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Effects of Diversispora spurca Inoculation on Growth, Root System Architecture and Chlorophyll Contents of Four Citrus Genotypes.

Byline: Yan Li, Ying-Ning Zou and Qiang-Sheng Wu

Abstract

A pot experiment was conducted to study the effects of arbuscular mycorrhizal fungi, Diversispora spurca, on growth, root system architecture (RSA), and chlorophyll contents of four citrus genotype plants, namely, kumquat (Fortunella margarita), red tangerine (Citrus tangerine), sour orange (C. aurantium) and trifoliate orange (Poncirus trifolaita), in order to assess whether the effects are dependent on host genotype. After 180 days of mycorrhizal inoculation, the order of root mycorrhizal colonization was kumquat Greater than sour orange Greater than trifoliate orange Greater than red tangerine, and mycorrhizal dependency kumquat Greater than trifoliate orange Greater than sour orange Greater than red tangerine.

Mycorrhizal colonization significantly increased plant height, stem diameter (except sour orange), leaf number of plant, shoot and root dry weights of all the plants, and mycorrhizas had host-specific differences in growth performance. Mycorrhizal inoculation markedly increased length, projected area, surface area, volume, branch and cross of the root systems, and the increases were kumquat Greater than red tangerine [?] trifoliate orange Greater than sour orange. Significantly higher chlorophyll a, chlorophyll b, and total chlorophyll contents were found in the mycorrhizal plants than in the non- mycorrhizal plants. Results suggested that inoculation with D. spurca promoted growth and improved both RSA and chlorophyll contents, while the defferences were host-specific. (c) 2013 Friends Science Publishers

Keywords: Arbuscular mycorrhizal fungi; Citrus; mycorrhizal dependency; Root

Introduction

Arbuscular mycorrhizal fungi (AMF), an ancient group of fungi belonging to the phylum of Glomeromycota, can establish mutualistic association with ~80 (Percent) of terrestrial plants, in order to obtain photosynthates of the host plant for their asexual life cycle (Harrison, 2005). In return, AMF are primarily responsible for water and nutrient transfer from soil to the host plant.

Citrus plants are broadly grown all over the world. Meanwhile, China is one of the important citrus producing countries, where the citrus industry is expanding. As a rule, citrus roots exhibit short and poorly distributed root hairs and thereby are highly dependent on arbuscular mycorrhizal symbiosis (Wu et al., 2011a). A large number of reports have shown that inoculation with AMF can have many beneficial effects on citrus plants. Inoculation with Glomus mosseae greatly increased growth of sour orange and rough lemon under the condition of a low soil phosphorus (P) content and also caused an increase of P and copper (Cu) in leaves (Krikun and Levy, 1980). In a low P soil, G. intraradices increased leaf 14CO2 incorporation by 67 (Percent) , total chlorophyll content by 28 (Percent) , and ribulose bisphosphate carboxylase activity by 42 (Percent) in sour orange seedlings (Nemec and Vu, 1990).

Additionally, in trifoliate orange seedlings, AMF colonization improved tolerance to drought stress (Wu et al., 2011b) and high temperature stress but not low temperature stress (Wu and Zou, 2010; Wu, 2011), and salt stresses (Wu et al., 2010). Therefore, arbuscular mycorrhizal symbiosis is critical for growth of citrus trees.

Although AMF specifically colonize the roots of the host plant and also exhibit low host specificity, the compatibility between AMF and the host plant is existent. Spatial arrangement of the root system architecture (RSA) in soil can determine the capacity of a plant to uptake water and mineral nutrients (de Dorlodot et al., 2007). It is well documented that improvement of RSA caused by AMF in trifoliate orange seedlings was dependent on AMF species (Wu et al., 2011a). However, to data, the information about RSA variation of AMF on citrus genotype is unclear. The present work was carried out to assess whether the effects of AMF on growth, RSA, and chlorophyll contents were dependent on citrus genotype.

Materials and Methods

Plant Culture

The experiment was performed in a plastic greenhouse of Yangtze University, Jingzhou, China. The citrus genotypes used here included kumquat (Fortunella margarita L. Swingle), red tangerine (Citrus tangerine ex. Tanaka), sour orange (C. aurantium L.) and trifoliate orange (Poncirus trifolaita L. Raf.). Seeds of citrus plants were sown in a plastic pot (20 cm upper mouth diameter x 15 cm bottom mouth diameter x 18 cm height) filled with 3.0 kg of autoclaved (121degC, 0.11MPa, 2 h) growth substrates of soil, vermiculite, and pearlite (5:1:1, v/v/v). The potted growth mixture had been inoculated with Diversispora spurca before transplant by placing 15 g of mycorrhizal inoculum in the rhizosphere. Non-AMF treatment was provided by the 15 g of sterilized mycorrhizal inoculum.

The mycorrhizal inoculum including spores, extraradical hyphae, and infected fragmentized roots was provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China. The seedlings were grown from March 27 to September 7, 2010 in a greenhouse.

Experimental Design

The experiment was a 2x4 factorial randomized block design including four citrus genotypes (kumquat, red tangerine, sour orange and trifoliate orange) and two mycorrhizal inoculations (with or without D. spurca), each with 3 replicates for a total of 24 pots (two seedlings per pot).

Determinations of Variables

After 180 days of AMF inoculation, the mycorrhizal and non-mycorrhizal citrus seedlings were harvested. Plant height, stem diameter, and leaf number per plant were recorded before the harvest. The seedlings from each pot were divided into shoot and root. The roots were rinsed with distilled water and scanned with the EPSON Flatbed Scanner V700. The root pictures were analyzed with the WinRHIZO Pro 2007b (Regent Instruments Inc., Quebec, Canada), and the RSA traits including branch, cross, surface area, projected area, volume, and total length were obtained. Leaf chlorophyll was extracted by 80 (Percent) of acetone, and its content was measured by the method of Lichtenthaler and Wellburn (1983).

A small quantity of 1-2 cm root segments were cleared with 10 (Percent) of KOH and stained with 0.05 (Percent) of trypan blue solution (Phillips and Hayman, 1970). Root mycorrhizal colonization was quantified based on the formula of Wu et al. (2007). Entry points, vesicles and arbuscules were observed with the LEICADME biomicroscope and expressed as each number per cm root.

Statistical Analysis

The data were analyzed by ANOVA with software of SAS 8.1 version. Significant differences of means were compared with the Least Significant Difference (LSD) at 5 (Percent) level.

Results

Mycorrhizal Colonization

Roots were not infected by the non-AMF treatment in the non-AMF citrus plants. All the citrus plants were well colonized by the D. spurca, and root mycorrhizal colonization in the inoculated plants ranged from 23 to 47 (Percent) (Fig. 1).

Plant Growth

Inoculation with D. spurca significantly enhanced plant height, stem diameter (except sour orange), leaf number of plant, shoot and root dry weights of all the citrus seedlings (Table 1). Additionally, the order of mycorrhizal dependency of the four citrus plants was kumquat (204 (Percent) Greater than trifoliate orange (136 (Percent) Greater than sour orange (131 (Percent) Greater than red tangerine (127 (Percent).

Root/shoot ratio of mycorrhizal kumquat and red tangerine plants was significantly higher than those of non- mycorrhizal plants (Table 1). However, in this study, we also found no significant differences of root/shoot ratio between mycorrhizal and non-mycorrhizal sour orange and trifoliate orange plants.

Root System Architecture

In the present work, inoculation with D. spurca markedly increased root length, projected area, surface area, root volume, root branch and root cross of the four citrus genotype plants, compared with non-AMF treatment (Table2; Fig. 2). The increases of RSA traits caused by AMF in the present work were the highest response to kumquat (91.3,94.5, 94.6, 100.0, 163.8 and 166.7 (Percent) , respectively), higher red tangerine (62.0, 71.6, 71.5, 82.3, 82.7 and 85.4 (Percent) , respectively) and trifoliate orange (57.8, 80.1, 80.1, 103. 4,120.4 and 70.0 (Percent) , respectively), and the lowest sour orange (28.7, 40.2, 40.3, 50.1, 59.3 and 33.6 (Percent) , respectively).

Chlorophyll

Our study showed that the mycorrhizal colonization had a conspicuous effect on chlorophyll content (Table 3). Significantly higher chlorophyll a content (38.6-66.3 (Percent) , chlorophyll b content (29.1-110.5 (Percent) , carotenoid content (29.1-56.1 (Percent) , and total chlorophyll content (37.5-70.6 (Percent) were observed in the four AMF citrus plants than in the non- AMF ones. The increased trends of chlorophyll a, chlorophyll b, and total chlorophyll contents caused by AMF were the highest in trifoliate orange, higher in kumquat and red tangerine, and the lowest in sour orange.

Discussion

In this study, we confirmed that different citrus genotypes varied widely in their dependency on D. spurca. The root colonization ranked as kumquat Greater than sour orange Greater than

Table 1: Effect of AIVIF (Diversispora spurca) on growth traits of four citrus genotype plants

Citrus genotype###AMF###Plant height Stem diameter Leaf number###Dry weight (g)###Root/shoot###Mycorrhizal

###inoculation###(cm)###(cm)###per plant###Shoot###Root###Total###ratio###dependency (Percent)

Kumquat

###AMF###18.0c,x###0.293c,x###18.9b,x###0.87b,x###0.47c,x 1.35c,x###0.54b,y###204

###Non-AMF###11.6c,y###0.233c,y###10.5c,y###0.32c,y###0.23c,y 0.55c,y 0.71ab,x

Red tangerine

###AMF###15.7c,x###0.308b,x###18.2b,x###0.95b,x###0.66b,x 1.61b,x###0.69a,y###127

###Non-AMF###13.2c,y###0.278b,y###12.7b,y###0.62b,y###0.52b,y 1.14b,y###0.84a,x

Sour orange

###AMF###23.3b,x###0.343a,x###16.0c,x###2.20a,x###1.38a,x 3.57a,x###0.63a,x###131

###Non-AMF###19.5b,y###0.356a,x###13.5b,y###1.77a,y###1.05a,y 2.83a,y###0.60b,x

Trifoliate orange

###AMF###32.3a,x###0.316b,x###25.Oa,x###0.94b,x###0.01b,x 1.56bc,x 0.65a,x###136

###Non-AMF###27.8a,y###0.288b,y###21.Oa,y###0.70b,y###0.45b,x 1. 15b,y 0.64b,x

Significance

Citrus genotype

AMF

Citrus genotype X AMF###NS###NS

Table 2: Effects of AMF (Diversispora spurca) on RSA traits of four citrus genotype plants

Citrus genotype###AMF inoculation###Length (cm)###Projected area (cm2)###Surface area (cm2)###Volume (cm3) Branch number Cross number

Kumquat

###AMF###219.31c,x###14.90c,x###46.81c,x###0.80c,x###1076.0c,x###60.8c,x

###Non-AMF###114.64c,y###7.66c,y###24.05c,y###0.40c,y###407.8c,y###22.8b,y

Red tangerine

###AMF###508.45b,x###30.39b,x###95.46b,x###1 .44b,x###3986.0b,x###298.8ab,x

###Non-AMF###313.82b,y###17.71b,y###55.65b,y###0.79b,y###2181.7b,y###161.2a,y

Sour orange

###AMF###651.57a,x###47.78a,x###150.lla,x###2.78a,x###5269.0a,x###246.7b,x

###Non-AMF###506.33a,y###34.07a,y###107.02a,y###1.84a,y###3307.3a,y###184.7a,y

Trifoliate orange

###AMF###510.87b,x###27.92b,x###87.72b,x###1.20bc,x###3713.3b,x###344.3a,x

###Non-AMF###323.74b,y###15.50b,y###48.70b,y###0.59bc,y###1684.7b,y###199.0a,y

Significance

Citrus genotype###NS

AMF###NS

Citrus genotype x AMF###NS###NS###NS###NS###NS###NS

Note: Means followed by different letters (a, b, c, etc.) in a column in AMF or non-AMF citrus plants or different letters (x, y) in a column between AMF and non-AMF citrus plants are significantly different (LSD, P Less than 0.05); NS - not significant; P Less than 0.05. P Less than 0.01

trifoliate orange Greater than red tangerine, suggesting that host genotype is an important factor controlling the mycorrhizal colonization (Graham and Eissenstat, 1994).

It is well known that AMF application to horticultural plants has been studied and achieved the enhancing effects (Ortas, 2010), just like the data listed in Table 1. The AM fungal enhancement of citrus growth may be attributed to enhanced mineral nutrition of AM plants under the low P supply but not high soil P conditions (Eissenstat et al., 1993; Peng et al., 1993). The order of growth increases in the four citrus plants treated by exogenous AMF was kumquat Greater than trifoliate orange Greater than red tangerine Greater than sour orange, suggesting that the enhancing effect of AMF on growth was dependent on citrus genotype.

This result is in agreement with the report of Youpensuk et al. (2009), who found that growth performance was increased by mixed AMF in lime, pomelo, and tangerine varieties, but little or none in cleopatra, troyer and sweet orange. So obviously, AMF have host-specific differences in growth performance, though they can associate with all host plant generally.

Roots are a major organ for plants to absorb moisture

Table 3: Effects of AIVIF (Diversispora spurca) on chlorophyll a, b, carotenoid and total chlorophyll contents of four citrus genotype plants

Citrus genotype###AMF inoculation###Chlorophyll a (mg/g)###Chlorophyll b (mglg) Carotenoid (mg/g)###Total chlorophyll (mglg)

Kumquat

###AMF###2.79a,x###0.73a,x###0.78a,x###3.52a,x

###Non-AMF###1.87a,y###0.55a,y###0.58a,y###2.42a,y

Red tangerine

###AMF###1.50c,x###0.28c,x###0.50c,x###1.78c,x

###Non-AMF###1.02b,y###0.l5cb,y###0.38b,y###1.17c,y

Sour orange

###AMF###2.19b,x###0.44b,x###0.89a,x###2.64b,x

###Non-AMF###1.59a,y###0.34b,x###0.57a,y###1.92b,y

Trifoliate orange

###AMF###2.91a,x###0.40b,x###0.66b,x###3.31a,x

###Non-AMF###1.75a,y###O.19c,y###0.51a,y###l.94b,y

Significance

Citrus genotype

AMF###

Citrus genotype x AMF###NS###NS###NS

Note: Means followed by different letters (a, b, c, etc.) in a column in AIvIF or non-AMF citrus plants or different letters (x, y) in a column between AMF and non-AMF citrus plants are significantly different (LSD, Pc0.05); NS - not significant; P Less than 0.05. Pc 0.01

and nutrients. RSA is an important parameter to judge the uptake capacity of the plant (Wu et al., 2011c). Our result showed that inoculation with D. spurca significantly enhanced various traits of RSA including length, surface and projected areas, volume, cross and branch, irrespectively of citrus genotype. The result is in agreement with the report conducted by Wu et al. (2011a). As stated in Table 2, among the four citrus genotypes, the highest root characteristics was sour orange, whereas the increase rate of RSA induced by D. spurca was the highest to kumquat compared with non-AMF plants. The alteration may be due to either the host plant's RSA status or the responses of host plant to specific mycorrhizal fungi.

Similarly, in the present work, colonization by D. spurca significantly increased leaf chlorophyll a, chlorophyll b, carotenoid, and total chlorophyll contents irrespectively of citrus genotype, whereas the increases of chlorophyll contents induced by D. spurca was dependent on host genotype. The chlorophyll alteration may be attributed to the increased mineral nutrients (Misra et al.,2005). In general, leaf chlorophyll content is closely related to photosynthetic ability in plants (Dong et al., 2007). Since higher chlorophyll content was in the mycorrhizal citrus plants, it seems that the AMF citrus plants might maintain better photosynthetic characteristics than the non-AMF plants, thereby providing more photosynthetic production for sustaining mycorrhizal development and root growth.

In conclusion, inoculation with D. spurca significantly enhanced growth performance, RSA, and chlorophyll contents of citrus plants in different genotypes, and the effects were obviously dependent on the host genotype.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31101513).

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For correspondence: zouyingning@163.com

To cite this paper: Li, Y., Y.N. Zou and Q.S. Wu, 2013. Effects of Diversispora spurca inoculation on growth, root system architecture and chlorophyll contents of four citrus genotype. Int. J. Agric. Biol., 15: 342-346
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Author:Li, Yan; Zou, Ying-Ning; Wu, Qiang-Sheng
Publication:International Journal of Agriculture and Biology
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Geographic Code:9CHIN
Date:Apr 30, 2013
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