter above the bars are not significantly different among treatments at P < 0.05.
hairs, and indirectly at the fungal–root interface through external AM hyphae (Garg et al., 2006; Requena et al., 2007). Our results indicated that the major phosphatase form in the citrus rhizosphere was acid phosphatase, which was significantly increased in the mycorrhizal colonized seedlings and/or under DS. Soil acid phosphatase activity was positively correlated to soil water content and was also increased by the sole mycorrhizal inoculation (Chethan Kumar et al., 2008; Sardans et al., 2008) and/or the dual inoculation of G. intraradices and Sinorhizobium meliloti (Stancheva et al., 2008). As a consequence, more soil available P could be released with the increase of soil acid phosphatase mediated by AMF (Stancheva et al., 2008) and might thus partially alleviate plant drought stress.
5. Conclusion Our results showed that greater fungal activities in AM hyphae, soil acid and total phosphatase activities, and leaf and root P contents in AMF colonized trifoliate orange seedlings, particularly in G. mosseae-colonized seedlings, would result in a better growth of host plants under drought stress. An enhanced P nutrition through soil acid phosphatase and hyphal activities mediated by mycorrhizal fungi might be a basis for enhancing drought tolerance in host plants. Acknowledgements This research was supported by the National Natural Science Foundation of China (30800747), the Key Project of Chinese Ministry of Education (No.: 211107), the Science-Technology Research Project for Excellent Middle-aged and Young Talents of Hubei Provincial Department of Education, China (No.: Q20111301), and partially by the Ministry of Agriculture Key Laboratory of Crop Nutrition and Fertilization and the Director’s Research Foundation, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, China (09-04 and 102-43). References
Fig. 2. Effect of mycorrhizal colonization on soil available P content of Poncirus trifoliata seedlings under well-watered (WW) and drought-stressed (DS) conditions. Data (means ± SE, n = 6) followed by the same letter above the bars are not significantly different among treatments at P < 0.05.
Abdel-Fattah, G.M., 2001. Measurement of the viability of arbuscular-mycorrhizal fungi using three different stains; relation to growth and metabolic activities of soybean plants. Microbiol. Res. 156, 359–367. Allen, M.F., 2006. Water dynamics of mycorrhizas in arid soils. In: Gadd, G.M. (Ed.), Fungi in Biogeochemical Cycles. Cambridge University Press, British Mycological Society, pp. 74–97. Allen, M.F., 2007. Mycorrhizal fungi: highways for water and nutrients in arid soils. Vadose Zone J. 6, 291–297. Allen, M.F., 2009. Bidirectional water flows through the soil-fungal-plant mycorrhizal continuum. New Phytol. 182, 290–293. Augé, R.M., 2004. Arbuscular mycorrhizae and soil/plant water relations. Can. J. Soil Sci. 84, 373–381. Bhusal, R.C., Mizutani, F., Rutto, K.L., 2002. Selection of rootstocks for flooding and drought tolerance in citrus species. Pak. J. Biol. Sci. 5, 509–512.
298
Q.-S. Wu et al. / Scientia Horticulturae 129 (2011) 294–298
Brundrett, M.C., Abbott, L.K., 2002. Arbuscular mycorrhizas in plant communities. In: Sivasithamparam, K., Dixon, K.W., Barrett, R.L. (Eds.), Microorganisms in Plant Conservation and Biodiversity. Kluwer Academic Publishers, Secaucus, NJ, pp. 151–193. Chapman, H.D., Pratt, P.F., 1961. Methods of Analysis for Soils Plants and Waters. University of California, Riverside, CA, USA. Chethan Kumar, K.V., Chandrashekar, K.R., Lakshmipathy, R., 2008. Variation in arbuscular mycorrhizal fungi and phosphatase activity associated with Sida cardifolia in Karnataka. World J. Agric. Sci. 4, 770–774. Farahani, A., Lebaschi, H., Hussein, M., Hussein, S.A., Reza, V.A., Jahanfar, D., 2008. Effects of arbuscular mycorrhizal fungi, different levels of phosphorus and drought stress on water use efficiency, relative water content and proline accumulation rate of cariander (Coriandrum sativum L.). J. Med. Plants Res. 2, 125–131. Garg, N., Jali, G., Kaur, A., 2006. Arbuscular mycorrhiza: nutritional aspects. Arch. Agron. Soil Sci. 52, 593–606. Graham, J.H., Syvertsen, J.P., 1984. Influence of vesicular-arbuscular mycorrhiza on the hydraulic conductivity of roots of two citrus rootstocks. New Phytol. 97, 277–284. Graham, J.H., Syvertsen, J.P., Smith, M.L., 1987. Water relations of mycorrhizal and phosphorus-fertilized non-mycorrhizal Citrus under drought stress. New Phytol. 105, 411–419. Guillemin, J.P., Orozco, M.O., Gianinazzi-Pearson, V., Gianinazzi, S., 1995. Influence of phosphate fertilization on fungal alkaline phosphatase and succinate dehydrogenase activities in arbuscular mycorrhiza of soybean and pineapple. Agric. Ecosys. Environ. 53, 63–69. Hardie, K., 1985. The effect of removal of extraradical hyphae on water uptake by vesicular-arbuscular mycorrhizal plants. New Phytol. 101, 677–684. Huang, H., Zhang, S., Wu, N., Luo, L., Christie, P., 2009. Influence of Glomus etunicatum/Zea mays mycorrhiza on atrazine degradation, soil phosphatase and dehydrogenase activities, and soil microbial community structure. Soil Biol. Biochem. 41, 726–734. Joner, E.J., Johansen, A., 2000. Phosphatase activity of external hyphae of two arbuscular mycorrhizal fungi. Mycol. Res. 104, 81–86. Joner, E.J., van Aarle, I.M., Vosatka, M., 2000. Phosphatase activity of extra-radical arbuscular mycorrhizal hyphae: a review. Plant Soil 226, 199–210. Karandoshov, V., Bucher, M., 2005. Symbiotic phosphate transport in arbuscular mycorrhizas. Trends Plant Sci. 10, 22–29. Khade, S.W., Rodrigues, B.F., Sharma, P.K., 2010. Arbuscular mycorrhizal status and root phosphatase activities in vegetative Carica papaya L. varieties. Acta Physiol. Plant. 32, 565–574. Khalvati, M.A., Hu, Y., Mozafar, A., Schmidhalter, U., 2005. Quantification of water uptake by arbuscular mycorrhizal hyphae and its significance for leaf growth, water relations, and gas exchange of barley subjected to drought stress. Plant Biol. 7, 706–712. Koide, R., 1993. Physiology of the mycorrhizal plant. Adv. Plant Path. 9, 33–54. Kojima, T., Saito, M., 2004. Possible involvement of hyphal phosphatase in phosphate efflux from intraradical hyphae isolated from mycorrhizal roots colonized by Gigaspora margarita. Mycol. Res. 108, 610–615. Levy, Y., Krikun, J., 1980. Effect of vesicular-arbuscular mycorrhiza on Citrus jambhiri water relations. New Phytol. 85, 25–31. Muok, B.O., Ishii, T., 2006. Effect of arbuscular mycorrhizal fungi on tree growth and nutrient uptake of Sclerocarya birrea under water stress, salt stress and flooding. J. Jpn. Soc. Hort. Sci. 75, 26–32. Nelsen, C.E., Safir, G.R., 1982. Increased drought tolerance of mycorrhizal onion plants caused by improved phosphorous nutrition. Planta 154, 407–413. Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., 1954. Estimation of Available Phosphorus in Soils by Extraction with Sodium Bicarbonate. United States Department of Agriculture Circular No. 939, Washington, DC, pp. 1–9.
Parniske, M., 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat. Rev. Microbiol. 6, 763–775. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161. Requena, N., Serrano, E., Ocón, A., Breuniger, M., 2007. Plant signals and fungal perception during arbuscular mycorrhiza establishment. Phytochemistry 68, 33–40. ˜ Sardans, J., Penuelas, J., Ogaya, R., 2008. Experimental drought reduced acid and alkaline phosphatase activity and increased organic extractable P in soil in a Quercus ilex Mediterranean forest. Eur. J. Soil Biol. 44, 509–520. Smith, S.E., Gianinazzi-Pearson, V., 1988. Physiological interactions between symbionts in vesicular-arbuscular mycorrhizal plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 221–244. Smith, S.E., Gianinazzi-Pearson, V., 1990. Phosphate uptake and vesicular-arbuscular activity in mycorrhizal Allium cepa L.: effect of photo irradiance and phosphate nutrition. Aust. J. Plant Physiol. 17, 177–188. Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, 3rd ed. Academic Press, San Diego. Stancheva, I., Geneva, M., Djonova, E., Kaloyanova, N., Sichanova, M., Boychinova, M., Georgiev, G., 2008. Response of alfalfa (Medicago sativa L.) growth at low accessible phosphorus source to the dual inoculation with mycorrhizal fungi and nitrogen fixing bacteria. Gen. Appl. Plant Physiol. 34, 319–326. Tang, M., Chen, H., 1999. Effects of arbuscular mycorrhizal fungi alkaline phosphatase activities on Hippophae rhamnoides drought-resistance under water stress conditions. Trees 14, 113–115. Tawaraya, K., Naito, M., Wagatsuma, T., 2006. Solubilization of insoluble inorganic phosphate by hyphal exudates of arbuscular mycorrhizal fungi. J. Plant Nutr. 29, 657–665. Tisserant, B., Gianinazzi-Pearson, V., Gianinazzi, S., Collotte, A., 1993. In plant histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections. Mycol. Res. 97, 245–250. Trouvelot, A., Kough, J.L., Gianinazzi-Pearson, V., 1986. Mesure du taux de mycorrhization VA d’un système radiculaire. Recherche de méthodes d’estimation ayant une signification fonctionnelle. In: Gianinazzi-Pearson, V., Gianinazzi, S. (Eds.), Physiological and Genetical Aspects of Mycorrhizae. INRA Press, Paris, pp. 217–221. Vivas, A., Marulanda, A., Gomez, M., Azcon, R., 2003. Physiological characteristics (SDH and ALP activities) of arbuscular mycorrhizal colonization as affected by Bacillus thuringiensis inoculation under two phosphorus levels. Soil Biol. Biochem. 35, 987–996. Wang, F.Y., Lin, X.G., Yin, R., Wu, L.H., 2006. Effects of arbuscular mycorrhizal inoculation on the growth of Elsholtzia splendens and Zea mays and the activities of phosphatase and urease in a multi-metal-contaminated soil under unsterilized conditions. Appl. Soil Ecol. 31, 110–119. Wu, Q.S., Levy, Y., Zou, Y.N., 2009. Arbuscular mycorrhizae and water relations in citrus. In: Tennant, P., Benkeblia, N. (Eds.), Citrus II. Tree and Forestry Science and Biotechnology, vol. 3, pp. 105–112 (Special Issue 1). Wu, Q.S., Xia, R.X., Zou, Y.N., 2008. Improved soil structure and citrus growth after inoculation with three arbuscular mycorrhizal fungi under drought stress. Eur. J. Soil Biol. 44, 122–128. Wu, Q.S., Xia, R.X., Zou, Y.N., Wang, G.Y., 2007. Osmotic solute responses of mycorrhizal citrus (Poncirus trifoliata) seedlings to drought stress. Acta Physiol. Plant. 29, 543–549. Wu, Q.S., Zou, Y.N., 2009. Mycorrhiza has a direct effect on reactive oxygen metabolism of drought-stressed citrus. Plant Soil Environ. 55, 436–442. Zhao, L.P., Jiang, Y., 1986. Research of determination of soil phosphatase activity. Chin. J. Soil Sci. 17, 138–141 (in Chinese with English abstract). Zlatev, Z.S., Yordanov, I.T., 2004. Effects of soil drought on photosynthesis and chlorophyll fluorescence in bean plants. Bulg. J. Plant Physiol. 30, 3–18.