TY - JOUR
AU - Zhang,, Zhengqun
AB - Abstract The tea green leafhopper, Empoasca onukii Matsuda (Hemiptera: Cicadellidae), is an economically important pest of tea crops, Camellia sinensis (L.) O. Kuntze (Ericales: Theaceae), in China. The morphological, physiological, and biochemical changes of two tea cultivars, the normal green tea cultivar ‘Fudingdabai’ and the novel chlorophyll-deficient albino cultivar ‘Huangjinya’, infested by E. onukii were investigated to determine the tolerance of different tea cultivars to E. onukii attack. E.onukii infestation affected the growth of tea plants, and decreased the shoot length, leaf area, leaf thickness, and stem diameter. Also, E. onukii infestation lowered the thicknesses of upper epidermis, palisade tissue, and spongy tissue of leaves, and the parenchyma tissue thickness and pith diameter of stem internode. E.onukii infestation reduced the chlorophyll a, b and carotenoid contents within the leaves of ‘Huangjinya,’ which further influenced the photosynthetic rate. The maximum quantum yield and actual photochemical efficiency of photosystem II, and non-photochemical quenching in ‘Huangjinya’ were inhibited under E. onukii infestation. Peroxidase activity of E. onukii-infested ‘Huangjinya’ increased more than superoxide dismutase and catalase. In addition, E. onukii feeding changed the contents of free amino acids, tea polyphenols, caffeine, and catechins in leaves of ‘Huangjinya’. Overall, the light-induced albino cultivar ‘Huangjinya’ was susceptible to E. onukii while ‘Fudingdabai’ was resistant. Empoasca onukii, plant growth, photosynthesis, chlorophyll fluorescence, antioxidative enzyme In the natural environment, plants trigger a wide range of morphological, physiological, and biochemical defences against biotic stresses such as herbivorous insects and pathogens(Atkinson and Urwin 2012;Truong et al. 2015). Investigation of plant growth and anatomic structural parameters is a preliminary to understanding and interpreting the influence of herbivores feeding on plants. The susceptible crop varieties infested by piercing–sucking insects showed greater variations than resistant ones in growth parameters, such as the plant height, internode length, and plant dry weight(McAuslane et al. 2004, Li et al. 2013). Furthermore, the anatomical structure of leaves and stems(e.g., the thicknesses of upper epidermis and palisade tissues) would be also altered against insect-feeding even among plant species and varieties within species (Tort 2004). The chlorophyll content, photosynthetic activity, and chlorophyll fluorescence parameters in plant tissue are the primary physiological indicators involved in the interactions between plants and herbivores (Goławska et al. 2010, Golan et al.2015). In host plants, decline in photosynthetic rates induced by insect pest injuries is usually associated with decreases in the chlorophyll content, photosynthetic capacity, and stomatal conductance (Haile and Higley 2003, Li et al. 2013). Moreover, some antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) play an important role in maintaining the balance between the generation and quenching of reactive oxygen species (ROS) in plant growth process, especially in a number of stress states (Upadhyaya et al.2008). For example, Bemisia tabaci infestation significantly increased POD and SOD activities in cucumber seedlings (Zhang et al. 2008). The SOD and POD activities greatly increased when Therioaphis trifolii attacked alfalfa leaves (Liu and Lan 2009). Furthermore, insect infestation induced changes in the biochemical composition of host plant leaves. For example, phenolics are secondary metabolites involved in the plant defense against pests and pathogens, and their increased accumulation after infection could be related to the resistance mechanism of the host(Bennett and Wallsgrove 1994, Anjani et al. 2010). Tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important woody crop, which has been grown worldwide to produce nonalcoholic beverage (Saha et al. 2012). Empoasca onukii Matsuda (Hemiptera: Cicadellidae) is one of the most common and economically important piercing–sucking herbivores of tea plants (Zhang et al. 2017). Adults and nymphs suck the sap of tender shoots and buds, causing leaf edge yellowing, redden vein, and wrinkled leaf (Jin et al.2012). However, relatively few studies have focused on varietal performance of tea plants against E. onukii. We found that, in the tea plantation, the densities of E. onukii on the normal green tea cultivar ‘Fudingdabai’ were significantly lower than the densities on the novel chlorophyll-deficient albino cultivar ‘Huangjinya’ (Zhengqun Zhang, unpublished data). Hence, we compared the morphological, physiological, and biochemical changes of these two tea cultivars infested by E. onukii to determine whether E. onukii has the same effects on both cultivars. Understanding the differences in E. onukii-induced responses between different tea cultivars is necessary to reveal the mechanisms involved in the tolerance of tea plants to E. onukii. Materials and Methods Insects, Plant Material and Treatments All experiments were conducted using healthy 3-yr-old ‘Huangjinya’ and ‘Fudingdabai’ cultivar seedlings (20–25 cm in height). Tea plants were grown in approximately 2.0-liter plastic pots containing acidic peat soil in a climate-controlled room (25°C and 70% RH,with a photoperiod of 14:10 (L:D) h) and watered twice weekly. No pesticides were aprayed at any stage of plant growth. E.onukii adults of mixed age and sex and of unknown mating status were collected from new shoots of tea plants using sweep nets at the Tea Experimental Plantation of Shandong Agricultural University in Tai’an, Shandong Province, China. The leafhoppers were aspirated from the sweep nets after collection in the field and reared on the tea cultivar ‘Fudingdabai’ in ventilated cages (50 × 50 × 50 cm). A colony of E. onukii for use in the infestation experiments was established in our laboratory. The cages were maintained in a climate-controlled chamber at 25 ± 2°C and 70 ± 5% RH, with a photoperiod of 14:10 (L:D) h.The infestation experiments were performed in the climate-controlled chambers described above. Each similar-sized ‘Huangjinya’ or ‘Fudingdabai’ seedling was placed in a nylon screen cage (50 × 50 × 50 cm), and 20 third and fourth nymphal instars of E. onukii per plant were introduced into the nylon cages. All measurements of morphological, physiological, and biochemical parameters were carried out on both infested and uninfested plants of two cultivars at 10 d of continuous exposure to leafhoppers. Determination of Shoot Length and Leaf Area After E. onukii being removed at 10 d, the tea shoot lengths were measured from the buds to the second leaves (from the bottom).The areas of the third leaves on tea shoots were determined using a portable leaf area measurer (CI-202, CID Bio-ScienceInc., Camas, WA). Anatomic Structures of Leaves and Stem Segments Measurement The third mature leaf on each tea shoot was selected to be cut to a 5 × 5 mm section, and fixed in FAA (formaldehyde:acetic acid:50% ethanol = 5:5:90). The samples were dehydrated in an increasing ethanol gradient, embedded in paraffin wax, sectioned using an ultra microtome (12 μm thick), and stained with safranine–fastgreen. Stem internodes between the second and third leaves on tea shoots were cut to study the anatomical structure. The internode segments(1 cm) were stored in FAA retaining the orientation of stems. Before sectioning, the samples were dehydrated in a graded series of ethanol and then embedded in paraffin wax. Longitudinal and cross sections of 8 μm thickness were cut by microtome and double stained with safranine–fastgreen. All anatomical characteristics of leaves and stem internode segments were measured using pannoramic viewer software with 20 replicates. The Photosynthetic PigmentContents Pigment extract was prepared from 0.2 g fresh leaves (the third leaf on each tea shoot) by grinding in a tissue homogenizer together with 60 ml of 80% acetone for 36 h in dark until the leaves became completely colorless. The measurement of absorbance was performed with three wavelengths, 663, 646, and 470 nm, using a spectrophotometer (UV-2450, Shimadzu, Japan). The concentrations of chlorophyll a and b and carotenoids were calculated according to the following equations: Cchl. a(mg g−1 FW) = (12.21 ×OD663−2.81×OD646)/(1,000 ×W) ×V Cchl. b(mg g−1 FW) = (20.13 ×OD646−5.03×OD663)/(1,000 ×W) ×V Ccar.(mg g−1FW) = (1,000 ×OD470−3.27 ×Cchl. a−104 ×Cchl. b)/(229 × 1,000 ×W) ×V, where V is the volume of extract in ml, and W is the fresh weight (FW) of leaf sample in grams; OD663, OD646, and OD470 represent the absorption at 663, 646, and 470 nm, respectively. Leaf Gas Exchange and Chlorophyll Fluorescence Measurements Leaf gas exchange measurements were conducted using a portable photosynthesis system(CIRAS-2, PP Systems International, Amesbury, MA) in environmentally controlled conditions. Photosynthetic photon flux density (PPFD) was maintained at 800 μmol m−2 s−1, and the leaf temperature was maintained at 25 ± 1°C inside the leaf chamber. The CO2 concentration within the leaf measurement chamber was maintained at 350–400 μmol m−2 s−1 using a gas buffer box. The third leaves of tea shoots on E. onukii-infested and uninfested plants of two cultivars were selected to measure gas exchange parameters, including photosynthesis rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (E) with five replicates. An analysis of chlorophyll fluorescence was conducted using an fluorescence monitoring system-II portable pulse modulation fluorescence analyzer (Hansatech, King’s Lynn, Norfolk, UK). The measurements in five replicates were made on the third leaves on different tea shoots located on E. onukii-infested and uninfested plants of two cultivars and with a similar orientation toward the light. Before the measurement, the leaves were shaded for about 20 min by means of manufactured clips. After dark acclimation, the maximum fluorescence (Fm) and the maximum quantum yield of photosystem II(Fv/Fm) were determined applying a saturating light pulse (PPFD ~3,000 μmol m−2 s−1). Light sufficient to drive photosynthesis (actinic light, PPFD = 1,200 μmol m−2 s−1) was then applied, and after 15 min, the actual photochemical efficiency of photosystem II (ΦPSII) and maximum fluorescence after steady-state conditions (F′m) were determined by applying pulses of the saturated white light every 60 s when the actinic light was on. Non-photochemical quenching (NPQ) was calculated according to the following formula: NPQ = (Fm − F′m)/F′m. Determination of Antioxidant Enzyme Activity The third leaves of tea shoots on E. onukii-infested and uninfested plants of two cultivars were selected to measure the activities of CAT, SOD, and POD with five replicates. For each treatment, 0.5 g of fresh leaf sample was homogenized with a mortar and pestle with 5 ml of 50 mM precooled phosphate buffer (pH 7.8) containing 1 mM ethylenediamine tetraacetic acid and 5% polyvinylpyrrolidone under liquid nitrogen. The crude homogenate was subjected high-speed centrifuge at 16,000 rpm for 15 min at 4°C. The supernatant was used to measure the activities of CAT, SOD, and POD, and to determine total protein content.The absorbance was read using a spectrophotometer (UV-2450, Shimadzu, Japan). The SOD, CAT, and POD activities were detected using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s manual. The SOD activity was assayed by its ability to inhibit photochemical reduction of nitroblue tetrazolium (NBT) at 550 nm. About 50% reduction of NBT was considered as one unit of enzyme activity. CAT activity was determined by monitoring the disappearance of H2O2 at 405 nm. One unit of CAT activity was defined as the amount that resolved the level of H2O2 by lmol mg−1 protein per second. POD activity was measured at 420 nm. One unit of POD activity was defined as the amount that catalyzed l μg substrate per minute per mg protein at 37°C. Protein concentrations were determined with the Bradford protein assay (Bradford 1976) using bovine serum albumin as the standard. Determination of Biochemical Components The tea shoots with two leaves and a bud on E. onukii-infested and uninfested plants of two cultivars were selected to measure biochemical components with five replicates. The tea shoots were steamed for 5 min, dried at 80°C, and finely ground into powder using a blender. The dried leaf samples were stored at −20°C until used for determination of biochemical components. Free Amino Acids The content of free amino acids was determined using the ninhydrin colorimetric method according to the State Standard of the People’s Republic of China, Determination of free amino acids content (GB/T 8314-2013, China).About 3 g of the dried leaf sample was transferred into a flask and 450 ml of H2O was added. This mixture was then extracted in a boiling water bath for 45 min with shaking once every 10 min. After filtration, the volume of filtrates was increased to 500 ml by adding H2O. Then, 1 ml of the mixture was transferred to a 25 ml flask, and 0.5 ml of buffer (containing 63 mM Na2HPO4 and 3 mM KH2PO4, pH 8.0)and 0.5 ml of a 2% ninhydrin solution (2 g ninhydrin and 80 mg SnCl2·2H2O dissolved in 100 ml of water) was added. The flask was incubated in a boiling water bath for 15 min and the volume was increased to 25 ml with H2O. After a 10 min settling period, the absorption values of the solution was read at 570 nm. The content of free amino acids was calculated from a standard curve generated with varying concentrations of glutamine. Tea Polyphenols Tea polyphenols were measured according to the State Standard of the People’s Republic of China, Determination of total polyphenols and catechins content in tea (GB/T 8313-2008, China);0.2 g of the dried sample was put into a 10 ml centrifuge tube and 5 ml of 70% methanol preheated in 70°C was added.The tubes were placed in a water bath at 70°C for 10 min, andthe mixture was centrifuged at 3,500 rpm for 10 min. Then, 1.0 ml of the supernatant was transferred into the cuvette, and 5 ml of 10% folin-ciocalteu reagent was added. After 5 min, 4 ml of 7.5% Na2CO3 was added into the cuvette. The mixture was tested via spectrophotometry at 765 nm absorbance. The content of tea polyphenols was calculated from a standard curve generated with a series of gallic acid concentrations. Catechins The content of catechins was determined using the vanillin-hydrochloric acid method (Zhang 2009). About 0.2 g of thedried sample was transferred into a flaskand 20 ml of 95% ethyl alcohol was added. The mixture was extracted in a water bath at 80°C for 30 min. After filtration, the volume of the filtrates was increased to 25 ml by adding 95% ethyl alcohol. Then, 10 µl of the solution was transferred into a centrifuge tube containing 95% ethyl alcohol, and 5ml of 1% the vanillin-hydrochloric acid reagent was added. After a 40 min settling period, the absorption values of the solution were read at 500 nm against a reagent blank. Caffeine The content of caffeine was determined using plumbous subacetate reagent method according to the State Standard of the People’s Republic of China,Determination of caffeine content(GB/T 8312-2013, China); 3 g of the dried sample was extracted in a boiling water bath for 45 min in 450 ml of H2O (with shaking once every 10 min). After filtration, the volume of the filtrates was increased to 500 mlby adding H2O. Then, 10 ml of the solution was transferred into a 100-ml centrifuge tube, and 4 ml of 0.01 mol/liter hydrochloric acid and 1 ml 0.5 g/ml lead subacetate were added. The volume was increased to 100 ml with H2O. After a 30-min settling period, 25 ml of the supernatant was transferred to a 50 ml flat-bottomed flask, and 0.1 ml of 4.5 mol/liter sulfuric acid solution was added. Then, the volume was increased to 25 ml with H2O. After filtration, the absorbance of the extract was read at 274 nm against a reagent blank. The caffeine content was calculated from a standard curve generated with a series of caffeine concentrations. Soluble Sugars One gram of the dried sample was extracted in boiling water for 30 min in 80 ml of H2O. The mixture was filtered immediately, and the filtrates were increased to 500 ml by adding H2O. Then, 1 ml of the mixture was transferred into the cuvette and 8 ml of anthrone reagent was added. The absorbance of the mixture was read at 620 nm against a reagent blank(Dubois et al.1956). Statistical Analysis All statistical analyses were carried out using SPSS statistical software (version 18.0, SPSS Inc., Chicago, IL). Statistically significant mean values were compared using one-way analysis of variance tests followed by Tukey’s honestly significant difference (HSD) method (P < 0.05). Independent samples t-test was applied to evaluate the differences between the E. onukii-infested and uninfested plants of the same cultivar for all parameters (P < 0.05). Results Effects of E. onukii Feeding on Plant Growth E.onukii infestation inhibited the growth of tea plants compared with the control. The lengths of tea shoots (the buds to the second leaves) of ‘Huangjinya’ and ‘Fudingdabai’ were significantly decreased by 45.0 and 39.9% on 10 d after E. onukii infestation compared with the uninfested tea plants, respectively (Huangjinya: t = 6.956, df = 29, P< 0.001; Fudingdabai: t = 6.545, df = 29, P < 0.001)(Fig. 1A and C). The areas of the third leaves on tea shoots of two tea cultivars also decreased by 73.7 and 71.6% after E. onukii infestation, respectively (Huangjinya: t = 5.587, df = 9, P < 0.001; Fudingdabai: t = 7.687, df = 9, P < 0.001) (Fig. 1B and D). Fig. 1. Open in new tabDownload slide Effects of E. onukii feeding on average lengths of tea shoots (from the buds to the second leaves) (A and C) and average areas of the third leaves (B and D) on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 4) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P< 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (Independent samples t-test, P < 0.05). Fig. 1. Open in new tabDownload slide Effects of E. onukii feeding on average lengths of tea shoots (from the buds to the second leaves) (A and C) and average areas of the third leaves (B and D) on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 4) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P< 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (Independent samples t-test, P < 0.05). For uninfested tea plants, the thicknesses of palisade and spongy tissue of leaves of ‘Fudingdabai’ were less than those of leaves of ‘Huangjinya’ (P < 0.05). E.onukii infestation caused a decrease in the leaf thickness of ‘Huangjinya’, and reduced the thicknesses of upper epidermis and palisade and spongy tissue, with decreases of8.20, 24.69, and 27.17%, respectively (t = −2.238, df = 19, P = 0.037; t = –4.848, df = 19, P < 0.001; t = −5.409, df = 19, P < 0.001).Compared with control, the thicknesses of palisade and spongy tissue of leaves of ‘Fudingdabai’ also decreased under E. onukii infestation, exhibiting 21.16 and 20.04% reductions, respectively (t = −6.281, df = 19, P < 0.001; t = −4.378, df = 19, P < 0.001)(Table 1;Fig. 2). Moreover, E. onukii infestation obviously lowered the diameters of stem internode of tea shoots of two tea cultivars (Huangjinya: t = −6.616, df = 19, P < 0.001; Fudingdabai: t = −7.751, df = 19, P < 0.001). The parenchyma thickness and pith diameter of stem internode of E. onukii-infested tea plants were lower than those of control tea plants without E. onukii infestation (P < 0.05)(Table 2;Fig. 2). Table 1. Effects of E. onukii feeding on leaf anatomic characteristics of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’ Tea cultivar Treatment Leaf thickness (μm) Stratum corneum (μm) Upper epidermis (μm) Palisade tissue (μm) Spongy tissue (μm) Mesophyll tissue (μm) Lower epidermis (μm) Huangjinya EITP 149.58 ± 5.63d* 2.07 ± 0.15a 12.09 ± 0.39b* 48.07 ± 2.64d* 74.85 ± 3.55d* 118.63 ± 7.65d* 11.83 ± 0.66b UTP 194.94 ± 3.58c 1.71 ± 0.11a 13.17 ± 0.35b 63.83 ± 1.38c 102.78 ± 2.69c 166.02 ± 3.08c 12.88 ± 0.37b Fudingdabai EITP 248.82 ± 4.85b* 2.18 ± 0.13a 17.22 ± 0.49a* 94.90 ± 2.60b* 120.28 ± 3.23b* 206.39 ± 10.61b* 13.13 ± 0.32b* UTP 307.26 ± 10.66a 2.04 ± 0.16a 18.95 ± 0.62a 120.37 ± 5.26a 150.43 ± 5.76a 270.51 ± 9.92a 15.03 ± 0.57a F 102.225 2.073 46.741 96.058 63.276 59.351 7.159 P <0.001 0.111 <0.001 <0.001 <0.001 <0.001 <0.001 Tea cultivar Treatment Leaf thickness (μm) Stratum corneum (μm) Upper epidermis (μm) Palisade tissue (μm) Spongy tissue (μm) Mesophyll tissue (μm) Lower epidermis (μm) Huangjinya EITP 149.58 ± 5.63d* 2.07 ± 0.15a 12.09 ± 0.39b* 48.07 ± 2.64d* 74.85 ± 3.55d* 118.63 ± 7.65d* 11.83 ± 0.66b UTP 194.94 ± 3.58c 1.71 ± 0.11a 13.17 ± 0.35b 63.83 ± 1.38c 102.78 ± 2.69c 166.02 ± 3.08c 12.88 ± 0.37b Fudingdabai EITP 248.82 ± 4.85b* 2.18 ± 0.13a 17.22 ± 0.49a* 94.90 ± 2.60b* 120.28 ± 3.23b* 206.39 ± 10.61b* 13.13 ± 0.32b* UTP 307.26 ± 10.66a 2.04 ± 0.16a 18.95 ± 0.62a 120.37 ± 5.26a 150.43 ± 5.76a 270.51 ± 9.92a 15.03 ± 0.57a F 102.225 2.073 46.741 96.058 63.276 59.351 7.159 P <0.001 0.111 <0.001 <0.001 <0.001 <0.001 <0.001 *Significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P< 0.05). EITP, E. onukii-infested tea plants; UTP, uninfested tea plants. Open in new tab Table 1. Effects of E. onukii feeding on leaf anatomic characteristics of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’ Tea cultivar Treatment Leaf thickness (μm) Stratum corneum (μm) Upper epidermis (μm) Palisade tissue (μm) Spongy tissue (μm) Mesophyll tissue (μm) Lower epidermis (μm) Huangjinya EITP 149.58 ± 5.63d* 2.07 ± 0.15a 12.09 ± 0.39b* 48.07 ± 2.64d* 74.85 ± 3.55d* 118.63 ± 7.65d* 11.83 ± 0.66b UTP 194.94 ± 3.58c 1.71 ± 0.11a 13.17 ± 0.35b 63.83 ± 1.38c 102.78 ± 2.69c 166.02 ± 3.08c 12.88 ± 0.37b Fudingdabai EITP 248.82 ± 4.85b* 2.18 ± 0.13a 17.22 ± 0.49a* 94.90 ± 2.60b* 120.28 ± 3.23b* 206.39 ± 10.61b* 13.13 ± 0.32b* UTP 307.26 ± 10.66a 2.04 ± 0.16a 18.95 ± 0.62a 120.37 ± 5.26a 150.43 ± 5.76a 270.51 ± 9.92a 15.03 ± 0.57a F 102.225 2.073 46.741 96.058 63.276 59.351 7.159 P <0.001 0.111 <0.001 <0.001 <0.001 <0.001 <0.001 Tea cultivar Treatment Leaf thickness (μm) Stratum corneum (μm) Upper epidermis (μm) Palisade tissue (μm) Spongy tissue (μm) Mesophyll tissue (μm) Lower epidermis (μm) Huangjinya EITP 149.58 ± 5.63d* 2.07 ± 0.15a 12.09 ± 0.39b* 48.07 ± 2.64d* 74.85 ± 3.55d* 118.63 ± 7.65d* 11.83 ± 0.66b UTP 194.94 ± 3.58c 1.71 ± 0.11a 13.17 ± 0.35b 63.83 ± 1.38c 102.78 ± 2.69c 166.02 ± 3.08c 12.88 ± 0.37b Fudingdabai EITP 248.82 ± 4.85b* 2.18 ± 0.13a 17.22 ± 0.49a* 94.90 ± 2.60b* 120.28 ± 3.23b* 206.39 ± 10.61b* 13.13 ± 0.32b* UTP 307.26 ± 10.66a 2.04 ± 0.16a 18.95 ± 0.62a 120.37 ± 5.26a 150.43 ± 5.76a 270.51 ± 9.92a 15.03 ± 0.57a F 102.225 2.073 46.741 96.058 63.276 59.351 7.159 P <0.001 0.111 <0.001 <0.001 <0.001 <0.001 <0.001 *Significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P< 0.05). EITP, E. onukii-infested tea plants; UTP, uninfested tea plants. Open in new tab Fig. 2. Open in new tabDownload slide Anatomical comparison of stems and leaves of E. onukii-infested and uninfested tea plants in two cultivars ‘Huangjinya’ and ‘Fudingdabai’. (A, D, G and J) The stem transverse sections; (B, E, H and K) the stem longitudinal sections; (C, F, I and L) the leaf transverse sections. Co, collenchyma; Par, parenchyma; Pi, pith; Ue, upper epidermis; Pal, palisade tissue; Sp, spongy tissue; Le, lower epidermis. Fig. 2. Open in new tabDownload slide Anatomical comparison of stems and leaves of E. onukii-infested and uninfested tea plants in two cultivars ‘Huangjinya’ and ‘Fudingdabai’. (A, D, G and J) The stem transverse sections; (B, E, H and K) the stem longitudinal sections; (C, F, I and L) the leaf transverse sections. Co, collenchyma; Par, parenchyma; Pi, pith; Ue, upper epidermis; Pal, palisade tissue; Sp, spongy tissue; Le, lower epidermis. Table 2. Effects of E. onukii feeding on stem anatomic characteristics of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’ Tea cultivar Treatment Stem diameter (μm) Epidermis (μm) Collenchyma (μm) Parenchyma (μm) Phloem (μm) Cambium (μm) Xylem (μm) Pith diameter (μm) Huangjinya EITP 1457.04 ± 36.86bc* 14.29 ± 0.40a 67.31 ± 1.63bc 121.98 ± 2.83bc* 45.57 ± 1.68b* 51.73 ± 2.02a 59.98 ± 3.52a 688.90 ± 35.29c* UTP 1813.38 ± 47.97a 13.56 ± 0.51a 72.69 ± 2.55b 148.20 ± 11.17a 54.06 ± 2.81a 49.01 ± 2.27a 54.11 ± 3.16ab 1066.78 ± 35.36a Fudingdabai EITP 1335.75 ± 13.74c* 14.50 ± 0.62a* 62.86 ± 2.59c* 112.92 ± 3.90c* 41.83 ± 1.69b 36.56 ± 1.37b 45.31 ± 1.70bc 671.24 ± 5.74c* UTP 1565.43 ± 25.62b 12.82 ± 0.33a 83.12 ± 3.10ab 144.43 ± 6.30ab 42.98 ± 1.94b 32.65 ± 1.67b 42.59 ± 2.26c 860.19 ± 10.92b F 36.684 2.520 11.955 6.258 7.052 24.976 8.421 51.268 P <0.001 0.064 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 Tea cultivar Treatment Stem diameter (μm) Epidermis (μm) Collenchyma (μm) Parenchyma (μm) Phloem (μm) Cambium (μm) Xylem (μm) Pith diameter (μm) Huangjinya EITP 1457.04 ± 36.86bc* 14.29 ± 0.40a 67.31 ± 1.63bc 121.98 ± 2.83bc* 45.57 ± 1.68b* 51.73 ± 2.02a 59.98 ± 3.52a 688.90 ± 35.29c* UTP 1813.38 ± 47.97a 13.56 ± 0.51a 72.69 ± 2.55b 148.20 ± 11.17a 54.06 ± 2.81a 49.01 ± 2.27a 54.11 ± 3.16ab 1066.78 ± 35.36a Fudingdabai EITP 1335.75 ± 13.74c* 14.50 ± 0.62a* 62.86 ± 2.59c* 112.92 ± 3.90c* 41.83 ± 1.69b 36.56 ± 1.37b 45.31 ± 1.70bc 671.24 ± 5.74c* UTP 1565.43 ± 25.62b 12.82 ± 0.33a 83.12 ± 3.10ab 144.43 ± 6.30ab 42.98 ± 1.94b 32.65 ± 1.67b 42.59 ± 2.26c 860.19 ± 10.92b F 36.684 2.520 11.955 6.258 7.052 24.976 8.421 51.268 P <0.001 0.064 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 *Significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). EITP, E. onukii-infested tea plants; UTP, uninfested tea plants. Open in new tab Table 2. Effects of E. onukii feeding on stem anatomic characteristics of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’ Tea cultivar Treatment Stem diameter (μm) Epidermis (μm) Collenchyma (μm) Parenchyma (μm) Phloem (μm) Cambium (μm) Xylem (μm) Pith diameter (μm) Huangjinya EITP 1457.04 ± 36.86bc* 14.29 ± 0.40a 67.31 ± 1.63bc 121.98 ± 2.83bc* 45.57 ± 1.68b* 51.73 ± 2.02a 59.98 ± 3.52a 688.90 ± 35.29c* UTP 1813.38 ± 47.97a 13.56 ± 0.51a 72.69 ± 2.55b 148.20 ± 11.17a 54.06 ± 2.81a 49.01 ± 2.27a 54.11 ± 3.16ab 1066.78 ± 35.36a Fudingdabai EITP 1335.75 ± 13.74c* 14.50 ± 0.62a* 62.86 ± 2.59c* 112.92 ± 3.90c* 41.83 ± 1.69b 36.56 ± 1.37b 45.31 ± 1.70bc 671.24 ± 5.74c* UTP 1565.43 ± 25.62b 12.82 ± 0.33a 83.12 ± 3.10ab 144.43 ± 6.30ab 42.98 ± 1.94b 32.65 ± 1.67b 42.59 ± 2.26c 860.19 ± 10.92b F 36.684 2.520 11.955 6.258 7.052 24.976 8.421 51.268 P <0.001 0.064 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 Tea cultivar Treatment Stem diameter (μm) Epidermis (μm) Collenchyma (μm) Parenchyma (μm) Phloem (μm) Cambium (μm) Xylem (μm) Pith diameter (μm) Huangjinya EITP 1457.04 ± 36.86bc* 14.29 ± 0.40a 67.31 ± 1.63bc 121.98 ± 2.83bc* 45.57 ± 1.68b* 51.73 ± 2.02a 59.98 ± 3.52a 688.90 ± 35.29c* UTP 1813.38 ± 47.97a 13.56 ± 0.51a 72.69 ± 2.55b 148.20 ± 11.17a 54.06 ± 2.81a 49.01 ± 2.27a 54.11 ± 3.16ab 1066.78 ± 35.36a Fudingdabai EITP 1335.75 ± 13.74c* 14.50 ± 0.62a* 62.86 ± 2.59c* 112.92 ± 3.90c* 41.83 ± 1.69b 36.56 ± 1.37b 45.31 ± 1.70bc 671.24 ± 5.74c* UTP 1565.43 ± 25.62b 12.82 ± 0.33a 83.12 ± 3.10ab 144.43 ± 6.30ab 42.98 ± 1.94b 32.65 ± 1.67b 42.59 ± 2.26c 860.19 ± 10.92b F 36.684 2.520 11.955 6.258 7.052 24.976 8.421 51.268 P <0.001 0.064 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 *Significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). EITP, E. onukii-infested tea plants; UTP, uninfested tea plants. Open in new tab Effects of E. onukii Feeding on Photosynthetic Pigments In ‘Huangjinya’, E. onukii infestation altered the contents of chlorophyll a and b and carotenoids of the third leaves on tea shoots (chlorophyll a: t = 5.691, df = 3, P = 0.030; chlorophyll b: t = 4.700, df = 3, P = 0.042; carotenoids: t = 6.408, df = 3, P = 0.023). But, the chlorophyll and carotenoid content of the third leaves on tea shoots of ‘Fudingdabai’ were not obviously affected by the E. onukii infestation compared with the control (P > 0.05) (Fig. 3). Fig. 3. Open in new tabDownload slide Effects of E. onukii feeding on the content of chlorophyll a (A), chlorophyll b (B), and carotenoids (C) of the third leaves on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 4) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Fig. 3. Open in new tabDownload slide Effects of E. onukii feeding on the content of chlorophyll a (A), chlorophyll b (B), and carotenoids (C) of the third leaves on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 4) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Effects of E. onukii Feeding on Photosynthetic Characteristics For ‘Huangjinya’ and ‘Fudingdabai’, Pn markedly decreased by 47.07 and 33.60% in E. onukii-infested tea plants when compared with the control, respectively (Huangjinya: t = 4.469, df = 4, P = 0.011; Fudingdabai: t = 2.767, df = 4, P = 0.048) (Fig. 4A). Compared with the uninfested tea plants, E. onukii in the third leaves on the tea shoots of ‘Huangjinya’ and ‘Fudingdabai’ also decreased significantly under E. onukii infestation, exhibiting 60.17 and 58.72% reductions, respectively (Huangjinya: t = 3.717, df = 4, P = 0.021; Fudingdabai: t = 3.011, df = 4, P = 0.040)(Fig. 4D). For ‘Fudingdabai’, gs in E. onukii-infested tea plants was lower than that in uninfested tea plants (t = 2.767, df = 4, P < 0.001)(Fig. 4B). There was no significant difference in Ci between the E. onukii-infested tea plants and the controls (P> 0.05)(Fig. 4C). For ‘Huangjinya’, Fv/Fm andΦPSII decreased significantly in E. onukii-infested tea plants when compared with the controls (Fv/Fm: t = 6.333, df = 4, P = 0.024; ΦPSII: t = 19.503, df = 4, P = 0.003) (Fig. 4E and F). For ‘Fudingdabai’, there were no significant differences in Fv/Fm and ΦPSII of the third leaves on tea shoots between control and E. onukii feeding (P> 0.05). E. onukii infestation increased NPQ of leaves of two tea cultivars (Huangjinya: t = –21.703, df = 4, P = 0.002; Fudingdabai: t = –6.581, df = 4, P = 0.022) (Fig. 4G). Fig. 4. Open in new tabDownload slide Effects of E. onukii feeding on photosynthesis of the third leaves on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’: photosynthetic rate (Pn) (A), stomatal conductance (gs) (B), intercellular CO2 concentration (Ci) (C), transpiration rate (E) (D), the maximum quantum yield of photosystem II (Fv/Fm) (E), PSII actual photochemical efficiency (ΦPSII) (F), and NPQ (G). Camellia sinensis individuals (n = 5) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Fig. 4. Open in new tabDownload slide Effects of E. onukii feeding on photosynthesis of the third leaves on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’: photosynthetic rate (Pn) (A), stomatal conductance (gs) (B), intercellular CO2 concentration (Ci) (C), transpiration rate (E) (D), the maximum quantum yield of photosystem II (Fv/Fm) (E), PSII actual photochemical efficiency (ΦPSII) (F), and NPQ (G). Camellia sinensis individuals (n = 5) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Effects of E. onukii Feeding on Activities of Antioxidative Enzymes The activity of POD in infested tea plants increased compared with that in the control in ‘Fudingdabai’(t = –16.650, df = 4, P = 0.004) (Fig. 5A). Although SOD level was elevated in the E. onukii-infested tea plants, the induction was not statistically different from those in the control (Huangjinya: t = –1.854, df = 4, P = 0.205; Fudingdabai: t = –0.600, df = 4, P = 0.609) (Fig. 5B). In addition, CAT activity did not differ between the E. onukii-infested and uninfested tea plants (Huangjinya: t = 2.862, df = 4, P = 0.103; Fudingdabai: t = 0.664, df = 4, P = 0.575) (Fig. 5C). Fig. 5. Open in new tabDownload slide Effects of E. onukii feeding on the activity levels of POD (A), SOD (B), and CAT (C) of the third leaves on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 5) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Fig. 5. Open in new tabDownload slide Effects of E. onukii feeding on the activity levels of POD (A), SOD (B), and CAT (C) of the third leaves on tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 5) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Effects of E. onukii Feeding on Biochemical Components For ‘Huangjinya’, a greater difference existed in the contents of biochemical components of E. onukii-infested tea plant and uninfested tea plant leaves, including free amino acids (t = 18.939, df = 4, P = 0.003, Fig.6A), tea polyphenols (t = 5.183, df = 4, P = 0.035, Fig.6B), caffeine (t = –7.546, df = 4, P = 0.017, Fig.6C), and catechins(t = 6.605, df = 4, P = 0.022, Fig.6D). For ‘Fudingdabai’, except for tea polyphenols, there were no significant differences in the contents of other biochemical components between the E. onukii-infested tea plants and the controls (P> 0.05) (Fig. 6). Fig. 6. Open in new tabDownload slide Effects of E. onukii feeding on the content of free amino acids (A), tea polyphenols (B), caffeine (C), catechins (D), and soluble sugars (E) of tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 5) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Fig. 6. Open in new tabDownload slide Effects of E. onukii feeding on the content of free amino acids (A), tea polyphenols (B), caffeine (C), catechins (D), and soluble sugars (E) of tea shoots of two tea cultivars ‘Huangjinya’ and ‘Fudingdabai’. Camellia sinensis individuals (n = 5) were infested by E. onukii for 10 d compared with uninfested tea plants. Different letters indicate significant differences between the different treatments (Tukey’s HSD test, P < 0.05). Asterisks designate a significant difference between control and E. onukii-infested plants of the same cultivar (independent samples t-test, P < 0.05). Discussion E.onukii feeding suppressed the growth of tea plants. The tea cultivars ‘Huangjinya’ and ‘Fudingdabai’ both exhibited a decrease in tea shoot length and leaf area in response to E. onukii feeding. Infestation of piercing–sucking pests can reduce the growth traits of host plants such as plant height, internode length, plant dry weight, and petiole length (McAuslane et al. 2004; Li et al. 2013). Although uninfested tea plants of different cultivars inherently differed in biomass, they showed similar reductions in some physical characteristics in response to E. onukii infestation. We can conclude that the light-induced albino cultivar ‘Huangjinya’ and the normal cultivar ‘Fudingdabai’ were not different in stunting associated with infestation by E. onukii. There is a negative correlation between the population densities of E. onukii and the thicknesses of palisade and spongy tissue of tea plant leaves (Zhang and Tan 2004). The thicknesses of palisade and spongy tissue in leaves are key factors for determining the tolerance of tea cultivars to E. onukii. The susceptible cultivars exhibit thin palisade and spongy tissue in leaves (Zhu 1992). So, compared with ‘Fudingdabai’, ‘Huangjinya’ has relatively thinner palisade and spongy tissue in leaves, suggesting its lower tolerance to E. onukii attack. For ‘Huangjinya’, E. onukii feeding caused 24.69 and 27.17% reduction in the thicknesses of the palisade and spongy tissue in leaves, whereas the thicknesses of the palisade and spongy tissue in leaves of ‘Fudingdabai’declined slowly in response to E. onukii feeding, exhibiting 21.16 and 20.04% reductions, respectively. The changes in the thicknesses of both the palisade and the spongy tissue further demonstrate the susceptibility of ‘Huangjinya’ to E. onukii attack. Piercing–sucking pest feeding causes varying degrees of destruction to chloroplasts, which leads to the reduction of overall chlorophyll content. For example, when B. tabaci nymphs infested tobacco for different periods of time, the levels of chlorophyll a and b and carotenoids in infested leaves were reduced beginning at 8 d of infestation (Li et al. 2013). In our study, after the infestation of E. onukii for 10 d, the systemic leaves of tea plants showed clear symptoms of a physiological disorder, exhibiting yellowing leaves with reddening veins. E. onukii infestation diminished the levels of chlorophyll a and b and carotenoids within the leaves of ‘Huangjinya’. Chlorophyll content in leaves of ‘Fudingdabai’ was not affected by E. onukii infestation. Moreover, leaf chlorophyll content is an important factor in determining the photosynthetic rate, which represents both potential photosynthetic productivity and general plant vigor (Golan et al. 2015). So, the markedly reduced Pn in ‘Huangjinya’ induced by E. onukii was associated with the most obvious decrease of chlorophyll content (Buntin et al. 1993; Li et al. 2013). In addition,the obvious decrease in Pn and E in tea plants infested with E. onukii seemed to be caused by lower stomatal conductance, which inhibits the exchange of CO2 and H2O (Hsu et al. 2015). E. onukii infestation was shown to reduce the value of some of the analyzed photosynthesis indicators in ‘Huangjinya’ leaves such as Fv/Fm, ΦPSII, and NPQ. Fv/Fm reflects the maximal efficiency of excitation energy capture by open PSII reaction centers (Retuerto et al. 2006). Therefore, the decrease in Fv/Fm suggests that the loss in photosynthesis was due to damage of the photosynthetic apparatus by E. onukii feeding. Blanco et al. (1992) showed that the relative decrease in Fv/Fm has also been used in the rapid assessment of plant susceptibility or resistance to aphids (Blanco et al. 1992). A greater decline in Fv/Fm in ‘Huangjinya’ can to a certain extent explain the susceptibility of this light-induced albino cultivar to E. onukii. In contrast, due to the relatively stable Fv/Fm in leaves after E. onukii injure, ‘Fudingdabai’ was capable of compensating for the losses and balancing the negative effect of E. onukii (Thomson et al. 2003). Under normal circumstances, the generation and scavenging of ROS maintain a dynamic equilibrium in cellular metabolism of tea plants. The mechanisms that inhibit ROS from damaging the bodies of tea plants are thought to play important roles in imparting tolerance in tea plants to E. onukii stress, and three major enzymes, SOD, POD, and CAT, are the most important components in protection from ROS (Zhang et al. 2013). POD activity exhibited various increases in many plants due to herbivore attack. For example, POD activity in B. tabaci-infested cabbage plants increased more than SOD and CAT (Zhang et al. 2013). After infested by E. onukii, POD activity showed a significant increase in ‘Huangjinya’, whereas SOD and CAT activities were not statistically different compared with the control. Expression levels of POD genes also increased in tea plants after insect infestation (Wang et al. 2016). Moreover, Chen et al. (2009) showed that resistant corn germplasm line had greater POD activity than susceptible corn germplasm line, and suggested that high inducibility of POD might be an indicator of Spodoptera frugiperda susceptibility of corn plants. Therefore, high inducibility of POD in ‘Huangjinya’ indicated that this tea cultivar might be susceptible to E. onukii. Plant nutritional levels often affect plant suitability and resistance to herbivory. For example, amino acids are major sources of nitrogen for phytophagous insects, and herbivores provided with added nitrogen typically develop faster and survive and reproduce better (Chen et al. 2009). The levels of free amino acids markedly decreased in ‘Huangjinya’ and remain unchanged in ‘Fudingdabai’ after E. onukii feeding confirmed that ‘Huangjinya’ was susceptible while ‘Fudingdabai’ was resistant. Phenolic compounds play an important role in the plant defense. One of the reasons for the tolerance of ‘Fudingdabai’ to E. onukii attack could be their ability to maintain higher levels of tea polyphenols in face of attack than ‘Huangjinya’(Chakraborty et al. 2005). Catechins, a class of polyphenols, are the major phenolic components of tea leaves. E. onukii infestation obviously reduced the content of catechins in ‘Huangjinya’, which may further weaken the resistance of this cultivar to E. onukii. In addition, lower content of catechins in E. onukii-infested ‘Huangjinya’resulted in the tenderness of tea shoots decreasing. Caffeine is an alkaloid that plays an active role in the taste of tea. E. onukii infestation significantly raised the caffeine content in ‘Huangjinya’, which would increase the bitter taste of commercial tea. Wang et al. (2016) showed that six tea caffeine synthase genes were upregulated in response to infestation by Ectropis obliqua. The changes in the biochemical composition of ‘Huangjinya’ leaves would have a negative effect on grade and quality of tea. In conclusion, E. onukii infestation affected the growth of tea plants, reduced the photosynthetic pigments, influenced the photosynthetic characteristics, and induced higher POD activity in ‘Huangjinya’ than in ‘Fudingdabai’. So, the light-induced albino cultivar ‘Huangjinya’ was susceptible to E. onukii while ‘Fudingdabai’ was resistant. Recently, new evidence shows that many crucial genes involved in defense responses of tea plants against the defoliators such as E. obliqua (Deng et al. 2013; Jeyaraj et al. 2017; Zhou et al. 2017). The molecular mechanisms of the response of tea plants to E. onukii infestation need further research. Acknowledgments We thank two anonymous reviewers for their helpful comments and suggestions on the manuscript. 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TI - Morphological, Physiological, and Biochemical Responses of Two Tea Cultivars to Empoasca onukii (Hemiptera: Cicadellidae) Infestation
JF - Journal of Economic Entomology
DO - 10.1093/jee/toy011
DA - 2018-04-02
UR - https://www.deepdyve.com/lp/oxford-university-press/morphological-physiological-and-biochemical-responses-of-two-tea-rvjscPZ6hI
SP - 899
VL - 111
IS - 2
DP - DeepDyve
ER -