Romanian Biotechnological Letters Copyright 2010 University of Bucharest

Vol. 15, No.3, 2010 Printed in Romania. All rights reserved ORIGINAL PAPER

The effect of mycorrhiza in nutrient uptake and biomass of cherry rootstocks during acclimatization
Received for publication, October 10, 2009 Accepted, May 15, 2010 YILDIZ AKA-KAAR1,2*, ADA AKPINAR3, ASLIHAN AGAR2, YEM YALIN-MEND1,2, SEDAT SERE4, BRAHM ORTA2,3 1 Department of Horticulture, Faculty of Agriculture, ukurova University, Balcal, 01330, Turkey 2 Institute of Basic and Applied Sciences, Biotechnology Department University of ukurova, Adana, Turkey 3 Department of Soil Science, Faculty of Agriculture, ukurova University, Balcal, 01330, Turkey 4 Department of Horticulture, Faculty of Agriculture, Mustafa Kemal University, Antakya, Turkey *Correspondence to: ykacar@cu.edu.tr

Abstract
The effect of arbuscular mycorrhizal fungi (AMF) on growth and nutrient uptake of micropropagated cherry rootstocks was evaluated during acclimatization and plant establishment. Two commonly used cherry rootstocks, Edabriz and Gisela 5, were propagated through tissue culture and grown in a greenhouse for 16 weeks. Plantlets were inoculated with Glomus clarum, Glomus caledonium, Glomus etunicatum, Glomus intraradices, Glomus mosseae, cocktail (mixture of these species) and indigenous mycorrhiza into three different substrate mixtures. All micropropagated cherry plantlets survived transplanting. After 16 weeks, mycorrhizal plantlets had greater nutrient uptake than non-mycorrhizal plantlets. Roots of inoculated cherry plantlets were heavily colonized with AMF. These results indicated that mycorrhizal inoculation during transplantation from in vitro to ex vitro culture can induce growth responses. The experiments also showed that the mycorrhizal cherry rootstocks were healthier and had higher Zn and P contents when compared to controls for both rootstocks. G. mosseae was one of the most efficient AMF species. Indigenous AMF isolated from ukurova region also significantly increased the plant growth and nutrient uptake. Gisela 5 rootstocks had significantly higher P and Zn contents than Edabriz. Taken together, our results indicate that AMF inoculations enhance growth and development of micropropagated plants which would be beneficial to improve cherry rootstock production.

Keywords: mycorrhiza species, micropropagated plants, sweet cherry, Prunus avium L., nutrient uptake.

Introduction
Micropropagation techniques increase the scale, speed of production and yield of healthier plants for fruit tree propagation. However, several problems limit the widespread use of micropropagation, of which transfer of in vitro plantlets to ex vitro conditions is the most critical. High rate of mortality is often observed upon transfer to ex vitro conditions because the cultured plants have functionally impaired stomata and poorly developed cuticle and root systems. Although some plant species acclimatize quickly due to faster root and shoot growth, others do not lead to an extended weaning stage, often accompanied by high losses and a large increase in use of fertilizer, pesticides and other chemicals (Gaur and Adholeya 1999). The major problem of commercial micropropagated plant production is low survival and poor growth while shifting these plantlets to field conditions. Plant loss is associated with the fact that micro-propagated plants have functionally less rhizosphere microorganisms such as mycorrhizae. Plant production through this technique can benefit from the utilization of mycorrhizae. Improved performance of endomycorrhizal plants is usually due to enhanced uptake of nutrients, especially phosphorous (Monticelli et al. 2000). Rai (2001) indicated that AMF improves bio-priming of micro propagated plantlets and plays a significant role in ensuring the health of plantlets. Mycorrhizae can act as bio-regulators, bio-fertilizers and bioprotectors, making possible the production of healthy, high-quality plants with low chemical inputs (Lovato et al. 1996). It has been reported that inoculation of arbuscular mycorrhizal AMF to the roots of micro propagated plantlets plays a beneficial role in their post-transplanting performance (Kapoor et al. 2008). Salamanca et al. (1992) demonstrated that AMF reduced the length of
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the micro propagated plant production cycle from 18 to 10 weeks. Fortuna et al. (1996) conclude that AMF can be used as a biotechnological tool to overcome blocked apical growth and to reduce chemical inputs, especially P, to micro propagated fruit trees. Mycorrhiza can also improve transplant viability (Biermann and Linderman 1983). Mycorrhizae form symbiotic associations between plant roots and certain soil fungi which play a key role in nutrient cycling in the ecosystem and also protect plants against environmental and cultural stress (Ortas and Varma 2007; Ortas 2008). Most of the major plant families are able to form mycorrhizal associations, the AMF association being the most common mycorrhizal type involved in agricultural systems (Azcn-Aguilar and Barea, 1997; Ortas 2008). Many studies on plants such as cherimoya (Azcon-Aguilar et al., 1996; Padilla and Encina, 2005), walnut (DolcetSanjuan et al., 1996), chestnut (Martins et al. 1997), banana (Yano-Melo et al. 1999), guava (Estrada-Luna et al. 2000), red raspberry (Taylor and Harrier 2000), strawberry (Taylor and Harrier 2001), pepper (Estrada-Luna and Davies 2003), Ficus benjamina (Srinath et al. 2003) and grape (Krishna et al. 2005) have utilized AMF to increase the growth rate and reduce mortality of plantlets acclimatization. Sweet cherry (Prunus avium L.) is an economically important crop in the world, especially in Turkey. Edabriz and Gisela 5 are frequently used rootstocks for sweet cherry and their clonal propagation is difficult by conventional methods. Grange et al. (1997) reported the beneficial effect of ectomycorrhizal fungus Hebeloma cylindrosporum during the in vitro rooting of micro propagated cherry and sweet cherries. The present study was conducted to determine the role of mycorrhization in nutrient uptake and biomass of two cherry rootstocks in three substrates during acclimatization period.

Materials and methods

Plant Material, Micropropagation Procedures and Plant Acclimatization Plantlets of cherry rootstocks Edabriz and Gisela 5 were propagated through in vitro shoot tip cultures. . The explant sources were surfaced disinfected with 70% ethanol for 3 min., soaked in 10% (v/v) NaOCl solution with few drops of Tween 20 for 10 min., followed by three rinses with sterile distilled water. This step was repeated twice. The shoot tips were cultured on the initiation medium. The cultures were grown under sterile conditions on a basal salt mixture and vitamin medium (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. The growth regulators used were BA for the initiation and multiplication stage (1 mg/L)) and IBA for the rooting stage (1 mg/L)). All media were adjusted to pH 5.7 with 0.1 N NaOH or HCl and were autoclaved at 1.05 kg/cm2 and 121 C for 15 min. All cultures were incubated at 25 1 C with a 16/8 h light/dark photoperiod illuminated by a cool-white fluorescent light (50 mol m-2 s-1). The cultures were transferred on fresh media every 4 weeks until a sufficient number of plantlets were obtained. After rooting and before acclimatization, 6-week-old uniform plantlets were selected from in vitro cultures and washed with tap water to remove residues of agar from the roots. Plantlets were then transplanted to individual 300 mL plastic pots containing a sterile substrate mixture (see below). They were inoculated with approximately 1000 spores (mix of spore, root, hyphae) before inserting the roots of the plantlets into the substrate. Noninoculated plants received the same amount of mycorrhizae spore free. Growth Medium Properties Three different substrates were used as a growth medium (GM) : GM1 = andesitic tuff : perlite : peat (1:1:1) (v/v/v), GM 2 = andesitic tuff : soil: compost (6:3:1), GM 3 = andesitic tuff : peat (1:1). All experiments were conducted under greenhouse conditions. The physical
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and chemical characteristics of the growth medium (soil, compost, peat and andesitic tuff) were measured according to Page et al. (1982) in the Rhizosphere Laboratory of ukurova University, Adana, Turkey. Soil material was collected from surface horizons of clay loam Menzilat soil series (0 - 20cm) (Typic Xerofluvents) in ukurova Basin with, 7.98 pH, and 0.5 M NaHCO3 (pH = 8.5) extractable 8.76 kg da-1 P. Compost had the following characteristics; 7.91 pH, 54 % organic matter, 1.13 % N, 0.18 % P while Andesitic tuff had 4.3 % K, 0.03 % P. Inoculum source The AM fungus treatments used in this study are non-mycorrhizal (NAM) control, Glomus mosseae (Nicolson and Gerdemann) Rothamsted Isolate, UK; G. etunicatum (Becker and Gerdemann) Nutri-Link Isolate; USA, G. intraradices (Schenck and Smith) Nutri-Link Isolate, USA; G. clarum (Nicolson and Schenk) Nutri-Link Isolate, USA, G. caledonium (Nicolson and Gerdermann) Rothamsted Isolate, UK; Cocktail (mixture of five AM species); and a mixture of indigenous mycorrhiza (isolated from ukurova Region, Turkey). Maize plants were used as stock plant for mycorrhizae spore propagation. Growth conditions After transplanting, the inoculated and non-inoculated plantlets were acclimatized in a controlled glasshouse with day-night temperatures about 27 1 C. Plantlets were maintained under 16 h photoperiod with cool white fluorescent lamps with about 50 m2 s1. The relative humidity was of 70 to 80% at night and of 80 to 85% at day period. Distilled water was added daily to maintain moisture at 80% of field capacity. Biomass assessment and nutrient analysis At the harvest of each pot, total plant biomass (dry weight of root and shoot), and plant height were recorded. Dried material from each pot was grounded with a Tema mill. Then, 0.2 g of grounded plant material was ashed at 550 C followed by dissolution in 3.3% HCl. After the digestion of the plant material, the concentration of P in this solution was determined colorimetrically (Murphy and Riley 1962). Atomic absorption spectrophotometer was employed to determine Zn contents of the plant samples. Shoots were separated from roots at 0.5 cm above the soil surface at harvest. Roots were separated from the soil by washing with running tap water and distilled water. Before drying the roots at 70 C for 48 hours, small sub-samples were taken and preserved in a mixture of ethanol, glacial acetic acid and formalin, for the determination of mycorrhizal infection. Portions of preserved roots were stained by the method of Koske and Gemma (1989). Mycorrhizal fungus colonization was determined using a gridline-intersect method of Giovannetti and Mosse (1980). Experimental design and statistical analysis Statistical analyses were carried using SAS (SAS, 2005). The two sweet cherry rootstocks were analyzed separately since the initial combined analyses indicated that the cultivar and their interactions were significant for most of the traits evaluated. The ANOVAs were constructed using a factorial model and the means of the main factors were separated by Duncans Multiple Range test at 5% using GLM. TABULATE was used to compare the means and the standard deviations. The data for the inoculation (%) traits were a sin transformed to increase normality although the untransformed data were used to calculate means.

Results and Discussion

Plant Dry Weight Growth media significantly affected dry weights of both Edabriz and Gisela 5 (Table 1). GM 1 produced significantly greater dry weight of Edabriz than GM 2 and GM 3 while GM 2 produced best growth of Gisela 5. AMF and their interactions with the
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substrates were not significantly different for either of the rootstocks tested. These results indicate that the rootstocks have differential preference for growing media. It also shows that mycorrhizal fungi did not have an effect on the shoot dry weights of the rootstocks. Although there was no effect of mycorrhizal fungus species on plant growth, this may be related to interaction between growth medium and plant genotypes. Root Dry Weight The ANOVAs indicated that all factors, media, treatments and their interactions were significantly different for both rootstocks tested (Table 1). As with shoot dry weight, the greatest root dry weights were recorded in GM 1 for Edabriz and in GM 2 for Gisela 5 (Table 2). Although some AMF species produced greater root dry weight than the control, no apparent and consistent patterns were observed (Fig. 1). For example, G. intraradices had much higher root dry weight than that of control for Edabriz, while being in the same mean group with control for Gisela 5. This is a very good example of the typical genotype difference in response to mycorrhizae species. Kafkas and Ortas (2009) found similar results with root dry weight of Pistachio genotypes inoculated with different mycorrhizae species. Colonization of Roots by AMF After 16 weeks of growth all seven treatments with AMF inocula had detectable levels of colonization. For both cultivars, all main effects and interactions were significant (Table 1). Similar to shoot and root dry weights, the plant root colonization varied with type of the substrate. Colonization of Edabriz ranged from 49.0 to 53.2% with GM 2 having the greatest value while the ranges for Gisela 5 were 42.3 to 61.5% with GM 3 being highest (Table 2). As expected, all AMF treatments had much higher root colonization than control for both cultivars. The AMF treatments had differential responses to media. For example, G. mosseae was not greatly affected by growth media and gave similar averages on all three growth media for Edabriz. G. clarium, G. caledonium and natural fungi treatments had less colonization in GM 2 when compared to the other two growth media. Differential patterns were apparent for other fungi for both rootstocks. These results suggest that the fungi not only have a preference for some cherry rootstocks, but also for the media in which the plants were grown. Mineral Nutrient Concentration Growth media, inoculation treatments and their interactions had significant effects upon mineral nutrient concentration for both cultivars (Table 1). The greatest P concentrations occurred with GM 3 (0.39 mg kg-1 for Edabriz and 0.45 mg kg-1 for Gisela 5). Since growth media are different in terms of nutrient content, it is possible plant grown on different medium have a differential response. For Zn concentrations, the media effect was different for rootstocks tested. While the Zn concentration was lowest on GM 1 for Edabriz (20.8 mg kg-1), the same treatments had the greatest mean (31.9 mg kg-1) for Gisela 5 (Table 2). This is possibly caused by the genotypic effect on Zn uptake. Uptake of P and Zn was significantly enhanced by mycorrhizal fungus inoculation. Mycorrhizal plantlets had greater elemental concentration of P and Zn than non mycorrhizal plants (Table 2). Ortas (2008) reported under field and greenhouse condition that several plants species inoculated with mycorrhizae have high P and Zn availability than non inoculated plants. Ortas et al. (2002) demonstrated that mycorrhizae significantly increased Zn and P uptake in Citrus. Although the tested fungi displayed differential responses according to the growth media, all fungi treatments had higher means than the control. G. etunicatum strains inoculated in both rootstocks accumulated more than two times P compared to control. Effectiveness of AMF in increasing P uptake has been related to the speed and extent of root colonization by AMF (Gaur and Adholeya 1999; Marschner 1995).
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Table 1. The mean and mean separations of several parameters for the effects of different arbuscular mycorrhizal species on the sweet cherry rootstocks grown on three growth media during their acclimations and establishments. Source Edabriz Media (M) Treatment (T) MxT Error Gisela 5 Media (M) Treatment (T) MxT Error z indicates significance at 5%. Dry weight (g plant-1) 23.5*z 5.3 4.8 4.3 2.73* 0.04 0.08 0.01 Root dry weight (g plant-1) 0.66* 0.18* 0.07* 0.02 0.57* 0.10* 0.05* 0.01 Colonization (%) 703.5* 20554.2* (?) 2140.9* 194.6 2166.9* 7660.1* 405.4* 60.7 P concentration (mg kg-1) 0.22* 0.20* 0.02* 0.00 0.61* 0.16* 0.02* 0.01 Zn concentration (mg kg-1) 1071.8* 509.0* 170.4* 7.7 235.8* 400.9* 71.1* 9.8

Table 2. The mean and mean separations of several parameters for the effects of different arbuscular mycorrhizal species on the sweet cherry rootstocks grown on three growth media during their acclimations and establishments.
Dry weight (g plant-1) 1.18 ay 0.35 b 0.30 b 0.53 0.44 0.53 0.43 0.52 1.47 0.36 0.49 0.58 Root dry Colonization P Zn Dry weight weight (%) concentration concentratio (g plant1 -1 -1 (g plant ) (%) n (mg kg ) )
Edabriz

Treatment
GMz

Root dry Colonization P Zn weight (%) concentration concentration -1 (g plant ) (%) (mg kg-1)
Gisela 5

GM 1 GM 2 GM 3 Treatment Indigenous G. caledonium G. clarium G. etunicatum G. intraradices G. mosseae Cocktail Control Mean
z y

0.47 a 0.28 c 0.34 b 0.40 ab 0.32 c 0.40 ab 0.33 c 0.46 a 0.32 c 0.23 d 0.34 bc 0.36

49.0 b 54.8 a 53.2 ab 65.9 d 38.0 e 81.4 a 58.0 cd 74.3 b 51.4 d 60.6 d 0.6 f 52.6

0.29 c 0.37 b 0.39 a 0.37 c 0.36 c 0.38 b 0.41 a 0.35 d 0.39 b 0.38 b 0.20 e 0.35

20.8 b 27.0 a 26.5 a 27.7 a 23.5 d 25.2 bc 28.7 a 24.8 c 26.1 b 28.8 a 17.8 e 25

0.35 c 0.76 a 0.45 b 0.58 0.46 0.50 0.43 0.48 0.50 0.56 0.44 0.49

0.39 b 0.59 a 0.42 b 0.53 a 0.38 c 0.42 bc 0.46 b 0.41 bc 0.58 a 0.43 bc 0.43 bc 0.45

42.3 c 58.7 b 61.5 a 67.7 a 55.0 b 64.1 a 61.5 a 48.5 b 62.6 a 67.8 a 1.2 c 53.21

0.43 b 0.27 c 0.45 a 0.37 c 0.39 bc 0.47 a 0.49 a 0.41 b 0.42 b 0.39 bc 0.22 d 0.4

31.9 a 30.1 b 29.4 b 34.0 ab 29.0 d 32.5 bc 31.0 c 28.2 d 35.2 a 32.9 b 22.6 e 30.6

Growth Medium: 1 = andesitic tuff : perlite : peat (1:1:1); GM 2: andesitic tuff : soil: compost (6:3:1); GM 3: (tuff : peat (1:1). Different letters represents significance differences by Duncan test at 5% level. The tests were conducted within each group separately.

Figure. 1. Mean of root weight (A), all fungus colonization (B), P (C) and Zn (D) concentrations of sweet cherry rootstocks inoculated with different arbuscular mycorrhizal fungi species grown on three growth media (GM) during acclimation and establishments. GM 1 = andesitic tuff : perlite : peat (1:1:1); GM 2: andesitic tuff : soil: compost (6:3:1); GM 3: (tuff : peat (1:1). Bars represent standard deviations.

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Zn concentration was the highest in Edabriz plantlets colonized by indigenous (27.7 mg kg-1), G. etunicatum (28.7 mg kg-1) and cocktail (28.8 mg kg-1) while G. mosseae determined the greatest for Gisela 5 (Table 1). It has been reported that AMF in horticultural plants can improve growth by increasing the uptake of P, Zn and other minerals (Pearson and Jakobsen 1993; Ortas and Varma, 2007). In general, elements with low mobility in the soil, such as P and Zn are absorbed in higher amounts by mycorrhizal than by non-mycorrhizal plants (Yano-Melo et al. 1999). The role of AMF in plant Zn acquisition highlights the complex set of tradeoffs that plants face in forming and maintaining AMF. AMF has beneficial effects on plant growth and health. It also acts as bio-fertilizers and bio-protectors. Thus, the appropriate management of this symbiosis would permit a satisfactory reduction of chemical fertilizer and pesticide inputs where they are the key aspects for sustainable horticultural plant production. Our results indicate that the maximum benefit from AMF will only be obtained from a careful selection of compatible host/fungus/substrate combinations. The performance of micropropagated plants may be greatly improved by ensuring a suitable mycorrhizal establishment at planting. In particular, woody horticultural plants, which are difficult to root in vitro, have been shown to exhibit greater survival and quality when inoculated with AMF. In vitro propagated plants are vulnerable and lacking vigor to survive transplant shock with great losses frequently observed (Moraes et al. 2004). Mycorrhization of in vitro-propagated plantlets has a positive impact on post transplanting performance (Rai 2001; Kapoor et al. 2008). Interactions between AMF and rhizobacteria have the potential to be useful biotechnological tools for benefiting plant development and health. AMF inocula production techniques need to be improved for the proper application of AMF biotechnology in commercial horticultural plant production systems (Azcn and Barea 1997). Conclusions Micro propagated cherry rootstocks were successfully inoculated with several AMF species and mixed species inocula under several growth substrates. G. mosseae was particularly efficient for both genotypes and growth media. Also, indigenous mycorrhizae had significant influence on plant development and nutrient uptake. Although there was no significant difference in overall shoot growth due to inocula, mycorrhizal plants had higher Zn and P concentrations than non-inoculated plants. Gisela 5 rootstocks had higher P and Zn concentrations than Edabriz rootstock. Edabriz rootstocks grew best in GM 1 and Gisela 5 less in GM 2. It may be useful to work more on similar plant species using several levels of mycorrhizal fungus inoculum and to follow the effect of colonization upon establishment in the field. Acknowledgment This work was supported by funding from the Prime Ministry State Planning Organization (Turkey) (Project no.: 2003 K-120-E).

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