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INCREASED TOLERANCE OF TRIFOLIATE ORANGE (PONCIRUS TRIFOLIATA) SEEDLINGS TO WATERLOGGING AFTER INOCULATION WITH ARBUSCULAR MYCORRHIZAL FUNGI.

Byline: Y. N. Zou A. K. Srivastava1 Q. S. Wu and Y. M. Huang

: ABSTRACT

Growth suppressive role of waterlogging (WL) as an important abiotic stress to perennial plants is well known. A pot experiment was conducted to study a potential contribution of arbuscular mycorrhizal fungi (AMF Diversispora spurca) on WL tolerance of trifoliate orange (Poncirustrifoliata L. Raf.) seedlings on the basis of growth root system architecture and antioxidant enzymes. Four-month-old seedlings inoculated with or without D. spurca were subjected to WL with non-waterlogging (NWL) as control treatment. Root mycorrhizal (MY) colonization and vesicles notably decreased but entry points significantly increased after 36 days of WL. The WL restricted plant growth performance and numbers of different order lateral roots but the MY symbiosis exhibited theameliorating roles on plant growth root morphology (total length surface area projected area and volume) and numbers of different order lateral roots.

The AMF inoculation significantly increased superoxide dismutase and catalase activities in leaf and root under both NWL and WL thereby resulting in lower oxidative damage in terms of malondialdehide concentration. These results suggest that the MY seedlings were biochemically and morphologically better prepared to tolerate WL compared to the NMY seedlings.

Keywords: Antioxidant enzymes; Arbuscular mycorrhiza; Root system architecture; Trifoliate orange; Waterlogging

INTRODUCTION

Soil waterlogging (WL) one of the important abiotic stresses impairs with plant growth and productivity (Yin et al. 2009) by blocking the oxygen supply to the roots and affecting several metabolic processes of plants (Sairam et al. 2009).

Arbuscular mycorrhizal fungi (AMF) are the important component of crop production system as they can form mutualistic associations with roots of most terrestrial plants (Bainard et al. 2011). The characteristic of the mycorrhizal (MY) symbiosis is the transfer of sugars from the host plant to the fungus and of both nutrients and water from the fungus to the host plant partner (Fitter et al. 2011). Many studies have confirmed that inoculation with AMF increased the degree of tolerance in citrus plants exposed to various abiotic stresses like drought (Wu and Zou 2010) high temperature (Wu 2011) salt stress (Wu et al. 2010) and elevated CO2 concentration (Jifon et al. 2002). Fougnies et al. (2007) earlier reported that colonization of Pterocarpus officinalis by Glomus intraradices enhanced its tolerance to WL through improvements in plant growth and P-uptake. Better tolerance to WLcaused by

AMF was previously reported in two semiaquatic grasses Panicum hemitomon and Leersia hexanda (Miller and Sharitz 2000) suggesting potential beneficial contribution of AMF inoculation to host plant possibly hold some promise for WL tolerance.

Citrus in southern region of China is extensively grown under various soil habitats including periodic soil waterlogged conditions. The waterlogged condition seriously inhibits tree growth and frequently declines in orchard productivity. However the limited efforts have been made to understand the responses of citrus plants to waterlogged conditions through MY inoculation. In this background the present study was carried out to study the effects of AMF on biochemical and morphological traits of citrus seedlings under WL conditions.

MATERIALS AND METHODS

Experimental design: A total of four treatments in 22 randomized factorial design comprising MY inoculations (with or without Diversispora spurca) and waterlogged treatments (WL and NWL) were tested through a pot experiment under controlled greenhouse conditions .

Plant culture: Trifoliate orange (Poncirus trifoliata L. Raf.) was used as the experimental plant. Seeds of trifoliate orange were sown into the sterilised mixture of yellow-brown soil and vermiculite mixture (1:1 v/v). The five-leaf-old seedlings without root MY colonization were transplanted into the plastic pots(17.5 cm A- 13 cm A- 11 cm) containing a sterilized (121C 0.11Mpa 2 h) yellow-brown soil from a campus citrus orchard of Yangtze University. At the time of transplanting 60 g of Diversisporaspurca inocula (12 spores/g) was inoculated into the rhizosphere of the citrus seedlings. The NMY seedlings received 60 g of autoclaved (121C 0.11Mpa 2 h) inocula plus 10 mL of 60 g inoculum filtrates through a 25 m filter to account only the differences of D. spurca spores between MY and NMY seedlings. The mycorrhizal inocula were propagated through the use of D. spurca spores based on pot culture

inoculated in white clover (Trifoliumrepens) and grew 13 w at a controlled growth charmber (PQX Life Apparatus Ningbo Life Science and Technology Ltd. China) under the conditions of 16:8 photoperiod 25/19C day/night temperature 80% relative humidity and light intensity 1700 Lx. The mycorrhizal fungus was isolated from the rhizosphere of Lycopersicon esculentum in Shougguang China and exhibited better tolerance of trifoliate orange to salinity stress (Zou and Wu 2011). All the seedlings were placed in a plastic greenhouse at the Yangtze University campus from May to October 2011. No exogenous application of nutrients was undertaken.

Waterlogged treatment: One hundred and seventeen days after acclimation for establishing root mycorrhizal colonization the seedlings were subjected to WLand NWL treatments. Waterlogging treatment was given by placing the pots into a larger plastic container (28 cm A- 17 cm A- 20 cm) filled with tap water to 2 cm above the pot. The seedlings of NWL treatment maintained ~25% soil water content (corresponding to field water capacity) by gravimetry. Trifoliate orange seedlings were subjected to WL and NWL treatments for 36-days and the WL seedlings exhibited a small amount of leaf tip yellowing at 36 days.

Measurements of plant growth root mycorrizal colonization and root system architecture (RSA): All the seedlings were harvested 36 days after WL and NWL treatments and fresh shoot and root weights (g) plant height (cm) and stem diameter (cm) were determined and recorded.

The taproot length per seedling was determined by the vernier caliper. The number of first second and third order lateral roots were manually mounted. The intact root system per seedling was scanned by an Epson Flatbed Scanner Epson Perfection V700 Photo Dual Lens System (J221A Indonesia). The image toeach root system was analyzed by a professional WinRHIZO software in 2007 version (Regent Instruments Inc. Quebec Canada) automatically obtaining root morphological traits.

The root MY staining was carried out by the procedure of Phillips and Hayman (1970). The root MY colonization was assessed using the method as described by Wu et al. (2008). Analysis of antioxidant enzymes and soluble protein: A 0.2 g frozen leaf or root sample was homogenized with 7 mL of 100 mM ice-cold phosphate buffer (pH 7.8). The homogenate was centrifuged at 4000 g for 10 min at 4C and the supernatant was used in the determination of protein and antioxidant enzymes. The soluble protein concentration of leaf and root was estimated using bovine serum albumin as a standard (Bradford 1976). The leaf and root SOD activity assay was carried out by the method of Giannopolitis and Ries (1977). One unit of SOD activity was defined as the quantity of SOD that brought a 50% inhibition in photochemical reduction of nitro blue tetrazolium at 560 nm. The leaf and root CAT activity was assayed according to the potassium permanganate titrimetric method previously described by Wu et al. (2010).

Lipid peroxidation: Lipid peroxidation was assessed by measuring the amount of malondialdehide (MDA) concentrations according to thiobarbituric acid reaction as described by Sudhakar et al. (2001).

Statistical analysis: Data were analyzed using two-way ANOVA carried out through SAS software (v8.1). Significant differences among means were assessed based on the Duncan's multiple range test at 5% level.

RESULTS AND DISCUSSION

Mycorrhizal development: Our study showed that the WL treatment significantly decreased root colonization by 29% (Table 1) which is consistent with Lotus glaber colonized by native AMF exposed to 35 days of WL (Mendoza and Garcia 2007). On the other hand the WL significantly increased number of entry point by 95% but decreased number of vesicleby 78%. In contrast earlier studies showed an increase of vesicles in roots of Lotus glaber subjected to WL (Mendoza and Garcia 2007). Interestingly under WL conditions the hyphal density and root colonization of trifoliate orange by Gigaspora margarita were increased when Paspalum notatum was intercropped (Matsumura et al. 2008).

Growth performance: The present study showed that the WL markedly restricted all the growth parameters viz. plant height stem diameter leaf number per plant and shoot and root fresh weights but the AMF inoculation significantly increased these growth variables under both NWL and WL conditions (Table 1). The observation provides a strong clue about the role of MY inoculation in improvement of plant growth under WL stress.

Root system architecture: RSA so-called the spatial configuration throughout the root system determines the acquisition of nutrients and water from soil (Hodge et al. 2009). In the present work the WL induced no significant change in RSA properties viz. taproot length root total length root projected area root surface area and root volume but significantly decreased the numbers of first- second- and third-order lateral roots (Table 2). On the other hand the seedlings colonized by AMF recorded significantly higher total root length root projected area and surface area root volume and numbers of first- second- and third-order lateral roots than the non-AMF seedlings regardless of WL and NWL. Since RSA is a major factor to affect tolerance to abiotic stresses (Remans et al. 2012) greater RSA of the MY seedlings in the present study would benefit nutrient uptake of plants thereby increasing tolerance of the MY seedlings to WL. Andioxidant enzyme system: WL stress usually induces root oxygen deficiency (axonsis) triggering further the photoxidative damage via an increased generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide (Yin et al. 2009). In our study the WL significantly increased MDA concentration of leaf and root in the AMF and the non-AMF seedlings whereas the AMF seedlings recorded significantly lower MDA concentration: 17% in leaf and 23% in root under the NWL and 16% in leaf and 16% in root under the WL as compared with the non-AMF controls (Fig. 1; Table 3). The result implies that oxidative damage was higher in the WL than in the NWL seedlings but lower oxidative damage was in the MY than in the NMY seedlings.

Table 1: Effects of WL treatment on MY development and plant growth of trifoliate orange (Poncirus trifoliata) seedlings

Waterlogged###AMF###Mycorrhizal attributes###Plant growth parameters

treatment###status

###Root###Entry###Vesicles###Height###Stem###Leaf###Shoot###Root

###colonizat###points###(num/cm###(cm)###diameter###number###fresh###fresh

###ion###(num./cm###root###(cm)###of plant###weight###weight

###(%)###root)###(g)###(g)

###NWL###Non-###00c###00c###00c###21.50.2c###0.260.01c###21.80.8b###1.60.1b###1.40.1b

###AMF

###AMF###48.90.0a###4.21.8b###7.70.9a###28.91.1a###0.320.01a###27.20.8a###1.90.0a###1.70.1a

###WL###Non-###00c###00c###00c###19.00.3d###0.230.01d###15.72.8c###1.20.1c###1.10.0c

###AMF

###AMF###34.70.1b###8.22.3a###1.70.8b###27.20.2b###0.290.01b###22.81.5b###1.70.1b###1.40.1b

Significance

###WL###

###AMF###

WL A-AMF###NS###NS###NS###NS

The antioxidant enzymes such as SOD CAT and peroxidase play scavenging effect toward ROS under WL stress (Ahmed et al. 2002). The SOD activity of leaf and root and CAT activity of leaf decreased but CAT activity of root increased on account of WL stress (Fig. 2 3; Table 3) suggesting that the MY and NMY seedlings suffered from a serious burst of ROS under WL and the antioxidant enzymes could not absolutely eliminate excess ROS. Our results also showed that in leaf SOD activity was 25% and 38% higher in the AMF than in the non-AMF seedlings under the NWL and WL conditions; respectively (Fig. 2; Table 3). While in root 12% and 15% higher SOD activity was observed in the AMF seedlings than in the non-AMF seedlings under the NWL and WL conditions respectively. Under the NWL conditions the AMF infection significantly decreased CAT activity of leaf by 8% but increased CAT activity of root by 14% (Fig. 3; Table 3).

Under the WL conditions inoculation with AMF significantly increased CAT activity of leaf and root by 27% and 15% respectively. Wu (2011) reported similar results in SOD and CAT activity of Glomus mosseae-colonized trifoliate orange exposed to high temperature. In an experiment performed by Fester and Hause (2005) hyphae and arbuscules of mycorrhizas could accumulatea certain amount of ROS such as hydrogen peroxide (H2O2) in roots. On the other hand MY symbiosis (colonized by G. intraradices) in combination with drought stress considerably increased the expression of the Mn-sod II gene (Ruiz-Lozano et al. 2001). The higher SOD and CAT activities of leaf and root caused by AMF are further accountable to both MY accumulation and the expression of specific genes. Our study also indicated that the AMF colonization significantly increased soluble protein concentration in leaf by 28% and 21% and in root 27% and 30% by under the NWL and WL conditions (Fig. 4; Table 3) respectively suggesting that some stressed proteins are strongly induced to tolerate WL conditions.

With regard to these reactions further studies are needed to unravel the mechanism involved at cellular and molecular levels. The higher antioxidant enzyme activities of the MY seedlings were associated with lower levels of ROS in the plant tissues thus decreasing extent of membrane lipid peroxidation namely lower MDA concentration. These results suggested metabolically better MY plant preparedness to combat WL stress.

Table 2: Effects of AMF and waterlogged stress on root morphological traits and numbers of different order lateral roots of trifoliate orange seedlings

WL###AMF###Root morphology parameters###Lateral roots characteristics

treatment###status###Taproot###Total###Projected###Surface###Root###First###Second###Third

###length###Length###area (cm2) area (cm2)###volume###order###order###order

###(cm)###(cm)###(cm3)

NWL###Non-AMF###31.17.6a###256.84.3b###13.90.1b###43.70.5b###0.430.01c###401b###1526b###212b

###AMF###34.02.8a###503.915.0a###20.30.6a###63.61.8a###0.640.02a###451a###1865a###281a

WL###Non-AMF###33.52.9a###321.115.1b###13.90.4b###43.71.2b###0.480.01b###371c###1074c###140c

###AMF###33.30.5a###499.137.1a

###62.74.2a###20.01.4a###15513b###0.630.04a###411b###201b

Significance

WL###NS###NS###NS###NS###NS###

AMF###NS###

WL A-AMF###NS###NS###NS###NS###NS###NS###

Table 3: Significance of the sources of variation for biochemical variables in mycorrhizal and non-mycorrhizal trifoliate orange seedlings exposed to waterlogged stress

Sources of###Soluble protein###SOD###CAT###MDA

variation###Leaf###Root###Leaf###Root###Leaf###Root###Leaf###Root

WL

AMF###NS

WL A-AMF###NS###NS###NS###NS

Conclusion: The WL stress restricted the plant growth RSA and SOD and CAT activities of the trifoliate orange seedlings. These adverse effects were significantly alleviated by MY inoculation enhancing SOD and CAT activities and collectively imparting higher tolerance of MY seedlings under WL stress.

Acknowledgements: This study was supported by the National Natural Science Foundation of China (31101513) and the Excellent Young Teacher Research Support Program of Yangtze University (cyq201326).

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Publication:Journal of Animal and Plant Sciences
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