Effect of arbuscular mycorrhizal fungal inoculation on root system architecture of trifoliate orange (Poncirus trifoliata L. Raf.) seedlings

Effect of arbuscular mycorrhizal fungal inoculation on root system architecture of trifoliate orange (Poncirus trifoliata L. Raf.) seedlings

Scientia Horticulturae 121 (2009) 458–461 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate...

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Scientia Horticulturae 121 (2009) 458–461

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Effect of arbuscular mycorrhizal fungal inoculation on root system architecture of trifoliate orange (Poncirus trifoliata L. Raf.) seedlings Q. Yao a,*, L.R. Wang a,1, H.H. Zhu b, J.Z. Chen a a b

College of Horticulture, South China Agricultural University, Guangzhou, 510642, China Guangdong Institute of Microbiology, Guangzhou, 510070, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 November 2008 Received in revised form 6 March 2009 Accepted 9 March 2009

Plant root system architecture is essential characteristics in relation to nutrient acquisition by root system from soil volume. Many environmental factors can affect the establishment of root system architecture, e.g. arbuscular mycorrhizal (AM) fungi. We inoculated the trifoliate orange (Poncirus trifoliata L. Raf.) seedlings with four AM fungal species in rhizoboxes, with non-inoculated seedlings as control. Using the WinRHIZO1 image analysis system, the root system architecture of seedlings was characterized. Results indicated that AM colonization did not affect the tap root length, the average root diameter, the basal root growth angle in spite that four AM fungal species exerted differential influence on the plant growth. Contrastingly, AM colonization significantly reduced the total root length, the root volume, the root surface area, but promoted the formation of lateral roots of high order. In addition, AM colonization induced more fine roots and less coarse roots. To our knowledge, it is the first report on the influence of AM fungi on the distribution of root diameter size classes. The mechanisms and implication of AM fungi on root system architecture is discussed. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Poncirus trifoliate L. Raf. Arbuscular mycorrhizal fungi Root system architecture Distribution of root diameter size classes

1. Introduction Arbuscular mycorrhizal (AM) fungi are obligate symbiotic soil fungi, and habitat almost all types of agricultural soil (Smith and Read, 1997). As promising biofertilizer and biopesticide (Azc´onAguilar and Barea, 1997), AM fungi have been attracting much attention from agronomist and horticulturist for decades. In symbiosis with citrus plants, the contributions of AM fungi to host have been intensively studied, involving the mycorrhizal dependency of several rootstocks (Graham and Syvertsen, 1985), the promoted nutrient uptake (Usha et al., 2004), the allocation of carbohydrate (Graham and Eissenstat, 1998), the application of AM fungi in citrus orchard (Ishii et al., 1998), and etc. Root system architecture is important morphological feature and plays a significant role in acquiring nutrients from soil volume (Lynch, 1995). In response to environmental stresses (nutrient limitation in particular), root system architecture can be modified to promote the nutrient-acquiring capacity (Sorgona et al., 2007). In addition to the abiotic factors, biotic factors (for instance AM fungi) also alter the root system architecture of host (Gamalero et al.,

* Corresponding author at: Department of Pomology, College of Horticulture, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou, 510642, China. Tel.: +86 20 85286902; fax: +86 20 85280228. E-mail address: [email protected] (Q. Yao). 1 Present address: Institute of Horticultural Crops, Yunnan Academy of Agricultural Sciences, Kunming, 650205, China. 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.03.013

2004). Atkinson et al. (1994) reviewed the literature on the influence of AM fungi on root system architecture, and indicated that the modification of root system architecture by AM fungi can contribute to the increased nutrient uptake and pathogen resistance of host. In a tropical woody fruit species Annona cherimola Mill, AM fungal colonization was confirmed to exert significant influence on the root system morphology, including the increased intensity of branching of the first-order laterals (Azco´n-Aguilar et al., 1996), the increased number of roots and root length (Padilla and Encina, 2005). These alterations in root morphology were linked to the improved uptake of nutrients from soil (Padilla and Encina, 2005). In citrus plants, although the increased nutrient uptake by AM fungi have been repeatedly reported (Usha et al., 2004), no information about the influence of AM fungi on root system architecture is available presently. In this study, we inoculated citrus seedlings with four AM fungal species, and investigated the changes in root system architecture in comparison with control seedlings. This can be of great significance in elucidating the mycorrhizal benefits to the promoted nutrient uptake in citrus species and in understanding the interaction between AM fungi and root system architecture. 2. Material and methods Trifoliate orange (Poncirus trifoliata L. Raf.) was selected as host plants. Seeds were surface sterilized with 10% sodium hypochlorite for 60 min, followed by several rinse in distilled water, and then

Q. Yao et al. / Scientia Horticulturae 121 (2009) 458–461

sown in autoclaved vermiculite for germination in glasshouse. After emergence, 1/2 strength Hoagland’s solution was applied to supply nutrients. At 3–4 leaf stage, seedlings were transplanted to specially designed rhizoboxes of acrylic PVC plates, with one seedling in each rhizobox. For the convenient analysis of root system architecture, a thin rhizobox was designed in a special dimension (35 cm height  21 cm width  3 cm depth) so that seedling roots can grow in a ‘‘flat’’ style. Four AM treatments were established with a non-inoculated treatment as control. For AM treatments, autoclaved substrates (the mixture of orchard soil and sand, 1:1 in volume) were inoculated with AM fungal inoculum of four fungal species, e.g. Glomus intraradices, Glomus caledonium, Gigaspora margarita, and Glomus versiforme. The inoculum was the mixture of mycorrhizal soil, colonized root fragments, AM hyphae and spores, with clover (Trifolium ripense L.) as host. In each rhizobox, 400 g inoculum was applied while control was free of inoculum. In each rhizobox, the total substrate was 2150 g. Seedlings were grown in glasshouse for 4 months with natural daylight from April to August. The average light intensity was 983 mmol m 2 s 1. The day and night temperature ranged from 14 8C to 36 8C and 11 8C to 25 8C, respectively. To minimize the possible effect of high temperature on root growth, rhizoboxes were inserted into a moistured soil pile (30 cm in height) to mimic the field environmental conditions throughout the experimental period. The average day and night temperatures inside the soil pile were 22 8C to 29 8C and 16 8C to 20 8C, respectively. Rhizoboxes were dissected at harvest, roots were carefully cleared of substrates with tap water and further rinsed with distilled water. Fresh weight was recorded after the shoots and roots were separated. Roots were then carefully arranged for the image capture using a scanner, followed by the analysis of root system architecture with the WinRHIZO1 image analysis system (V4.1c, Re´gent Instruments, Quebec, Canada) (Rillig et al., 2008). Using this system, total root length, root surface area, root volume, average root diameters of different sizes were automatously analyzed. Those roots with diameter of 0–0.4 mm and 0.4– 1.2 mm were defined as fine roots and coarse roots, respectively thereafter. The tap root length and the basal root growth angle were measured manually. The numbers of the 1st order roots (LR1) and the 2nd order roots (LR2) were recorded. Fine roots were sheared and further stained with trypan blue for the measurement of mycorrhizal colonization (Giovannetti and Mosse, 1980). According to the biomass, mycorrhizal dependency was calculated as previous report (Yao et al., 2005). For statistical analysis of data, SPSS v13.0 was employed for one-way ANOVA with Duncan’s multiple range test (DMRT). Data in percentage were subjected to arcsin transformation before the DMRT. 3. Results 3.1. AM fungal colonization and its effects on the plant growth The root colonization of the seedlings inoculated with AM fungi varied from 31.7% to 49.5%, without significant difference between fungal species (Table 1). No AM fungal structure was observed in the roots of control seedlings. Biomass measurement demonstrated that AM fungal inoculation exerted differential influence on the shoot biomass and the root biomass. In general, AM fungi promoted the shoot fresh weight except for G. caledonium, but reduced the root fresh weight except for G. versiforme, which resulted in a significantly decreased R/S ratio for inoculated seedlings (Table 1). In detail, G. margarita and G. versiforme significantly promoted the shoot fresh weight, while G. mosseae and G. caledonium significantly reduced the root fresh weight. This reflected the different effects

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Table 1 Influence of AM fungal inoculation on the biomass and root colonization of trifoliate orange seedlings. Treatments

Colonization (%)

Shoot fresh weight (g/plant)

Root fresh weight (g/plant)

R/S ratio

Non-inoculation G. margarita G. mosseae G. versiform G. caledonium

0b 49.5 42.6 34.0 31.7

0.33 0.48 0.37 0.45 0.30

0.70 0.59 0.43 0.70 0.42

2.11 1.24 1.15 1.57 1.35

a a a a

c a bc ab c

a ab b a b

Mycorrhizal dependency (%) b a a a a

– 3.9% 22.3% 11.7% 30.1%

Note: Data with different letters in each column are significantly different (DMRT0.05).

Table 2 Influence of AM fungal inoculation on the root system architecture of trifoliate orange seedlings. Treatments

Tap root length (cm)

Total root length (cm)

Root surface area (cm2)

Root volume (cm3)

Average root diameter (mm)

Basal root angle (degree)

Non-inoculation G. margarita G. mosseae G. versiform G. caledonium

39.7 37.0 39.0 39.9 35.1

390.9 323.0 232.9 402.0 212.9

104.4 a 90.4 ab 62.4 bc 113.5 a 54.8 c

2.2 2.0 1.3 2.6 1.1

0.85 0.89 0.86 0.89 0.82

69.3 61.3 71.5 60.3 76.8

a a a a a

a ab bc a c

a ab bc a c

a a a a a

a a a a a

Note: Data with different letters in each column are significantly different (DMRT0.05).

between fungal species to promote the seedling biomass, which can be quantified using the parameter of mycorrhizal dependency. According to mycorrhizal dependency (Table 1), G. versiforme and G. margarita were promotive while G. mosseae and G. caledonium were inhibitive, in relation to the plant growth. 3.2. Effects of AM fungal inoculation on the root system architecture The tap root length, the average root diameter and the basal roots growth angle of seedlings were 35.1–39.9 cm, 0.82–0.89 mm and 60.38–76.88, respectively (Table 2), with no significant influence from AM fungi. In contrast, AM fungi exerted great influences on the total root length, the root surface area and root volume. In general, AM fungi reduced these parameters to certain extent, with the exception of G. versiforme. The total root length, the root surface area and the root volume were slightly decreased by G. margarita, but significantly decreased by G. mosseae and G. caledonium (Table 2). In particular, these parameters of seedlings inoculated with G. caledonium were only about 50% of the control. These data revealed not only the difference in modifying the root system architecture between fungal species, but also the differential effects on these parameters exerted by a particular fungal species, G. caledonium, for instance. Table 3 indicated that AM fungal inoculation tended to decrease LR1, but there was no significant difference between control and mycorrhizal seedlings. In contrast, LR2 was increased by AM fungi with the exception of G. mosseae (Table 3). G. margarita and G. versiforme, in particular, significantly increased LR2 by nearly 100%. This differential effect of AM fungi on LR1 and LR2 resulted in much higher ratio of LR2 to LR1 for mycorrhizal seedlings than control (Table 3). 3.3. Effect of AM fungal inoculation on the distribution of root diameter size classes Although AM fungal inoculation decreased the total root length, the root length of particular diameter size was differentially

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460

Table 3 Influence of AM fungal inoculation on the number of lateral roots of trifoliate orange seedlings. Treatments

Numbers of LR1 per plant

Numbers of LR2 per plant

LR2/LR1 ratio

Non-inoculation G. margarita G. mosseae G. versiform G. caledonium

68.3 66.5 67.5 44.5 46.5

54.0 97.5 45.0 93.3 67.0

0.79 1.47 0.67 2.10 1.44

a a a a a

bc a c ab abc

Note: Data with different letters in each column are significantly different (DMRT0.05).

Table 4 Influence of AM fungal inoculation on the distribution of root diameter size classes of trifoliate orange seedlings in terms of root length. Treatments

Root length (cm) Non-inoculation G. margarita G. mosseae G. versiform G. caledonium

0–0.2 mm

0.2–0.4 mm

0.4–0.6 mm

0.6–0.8 mm

0.8–1.2 mm

6.6 7.1 6.1 9.7 3.5

80.7 b 90.1 ab 64.2 b 107.2 a 65.1 b

87.1 65.8 49.7 87.6 50.4

ab bc c a c

137.2 a 83.6 bc 63.5 c 109.2 ab 51.1 c

79.4 76.3 49.4 88.4 39.7

a ab bc a c

20.6 28.2 27.3 27.2 30.9

22.3 20.1 21.2 21.9 24.1

ab b ab ab a

35.2 25.8 27.5 26.9 24.4

20.2 23.7 21.3 21.6 18.9

a a a a a

abc ab bc a c

Proportion in total length (%) Non-inoculation 1.7 b G. margarita 2.2 ab G. mosseae 2.6 a G. versiform 2.4 ab G. caledonium 1.7 b

b a a a a

a b b b b

Note: Data with different letters in each column are significantly different (DMRT0.05). Table 5 Influence of AM fungal inoculation on the proportion of the fine roots and the coarse roots in terms of root length. Treatments

0–0.4 mm

0.4–1.2 mm

Non-inoculation G. margarita G. mosseae G. versiform G. caledonium

22.3 30.1 30.2 29.1 32.7

77.7 69.9 69.8 70.9 67.3

b a a a a

a b b b b

Note: Data with different letters in each column are significantly different (DMRT0.05).

affected. In general, the length of fine roots (diameter of 0–0.4 mm) was increased, while that of coarse roots (diameter of 0.4–1.2 mm) was decreased (Table 4). This trend was more evident when the proportion of root length of particular diameter size was presented (Table 4). The proportion of 0–0.2 mm roots was increased from 1.7% to 1.7%–2.6% by AM fungi, with significant difference for G. mosseae, and that of 0.2–0.4 mm roots was increased from 20.6% to 27.2%–30.9%, with significant difference for each fungal species. On the contrary, that of 0.6–0.8 mm roots was significantly lowered from 35.2% to 24.4%–27.5%. Totally, the proportion of fine roots was increased (from 22.3% to 29.1%–32.7%) while that of coarse roots was decreased (from 77.7% to 67.3%–70.9%) by AM inoculation (Table 5). 4. Discussion In this study, we investigated the influence of AM fungal inoculation on the root system architecture of trifoliate orange seedlings. We found that that AM fungi manifestly affected the distribution of root diameter classes, increasing the proportion of fine roots (0–0.4 mm) and decreasing the proportion of coarse roots (0.6–1.2 mm), although the average root diameter was not

affected. To our knowledge, this is the first report in this field. Other data strongly demonstrated that AM fungi significantly reduced the total root length, the root surface area, and the root volume, but did not affect the tap root length, average root diameter, and the basal root growth angle. The alterations of root system characteristics by AM fungal colonization have been intensively reported, with inconsistent results for different plant and/or fungal species. For example, a decrease in total root length was recorded for the herbaceous species rice plants in symbiosis with G. intraradices (Herdlera et al., 2008), but an increase was recorded for woody species Garcinia mangostana L. (Masri and Azizah, 1998) and Annona cherimola Mill. (Padilla and Encina, 2005). The decrease in root diameter was also recorded for rice plants (Herdlera et al., 2008). This inconsistence probably reflects the difference between herbaceous plants and woody plants, whose root anatomy and carbon distribution between shoots and roots are of different pattern. In addition, the increase in LR2 but not LR1 in mycorrhizal seedlings in this study was consistent with the previous reports (Citernesi et al., 1998; Masri and Azizah, 1998; Zai et al., 2007). The increase in the number of higher order lateral roots may contribute much to the higher proportion of fine roots. However, AM fungi could promote the number of 1st order roots in A. cherimola (Azco´n-Aguilar et al., 1996; Padilla and Encina, 2005). This complexity indicates that a further and in-depth exploration in this aspect is needed in the future. The alteration of root system architecture by AM fungi can be probably interpreted in two aspects: (1) the modified nutritional status (Citernesi et al., 1998), and (2) the altered phytohormone level (Yao et al., 2005). In the present experiment, G. margarita and G. versiforme positively but G. mosseae and G. caledonium negatively influenced the growth of seedlings, indicating their respective influences on the nutritional level of the seedlings. This pattern is consistent with the AM fungal influences on the total root length, the root surface area and root volume. The nutritional mechanism has also been proposed by Atkinson et al. (1994). The modifications of root system architecture induced by mineral nutrition, phosphorus in particular, have been repeatedly reported in many plant species (see the review article by Lo´pez-Bucio et al., 2003). In our experiment, however, the alterations induced by AM fungi in the number of lateral roots of higher order and the distribution of root diameter size were not in parallel with the plant growth (nutritional status), indicating a non-nutritional mechanism involved in. In previous experiments, the phytohormone mechanism has been proposed to explain the alteration in root system of litchi (Litchi chinesis Sonn.) (Yao et al., 2005) and maize (Zea mays L.) (Kaldorf and Ludwig-Mu¨ller, 2000) induced by AM fungi. The relationship between root system architecture and phytohormones has been well established (Perez-Perez, 2007), because phytohormone are key signaling factors conferring the effects of environmental cues on the root system architecture (Jiang et al., 2007). The alteration in root system architecture of citrus seedlings induced by AM inoculation is of certain significance in relation to nutrient uptake and the plant growth, because the roots of trifoliate orange is typical of coarse roots and sparse feeder roots (Spiegel-Roy and Goldschmidt, 1996). The higher percentage of fine roots represents increased capacity to take up nutrients, while more high order lateral roots represent more soil volume accessible. In fact, these alterations are independent of plant growth response to AM fungal inoculation, and thus of special significance in spite of AM fungal species. In our experiment, it is obvious that the effects on some parameters of root system architecture are dependent on the AM fungal species. The fungal species-dependent effects have previously reported (Zai et al., 2007), probably due to their differential influences on nutritional status and/or phytohormone level. It can

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