Impact of rootstock and interstems on fine root survivorship and seasonal variation in apple

Scientia Horticulturae 148 (2012) 169–176

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Impact of rootstock and interstems on fine root survivorship and seasonal variation in apple Changwei Hou, Li Ma, Feixiong Luo, Yi Wang, Xinzhong Zhang, Zhenhai Han ∗ Institute for Horticultural Plants, China Agricultural University, 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China

a r t i c l e

i n f o

Article history: Received 27 April 2012 Received in revised form 8 October 2012 Accepted 10 October 2012 Keywords: Malus domestica Borkh. Dwarfing rootstock Fine root Minirhizotron

a b s t r a c t Both the production and longevity of fine roots have great impacts on the growth and development of fruit trees. To determine whether different dwarfing rootstocks and interstems could affect fine root longevity, root diameter, and peak-production season, minirhizotrons were used to examine root production, senescence, and mortality over two years in six apple (Malus domestica Borkh.) rootstock/interstem combinations. Fine root longevity was significantly affected by diameter, season of born and rootstock/interstem combinations. Fine roots on three dwarfing rootstock combinations, Dwarfing-Red Fuji/M9, Dwarfing-Red Fuji/SH40, and Dwarfing-Red Fuji/Malus xiaojinensis, had smaller diameters and shorter lifespans than the Vigorous-Red Fuji/Baleng Crab combination. Insertion of a dwarfing interstem in the Red Fuji/M9/Baleng Crab and Red Fuji/SH40/Baleng Crab combinations did not significantly change the diameter or longevity of fine roots. The dwarfing rootstock combination Dwarfing-Red Fuji/SH40 did not have an autumn peak like others. The lifespan of fine roots differed significantly among seasons. Fine roots produced in autumn had longer lifespans regardless of rootstock/interstem combination. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Fine roots (<2 mm in diameter) play a critical role in the growth and development of fruit trees. Fine roots are the major root type to absorb water and minerals from the soil solution (Vogt et al., 1986). The production of a large amount of fine roots represents the allocation of plant photosynthates below the ground (Jackson et al., 1997). Additionally, fine roots are ephemeral and their turnover may cause greater carbohydrate losses than leaf litter (Raich and Nadelhoffer, 1989; Vogt et al., 1986). For an individual plant, the amount and lifespan of its fine roots are critical to its acquisition of belowground resources and partitioning of assimilates. Fine root longevity can be affected by many factors, including species, root diameter, and environmental conditions. Fine roots of citrus (Citrus sinensis) have longer lifespan than kiwifruit (Actinidia chinensis) or apple (Eissenstat and Yanai, 2002). The roots of the citrus rootstock ‘Trifoliate orange’ lived for 90 d, but another rootstock ‘Volkamer lemon’, had a 152-d root lifespan (Eissenstat and Yanai, 1997). The lifespan of fine root increased with increasing diameter among plant species (Eissenstat et al., 2000). The median fine root lifespan varied from 0.5 to 2.5 years, and the longevity of fine root increased with diameter among temperate tree species (Withington et al., 2006). Very similarly, fine root lifespan increased

∗ Corresponding author. Tel.: +86 10 62732467; fax: +86 10 62734391. E-mail address: [email protected] (Z. Han). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.10.008

with root order in Pinus and Quercus species, in fact however, root diameter generally increased with root order (Espeleta et al., 2009). Also, the lifespan of fine root positively correlated with their diameter within a single species as well; thinner fine roots usually have shorter lifespans in many cultivated fruit species, such as Prunus avium (Baddeley and Watson, 2005), peach (Prunus persica) (Wells et al., 2002), and apple (Wells and Eissenstat, 2001; Yao et al., 2009). Live fine roots <0.5 mm had more rapid turnover than 0.5–2 mm roots in a 14 C-labeled hardwood forest (Joslin et al., 2006). However, in fresh ectomycorrhizal roots of ponderosa pine, diameter did not correlate with root lifespan (Gaudinski et al., 2010). Thus, longevity may or may not correlate to diameter, depending on the species (Guo et al., 2008). The longevity of fine roots produced during different seasons differed significantly in fruit trees (Anderson et al., 2003; Baddeley and Watson, 2005). Fine root longevity is affected by soil temperature, shoot management, nutritional status and allocation, and atmospheric CO2 concentration (Comas et al., 2005; Farrar and Jones, 2000; Graefe et al., 2008; Stover et al., 2010). Dwarfing rootstocks are used in the apple industry worldwide because they can optimize vegetative growth and maximize fruit yield and quality. Dwarfing interstems in apple trees have been proven to reduce shoot growth and improve yield (Ferree and Knee, 1997; Samad et al., 1999). Apple dwarfing rootstocks have significant impacts on root production, growth, and architecture (Atkinson and Else, 2001; De Silva et al., 1999). For example, dwarfing rootstocks had fewer fine roots than vigorous rootstocks

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(Neilsen et al., 1997), and the dry weight of roots in M9 rootstocks was lower than in semi-dwarfing rootstocks MM106 and M793 (Hooijdonk et al., 2011). Five-year-old apple trees grafted onto dwarfing ‘Mark’ rootstock had lower mean root length density (RLD), and shallower vertical root distributions than those grafted onto M26 (semi-dwarfing) or MM106 (semi-vigorous) rootstocks (De Silva et al., 1999). Apple root growth has been commonly reported as having a bimodal pattern with distinct flushes of fine roots (Cripps, 1970; Head, 1966). However, a study using minirhizotrons on young ‘Mutsu’/M9 trees over two years indicated a single peak of root production, primarily in June and July, compared to old apple trees, with little root production in either the spring or fall in these young apple trees (Psarras et al., 2000). Whether the peak initiation season of fine roots differs among rootstocks is not clear. In general, apple roots are relatively small in diameter with low tissue density and short longevity (Eissenstat et al., 2000). The lifespans of fine roots in apple trees ranged from 14 d (Head, 1966) up to 130 d (Psarras et al., 2000; Yao et al., 2009). Preliminary data on two new hybrid apple cultivars showed that root lifespans decreased by being grafted onto dwarfing rootstock M9, compared to that grafted onto vigorous rootstocks (García-Villanueva et al., 2002). Under similar soil conditions, dwarfing rootstocks G30, which had similar lifespan with semi-dwarfing rootstock M7, had a significantly shorter root lifespan than another semi-dwarfing rootstock Cornell-Geneva 6210 (Yao et al., 2006). Thus dwarfing rootstocks may be more likely to have shorter fine root lifespans, although there is scant data. Fewer fine roots with shorter lifespans may indicate lower levels of root consumption of photosynthates and may benefit fruit yield and quality; on the other hand, well-timed initiation of fine roots ensures healthy tree development. Lifespans of fine roots that are produced in different seasons could be predicted if fine root dynamics show seasonal patterns. Timing of root initiation has important implications in fruit crops management, including timing of fertilization and factors influencing carbohydrate and nutrient sink activity. Dwarfing rootstock M9 and its strains are most commonly used in the apple industry worldwide (Zhu et al., 2001). Another dwarfing rootstock, SH40, is a winter-hardy dwarfing rootstock increasingly used as interstems in China, and interstems of SH40 produce dwarf scions (Jin et al., 2010). Malus xiaojinensis is an ironefficient dwarfing rootstock selected in China (Dong, 1994; Han et al., 1994). In this paper, several fine root traits of different combinations of dwarfing rootstocks and interstem were measured using minirhizotrons, which allows the non-destructive in situ examination of roots. The dwarfing rootstocks were M9, SH40 and Malus xiaojinensis, and the vigorous rootstock was Baleng Crab. The objectives of this study were to determine whether different dwarfing rootstocks and interstems affect fine root longevity, fine root diameters, and root emergence season.

2. Materials and methods 2.1. Facilities and equipment The experiment was conducted in a greenhouse on the campus of China Agricultural University, Beijing, China. Twelve 1.5 × 1.5 × 3 m (length × width × height) pits were built below the ground level, which will henceforth be referred to as Rhizo pits. The bottom and side walls of each Rhizo pit were made of reinforced concrete, and eight drains were built into the bottom of each Rhizo pit to avoid flooding due to unintended over-irrigation. The roofs of the Rhizo pits were left open, and the tops of the side walls were 10 cm above the ground level.

Minirhizotron tubes (178 cm in length, 5.6 cm in diameter) were pre-installed horizontally at five depth levels: −20 cm, −60 cm, −100 cm, −150 cm and −200 cm, respectively, from the greenhouse ground. At each depth there were four parallel tubes at 37.5 cm intervals. Therefore, the two tubes on the same side of the tree were 18.75 cm and 56.25 cm from the trunk of the tree, respectively. The closed end of a minirhizotron tube was inserted through a predrilled hole in the wall and its open terminus was covered with an opaque lid. After the pre-installation of the minirhizotron tubes, the Rhizo pits were filled with medium composted garden soil (the 0–30 cm layer soil of a vegetable garden, the soil was brown soil in type and clay loam in texture), peat, and vermiculite (3:1:1, v/v/v). The organic matter, total nitrogen, and available phosphorus and potassium in the mixed medium were 44.5 g kg−1 , 1.4 g kg−1 , 40.8 mg kg−1 , and 92.0 mg kg−1 respectively. 2.2. Plant materials and management Scions of Red Fuji were grafted onto the dwarfing rootstocks in the combinations Red Fuji/M9 (D-RF/M9), Red Fuji/SH40 (DRF/SH40), and Red Fuji/Malus xiaojinensis (D-RF/MX) and onto dwarfing interstems in the combinations Red Fuji/M9/Baleng Crab (D-RF/M9/BC) and Red Fuji/SH40/Baleng Crab (D-RF/SH40/BC). Red Fuji on the vigorous seedling rootstock Baleng Crab (V-RF/BC) served as the vigorous control. The rootstocks and interstem sections were 10 cm and 25 cm, respectively (Richards et al., 1986). All trees were grafted and potted on March, and were transplanted into Rhizo pits on May, 2009. A single tree was planted in each pit, and two replicates were designed for each rootstock/interstem combination. The entire interstem piece was left above ground when planting. The trees were pruned as thin spindles. Conventional disease and pest management was used. The temperature was maintained at 15–25 ◦ C/10–20 ◦ C (day/night) during the growing seasons (May–November) and at a constant 5–15 ◦ C during dormancy (December–next April). The trees were drip irrigated on the soil surface once every 2 weeks during the summer (beginning in June) and once every 3 weeks during the other three-month seasons, which ensured enough water supply for each tree. The volume per irrigation was about 80 L each time for each Rhizo pit. 2.3. Data collection Fine roots (<2.0 mm) growing against the surface of the minirhizotron tubes were observed using an ET100 Root Observation System (Bartz Technology Corp, Santa Barbara, CA, USA). The view field of the digital camera on the ET100 system was 1.8 × 1.35 cm. Images were captured in a single vertical up direction with a 60◦ angle facing the tree. Pictures were taken every 1.35 cm along the length of the tube for a total of 98 images per tube. Fine root images were collected between 2 August 2009 and 1 August 2011 with an interval of approximately 10 d. All images of individual fine roots were analyzed with WinRHIZO Tron MF software (Regent Instruments Inc., Quebec City, Canada). For each fine root or portion of one, the date of appearance, date of death, and root diameter were recorded. The date of first appearance was regarded as the birth date of each new root. Root death was defined as either (a) disappearance or (b) a darkened and shriveled appearance (Comas et al., 2000; Anderson et al., 2003). The number of days between the birth and death of individual fine roots was regarded as the root’s longevity (Baddeley and Watson, 2005; Anderson et al., 2003). The diameter of each root was only measured at its first appearance, because we examined none of the roots increased in diameter with time. The fine roots already existing at the beginning of the study were not included in the analyses, because it was impossible to determine the dates of their first appearance. The cumulative

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number of roots and the total number of newly produced roots per 3-month season for each combination were calculated. Roots that disappeared abruptly from the view field without being considered dead were omitted from analyses, and roots that persisted to the end of study were classified as “censored”. 2.4. Statistical analyses of minirhizotron data Fine roots were assigned one of three subgroups according to their diameters: less than 0.2 mm, 0.2–0.4 mm, or 0.4–2.0 mm from direct measurements of images on computer screen. The number of roots observed on the surface of tubes varied from 250 to 420 among the six rootstock combinations. Variation in mean root diameter, distributions of root diameter classes and proportion of roots produced by season for the rootstock combinations were estimated using analysis of variance and least significant difference multiple-comparison (LSD) (P < 0.05). Kaplan–Meier survival analysis (Kaplan and Meier, 1958) was plotted against time to generate root survival curves for all roots of the two replicates in each combination (Baddeley and Watson, 2005; Pritchard and Strand, 2008; Tierney and Fahey, 2001). Differences in survivorship curves among the three diameter classes and seasons were assessed by making pairwise comparisons in all combinations using the Wilcoxon test (Wells and Eissenstat, 2001) using the statistical software package SPSS 16.0 (IBM Corporation, Armonk, NY, USA); differences were considered statistically significant when P < 0.05. The significance of influences of season, diameter and rootstock combination on fine root lifespan was identified with Cox proportional hazards regression using the statistical software package SPSS 16.0 (IBM Corporation, Armonk, NY, USA) (Wells and Eissenstat, 2001; Anderson et al., 2003; Baddeley and Watson, 2005). Median survival time was defined as the first time point at which survival probability was ≤50% (Kaplan and Meier, 1958). 3. Results 3.1. Fine root diameter in different rootstock/interstem combinations There was a significant difference in mean root diameter between the vigorous rootstock combination and the dwarfing rootstock combinations (Fig. 1). The fine roots of the vigorous combination V-RF/BC had a significantly thicker mean diameter than those of the dwarfing combinations D-RF/M9, D-RF/SH40, and DRF/MX (P < 0.05). Mean root diameters of the dwarfing interstem combinations D-RF/M9/BC and D-RF/SH40/BC were similar to that of V-RF/BC (P > 0.05). 3.2. Distributions of fine roots in different diameters and produced during different seasons The majority of observed roots (>80%) were less than 0.4 mm in diameter in all combinations (Fig. 2A). The proportion of fine roots less than 0.2 mm in diameter in the three dwarfing rootstocks was significantly higher than in the other combinations (P < 0.05). Almost half of all fine roots produced in the vigorous rootstocks and interstem combinations were between 0.2 and 0.4 mm in diameter, comparing with approximately 40% of fine roots in the dwarfing rootstocks. There are no significant differences in distributions of fine root diameters between V-RF/BC and the two interstem combinations (P > 0.05) (Fig. 2A). The proportion of fine roots produced during different seasons varied among rootstock combinations (Fig. 2B). For five of the six combinations, more than 68% of fine roots were born in summer and fall, and root numbers decreased significantly in winter (Fig. 2B). The proportion of new fine roots was highest in fall, except

Fig. 1. Box plots of root diameters of six rootstock/interstem combinations of Malus domestica Borkh. Dwarfing-Red Fuji/M9 (D-RF/M9), Dwarfing-Red Fuji/SH40 (D-RF/SH40), Dwarfing-Red Fuji/Malus xiaojinensis (D-RF/MX), VigorousRed Fuji/Baleng Crab (V-RF/BC), Red Fuji/M9/Baleng Crab (D-RF/M9/BC), and Red Fuji/SH40/Baleng Crab (D-RF/SH40/BC). Root data were collected every 10 d between 2 August 2009 and 1 August 2011. The horizontal line in each box represents the mean. The bottom and top edges of each box represent the 25th and 75th percentiles, respectively. The top and bottom error bars represent the 90th and 10th percentiles, respectively. The black dots represent outliers beyond the 95th percentile. Mean root diameters from two replications of each rootstock/interstem combination were compared using least significant difference multiple-comparisons. Different lowercase letters indicate significant differences (P < 0.05, LSD) among combinations.

for D-RF/SH40 and V-RF/BC. D-RF/SH40, which produced 62.8% of fine roots in spring and summer, did not have an autumn peak like other cultivars. About 12% of the fine roots of dwarfing rootstocks were produced in winter, compared with 7% of all fine roots in interstem combinations and in V-RF/BC, which had the greatest percentage of fine roots produced in summer (45.6%) (Fig. 2B). The total number of fine roots varied among rootstock combinations (Fig. 2C). For six combinations, D-RF/MX produced the least number of fine roots, while the total number of fine roots in V-RF/BC was the most. 3.3. Variations in fine root longevity as affected by diameter, season of production and rootstock/interstem combinations The median lifespan of fine roots varied significantly among rootstocks/interstems, diameter classes and seasons as well (Table 1). The results of Cox proportional hazards regression analyses were shown in Table 2. Fine root longevity was significantly affected by fine root diameter, season of fine root production and rootstock/interstem combinations (the P values were <0.001, <0.001 and 0.008, respectively). Fine root lifespan was significantly (P = 0.008) affected by rootstock/interstem combination (Table 2). The fine root longevity of the six combinations ranged from 82 to 148 d, D-RF/M9 had the shortest median root lifespan, while that of D-RF/M9/BC was longest. The median root lifespans of D-RF/M9, D-RF/SH40, and DRF/MX were significantly shorter than that of V-RF/BC and the two interstem combinations (P < 0.05, Wilcoxon test). There was no significant difference between the two interstem combinations and the vigorous one (P > 0.05, Wilcoxon test) (Fig. 3). Fine root diameter had a significant effect on fine root lifespan in our experiment (Table 2, Fig. 4). Fine roots with greater diameters were associated with a significant increase in survival (Table 1, Fig. 4). Each millimeter increase in fine root diameter prolonged fine root lifespan by 48.6% according to the hazard ratio (Table 2). The median lifespan of fine roots with diameters less than 0.2 mm was 50–77 d, with an average of 65 d across the six root lifespans. The median longevity of fine roots with diameters from 0.2 to 0.4 mm was 126–173 d, with an average of 149 d. Lifespan for D-RF/M9, D-RF/SH40, and D-RF/MX with diameter from 0.2 to 0.4 mm was

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Fig. 2. Total number, diameter distribution and seasonal variation of fine roots in different rootstock/interstem combinations. (A) Distribution of fine roots by diameter classes (<0.2 mm, 0.2–0.4 mm, 0.4–2.0 mm) produced in six rootstock/interstem combinations. (B) Proportion of roots produced in different three-month seasons, where spring begins in March, for each of six rootstock/interstem combinations. (C) Total number of fine roots produced in six rootstock/interstem combinations ‘Dwarfing-Red Fuji/M9 (DRF/M9), ‘Dwarfing-Red Fuji/SH40 (D-RF/SH40), ‘Dwarfing-Red Fuji/Malus Xiaojinjenesis (D-RF/MX), ‘Vigorous-Red Fuji/Baleng Crab’ (V-RF/BC), ‘Red Fuji/M9/Baleng Crab’ (D-RF/M9/BC), and ‘Red Fuji/SH40/Baleng Crab’ (D-RF/SH40/BC). Root date was collected between 2 August 2009 and 1 August 2011 with every 10 d. Error bars represent one standard error of mean (N = 2). Lower case letter that differ between the six combinations indicated a significant difference (P < 0.05, LSD).

Table 1 Median root lifespan of the six rootstock/interstem combinations of Malus domestica Borkh. in different diameter classes and seasons. Covariate

Fine root lifespan (d) D-RF/M9b

D-RF/SH40

D-RF/MX

V-RF/BC

D-RF/M9/BC

D-RF/SH40/BC

Diameter <0.2 mm 0.2–0.4 mm 0.4–2.0 mm

64ac 128b 214c

50a 126b 284c

70a 141b 295c

61a 163b 263c

77a 173b 337c

66a 161b 354c

Seasona Spring Summer Autumn Winter

53A 60A 129B 108AB

66AB 106BC 142C 59A

62A 113B 137B 99AB

89AB 144BC 205C 62A

77A 170AB 182B 143AB

81A 134B 183B 74A

a

The 3-month season, where spring begins in March. ‘Dwarfing-Red Fuji/M9 (D-RF/M9), ‘Dwarfing-Red Fuji/SH40 (D-RF/SH40), ‘Dwarfing-Red Fuji/Malus Xiaojinjenesis (D-RF/MX), ‘Vigorous-Red Fuji/Baleng Crab’ (V-RF/BC), ‘Red Fuji/M9/Baleng Crab’ (D-RF/M9/BC), and ‘Red Fuji/SH40/Baleng Crab’ (D-RF/SH40/BC). c Lowercase letters within a combination that differ between diameter classes and seasons indicate a significant difference (P < 0.05; Wilcoxon test). b

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Table 2 Cox proportional hazards regressions on fine roots of Malus domestica Borkh. in relation to the covariates season, diameter and rootstock combination. Covariate

Cox regression coefficient

SE

Hazard ratio

2

P

Season Diameter Rootstock combination

−0.181 −0.666 0.052

0.037 0.048 0.020

0.835 0.514 1.053

23.348 191.58 7.061

<0.001 <0.001 0.008

Fig. 3. Kaplan–Meier estimates of survival probabilities for roots of six apple rootstock/interstem combinations. Dwarfing-Red Fuji/M9 (D-RF/M9), Dwarfing-Red Fuji/SH40 (D-RF/SH40), Dwarfing-Red Fuji/Malus xiaojinensis (D-RF/MX), Vigorous-Red Fuji/Baleng Crab (V-RF/BC), Red Fuji/M9/Baleng Crab (D-RF/M9/BC), and Red Fuji/SH40/Baleng Crab (D-RF/SH40/BC). Root date was collected between 2 August 2009 and 1 August 2011 with every 10 d. Median root lifespan (MRL) was defined as the first time point at which survival probability was ≤50%. ‘+’ datapoints were censored. The proportion of censored roots for all genotypes was less than 15.5%, in line with the requirements for the analysis (up to 50%). Whole survivorship curves followed by different lowercase letters in corners are significantly different (P < 0.05; Wilcoxon test) among combinations.

Fig. 4. Kaplan–Meier estimates of survival probabilities for roots of three diameter classes (<0.2 mm, 0.2–0.4 mm, and 0.4–2.0 mm). Dwarfing-Red Fuji/M9 (D-RF/M9), Dwarfing-Red Fuji/SH40 (D-RF/SH40), Dwarfing-Red Fuji/Malus xiaojinensis (D-RF/MX), Vigorous-Red Fuji/Baleng Crab (V-RF/BC), Red Fuji/M9/Baleng Crab (D-RF/M9/BC), and Red Fuji/SH40/Baleng Crab (D-RF/SH40/BC). (, <0.2 mm; , 0.2–0.4 mm; , 0.4–2.0 mm.) ‘+’ datapoints were censored.

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Fig. 5. Kaplan–Meier estimates of survival probabilities for roots produced in different 3-month seasons, where spring begins in March. Dwarfing-Red Fuji/M9 (D-RF/M9), Dwarfing-Red Fuji/SH40 (D-RF/SH40), Dwarfing-Red Fuji/Malus xiaojinensis (D-RF/MX), Vigorous-Red Fuji/Baleng Crab (V-RF/BC), Red Fuji/M9/Baleng Crab (D-RF/M9/BC), and Red Fuji/SH40/Baleng Crab (D-RF/SH40/BC). (, spring; , summer; × autumn; , winter.) Root date was collected between 2 August 2009 and 1 August 2011 with every 10 d. ‘+’ datapoints were censored.

more than 20 d shorter than V-RF/BC and interstems (Table 1). The median longevity of fine roots with diameters greater than 0.4 mm was 214–354 d and averaged 291 d across the six root lifespans; some of these roots survived for more than 600 d in the two years of this study. There were significant differences in longevity of fine roots produced during different seasons (Table 2, Fig. 5). Fine roots produced in spring had shorter median lifespans, ranging from 53 to 89 d. The longevity of fine roots born during winter was 59–143 d. Fine roots born during summer survived for more than 100 d, except in DRF/M9. Fine roots produced in fall had the longest lifespans in all combinations, ranging from 129 to 205 d. 4. Discussion The fine roots of apple trees on dwarfing rootstocks have smaller diameters and hence shorter longevities than those on vigorous rootstock. In this study, the three dwarfing rootstocks had smaller mean root diameters than the vigorous rootstock, and the proportion of fine roots less than 0.2 mm in diameter was significantly higher in the dwarfing rootstocks. Fine roots with smaller diameters may be more vulnerable to environmental stress such as parasitism (Graham, 1995). The lifespan of fine roots is known to be positively correlated with their diameters within a single species such as apple, peach, cherry and grapevines (Anderson et al., 2003; Baddeley and Watson, 2005; Wells and Eissenstat, 2001; Wells et al., 2002). Rootstock has been previously reported to affect the longevity of fine roots (Eissenstat and Yanai, 1997; GarcíaVillanueva et al., 2002; Yao et al., 2006). In our study, the average root lifespans of the three dwarfing rootstock combinations was only 91 d, much less than root lifespan of the vigorous rootstock combination. According to our data, smaller diameters might be correlated to the shorter lifespans of fine roots in dwarfing rootstocks. The shorter fine root lifespan in dwarfing rootstocks may cause reduced vegetative growth via insufficient water and mineral supplies, and apple cultivars on dwarfing rootstocks have less root redundancy so that their cumulative yield efficiency will be

increased (Webster, 2004). Based on these observations, the cultivation management, for example fertilizing or fertigating adult apple trees with dwarfing rootstocks, could be carefully scheduled (Neilsen et al., 1997; Perry et al., 2009) to reduce water and fertilizer supplies in order to increase their efficiency. Dwarfing interstems in apple trees are able to affect shoot and root growth by controlling the transportation of substances (Li et al., 2012). The limitation on basipetal flux of assimilates and endogenous auxin was regarded as one potential mechanism of dwarfing interstems effects (Richards et al., 1986; Li et al., 2012). However, distributions of fine root diameters in the two interstem combinations were similar to that of V-RF/BC. The total fine root longevity of the two dwarfing interstem combinations, D-RF/M9/BC and D-RF/SH40/BC, was more than 120 d, not significantly different from the vigorous graft combination V-RF/BC. Based on our findings, inserting a dwarfing interstem did not significantly change the diameter or the longevity of fine roots. Therefore, the limitation on carbohydrates and phytohormones is unlikely to alter the longevity and diameter of fine roots. From this point of view, the dwarfing mechanism of a given rootstock was quite different when it was used as interstem than as a rootstock (Li et al., 2012). The lifespan of fine roots differed significantly among seasons. Fine roots produced in autumn were longer lived, regardless of rootstock/interstem combination. However, for only three of the six combinations, fine roots produced during spring had the shortest median lifespans, and the lifespan of fine roots produced during winter in D-RF/SH40 and V-RF/BC was much less than that in DRF/M9/BC. In white poplar, the lifespan of the fine roots produced during May, was only 62 d, shorter than that of fine roots produced during July (Eissenstat and Yanai, 1997). Similarly, the fine roots of pear (Pyrus communis L.) and apple trees produced during spring had the shortest median lifespans, while fine roots produced during fall lived longest (Atkinson, 1985; Quartieri et al., 2002). However, in other tree species, like cherry (Prunus avium), no difference in lifespan was found between spring- and autumn-born fine roots (Baddeley and Watson, 2005). Thus, seasonal changes in fine root longevity might vary greatly depending on the plant species and

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rootstock types. Many environmental factors, such as soil temperature and moisture, can affect the seasonal longevity of fine roots (Anderson et al., 2003; Burke and Raynal, 1994). In our case, seasonal differences in fine root lifespan may have been associated with soil temperature in the greenhouse system that was different between seasons, rather varying than soil moisture, because consistent water management was applied year-round. The amount of fine roots produced during different seasons varied significantly in the apple rootstocks in our study. Rootstock had an impact on seasonal variations in apple root production (Head, 1966). Previous studies have reported that fine root systems in early spring were composed almost entirely of roots initiated the previous fall (Psarras et al., 2000; Wells and Eissenstat, 2001). In our experiment, approximately 40% of all fine roots were born during fall in all combinations except D-RF/SH40, and most fine roots produced in fall tended to survive until the next spring. However, up to 27% of fine roots were initiated between April and May on the dwarfing rootstock D-RF/SH40, which had an earlier peak of root production. This pattern was similar to our previous report that maximum total root length density of D-RF/SH40 occurred during May (Ma et al., unpublished data). In apple trees grafted on SH series interstems, the bud break and bloom was delayed by several days, but cessation of shoot growth and leaf fall was earlier (Shao et al., 1991). The dwarfing potential of SH40 was inherited from Malus honanensis rather than from the East Malling series rootstocks, such as M9, and that of M. xiaojinensis originated de novo (Dong, 1994). Whether there are any differences in dwarfing mechanism among rootstocks with different origins of dwarfism is not clear. Data from previous studies suggest that the fine root behavior and root architecture of SH40 differed from M9, and that SH40 interstem changed the root architecture in a different way than M9 interstems did (Ma et al., unpublished data). 5. Conclusion In this study, apple fine roots on dwarfing rootstocks had smaller diameters and shorter lifespans than on a vigorous rootstock. Inserting a dwarfing interstem did not significantly change the diameter or longevity of fine roots. Fine roots initiated in autumn lived longer than other seasons, regardless of rootstock/interstem combination. The amount of fine roots produced during different seasons varied significantly, and the dwarfing rootstock combination D-RF/SH40 did not have an autumn peak like other cultivars. Based on findings from this study, the dwarfing mechanism of a given apple rootstock may be quite different when it was used as interstem than as rootstock. There may be some differences in dwarfing mechanism among rootstocks with different origins of dwarfism. This conclusion should improve understanding of the dwarfing mechanism of rootstock. Acknowledgements This project was supported by the Special Fund for Agroscientific Research in the Public Interest (201203075); the Modern Agricultural Industry Technology System (Apple); the Key Laboratory of the Beijing Municipality of Stress Physiology and Molecular Biology for Fruit Trees; and the Key Laboratory of Biology and Genetic Improvement of Horticulture Crop (Nutrition and Physiology), Ministry of Agriculture, PR China. References Anderson, L.J., Comas, L.H., Lakso, A.N., Eissenstat, D.M., 2003. Multiple risk factors in root survivorship: a 4-year study in Concord grape. New Phytol. 158, 489–501. Atkinson, D., 1985. Spatial and temporal aspects of root distribution indicated by the use of a root observation laboratory. In: Fitter, A.H., Atkinson, D., Read, D.J.,

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