Mycol Progress (2015) 14: 84 DOI 10.1007/s11557-015-1108-1
ORIGINAL ARTICLE
Contributions of an arbuscular mycorrhizal fungus to growth and physiology of loquat (Eriobotrya japonica) plants subjected to drought stress Yan Zhang 1,2 & Qing Yao 1 & Juan Li 3 & Yong Wang 2 & Xiangyu Liu 1 & Youli Hu 1 & Jiezhong Chen 1
Received: 20 April 2015 / Revised: 24 August 2015 / Accepted: 25 August 2015 / Published online: 12 September 2015 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2015
Abstract Loquat (Eriobotrya japonica) is an evergreen tree with a shallow root system subjected to drought stress. We have found that AM fungi can alleviate drought stress by improving loquat nutrient uptake. However, the physiological mechanisms of improving drought tolerance have not been described so far in loquat mycorrhiza symbiosis. Funneliformis mosseae was used as arbuscular mycorrhizal fungus and loquat was selected as a model for an evergreen, woody plant. Thus, a pot experiment with four treatments was conducted. Growth, leaf water status, solute accumulation, oxidative damage to lipids, antioxidant activities, and phytohormones were evaluated by nonmycorrhizal (NM) and arbuscular mycorrhizal (AM) loquat plants growing under well-watered or drought-stressed conditions. Results showed that AM plants had higher dry-biomass production and leaf water potential than NM plants under drought-stressed conditions. The drought-stressed AM roots accumulated more proline than in NM roots, while not in leaves. Lipid peroxides of leaves and roots in drought-stressed AM plants were 26 and 61 % lower than in NM plants. The AM symbiosis may enhance osmotic adjustment in roots, contributing to maintaining a water potential gradient and water absorption from soil into the roots. The cumulative effects increased Section Editor: Franz Oberwinkler * Jiezhong Chen
[email protected] 1
College of Horticulture, South China Agricultural University, Guangzhou 510642, China
2
College of Agricultural, Guizhou University, Guiyang 550025, China
3
College of Horticulture and Landscape Architecture, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
the AM plant tolerance to drought stress. The results are compared to findings reported hitherto mainly from short-lived, herbaceous AM plants in the literature. Keywords Fruit trees . Glomeromycetes . Glomus mosseae . Pomiculture
Introduction Drought stress is a major abiotic stress, which often limits agricultural crop production worldwide (Farooq et al. 2009). It causes damage to cells through the excessive formation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radicals (·OH), singlet oxygen (1O2), superoxide anion radicals (O2·−), etc. (Impa et al. 2012). The improvement of stress tolerance is often related to enhanced contents of antioxidant compounds in plants (Hasanuzzaman et al. 2012). Given the toxicity of ROS, plants have appropriate detoxification systems, which allow rapid removal of these compounds. These systems include several antioxidant enzymes and other non-enzymatic compounds such as ascorbate (AsA) and glutathione (GSH) (Ma et al. 2008; Gill and Tuteja 2010; Impa et al. 2012). Arbuscular mycorrhizal (AM) fungi establish non-specific symbioses with most terrestrial plants (Peterson et al. 2004). AM plants have an improved ability for nutrient uptake and tolerance to biotic and abiotic stresses while the fungus acquires a protected ecological niche and plant photosynthates (Miransari et al. 2008; Daei et al. 2009; Audet 2012; Zhang et al. 2014). Furthermore, the symbiosis protects host plants against detrimental effects of drought (Augé 2001; RuizLozano 2003; Ruiz-Lozano and Aroca 2010). Recently, many studies demonstrated that large numbers of comparatively short-lived herbaceous plants inoculated with AM fungi
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increased the drought stress tolerance, such as soybean (Porcel and Ruiz-Lozano 2004), rice (Ruiz-Sánchez et al. 2010), lettuce (Subramanian et al. 2006) and other similar plants (Augé 2001). Few studies, however, include woody plants, namely citrus and Poncirus trifoliata (Fan and Liu 2011; Wu et al. 2011), strawberry tree (Navarro et al. 2011), the date palm (Benhiba et al. 2015), and Erythrina variegata (Manoharan et al. 2010). It has been currently accepted that the contribution of AM symbiosis to plant drought stress tolerance resulted from a combination of physical, nutritional, and cellular effects, but the precise mechanisms involved are still in debate (Ruiz-Lozano 2003; Birhane et al. 2012). The main mechanisms of AM symbiosis to alleviate drought stress are direct uptake and transfer of water through the hyphae to the host plant (Marulanda et al. 2003), changed water retention properties in soil (Rillig 2004), improved osmotic adjustment (Kubikova et al. 2001) and so on, indicating diverse mechanisms depending on the plant species. Loquat (family Rosaceae, subfamily Maloideae) is an evergreen fruit tree, native to southeastern China. This ancient fruit has become commercialized on a large scale in recent times (Janick 2007). It was only during the 19th century that selections of cultivars with larger fruits were performed for fruit production (Badenes et al. 2000, 2013; He et al. 2011). So far, loquat has been grown in more than 30 countries in subtropical and mild-temperate regions of the world (Lin et al. 1999; Feng et al. 2007; Ferreres et al. 2009). It is becoming an important industry in China as well as in Japan, India, Pakistan, the United States (mainly California and Florida), Brazil, Venezuela, the Mediterranean basin (mainly Spain, Italy, Turkey, and Israel) and Australia (Vilanova et al. 2001; Janick 2007; Badenes et al. 2013). Loquat is mainly consumed as fresh juicy fruit and has many medicinal uses (Hamada et al. 2004; Lin et al. 2007; Kim and Shin 2009). The loquat fruit is particularly popular for its succulent texture, tangy flavor, and unusual harvest season (late spring/early summer), when few cultivated plants produce palatable fruits (Qin et al. 2013). There are no Food and Agricultural Organization of the United Nations (FAO) statistics available for world loquat fruit production; however, China is the world’s largest producer of loquat with a cultivation area of 170,000 ha, producing more than 400,000 t of fresh fruits per annum (Lin 2007; Zhang et al. 2015). The loquat root is too shallow to resist environmental stresses, which represents a huge challenge in the production areas of China (Lin 2007). In these areas, most loquat orchards are established in hillsides without irrigation, and there are repeated seasonal droughts, which limits fruit yield and quality (Luo et al. 2007). Loquat plants used for fruit production in the orchards are usually over 2,000 years
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old (Lin et al. 1999; Zheng et al. 2015). In this longterm context, the AM symbiosis technique is a potential biotechnology to enhance the drought tolerance of loquat plants in these areas. To maximize the beneficial effects of AM fungi in loquat production, particularly under drought stress condition, much effort should be made to investigate the AM fungal effects on drought tolerance of loquat plants and reveal the corresponding mechanisms. We have demonstrated the dramatic effect of three AM fungal s p e c i e s A c a u l o s p o r a l a e v i s G e r d . & Tr a p p e , Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler (syn. Glomus mosseae), and F. caledonium (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler (syn. G. caledonium) on loquat growth by improving nutrient uptake under drought-stressed conditions (Zhang et al. 2014). The study of Torta and Costantino (1996) showed AM fungi of the genera Glomus, Acaulospora, and Gigaspora kept constant association with loquat, carob, and prickly pear. Then Wang et al. (2007) found that inoculation with exogenous AM fungus (F. mosseae) increased the infection rate of loquat root. Here the mechanisms responsible for improving drought tolerance inoculated with F. mosseae were explored, with special emphasis on the osmotic adjustment, non-enzymatic antioxidant compounds, antioxidant enzymes, and phytohormones.
Materials and methods Experimental design The experiment was a 2×2 factorial randomized complete block design with two inoculations (control non-mycorrhizal and mycorrhizal) and two water regimes (well-watered and drought-stressed). It thus produced four treatments: wellwatered control non-mycorrhizal (NM) plants; droughtstressed NM plants; well-watered arbuscular mycorrhizal (AM) plants; and drought-stressed AM plants. For each treatment, five replicates were set which produced 20 pots. The soil moistures in well-watered and drought-stressed pots were kept at 75 and 45 % of the field capacity, corresponding to 16.5 and 9.9 % gravimetric soil moisture, respectively. All data were the average of five replicates. Soil and biological materials The soil substrate in this study was mixed by red soil, peat soil and sand (3:1:1, v/v/v). Red soil, with a clay loam texture, was derived from the teaching experiment field in the college of horticulture, South China Agricultural University, Guangzhou, China, which has the main soil type in south China
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and has many limitations for crop production, especially low phosphorus (P) bioavailability (Zeng et al. 2014). All components were sieved (5 mm) and autoclaved (121 °C, 2 h). Substrate properties were pH = 4.98, 2.36 % organic matter, 33.63 mg·kg−1 alkali hydrolysable nitrogen, 1.78 mg·kg−1 available phosphorus, 30.53 mg·kg−1 available potassium. Loquat (E. japonica cv. ‘Zaozhong No. 6’) seeds were sterilized with 5 % sodium hypochlorite (NaClO) for 30 min, then washed several times with distilled water and finally placed on wet cotton cloth at 28 °C to germinate. Post-germinated seeds were sown in an autoclaved (121 °C, 2 h) mixture of peat soil and sand (3:1, v/v). One-month-old seedlings with similar size (5–6 leaves) were transferred to pots containing 2500 g of prepared substrate. Mycorrhizal inoculum or sterilized inoculum (300 g) was added near roots of each seedling at the center of an AM or a NM pot. Our research fungal strain, F. mosseae BGC BJ01 was provided by Beijing Agro-Forestry Academy and had been isolated from Osmanthus fragrans (Thunb.) Lour. in Shangrao city, Jiangxi province. Fungal inoculants were propagated with white clover (Trifolium repens) as hosts in an open-pot culture and consisted of sand, spores, mycelia, and colonized root fragments with a colonization efficiency of 85 %. Growth conditions Plants were grown in a controlled environmental chamber with 50−60 % relative humidity, 30/25 °C (day/night), and a photoperiod of 16 h at a photosynthetic photon flux density of 520 μmol·m−2 ·s−1 using a LI-6400 open-flow gas exchange system (LI-COR, Lincoln, NE, USA). Soil moisture was measured by the weighing method. During the beginning 2 months after transplanting, all pots were well watered (75 % of the field capacity). Thereafter, AM and NM seedlings were subjected to well-watered and droughtstressed treatments as described in the experimental design. Each pot was weighed daily at the end of the afternoon and then supplied with water to maintain the target relative water contents. Plants were maintained under such conditions for an additional 6 months (significant differences can be observed from the external morphology) before sampling. Parameters measured Morphological parameters and biomass production The leaf number, plant height, and basal stem diameter were recorded every 20 days after drought stress treatment, and the last time was on the harvest day. At the day before harvest, a Minolta SPAD-502 chlorophyll meter (Minolta Inc., Japan) was used to measure the leaf SPAD (soil plant analysis development) values from foliar discs around the mid-point of the
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second fully-expanded loquat leaf per plant. This instrument makes instantaneous and non-destructive chlorophyll measurements known as the relative chlorophyll index (SPAD units). Two SPAD readings were taken for each of the five replicates per treatment (Sommer et al. 2012; Vicente-Sánchez et al. 2014; de Andrade et al. 2015). Immediately before plant sampling, one newly mature leaf from each plant was cut for the determination of leaf water potential (leaf Ψ) using a pressure chamber (PMS Instruments Co., Corvallis, OR, USA) (Li 2000; Asrar et al. 2012). At the time of sampling, the shoots and roots were separated for calculating their fresh weight after being cleaned with tap water. Subsequently, three mature leaves and parts of fine roots from each plant were cut into small pieces, ten aliquots (0.5 g) were frozen in liquid nitrogen and stored at −74 °C prior to other measurements. Additional aliquots of fresh roots were taken for the determination of AM colonization. Other plant parts were weighed and oven-dried (70 °C, 2 days), and the dry weight was also measured. Symbiotic development AM colonization was estimated according to Giovannetti and Mosse (1980). The mycorrhizal contribution (%) to plant biomass was calculated as: (value for AM plant - mean value for NM plants) / value for AM plant×100 (Plenchette et al. 1983; Smith et al. 2003). Proline and total soluble sugars (SS) Free proline was extracted from 0.5 g fresh leaves or roots, and estimated by spectrophotometric analysis by ninhydrin reaction (520 nm). Soluble sugar was determined by the anthrone method using sucrose as Li (2000). Hydrogen peroxide content and oxidative damage to lipids Hydrogen peroxide content in 0.5 g leaves or roots was measured by Patterson’s method slightly modified by Aroca et al. (2003). Lipid peroxides were extracted from 0.5 g grinded leaves or roots kept in liquid nitrogen and homogenized with 10 ml of 100 mM potassium phosphate buffer (pH 7.0). Homogenates were centrifuged at 10,000×g for 20 min. Malondialdehyde (MDA) of extracts was determined by the thiobarbituric acid reaction described by Sudhakar et al. (2001). Enzyme assays For enzyme extracts, 0.5 g fresh leaves or roots were frozen in liquid nitrogen and then grounded in 10 ml solution, which contained 50 mM K-phosphate buffer (pH 7.8), 1 % (w/v) polyvinylpolypyrrolidone (PVPP). The homogenate was
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centrifuged at 10,000×g for 20 min (4 °C), and the supernatant was collected for enzyme assays. The activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and peroxidase (POD, EC 1.11.1.7) were measured according to Jimenez et al. (1997) and Li (2000). Ascorbate (AsA) and glutathione (GSH) content The 0.5 g fresh leaves or roots were homogenised in icecold 5 % (w/v) trichloroacetic acid (TCA) and then centrifuged at 10,000×g for 20 min at 4 °C. Supernatant was used for AsA, and GSH was determined according to method of Huang et al. (2007). Phytohormones The extraction, purification, and determination of endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), and zeatin riboside (ZR) by an enzyme-linked immunosorbent assay (ELISA) technique were performed as in Teng et al. (2006). Fresh leaves or roots (0.5 g) were ground into homogenates in liquid nitrogen and weak light. The absorbance of each well was measured at 490 nm using an auto microplate reader (infinite M200, Tecan, Austria).
Mycol Progress (2015) 14: 84 Table 1 Percentage of mycorrhizal colonization (%) in arbuscular mycorrhizal (AM) loquat plants cultivated under well-watered or drought-stressed conditions Treatment
Total (%) Hypha (%) Arbuscules (%) Vesicle (%)
Well-watered
72.5 *
Drought-stressed 44.7
72.3 * 44.7
13.6 ns 8.5
22.5 * 8.2
Means followed by the same letter within a column are not significantly different (P
