The tea seedling varieties used in this study were Camellia sinensis cv. Pingyang Tezao and Camellia sinensis cv. Ruixue, which were 1.5 years of age. Plants were purchased from Jufeng town, Rizhao city, Shandong Province, and planted in April 2018 at experimental bases in Weifang University of Science and Technology (Shouguang city, SG) in Pingjia village, Jufeng town, Rizhao city (Rizhao city, RZ). Plants were all managed using the same techniques. Plant leaves at the third and fourth leaf position were harvested from plants in October 2018. The experimental site at the Weifang University of Science and Technology had high soil pH (6.84) with a brown soil type; the experimental site at Jufeng town, Rizhao city, had a lower soil pH that is optimal for tea growth (4.56) and a brown loam soil type.

The leaf pigment content was determined using acetone extraction spectrophotometry, as previously described (4).

The fourth functional leaf of the new annual leaves of tea plants was selected and leaf cells were observed using a transmission microscope according to previously published methods (5).

Soil samples were taken from Rizhao and Shouguang experimental sites, and five points of soil (0–20 cm) were taken in the shape of an S and mixed well for use. The pH of a mixture containing soil and water in a ratio of 1:1 was measured using a pH meter (Orion 3 STAR, Thermo Fisher Scientific, Waltham, MA, USA). Soil organic matter (SOM) was measured using the potassium dichromate volumetric method, and total nitrogen was measured using a C/N elemental analyzer (Vario Max Elementar, Germany). Effective state elements were extracted from a water-to-soil mixture in a ratio of 10:1 using an M3 leaching agent, shaken for 5 min, and measured using an iCAP6300 inductively coupled plasma spectrometer (Thermoelectric Corporation, USA) (6).

Table 1 shows the soil nutrient grading standard of tea plantations (7).

The levels of chemical elements were determined by inductively coupled plasma emission spectroscopy (ICP-OES) (8).

The tea polyphenol content was determined using the ferrous tartrate colorimetric method (9); the free amino acid content was determined using the ninhydrin colorimetric method (10); the soluble sugar content was determined using the anthrone colorimetric method (11); the protein content was determined using the Coomassie Brilliant Blue method (11); and the water-soluble leachate content was determined using the full amount method (11).

After planting Shouguang tea seedlings (Pingyang Tezao and Ruixue), according to the results of the single-factor experiment of foliar spray fertilization, a set of optimized programs of foliar formula fertilization to improve the yellowing disease and albinism of tea leaves, 0.8% urea + 0.8% potassium dihydrogen phosphate + 0.1% manganese sulfate + 0.01% copper sulfate + 0.01% zinc sulfate, was sprayed every 15 days for 6 months, and control sprayed with water.

Microsoft Excel 2010 was used for data analysis and graph production, and SAS software was used for the LSD significance test (LSD method, p < 0.05).

Pingyang Tezao and Ruixue planted in Rizhao grew well, with green leaves (Figures 1A, C). The Pingyang Tezao planted in Shouguang had yellowing disease and albinism (Figure 1B), and the Ruixue planted in Shouguang had yellowing disease, with withered leaf margins and the development of black spots on the leaves (Figure 1D).

The chlorophyll and carotenoid contents of Pingyang Tezao and Ruixue grown in Rizhao and Shouguang differed greatly (Table 2). Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents of Rizhao-grown Pingyang Tezao and Ruixue were significantly higher and chlorophyll a/b was significantly lower than those grown in Shouguang. Specifically, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents of Pingyang Tezao grown in Rizhao were 84.5%, 110.7%, 91.1%, and 61.3% higher than those grown in Shouguang, respectively, while chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids of Ruixue grown in Rizhao were 84.5, 104.9, 90.3, and 90.0% higher than those grown in Shouguang, indicating that the chlorophyll synthesis in Pingyang Tezao and Ruixue was blocked when cultivated in Shouguang soil.

Chloroplast structures in the leaves of Pingyang Tezao and Ruixue plants grown in Rizhao and Shouguang are shown in Figure 2. Chloroplast structures of Pingyang Tezao and Ruixue grown in Rizhao were complete and elliptical (Figures 2A, C), with clearly observable chloroplast envelopes and cell membranes (Figures 2A, C, black arrow). Mitochondria were intact, with complete membrane structures and clear cristae (Figures 2A, C, black arrow). In contrast, chloroplasts of Pingyang Tezao and Ruixue leaves from plants grown in Shouguang were structurally disordered, with distorted vesicle lamellae, severe separation of the plastid wall, dissolved cell membrane (Figures 2B, D, black arrow), and cavitated mitochondria. Ruixue chloroplasts were broken by the periplasm (Figure 2D, black arrow). Given that chloroplasts are the site of chlorophyll formation, disruption of their structure results in decreased chlorophyll content.

The levels of quality-determining components of tea plants in Rizhao and Shouguang are shown in Table 3. Polyphenols, amino acids, and soluble extracts of the two varieties cultivated in Rizhao were significantly higher than those cultivated in Shouguang. Polyphenols in the leaves of Pingyang Tezao and Ruixue grown in Rizhao were 89.6 and 74.8% higher than their equivalents grown in Shouguang, respectively; amino acids in the leaves were 22.3 and 62.3% higher in plants grown in Rizhao compared with Shouguang, indicating that Shouguang soil reduced the synthesis of these major components of tea. Soluble sugar and protein contents were both higher in Shouguang planting than in Rizhao (Table 3).

The elemental contents of tea leaves in the two plantations differed greatly, as shown in Table 4. N, P, K, Mn, Zn, and Cu in the leaves of the two varieties planted in Rizhao were significantly higher than in Shouguang. Pingyang Tezao and Ruixue plants grown in Rizhao had a leaf N content that was 35.6 and 59.1% higher, respectively, compared with plants grown in Shouguang; leaf P was 191.7 and 275.7% higher; leaf K was 33.5 and 30.3% higher; leaf Mn was 270.7 and 628.0% higher; leaf Zn was 138.7 and 303.2% higher; and leaf Cu was 379.3 and 303.2% higher. In contrast, Ca, Mg, and B levels in the leaves of the two varieties planted in Rizhao were significantly lower than in Shouguang. Pingyang Tezao and Ruixue plants grown in Rizhao had a leaf Ca content that was 82. 1 and 82.8% lower in Rizhao, respectively, compared with plants grown in Shouguang; leaf Mg content was 43.3 and 38.3% lower; and leaf B content was lower at 39.2 and 17.4% lower. S and Fe contents of the two varieties did not differ significantly between the two locations.

Chloroplasts deficient in N, sulfur (S), magnesium (Mg), iron (Fe), Mn, Cu, and Zn become chlorotic. This is because N and Mg comprise chlorophyll, Fe is a component of iron–sulfur protein in the chloroplast photosynthetic electron transfer system of chloroplasts, and Mn, Cu, and Zn are activators of certain enzymes in the chlorophyll formation process. All of these functions are closely related to the formation of chlorophyll or chloroplasts. In the absence of Mn, vesicles, the basic structural unit of chloroplasts, cannot form lamellae, and with Zn deficiency, the inner membrane system of chloroplasts is disrupted (12–14). The two varieties grown in Shouguang had serious damage to the chloroplast structure (Figure 2), distortion and breakage of the cystoid, and destruction of the chloroplast membrane and cell membrane, resulting in the reduction of chlorophyll content. It is likely that the yellowing and albinism of tea plants in high-pH areas were caused by the element deficiencies we observed. In addition, a high concentration of Ca damages chloroplast lamellae and decreases chlorophyll content (15); Ca may also be an important cause of the yellowing and whitening of tea plants in high-pH areas.

The properties of soils in Rizhao and Shouguang differed greatly, as shown in Table 5. The pH of the soil in the Rizhao tea area was 4.56, which was within the suitable range for tea plant growth (pH 4.5–5.5), and the pH of Shouguang soil was 6.84, which was outside of this range (considered high pH). Referring to soil nutrient grading standards of tea plantations (Table 1) (7), the total N content and SOM content of Shouguang soil reached the grade I standard, and the total N content and SOM content of Rizhao soil reached the grade II standard. The total N content of Shouguang soil was 1.83 times higher than that of Rizhao soil, and the SOM content in Shouguang was 2.23 times higher than in Rizhao soil. The effective Zn, effective Cu, and effective Mn in both Shouguang and Rizhao soils reached the grade I standard, while the effective Zn and effective Cu in Shouguang soil were significantly higher than those in Rizhao soil, and the effective Mn of Shouguang soil was significantly lower than in Rizhao soil.

According to the soil nutrient grading standards (Table 1) (7), the effective P of both Rizhao and Shouguang soils reached class I standard, the effective K of Shouguang soil reached class II standard, and the effective K of Rizhao soil reached class I standard. The content of effective P and effective K in Shouguang soil was significantly lower than that in Rizhao soil, resulting in significantly lower P and K in the leaves of both varieties grown in Shouguang compared with Rizhao (Table 4). P facilitates the formation of polyphenols in tea leaves, and P deficiency affects the conversion of sugars into polyphenols, which leads to the accumulation of sugars in leaves (16). As shown in Table 3, the soluble sugar contents of the two tea varieties grown in Shouguang were more than two times that of the tea grown in Rizhao. As shown in Tables 4, 5, the effective K levels in Rizhao soil and Rizhao-grown tea leaves are both higher than those in Shouguang soil and Shouguang-grown tea leaves. The K content in tea leaves is positively correlated with total amino acid levels, and K⁺ ions regulate stomatal movement and promote photosynthesis, thus facilitating the formation of sugars and polyphenols, which likely explains the high tea polyphenol and amino acid contents in Rizhao-grown tea leaves compared with those grown in Shouguang.

The elemental content of tea leaves before and after foliar spray fertilization on Pingyang Tezao and Ruixue planted in Shouguang is shown in Table 6. The elemental contents of N, P, K, S, Mn, Zn, and Cu of the leaves were significantly higher after fertilization compared with a non-fertilized control, while Ca, Mg, Fe, and B contents were not significantly different. Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents of Pingyang Tezao and Ruixue were significantly higher after fertilization than in the non-fertilized control plants (Table 7). After foliar spray fertilization, Pingyang Tezao and Ruixue plants had greener leaves and good growth, and the preventative effect on yellowing diseases and albinism was clear (Figure 3). This suggests that higher soil pH reduced the uptake of N, Zn, Cu, and Mn by tea plants, resulting in the destruction of chloroplast structure and decreased chlorophyll content, resulting in yellowing and albinism.