Comparative Study on Pseudaulacaspis Pentagona Resistance of Four Different Cultivars of Kiwifruit

Background: Kiwifruit is a common and popular fruit around the world. However, white peach scale (Pseudaulacaspis pentagona) [Targioni-Tozzetti], a scale insect with a wide range of hosts, seriously affects the yield and quality of kiwifruit. To investigate the differences in resistance of different kiwifruit cultivars to Pseudaulacaspis pentagona, cellular structure and gene expression assays were used to explain the mechanism. Results: In this study, based on the stability of the rate of injury fruit, we selected four cultivars from fty kiwifruits for in-depth study, including “LC-04285”, “CF-3”, “DA-7B” and “Hayward”. By analyzing the differences in the anatomical structure of the canes of these cultivars, we found that the resistant cultivar "LC-04285" had thicker cuticle, denser epidermis and cortex. The real-time quantitative PCR data indicated that the expression levels of genes related to cuticle synthesis and formation of epidermis and cortex are also higher in “LC-04285”. Jasmonic acid (JA) is an important hormone involved in plant defense against many insect pests. In this study, we found that the expression levels of JA receptor COI1 were higher in “LC-04285”. However, the expression levels of AcJAZs, which played negative role in JA signaling, were higher in susceptible cultivar “Hayward”. Besides, the expression levels of AcICS, AcPAL4, AcPAL5, and AcNPRs, which were involved in salicylic acid (SA) synthesis and SA response, were also higher in “LC-04285”. Our results revealed the mechanism of kiwifruit resistance to P. pentagona at the molecular and cellular levels. This study provided useful guidance for breeding insect-resistant kiwifruit in future. great promise. In this study, we focuse on four kiwifruit cultivars with different P. pentagona-resistance ability after a large-scale screening. According to analyzing the difference of cell structure of cane and some JA/SA-related genes’ expression levels among these cultivars, we preliminarily analyzed the P. pentagona-resistance mechanisms of resistant kiwifruit cultivars. These results will provide new thoughts and methods for insect prevention in kiwifruit and breeding of new resistant kiwifruit cultivars. “LC-04285” has stronger SA/JA synthesis and response ability. These results provide more new ideas for the development of insect resistant cultivars

role in protecting and supporting plant growth. As the outermost barrier of plants, epidermis acts a pivotal part in protecting plants [9]. In the model plant rice, epidermis controls rice's growth process and protects rice against biotic and abiotic stress [10]. The epidermis differentiates into trichomes, which are closely related to plant resistance to insects [11,12]. Cortex is a thin-walled tissue between the epidermis and the vascular bundle [13]. Cortical cells usually contain resins, essential oils, tannins, which confer partially resistance to pest [14][15][16]. Cuticle, which mainly contained wax, coatings the outside of the epidermal cells. It has been well documented that thicker cuicle and epidermis contribute to insect resistance in cacti [17].
Jasmonic acid (JA) and salicylic acid (SA) are representative hormones for plant resistance [18][19][20]. Many studies have reported its defensive effects on pests [21][22][23]. The silencing of the ICS1 gene, which regulates SA biosynthesis, reduces the insect resistance of tobacco [24]. The expression of ICS1 and NPR1 in rice can be induced after infesting by white-backed planthopper [25]. SA accumulation in wheat is also increased when suffering the Russian wheat aphid [26]. In the JA signaling pathway, COI1 induces the degradation of JAZ, which in turn activates the defense response of plants. It has been shown that silencing the expression of NaCOI1 make tobacco more vulnerable to Manduca sexta [27]. Arabidopsis JAZ mutants display a high level of resistance to insect [28]. The JA defective mutant of arabidopsis is hyper-susceptible to Meloidogyne hapla [29]. Furthermore, exogenous application of JA or SA can signi cantly improve the insect resistance of plants [30,31]. The ability of pest resistance in plant can be regulated by changing the expression levels of JA/SA-related genes [32,33].
Although mineral oils and organophosphate sprays have been widely used for P. pentagona management, breeding of new resistant kiwifruit cultivars still shown great promise. In this study, we focuse on four kiwifruit cultivars with different P. pentagona-resistance ability after a large-scale screening. According to analyzing the difference of cell structure of cane and some JA/SA-related genes' expression levels among these cultivars, we preliminarily analyzed the P. pentagona-resistance mechanisms of resistant kiwifruit cultivars. These results will provide new thoughts and methods for insect prevention in kiwifruit and breeding of new resistant kiwifruit cultivars.

Growth conditions and the infestation rate measurement
In total fty kiwifruit cultivars were planted outside in the eld. The plants spaced 2 m apart in rows, and rows were placed 3 m apart. The kiwifruit plants had been mature and began to bear fruit in 2007. Then fruits of fty kiwifruit cultivars were harvested in late August 2008, 2010 and 2011, and infestation rate was counted. Forty plants of each cultivar were harvested and thirty fruits of each cultivar were randomly sampled .
Samples collection and pre-processing Page 4/20 P. pentagona was cultured in the insect rearing facility of The Sichuan Provincial Academy of Natural Resource Science. Twelve-hour newborn female eggs of P. pentagona on pumpkin or potato were collected before the test. One-year-old canes (approximately 1.0-1.5 m long and 1-2 cm in diameter), including "LC-04285", "CF-3", "DA-7B" and "Hayward", were collected in winter (middle of December in China). The canes were wrapped in black plastic and held in a cool store at 0-2℃, RH60-70%. The laboratory experiment was set up 1-2 months later (Jan. or Feb.) in a controlled environment room at kiwifruit laboratory, Chengdu. The environmental conditions were 25℃, 70±10% Relative Humidity (RH), with a 14 h light:10 h dark photoperiod regime.
The kiwifruit canes were removed from the cold store, marked and cut into 60 cm with removing the bottom of 10 cm. Each cane length was labelled and the buds were cut off. All wounds were sealed with Vaseline. Each cane was gently wrapped 6-9 turns with wool, providing a place for the eggs and crawlers to colonize [56]. Each kiwifruit cultivar had ve replicates.

Colonization rate and survival rate measurement
The female eggs (about 200) of P. pentagona were gently transfer to the sites which wrapped turns on branches using stroke pen. The branches were placed at an angle of 30-50 ° to ensure that the eggs would not fall. Additionally, a large number of male eggs were inoculated into the other two branches, so that the males could mate with the females on the branches after hatching. All branches were placed in an industrial climate box (23 ± 0.5 ° C, 60 ± 5% RH, 14 hours of light, and 10 hours of darkness). The water in the beaker was replaced every week. The buds were cut off and sealed with Vaseline weekly. The speci c number of transferred female eggs was recorded under a stereomicroscope. After 7 days of egg hatching, the wool was cut off, the number of colonies of the crawlers was recorded, and a marker was used to mark the sides. The number of live and dead pests on the branch were recorded when the eggs hatch for about 40 days (the pests were in the adult stage). And then, the survival rate was calculated.
The speci c measurement methods referred to Hill's report [56].

Scale area measurement
The scale's area in three stages, including early second instar stage, early adult stage and oviposition stage, was measured by a transparent template which had circular scale. The area of template was 0.4-1.4 mm 2 and increased by 0.2 mm 2 . The speci c measurement methods referred to Hill's report [56]. If the female scale's area was larger than 1.2 mm 2 , the software, ImageJ, was used to measure the area. Thirty female scales were measured each time.

Anatomic characterization
The sites of another canes, which are same as insect inoculation site, were chosen. The canes samples were cut into 2 mm thick slices using a scalpel. The samples were para n-embedded and sectioned as described previously [11]. A JS5BS stereo microscope was used to observe the samples. Culticle was measured under a microscope at 400-fold magni cation. The cell length of pith, cortex and epidermis were measured under a microscope at 40-fold, 100-fold or 400-fold magni cation, respectively. The number of epidermis and cortex cells in an area of 1.5 cm × 1.5 cm was counted under a microscope at ×100 magni cation.

RNA extraction and real-time quantitative PCR
Canes that were not infested by P. pentagona were used to extract RNA. RNAprep Pure Plant Plus Kit (TIANGEN) was used to isolate total RNA. SYBR Green Pro Taq HS (Accurate Biology) was used to qRT-PCR. The methods were performed as previously described [57]. The sequences of primers used for qRT-PCR are listed in Table S2.

Statistical analysis
All data in our study are presented as the mean ± SD of at least three independent experiments. SPSS software version was used to analyze the data. The Student's t-test was used to calculate the P value and analyze the signi cant differences between two groups of data. One-way ANOVA was used to calculate the P value and analyze the signi cant differences between multiple groups of data.

Different survival situations of P. pentagona in four kiwifruit cultivars
For the purpose of screening kiwifruit cultivars with excellent P. pentagona resistant trait and breeding potential, fruits of fty kiwifruit cultivars were collected outside and infestation rate by P. pentagona was counted respectively in 2008, 2010 and 2011. Considerable variability among cultivars for resistance to P. pentagona was revealed (Table S1). P. pentagona was not found in "JXFujun-CK-04227", "LC-04285", "SF-2", "CF-3" and "Me05-01" among these years, suggesting their potential resistance to P. pentagona. Based on the growth status, "LC-04285" (wild) and "CF-3" (wild) were used for further study. Meanwhile, the other two susceptible cultivars, "DA-7B" (wild) and "Hayward" (commercial), were also tested as controls.
To further investigate the P. pentagona resistant trait of these kiwifruit cultivars, the canes of these four cultivars were collected and inoculated with P. pentagona indoors. The colonization rate, survival rate and scale area were counted and used as indicators of the P. pentagona resistance. (Table 1). The canes infestation by P. pentagona was signi cantly different between resistant cultivars and susceptible cultivars. Average colonization rate was lower in "LC-04285" (9.92%), "CF-3" (7.48%), "DA-7B" (14.37%), and higher in "Hayward" (24.18%). After 40 days incubation, all nymphs in "LC-04285" did not survived. Although particial crawlers in "CF-3" and "DA-7B" survived, the scale area of all crawlers in "CF-3" was less than 1.2 mm 2 , investigating the potential inhibition of somatic development by "CF-3". "Hayward" had the highest survival rate of crawlers at 65.2%, and all of the crawlers had a scale area of more than 1.2 mm 2 . These results indicated that "LC-04285" and "CF-3" had signi cant P. pentagona resistant trait. "LC-04285" had thicker cuticle than other kiwifruit cultivars To investigate the reasons for the difference in resistance to P. pentagona among different kiwifruit cultivars, we performed anatomical analysis using para n sections of the canes of these four kiwifruit cultivars. As the outermost barrier for plants, cuticle protect plants from biotic stresses [34]. Interestingly, "LC-04285" had thicker cuticle than other cultivars (Fig. 1). Then, we analyzed the expression levels of AcSHN1, AcKCS3, AcKCS4, AcCER4, AcCER5 and AcGPAT1 in the canes of four kiwifruit cultivars by realtime quantitative PCR (qRT-PCR), which were involved in the synthesis of cuticle. As show in Fig. 2, the expression levels of these genes in "LC-04285" were higher than those of other kivifruit cultivars. These results showed that thicker cuticle conferred greater resistance to "LC-04285" against P. pentagona.
"LC-04285" had denser epidermis and cortex than other kiwifruit cultivars In order to further analyze the relationship between anatomical structures and P. pentagona resistance, we counted the number of cell layer, cell number and cell length. No difference in number of cell layer and cell length was detected among these four kiwifruit cultivars (Fig. 3a-c). However, the densities of epidermal and cortical cells in "LC-04285" were higher than the others (Fig. 3a, d). We also analyzed the expression levels of genes related to the formation of the epidermis and cortex. As show in Fig. 4, the expression level of positive regulator AcSHR1 in "LC-04285" was higher, while the expression levels of nagative regulators, AcAGO4 and AcSCL3, were lower. These results indicated that denser epidermis and cortex protected kiwifruit by preventing P. pentagona from sucking plants' sap.
Resistant cultivar "LC-04285" had a stronger response to JA.
Previous studies demonstrated that JA played an important role in pest resistance [22,35]. Changes in the expression of JA-related genes affected the pest resistant trait of plants [27]. COI1 was the key gene in response to JA signal. As show in Fig. 5a, the expression levels of AcCOI1 in "LC-04285" were higher than that of other cultivars. This result indicated that "LC-04285" could response to JA signal fast when being fed by P. pentagona. JAZ family genes were plant-speci c JA response genes and nagative regulation of JA signal. Suppression of JAZ family genes in plants could enhance the biotic stresses resistance [36]. In the above research, we had found that "Hayward" had the worst resistance to P. pentagona (Table 1). Similarly, the expression levels of AcJAZ8, AcJAZ11, AcJAZ12, AcJAZ13, AcJAZ14 and AcJAZ15 in "Hayward" were higher than those of other kiwifurit cultivars (Fig. 5b-h). In contrast, these genes were lowly expressed in "LC-04285", which had strong resistance to P. pentagona. The above results indicated that the strong response to JA conferred great P. pentagona resistance in resistant cultivar "LC-04285" .
Resistant cultivar "LC-04285" had higher expression levels of SA-related genes.
SA played an important role in pest resistance as it was involved in the inducing of various defensive responses [26]. SA is synthesized via two routes by two crucial enzymes, isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL) [19]. To determine whether more SA was synthesized in "LC-04285", we analyzed the expression levels of SA biosynthesis-related genes. As show in Fig. 6a-c, the expression levels of AcICS, AcPAL4 and AcPAL5 in "LC-04285" were higher than those in other kiwifurit cultivars. These results indicated that "LC-04285" had more SA accumulation and leaded to P. pentagona resistance. Further, NPR genes were SA receptor and regulated some resistant genes' expression [37]. As show in Fig. 6d-f, the expression levels of AcNPR1, AcNPR2 and AcNPR3 in "LC-04285" were higher than that in other kiwifurit cultivars. Collectively, the above results indicated that resistant cultivar "LC-04285" possessed a stronger ability to synthesize SA and a higher level of resistance mediated by SA.

Discussion
China is the origin of kiwifruit, and there are a large number of wild kiwifruits in the mountains. For the purpose of screening kiwifruit with insect resistance and breeding potential, we investigated the rate of fruit injury of 336 kiwifruits in 2008, 2010 and 2011, and sellected fty cultivars with stable tolerance performance to P. pentagona. In this study, we selected four representative cultivars for study, including "LC-04285", "CF-3", "DA-7B" and "Hayward". By analyzing their anatomical structure of cane, we found that resistant cultivars, "LC-04285", had thicker cuticle, denser epidermis and cortex (Fig. 1, 3). The qRT-PCR data indicated that the expression levels of some genes related to cuticle synthesis and formation of epidermis and cortex are higher in "LC-04285" (Fig. 2, 4). Some studies have reported that the cuticle have a certain relationship with plant insect resistance [38,39]. For example, Kosma et al. found that wheat with intact cuticles was more resistant to Hessian y larvae [40]. The olive trees with thicker epidermis and cuticle were more resistant to eriophyid mites [41]. As a result, thicker cuticle and denser epidermis and cortex prevent P. pentagona from sucking plant juice.
In our research, we found that the "CF-3" had yellow epidermal cells (Fig. 1a). We supposed the cells were rich in avonoids. Furthermore, the qRT-PCR results show that the expression levels of AcC4H, AcFLS7 and AcLDOX2 in "CF-3" were higher (Fig. S1). Flavonoids are present in various tissue cells of the plant, including epidermal cells. Among the petal epidermal cells of lisianthus owers, yellow-colored cells had higher levels of avonoids [42]. The orange color of tomato fruit was also due to the accumulation of avonoids [43]. Flavonoids were closely related to plant biological stress. Genistein was a kind of avonoid found mainly in legumes, which could help protect legumes from Piezodorus guildiniiIn [44]. Tomatoes with higher avonoids are more resistant to white y Bemisia tabaci [45]. However, it is still necessary to identify the structure and function of the avonoids in "CF-3" in the future work.
In this study, we also analyzed the expression level of JA/SA-related genes. we found that the genes involed in SA synthesis were expressed higher in "LC-04285", which has stronger resistance to P. pentagona (Fig. 6a). It has been reported that spraying the fruits with certain concentration of exogenous SA can increase the content of the endogenous SA in fruit and the resistance to pathogenes without affecting the quality of fruit [46,47]. In addition, the expression levels of NPR genes in "LC-04285" are still increased (Fig. 6b-f). NPR family genes are closely related to the response of plants to biological stress [37,48,49]. The resistances to various pathogens in different plants are increased through heterologously expressing NPR1 of Arabidopsis thaliana [50][51][52]. Studies have shown that overexpression of NPR1 of Arabidopsis thaliana in tobacco increases the resistance of tobacco to Spodoptera litura [53]. More interestingly, it is known that SA-signaling pathway can be elicited by many sap-sucking insects, and, simultaneously, the JA-signaling pathway is suppressed [54,55]. However, we found that the expression levels of AcCOI1 in "LC-04285" were higher than that in other kiwifurit cultivars (Fig. 5a). Furthermore, the expression levels of some JAZ repressors in "Hayward" were higher (Fig. 5b-h). These results indicate that in addition to SA, JA also plays a crucial role in plant resistance to P. pentagona. However, the mechanism of JA and SA against P. pentagona needs further investigation.

Conclusions
In general, "LC-04285" has a thicker cuticle and denser epidermis and cortex, which can resist the sucking of P. pentagona. Furthermore, "LC-04285" has stronger SA/JA synthesis and response ability. These  Asterisks indicate signi cant differences between "LC-04285" and other kiwifruit cultivars (**P<0.01), as determined by Student's t test. Scale bars, 10 μm.