Preference of Japanese field voles (Microtus montebelli) for apple rootstock genotypes: Correlations with bark characteristics

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Preference of Japanese field voles (Microtus montebelli) for apple rootstock genotypes: Correlations with bark characteristics

T

Takuya Shimadaa,∗, Shigeki Moriyab a b

Department of Wildlife Biology, Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, 305-8687, Japan Apple Research Station, Institute of Fruit Tree and Tea Science, NARO, 92-24 Nabeyashiki, Shimo-Kuriyagawa, Morioka, 020-0123, Japan

A R T I C LE I N FO

A B S T R A C T

Keywords: Cafeteria experiment Malus Nitrogen Phenolics Vole damage Vole resistance

Along with the expansion of dwarfing apple rootstocks in Japan, damage through bark-eating by the Japanese field vole (Microtus montebelli Milne-Edwards, 1872) in apple orchards has increased. The most prevalent cultivar, JM7, in particular, is highly susceptible to vole gnawing. Hence, it is a pressing task to reduce vole damage to apple rootstocks by developing and introducing a new dwarfing rootstock cultivar that is less susceptible to voles. In order to achieve this, it is critical to elucidate which rootstock characteristics underlie vole preference. Preferences of the Japanese field vole among seven apple rootstocks were examined by a cafeteria experiment using ten voles. Voles showed a clear preference for the JM7 cultivar. Nutrient and morphological analyses of these rootstocks revealed that voles preferred to eat rootstocks which were poor in total phenolics, rich in nitrogen, and had low edible ratios of shoots. Among the tested seven rootstocks, JM7 was richest in nitrogen, and poorest in total phenolics. Our results indicate that future breeding projects on vole-resistant dwarfing apple rootstock genotypes should aim to develop a cultivar that is rich in total phenolics and poor in nitrogen, while keeping the advantageous traits of the JM7 rootstock, such as high productivity, and ease of propagation by hardwood cutting.

1. Introduction

although a congeneric vole species (the Japanese field vole, Microtus montebelli Milne-Edwards) is widely distributed (Ohdachi et al., 2015). Prior to the 1980s, the most commonly used apple rootstock in Japan was ‘Marubakaido’ (Malus prunifolia Borkh.), which induces vigorous growth to its scion, has high rooting capability by hardwood cutting, and is well adapted to the climate, soil, diseases and pests of Japan (Arakawa and Komori, 2006; Soejima et al., 2010). However, after the Malling (M) series dwarfing rootstocks (Malus pumila var. paradisisiaca C.K.Schneid) were introduced to Japan in the 1980s, damage by voles increased (Kounuma, 1988). Recently, in order to integrate the advantageous characteristics of M. prunifolia described above and the dwarfing ability of the M series (Soejima et al., 2010, 2013) into the one genotype, the JM series dwarfing rootstocks (JM1, JM2, JM5, JM7, and JM8; M. prunifolia × M. pumila var. paradisisiaca) were developed, and these became available in the 2000s. Of these varieties, the planting area of the JM7 dwarfing rootstock has been rapidly increasing, due to its high productivity (Soejima et al., 2010, 2013). According to data collected in 2014, of the 1168 ha of apple orchards using dwarfing rootstocks in Japan, 9.3% adopted JM7 as rootstock (Ministry of Agriculture, Forestry and Fishery, 2017). Along with the expansion of the JM7 rootstocks, however, damage of the JM7 rootstocks by voles

Damage to apple trees by vole gnawing causes weakening and death of trees, if it is severe. In North America, serious damage to apple orchards by pine voles (Microtus pinetorum Le Conte) and meadow voles (Microtus pennsylvanicus Ord) has been repeatedly reported since the early 20th century (Lantz, 1905; Siegler, 1937; Hamilton Jr, 1940; Holm et al., 1959). Similar cases were also reported in Europe; for instance, damage by water voles (Arvicola terrestris L.) and common voles (Microtus arvalis Pallas) in Germany (Walther et al., 2008; Walther and Fuelling, 2010) and by montane water voles (Arvicola scherman Shaw) and Lusitanian pine voles (Microtus lusitanicus Gerbe) in Spain (Miñarro et al., 2012; Somoano et al., 2017). Voles consume the bark, phloem, cambium, and outer xylem of large roots and lower trunks in rootstocks, in which primary nutrients, such as carbohydrate and nitrogen, are accumulated (Hamilton Jr, 1940; Tromp, 1983; Tobin and Richmond, 1993; Merwin et al., 1999). Damage is most frequent in the dormant season, from late fall to early spring, when other food sources are relatively scarce, and snow cover protects foraging voles from avian and mammalian predators (Servello et al., 1984; Merwin et al., 1999). Similar damage, however, was not common in Japan until the 1980s,

∗

Corresponding author. E-mail addresses: tshmd@affrc.go.jp (T. Shimada), moriyas@affrc.go.jp (S. Moriya).

https://doi.org/10.1016/j.cropro.2018.06.007 Received 26 January 2018; Received in revised form 11 June 2018; Accepted 12 June 2018 0261-2194/ © 2018 Elsevier Ltd. All rights reserved.

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Science (Morioka, Iwate Prefecture, Japan). All rootstocks, with the exception of Sanashi 63, were cut back at 1-m height in every dormant season. Sanashi 63 was formed as free spindle. For the feeding experiment, one-year-old shoots were taken from each progeny in early to mid-December 2013, when the dormant season began. Whole shoots were immediately covered with a plastic sheet, and stored at 1 °C in a refrigerator until use. These shoots were prepared for cuttings in the next spring and, thus, still vital when they were used for the experiment.

has been increasing (Takano, 2011), although no official statistical information was available. The Japanese field vole is endemic to Japan, and is distributed in Honshu, Kyushu, and Sado Island in the Japanese archipelago (Ohdachi et al., 2015). It mainly lives in cultivated fields, meadows, conifer plantations, and riverbanks (Urayama, 1996). These voles predominantly eat plants, including leaves, stems, roots, bark, and seeds, and sometimes cause damage to crops and planted trees (Ito, 1975; Kawamoto et al., 2012). Unfortunately, dynamics of population density and damage caused by these voles in orchards and farmlands have not been monitored in Japan. It is suggested that vole density and damage to trees and crops may increase in relation with mast seeding of beech or dwarf bamboo (Ito, 1975; Miguchi, 1996). In addition, damage to trees, including orchard, is thought to be augmented in the winter with heavy snow. The bark of large roots and lower trunks in trees or rootstocks is most vulnerable, but, when snow cover helps voles to approach upper branches, they are also damaged (T. Shimada and S. Moriya, personal observations). As such, it is a pressing task to reduce the JM7 rootstock damage by voles. Habitat management (ex. frequent mowing of ground vegetation, setting barriers for vole migration) and rodenticide baits are the primary methods for controlling vole damage in apple orchards (Byers et al., 1976; Tobin and Richmond, 1993; Merwin et al., 1999; Pelz, 2003; Walther and Fuelling, 2010), but these methods require continuous efforts, and rodenticide has the risk of secondary poisoning of predators that eat voles (Merson et al., 1984; Takano, 2011; Coeurdassier et al., 2014). Thus, it is important from a medium to longterm perspective to develop a new rootstock genotype that is less susceptible to voles, whilst keeping the advantageous traits of the JM rootstocks. Such rootstock genotypes will be able to mitigate vole damage to apple orchards without requiring much management by orchard managers. In order to achieve this goal, it is critical to elucidate why voles prefer the JM7 rootstock. However, vole preferences for apple rootstocks have not been previously investigated in the Japanese field vole. In contrast, the preference of voles for different apple genotypes has been extensively investigated in North America (Byers and Cummins, 1977; Pearson et al., 1980; Wyslomerski and Byers, 1980). These studies show that voles have a clear preference among apple genotypes. However, the factors underlying such preferences remain unexplained. Elucidating these factors will expedite the development of apple rootstocks that are resistant or less palatable to vole gnawing. Therefore, the principal aim of the current study was to examine the preference of the Japanese field vole among several apple rootstocks in a cafeteria feeding experiment. We predict that the JM7 rootstock would be highly preferred. The second aim was to elucidate the factors underlying different rootstock preferences in voles by comparing their chemical and morphological characteristics.

2.2. Characteristics of rootstocks In order to examine which characteristics of rootstocks are related to vole preference, we measured edible ratio in the shoots, and contents of nitrogen, total non-structural carbohydrate (TNC), total phenolics, and condensed tannins. We selected these characteristics for the following reasons; edible ratio represents feeding efficiency, nitrogen (protein) and TNC are the major nutrients contained in plants, and total phenolics and condensed tannins are common defense chemicals. Approximately ten shoots from each genotype were sampled for this purpose. Sampled shoots were dissected into bark, cambium, and xylem. Bark and cambium of a rootstock shoot were defined as the edible part, because voles usually eat this part (Tobin and Richmond, 1993; Merwin et al., 1999). Thus, the edible ratio (%) was calculated for each shoot as (dry wt. of bark + dry wt. of cambium)/dry wt. of shoot × 100. Samples of edible parts were pooled for each genotype (ca. 5 g in dry wt.), then lyophilized, and milled for the chemical analyses. The nitrogen content of each sample was measured using an NC analyzer (SUMIGRAPH NC22-F, Sumika Chemical Analysis Service Ltd., Tokyo). The TNC content was spectrophotometrically determined using the perchloric acid extraction method (McCready et al., 1950), and the phenol-sulfuric acid method (Dubois et al., 1956), using Dglucose (Kanto Chemicals Inc., Tokyo) as the standard. The content of total phenolics was measured using the modified Price-Butler method (Hagerman, 2002), with tannic acid (Wako Pure Chemicals Inc., Kyoto) as the standard. The content of condensed tannins was determined using the acid butanol method (Hagerman, 2002), with cyanidin chloride (Wako Pure Chemicals Inc., Kyoto) as the standard. Chemical assays were conducted twice for each sample, and the mean values were used for the analyses. Averages of the coefficient of variation between the two assays were 1.52 ± 0.98% for nitrogen, 2.93 ± 3.06% for TNC, 3.10 ± 2.98% for total phenolics, and 2.29 ± 1.63% for condensed tannins, respectively. 2.3. Field voles In the current study, we used ten Japanese field voles (six adult females and four adult males) for the cafeteria experiment. This unbalanced sex composition was due to the limitation of available animals. These voles were captured with Sherman traps in early April 2014 at the orchard in the Apple Research Station (Morioka, Iwate Prefecture, Japan). The voles were housed separately in cages, and were allowed free access to distilled water and feed for herbivorous mammals (ZF, Oriental Yeast Co. Ltd., Tokyo) under regulated conditions (20 °C; photoperiod, 12L:12D) throughout the study at the Tohoku Research Center of the Forestry and Forest Products Research Institute (located next to the Apple Research Station). The photoperiod was determined to match the condition of the season, in which vole damages to apple rootstock may occur (early spring). All procedures in the experiments using the Japanese field voles were approved by the Committee of the Animal Experiments of the Forestry and Forest Products Research Institute (Permission No. H2614A-1).

2. Materials and methods 2.1. Plant materials We used the following seven genotypes of apple rootstocks for the feeding experiment: JM7, Mo 84a (M. prunifolia, a common rootstock in Japan), Sanashi 63 (Malus sieboldii (Regel) Rehder, used as a rootstock in Japan until about 100 years ago), and four F1 progenies (#32, #47, #64, and #100) derived from the cross JM7 × Sanashi 63 in 1994. JM7 is a dwarfing rootstock, whereas Mo 84a and Sanashi 63 are not. The dwarfing abilities of F1 progenies are under examination. JM7, Mo 84a, and all F1 progenies show high adventitious rooting from their hardwood cuttings, whereas Sanashi 63 does not (Soejima et al., 2010; Moriya et al., 2015). Sanashi 63 and all F1 progenies are resistant to crown gall disease, whereas JM7 and Mo 84a are not (Moriya et al., 2008, 2010). All plant materials were planted and maintained at the orchard of the Apple Research Station, NARO Institute of Fruit Tree

2.4. Cafeteria experiment The cafeteria experiment was conducted in mid-June 2014 using a 240

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medium-sized cage (31.5 × 41.0 × 15.0 cm) with a feeding tray placed in the center. Each vole was introduced to the cage, and then rootstock shoots (total three pieces selected from three different shoots of each genotype) were presented to each vole simultaneously. Pieces of rootstock shoots were presented as uniform pieces (1 cm long, and 5–7 mm diameter). All pieces, numbered on the cut end, were mixed and placed in the feeding tray. The cafeteria experiments were started at the beginning of the dark phase. The amount of food consumed was checked after two, six, and 10 h from the start of the experiment. Then the proportion of consumption for each genotype was estimated based on the surface area eaten by a vole at the 5% scale. Consumed area was measured using a ruler from four diagonal directions of each piece. The experiments were conducted two times (two trials) with each vole at the interval of two days. The results of the experiments were analyzed using the Rodgers's index of preference for cafeteria experiments (Rodgers and Lewis, 1985; Krebs, 1999). First, the cumulative consumption curve was drawn for each food item, and the area under the cumulative consumption curve was estimated. Then the Rodgers's index of preference for food item i (Ri) was calculated as follows:

Table 1 Characteristics of one-year-old shoots of the seven apple rootstocks used in the cafeteria experiment. Rootstock genotypes

Nitrogen %

TNC %

JM7 1.31 24.4 Mo 84a 1.18 27.2 Sanashi 63 1.23 20.0 F1 progenies (the cross JM7 × Sanashi #32 1.14 22.0 #47 0.98 20.4 #64 1.07 21.0 #100 1.07 23.9

Total phenolics % 10.3 13.6 17.2 63) 13.8 11.7 12.6 13.7

Condensed tannins %

Edible ratio %

1.74 2.21 2.96

26.7 25.5 19.9

2.21 2.09 1.91 2.76

29.8 28.1 31.5 21.3

Bark and cambium of a rootstock shoot were defined as the edible part, which was used for chemical analyses. Values are expressed on the basis of dry weight. TNC represents total non-structural carbohydrates. Table 2 Correlation coefficients between the five characteristics of apple rootstocks.

Ri = Ai/max(Ai),

TNC Total phenolics Condensed tannins Edible ratio

where Ai denotes area under the cumulative consumption curve for food item i, and max Ai does the largest value of the Ai. Rodgers's index ranges from 0 to 1, where 0 indicates avoidance, and 1 indicates preference.

Nitrogen

TNC

Total phenolics

Condensed tannins

0.357 0.104 −0.031 −0.295

−0.279 −0.200 −0.106

0.873* −0.606

−0.837*

TNC represents total non-structural carbohydrates. *P < 0.05.

were the lowest among the seven rootstocks, whereas the nitrogen content was the highest. In contrast, the Sanashi 63 rootstock had the highest levels of total phenolics and condensed tannins. Table 2 shows correlations among the five characteristics; condensed tannins were highly correlated with total phenolics (n = 7, r = 0.873, P = 0.010), and edible ratio (n = 7, r = −0.837, P = 0.019).

2.5. Data analyses Correlations among the five characteristics of rootstocks were analyzed by Pearson's correlation coefficients. Differences in vole preference among rootstocks (Ri) were examined separately for each sex. First, differences in Ri between the two trials for each sex were examined by Wilcoxon signed-rank tests, because values of Ri did not show normal distribution. As a result, no differences between the two trials were detected in both sexes. Then, the data of the two trials were combined as one dataset for each sex, and pairwise comparisons between rootstocks were made using Steel-Dwass tests in order to secure the sufficient sample size for each sex. To examine which characteristics of rootstocks are related to vole preference, we analyzed the relationships between them, separately for each sex, using generalized additive mixed models (GAMMs). GAMMs provide a flexible modeling approach that allows consideration of multiple smooth explanatory variables, thereby enabling evaluation of nonlinear effects of the explanatory variables (Rigby and Stasinopoulos, 2005). We set Ri as the response variable and the edible ratio, nitrogen content, TNC content, and total phenolics content as the explanatory variables with a beta error and logit-link function. All explanatory variables were smoothed with cubic splines. We combined the data from the two trials and designated vole ID as the random effect in the above model. The content of condensed tannins was removed from these models, because it was highly correlated with edible ratio (−0.837) and total phenolics (0.873). As total phenolics include condensed tannins, we think that this procedure can minimize the loss of the information on rootstock characteristics. All statistical procedures were performed using the GAMLSS package (Rigby and Stasinopoulos, 2005) implemented in R (R Core Team, 2017).

The tendency of vole preference for apple rootstocks did not change between the two trials in both sexes (Wilcoxon signed-rank test, female: V = 286, P = 0.141; male: V = 174, P = 0.502). Further, their preference was mostly constant throughout the time course of each trial. The voles showed a very clear preference for JM7, but there were slight differences in their preference between sexes. For female, the Ri of JM7 was nearly equal to 1 (1st trial, 0.99 ± 0.03 SD; 2nd trial, 0.87 ± 0.32), but those of the others ranged from 0.02 to 0.24. SteelDwass tests revealed that Ri of JM7 was significantly higher than those of the other rootstocks, and there were no differences among the others (Fig. 1a; JM7 vs. other rootstocks, t = 3.91–4.34, P < 0.01; between six genotypes other than JM7, t = 0.00–0.96, P = 0.96–1.00). For male, the Ri of JM7 was nearly equal to 1 (1st trial, 0.99 ± 0.03; 2nd trial, 0.90 ± 0.20), but those of the others ranged from 0.03 to 0.54. As is the case with female, the male voles most preferred JM7, but the differences between other genotypes were less clear. The difference in Ri between JM7 and Mo 84a was not statistically significant (Fig. 1b; Steel-Dwass test, t = 2.36, P = 0.218), but those between JM7 and the other five genotypes were significant (t = 3.45–4.53, P < 0.05). Further, differences among the six genotypes other than JM7 were not significant statistically (t = 0.05–2.14, P = 0.33–1.00).

3. Results

3.3. Factors affecting vole preference

3.1. Characteristics of rootstocks

Vole preference for the tested apple rootstocks was correlated with the contents of total phenolics and nitrogen, and the edible ratio of shoots, but not by the TNC content (Table 3). The results of GAMMs, in general, showed the same tendency between females and males (Table 3). The total phenolics and edible ratio had a negative relationship with Ri, whereas the nitrogen content had a positive one;

3.2. Vole preference for rootstock genotypes

The seven apple rootstocks exhibited large differences in their chemical and morphological characteristics (Table 1). JM7 was characterized by a low level of defense chemicals, and a high level of nutrients. The contents of total phenolics and condensed tannins in JM7 241

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seemed slightly weaker compared with those of total phenolics and nitrogen (Figs. 2 and 3). Comparing the absolute values of the estimates of partial coefficients between sexes (Table 3), the female estimates for nitrogen and total phenolics were smaller and larger than male, respectively. 4. Discussion Several studies in North America, which investigated voles' selectivity for various apples, found that pine and meadow voles have a clear preference for some genotypes (Byers and Cummins, 1977; Pearson et al., 1980; Wyslomerski and Byers, 1980). Most of these studies found that voles prefer Golden Delicious and M9 to other apple cultivars. However, factors underlying such preferences have not been examined. In the current study, the Japanese field vole, similar to pine and meadow voles in North America, had a distinct preference for a specific rootstock, in this case, the JM7 rootstock. This tendency was robust in both female and male voles, as indicated by the similar results in the two trials. In addition, we identified, for the first time, the characteristics of apple rootstocks related to vole preference. Our analyses indicate that vole preference for apple rootstocks were influenced by the nitrogen and total phenolic content, and edible ratio of rootstocks. Animals need to ingest proteins from foods to grow, reproduce, and maintain their bodies. Thus, protein-rich food is generally preferred by herbivores (Marquis and Batzli, 1989; Windley et al., 2016). In contrast, phenolic compounds are one of the most prevailing plant defense chemicals, which cause various negative effects in consumers (Waterman et al., 1994). Most herbivores, including voles, generally avoid consuming plants containing high levels of phenolic compounds (Lindroth et al., 1986; Roy and Bergeron, 1990; Hambäck et al., 2002; Dai et al., 2014). Therefore, it is understandable that the tested voles preferred the JM7 rootstock, as this rootstock was the richest in nitrogen and poorest in total phenolics among the tested rootstock varieties. These two characteristics of the JM7 rootstock are likely to be related to each other. Plants, in general, have trade-offs between defense and growth or reproduction, because anti-herbivore defense is costly (Coley, 1986; Herms and Mattson, 1992; Koricheva, 2002). One criterion for the selection of the JM7 rootstock was high productivity, which is likely to be accompanied with a high retention of nitrogen in plant tissues. Anti-herbivore defense of the JM7 rootstock, in turn, may be reduced as a result of the trade-off. Species of genus Microtus are thought to be relatively sensitive to negative effects of phenolic compounds, although the sensitivity differs among animal species. A number of previous studies have demonstrated that the level of total phenolics in plants is one of the most important regulating factors for food selection in species of genus Microtus (Roy and Bergeron, 1990; Hjältén and Palo, 1992; Hambäck et al., 2002; Dai et al., 2014). For instance, meadow voles show higher preferences for conifer seedlings containing less total phenolics (Roy and Bergeron, 1990). Further, food preference of root voles (Microtus oeconomus Pallas) in Qinghai, China is explained primarily by the level of total phenolics in plants (Dai et al., 2014). Species of genus Microtus ingesting high levels of dietary phenolics, in general, exhibit decreases in digestibility, and reductions in growth, reproduction, and survival (Lindroth and Batzli, 1984; Meyer and Richardson, 1993; Dietz et al., 1994). This is likely the reason why species of genus Microtus tend to avoid consuming plants rich in phenolics. Although, to our knowledge, there are no such studies on the Japanese field vole, this species may have a similar tendency for food selection. In addition to total phenolics and nitrogen content, we found that the edible ratio in a shoot had a negative effect on voles' food selection. Specifically, the tested voles tended to select rootstock shoots with relatively low edible ratios. It is not easy to interpret this relationship, because low edible ratios commonly lead to reduced feeding efficiency for herbivores. It is possible that other unmeasured characteristics, such as the hardness of barks, and/or ease of abrasion of edible parts might

Fig. 1. Vole preferences for the seven apple rootstocks in the cafeteria feeding experiments. Values are expressed as Rodgers's index of preference for cafeteria experiments (Ri): 0 = avoid, and 1 = prefer. The upper and lower panels represent the preference of a) females and b) males, respectively. Table 3 Summary of generalized additive mixed models for examining vole preferences for the apple rootstocks using shoot characteristics as explanatory variables. a) female Variables

Estimates

SE

t

P

(Intercept) cs(N) cs (TNC) cs (Total phenolics) cs (Edible.ratio)

5.45 7.01 −0.09 −0.65 −0.14

3.29 1.53 0.06 0.10 0.05

1.66 4.57 −1.44 −6.41 −2.98

0.10 < 0.0001 0.15 < 0.0001 < 0.01

Variables

Estimates

SE

t

P

(Intercept) cs(N) cs (TNC) cs (Total phenolics) cs (Edible.ratio)

−2.93 8.78 0.09 −0.51 −0.11

3.82 1.79 0.07 0.11 0.05

−0.77 4.90 1.24 −4.65 −2.10

0.44 < 0.0001 0.22 < 0.0001 0.04

b) male

TNC represents total non-structural carbohydrates. Smoothed terms are indicated as ‘cs( …)’.

namely, voles preferred rootstocks which were rich in nitrogen, and poor in total phenolics and edible ratio (Figs. 2 and 3). These effects were mostly monotonic. The effects of the edible ratio, however, 242

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Fig. 2. Partial response curves for female vole preferences for the apple rootstocks. The x-axes represent values of the model explanatory variables, and the y-axes show the additive contribution of each variable to the nonparametric generalized additive mixed model (GAMM) smoothing function. Gray zones show the 95% confidence interval around the estimated function.

order to mitigate vole damage to apple orchards, the development of a new rootstock cultivar resistant to vole gnawing is urgently required. Specifically, our results indicate that future breeding projects to develop vole-resistant cultivars should aim to develop a cultivar that is rich in total phenolics, whilst keeping the advantageous traits of the JM7 rootstock, such as high productivity, and high rooting capability by hardwood cutting. This may not be an easy goal, and achieving it will require a balanced approach, because trade-offs between defense and growth in plant resource allocation could occur in the process of breeding. Finally, the validity of the plant samples used in the cafeteria experiment should be considered from the two aspects. The first matter is that the shoots used for the experiment were not fresh (stored almost for six months). The stored shoots were vital, and thus we assumed that the tendency of chemical composition among seven genotypes would not change, even if the content itself varied slightly. It should be verified in future research. Second, although we conducted the cafeteria experiment using shoots of apple rootstocks to examine vole preference as a first approach, we believe future studies using bark from rootstock roots and trunks, which are the parts most susceptible to vole gnawing, are needed, because it is possible that vole preference may vary according to parts of rootstocks.

be correlated with the edible ratio, and thus further investigation is required. Diet selection of voles is also influenced by other plant characteristics that were not considered in this study, such as the content of fiber, silica, defense chemicals other than phenolics (Bucyanayandi et al., 1990; Hjältén et al., 1996; Massey and Hartley, 2006). Among them, we think that the role of acid detergent fiber (ADF) should be examined in the next step, because there is a report, in which ADF is demonstrated to have a more dominant effect on vole preference than nitrogen and total phenolics (Hjältén et al., 1996). Interestingly, we detected sex differences in vole preference. Although the general tendency of the preference did not differ between sexes, our results indicated that female voles had a firmer bias to avoid total phenolics and weaker one to select nitrogen content. Some previous studies on mammalian herbivores documented the presence of sex differences in diet selection (Hewson, 1976; Clutton-Brock et al., 1987), but there are also a lot of studies that failed to find sex differences (Iason and Waterman, 1988; Remis, 2002; Gould et al., 2009). It is a controversial topic, but studies examining sex differences in diet selection based on the chemical analyses of diets have been still limited. It would be an important consideration for future studies. Recently vole damages to JM7 rootstocks have become prevalent in Japan. Our finding that the Japanese field vole has a clear preference for JM7 is consistent with this situation. Bark of the JM7 rootstock is probably a favorable resource for these voles in apple orchards. Thus, in 243

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Fig. 3. Partial response curves for male vole preferences for the apple rootstocks. The x-axes represent values of the model explanatory variables, and the y-axes show the additive contribution of each variable to the nonparametric generalized additive mixed model (GAMM) smoothing function. Gray zones show the 95% confidence interval around the estimated function.

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