The Combined Use of Soil Conditioner and Foliar Sulfur Spray Successfully Prevents Dark Pericarp Disease Induced by Manganese Toxicity in Litchi

Abstract

Manganese toxicity is a major obstacle to agriculture in acid soils. Dark pericarp disease (DPD) is a newly spread physiological disorder induced by excess Mn in litchi, leading to undesirable fruit appearance and substantial economic loss. In this work, broadcast of alkaline soil conditioner in winter, followed by foliar sprays of ascorbic acid and sulfur solution at fruit development, was adopted to examine the effect of these combinations on DPD alleviation in a litchi orchard, with DPD morbidities of 70~85% in recent ten years. The combination of soil conditioner broadcast and foliar water spray was used as the control. At harvest, DPD incidence was significantly decreased by sulfur spray (3.3 ± 1.0%) and slightly reduced by ascorbic acid spray (10.7 ± 8.0%) compared to the control (12.9 ± 7.6%). Soil pH and available Mn were significantly increased and reduced by the soil conditioner broadcast. Sulfur spray significantly inhibited Mn uptake but enhanced the accumulation of Mg, Ca, sugars and cyanidin-3-rutinoside in the pericarp, leading to improved fruit pigmentation. Antioxidase activities were regulated to resist Mn stress by sulfur spray. The spray of ascorbic acid could not mitigate DPD as expected, probably due to the dose used. Conclusively, this study provides a practicable approach to mitigate Mn phytoavailability in acid soils.

1. Introduction

In acidic soils that account for one-half of the world’s potentially arable lands, manganese (Mn) stress is a limiting factor for plant growth [1]. Litchi (Litchi chinensis Sonn.) is a popular subtropical fruit with bright red fruit color and delicious and succulent flesh. Litchi is cultivated in more than 20 countries, and the plantation area and the yield of litchi in southern China account for 52% and 61% of the total plantation area and the total yield in the world, respectively [2]. Soil acidity in litchi orchards of South China was documented two decades ago [3,4] and further intensified in recent years [5].

Unfortunately, dark pericarp disease (DPD), a newly identified physiological disorder induced by Mn stress in litchi, has frequently broken out at the fruit swelling stage in South China in recent years [6,7]. Litchi fruits subject to DPD are characterized by uneven dark pericarp, which markedly differs from the postharvest browning in litchi [8].

Waterlogging promotes Mn bioavailability in soil and enhances Mn uptake by plants, thereby exacerbating Mn toxicity to plants [9,10,11]. Moreover, sunlight exposure aggravates Mn intoxication in plants as well [12]. As the Blue Book on Climate Change in China 2021 issued, the last twenty years have been the hottest period since the 20th century in China, with the mean annual temperature increasing by 0.26 °C per 10 years during 1951~2020, considerably higher than that of the world (0.15 °C per 10 years) [13]. Meanwhile, the average annual rainfall increased by 5.1 mm per 10 years during 1961–2020, with significantly decreased rain days but increased heavy rain days. In southwest China, the annual flood and heavy rain, especially from June to August, have been increasing in recent decades [14]. Therefore, soil acidity, abnormally heavy rain, and warmer and longer summers [6,15,16] are jointly responsible for the breakout of DPD in litchi in southern China.

DPD is frequently observed in more than twenty litchi cultivars in South China during 2022~2023, based on the surveys of litchi production executed by our research team. As a matter of fact, prior to the identification of DPD, this disorder had been treated as an infectious disease, and substantial bactericides had been used to prevent it. The litchi growers suffer from serious economic loss owing to the poor appearance of litchi fruit subject to DPD. Meanwhile, litchi fruit quality is impacted by the overuse of pesticides. Therefore, the prevention of DPD is an impending task in litchi production.

Liming is a useful method to decrease soil acidity and mitigate Mn stress to plants in acid soils [17,18]. Our previous work suggests that the formation of dark substances in the dark pericarp of DPD fruit might be catalyzed by peroxidase (POD) and polyphenol oxidase (PPO) [6]. Ascorbic acid is a potent inhibitor for POD and PPO in litchi fruit [19,20,21], and sulfur (S) depresses the activities of both these enzymes in plants as well [22,23]. The influence of S on plant growth is associated with its combination of amino acids such as cysteine and methionine, along with a range of defense compounds like glutathione and phytochelatins [24]. Consequently, S is widely utilized as a mineral nutrient and an antifungal agent in agriculture. The prominent effect of S on plant resistance to various stresses has been extensively reported [25,26,27,28]. However, the combined effect of liming or soil conditioner application and foliar toxinicides on Mn phytotoxicity is not clear yet to date.

Therefore, we speculate that DPD in litchi might be fundamentally prevented by liming in soil, as well as spraying ascorbic acid and sulfur. In this study, a field experiment was conducted to examine the effect of spraying ascorbic acid and sulfur on DPD in litchi, along with land application of an alkaline soil conditioner. We aim to put forward an effective approach to mitigate Mn toxicity in litchi.

2. Materials and Methods

2.1. Experimental Litchi Orchard and Tree Used

A field experiment was carried out in a plateaued litchi orchard (24°45′ N, 97°59′ E) in Yingjiang county of Yunnan province, southwest China, with an altitude of 1168 m. The experiment site belongs to the subtropical monsoon climate region, with a yearly temperature of 19.3 °C and sunshine of 2364.5 h. The annual precipitation is approximately 1552 mm, with a distinct rainy season from late May to late August. Soil in this orchard was a umbric ferralsol with low pH (4.54 ± 0.02) and abundant available Mn (657 ± 26 mg kg⁻¹).

The litchi cultivar used was Maguili, a typical late cultivar in China. The Maguili litchi in this orchard was grafted from another litchi cultivar, Huaizhi, in 2009, and started to bear fruit in 2011. Commonly, litchi fruit develops rapidly from late June to August, and matures from late August to mid-September in this orchard. Unfortunately, serious DPD appeared from July to August every year during 2012~2021 in this orchard. This symptom had been treated as a bacterial disease before 2022. The grower reported that the DPD incidence was within 70~85% in the last ten years, and it was approximately 75% based on our field observation in 2021 (Figure 1). No lime or soil conditioner was used in this orchard during the last ten years.

2.2. Soil Conditioner, Ascorbic Acid, Sulfur, S Adjuvant and Standard Materials

A soil conditioner supplied by a commercial factory in Guangdong province was used in this experiment. This soil conditioner consisted of dolomite and humus, containing 127.6 ± 3.7 g kg⁻¹ organic matter, 175.7 ± 5.6 g kg⁻¹ Ca and 26.7 ± 0.6 g kg⁻¹ Mg, with a pH of 12.3 ± 0.1.

The ascorbic acid used was a chemical pure agent (≥99.5%) bought from Shanghai Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. The sulfur (≥99.5%) was purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China), with the particle diameters of 665.7~810.9 nm as measured by granularity potentiometer (Zatasizer Nano ZS, Malvern, United Kingdom). The adjuvant for sulfur particle was a mixture containing 2% (w/w) sodium alginate, 10% (w/w) polyethylene pyrrolidone, 5% (w/w) NNO dispersant, and 0.1% (w/w) sodium dodecyl sulfonate, and prepared in the laboratory using chemical agents.

The standard substance of epicatechin was purchased from Macklin-Lab (Shanghai, China). The reference materials of quercetin-3-glucoside, malic acid, and α-ketoglutaric acid were bought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), and those of procyanidin A2, procyanidin B2, ferulic acid, rutin, kaempferol-3-glucoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside from Derick Biotechnology Co., Ltd. (Chengdu, China). The reference substances of sucrose and glucose were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), and those of quinic acid, fumaric acid and shikimic acid by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), and those of citric acid, ascorbic acid and acetic acid by Guangzhou Chemical Reagent Factory (Guangzhou, China). The reference substance of fructose was provided by Shanghai Boao Biotechnology Co., Ltd. (Shanghai, China), and that of galactose by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The standard material of oxalic acid was purchased from Tianjin Fuchen Chemical Reagent Co., Ltd. (Tianjin, China), and that of tartaric acid from Tianjin Kemi Ou Chemical Reagent Co., Ltd. (Tianjin, China).

2.3. Field Experiment Scheme

Twenty healthy trees with similar canopies (4 m in height and 5 m in diameter) were chosen and classified into four groups. Each group was assigned a foliar spray treatment with five replicates, and each tree was used as a replicate. The four foliar spray treatments included the control (spraying water), the ascorbic acid (1.0 mM), the sulfur solution (3.12 mM), and the adjuvant. Meanwhile, soil conditioner was evenly broadcast to the soils under the canopy of all the twenty litchi trees on 26 January 2022, with the use dose of 0.15 kg m⁻². Prior to the application of soil conditioner, soil samples with a depth of 0~50 cm were randomly collected from the soils under the canopy of the twenty trees. The first foliar spray was carried out on 27 May 2022, and the second and the third spray on 6 June and 26 July, respectively. In total, 75 L of solution were used for each spray of each treatment. All the trees in this orchard were managed as per traditional practice.

2.4. Fruit Harvest, Sample Collection and Preparation

The litchi fruit was harvested on 1 September 2022. All the fruits on each tree were picked and classified into both types, viz the normal fruit and the dark pericarp fruit (DPF). Both fruits were weighed separately, and the yields were recorded. The samples of both fruit types were separately gathered from each tree. All fruit samples were put into foam containers with ice packs and sealed. Simultaneously, soil samples with a depth of 0–50 cm were gathered with the same method again. All the samples were delivered to the laboratory soon by air.

The fruit samples were rapidly purged with tap water and made dry with paper tissue in the laboratory. The pericarp was peeled with a hand. Each pericarp sample was classified into both sections. The first section was put into liquid nitrogen rapidly while peeling. One-half of the first section was lyophilized, ground and then kept at −80 °C for the detection of phenolic compounds. The other half was preserved at −80 °C for evaluation of antioxidase activity and sugar components in the pericarp. The second section was oven-dried and ground for element analysis.

While peeling, the flesh was gathered and placed into liquid nitrogen immediately, ground into slurry at 4 °C, and then preserved at −80 °C for the measurement of sugar and organic acid components.

2.5. Quantification of Pericarp Element

Pericarp samples were digested with concentrated HNO₃ + HClO₄ (5:1, v/v) solution. The concentrations of Ca, Mg, Fe, and Mn in the digested solutions were detected by atomic absorption spectrophotometer, and those of S and Al were quantified by ICP-OES (Varian 710-ES, USA) as described by Lu [29]. Standard substance GBS 07603 (GSV-2) was used to assure the analysis quality.

2.6. Measurement of Antioxidase Activity in Pericarp

The activities of antioxidases, including superoxide dismutase (SOD), POD, PPO, and laccase, were assessed with the kits (Comin Biotechnology Co., Ltd., Suzhou, China) as the manufacturer’s guidance. The absorbance of SOD, POD, PPO, and laccase were detected by UV spectrophotometer (Yuanxi UV-5500, Shanghai, China) at 450, 525, 470, and 420 nm, respectively.

2.7. Detection of Phenolic Compounds in Pericarp

Phenolic compounds in fruit pericarp were extracted using the method described by Su et al. (2022) [6] and determined by HPLC (Agilent 1260 Infinity II, USA) equipped with a diode array detector (Agilent G7115A, USA). The mobile phase was comprised of 2% phosphoric acid (A) and acetonitrile (B) at 0.8 mL min⁻¹. The mobile procedure was allotted as: 0~5 min, 10% B; 5~10 min, 10~13% B; 10~20 min, 13~18% B; 20~25 min, 18% B; 25~30 min, 18~30% B; 30~31 min, 30~50% B, and 31~33 min, 50% B. The column temperature was set at 35 ℃, and the injection level was 20 μL.

2.8. Analysis of Sugar and Organic Acid Components in Pericarp and Flesh

Approximately 0.3 g of pericarp tissue and 2 mL of ultrapure water were mixed, then kept in boiled water bath for 8 min, followed by sonication of 20 min and centrifugation at 10,000× g for 10 min. The supernatant was leached using a C₁₈ cartridge (Waters Sep-Pak-1 cc, USA) and then filtered for the detection of sugar components. For the sugar components in litchi flesh, approximately 0.1 g of flesh slurry was weighed and then prepared the same as the pericarp sample but without the process of sonication. Sugar components in the filtrate were separated with a Cybele CCM column (Drogon, China) and detected using HPLC (Agilent 1260, USA) outfitted with a refractive index detector. The ultrapure water was used as the mobile at 0.5 mL min⁻¹. The column temperature remained at 80 °C, and the injection dose was 10 μL.

For the determination of organic acid components in the flesh, approximately 1 g of flesh slurry was mixed with 10 mL 0.2% (w/v) phosphate buffer solution. Then, the sample was ground at 4 °C for 1 min and centrifugated with 4000× g for 10 min. The supernatant was filtrated for detection by HPLC. The components of organic acids were separated by a C₁₈ column (Agilent Polaris C18-A 250 mm × 4.6 mm, USA) and then determined by HPLC (Agilent 1200LC, USA) coupled with a diode array detector at 210 nm. Then, 0.2% phosphate buffer was used as the mobile, at 0.4 mL min⁻¹. The column temperature was kept at 35 °C, and the input volume was 10 µL.

2.9. Determination of Soil Property

Soil pH was determined in a soil: water (1:2.5, w/v) suspension. Soil organic matter (OM) was quantified using the K₂Cr₂O₇ oxidation volumetric method. Soil available N was detected by alkali N-proliferation method. Soil available P was extracted using 0.025 M HCl + 0.03 M NH₄F and detected by the Mo-Sb colorimetric method. Soil was extracted with 0.1 M HCl for available Fe and Mn and with 1 M CH₃COONH₄ for exchangeable Ca and Mg. Then, the extracts were determined using an atomic absorption spectrophotometer (Hitachi ZA-3300, Japan), respectively. Soil available K was eluted by 1 M CH₃COONH₄, and analyzed with flame photometer. All the analysis methods for soil properties were depicted by Lu [29]. Reference substance GBW07458 (ASA-7) was used to control the quality of the analysis.

2.10. Data Analysis and Statistics

All the data were the means of at least three repeats calculated by Excel 2021 and expressed as mean ± STDEV. The morbidity of DPD was computed as the weight percent of the DPFs accounting for all the fruits in one plant as the previous method [6]. All the data of plant tissues were subjected to a two-factor analysis of variance (ANOVA) and then multiple comparisons with Duncan’s (p < 0.05) by SAS 9.2. The data of soil properties were performed one-factor ANOVA, with Duncan’s as well.

3. Results

3.1. The Morbidity of DPD in Litchi Fruit

The experimental trees yielded from 20.2 to 74.7 kg per plant. DPD incidence was significantly reduced by sulfur spray (3.3 ± 1.0%), whereas slightly inhibited by spraying ascorbic acid (10.7 ± 8.0%), compared to the control (12.9 ± 7.6%) (Figure 2). The adjuvant spray did not affect DPD morbidity (12.5 ± 8.1%). Specifically, the severity of DPD symptoms was markedly alleviated by sulfur spray, according to the field observation at harvest.

3.2. Pericarp Mn, Fe, Al, S, Ca, and Mg

The spray of ascorbic acid considerably decreased pericarp Mn, whereas it had no impact on pericarp Fe, Al, S, Ca, and Mg, compared to the control (

Table 1. The contents of Mn, Fe, Al, S, Ca, and Mg in litchi pericarp as influenced by treatment and fruit type.

Item	Mn (mg kg−1)	Fe (mg kg−1)	Al (mg kg−1)	S (g kg−1)	Ca (g kg−1)	Mg (g kg−1)
Treatment
Control	226.2 ± 70.4 A	34.1 ± 4.6 A	40.9 ± 6.6 a	0.83 ± 0.11 a	4.30 ± 1.03 ab	1.29 ± 0.27 B
Ascorbic acid	204.6 ± 37.8 AB	33.3 ± 6.7 A	40.2 ± 4.3 a	0.80 ± 0.06 a	4.86 ± 0.48 ab	1.42 ± 0.16 B
Sulfur	190.2 ± 34.3 B	28.2 ± 7.0 B	40.0 ± 6.2 a	0.80 ± 0.11 a	4.98 ± 0.63 a	1.63 ± 0.24 A
Adjuvant	213.3 ± 21.6 AB	34.0 ± 3.4 A	38.4 ± 5.0 a	0.81 ± 0.11 a	4.04 ± 0.99 b	1.34 ± 0.31 B
Fruit type
Normal fruit	186.8 ± 31.4 B	32.2 ± 5.1 a	39.6 ± 5.4 a	0.80 ± 0.09 a	4.33 ± 0.99 b	1.41 ± 0.27 a
DPF	230.4 ± 48.0 A	32.6 ± 7.0 a	40.2 ± 5.9 a	0.82 ± 0.10 a	4.47 ± 0.96 a	1.43 ± 0.29 a
Treatment × Fruit type	p < 0.05	p < 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05

Note: The lowercase or uppercase letters affiliated with the data in each column denote significance at 0.05 or 0.01 level, respectively.

). Pericarp Mn and Fe were significantly reduced, but pericarp Mg was significantly enhanced (p < 0.01), while pericarp Ca was insignificantly increased by sulfur spray. Sulfur spray did not affect pericarp Al and S. In contrast to the control, the spray of adjuvant slightly decreased pericarp Mn and had no influence on pericarp Fe, Al, S, Ca, and Mg. No significant variations were observed for pericarp Fe, Al, S, and Mg between the DPF and the normal fruit, with the exception of significantly higher pericarp Mn and Ca in the DPF (p < 0.05), supporting the previous result that DPD is induced by Mn toxicity in litchi pericarp [6]. Significant interaction was observed between treatment and fruit type for pericarp Fe and Al but not for pericarp S, Ca, and Mg.

3.3. Phenolic Compounds in Litchi Pericarp

Total nine phenolic compounds, including flavanols (epicatechin, proanthocyanin A2 and proanthocyanin B2), flavonols (rutin, quercetin-3-glucoside, kaempferol-3-glucoside), anthocyanidins (cyanidin-3-glucoside and cyanidin-3-rutinoside), and phenolic acid (ferulic acid) were detected in litchi pericarp (

Table 2. The contents of phenolic compounds in litchi pericarp as influenced by treatment and fruit type.

Item	Epicatechin (g kg−1)	Proanthocyanin A2 (g kg−1)	Proanthocyanin B2 (g kg−1)	Rutin (mg kg−1)	Ferulic Acid (mg kg−1)	Quercetin-3-Glucoside (mg kg−1)	Kaempferol-3-Glucoside (mg kg−1)	Cyanidin-3-Glucoside (mg kg−1)	Cyanidin-3-Rutinoside (mg kg−1)
Treatment
Control	6.5 ± 1.5 a	11.8 ± 0.5 ab	1.25 ± 0.24 a	907 ± 158 ab	76.9 ± 25.6 a	93.3 ± 25.7 a	235 ± 52 a	20.8 ± 5.1 a	343 ± 156 b
Ascorbic acid	7.8 ± 1.4 a	12.2 ± 0.6 a	1.20 ± 0.30 a	929 ± 89 ab	77.9 ± 28.9 a	86.2 ± 20.3 a	217 ± 57 a	21.4 ± 4.0 a	431 ± 140 ab
Sulfur	7.5 ± 1.1 a	12.1 ± 0.4 a	1.26 ± 0.22 a	1002 ± 79 a	87.9 ± 29.3 a	92.0 ± 15.2 a	210 ± 37 a	22.6 ± 2.2 a	511 ± 117 a
Adjuvant	6.5 ± 1.6 a	11.5 ± 0.9 b	1.04 ± 0.31 a	868 ± 80 b	67.7 ± 19.3 a	80.0 ± 13.0 a	191 ± 56 a	22.3 ± 4.8 a	400 ± 175 ab
Fruit type
Normal fruit	6.5 ± 1.5 a	11.7 ± 0.8 a	1.1 ± 0.3 a	915 ± 118 a	68.7 ± 22.1 a	78.1 ± 15.3 B	197 ± 50 b	22.8 ± 4.4 a	466 ± 183 a
DPF	7.2 ± 1.5 a	12.1 ± 0.5 a	1.2 ± 0.2 a	939 ± 115 a	86.1 ± 27.2 a	97.5 ± 18.3 A	229 ± 48 a	20.8 ± 3.0 a	384 ± 114 a
Treatment × Fruit type	p > 0.05	p > 0.05	p > 0.05	p > 0.05	p < 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05

Note: The lowercase or uppercase letters affiliated with the data in each column denote significance at 0.05 or 0.01 level, respectively.

). Proanthocyanin A2 was the primary phenolic component, followed by epicatechin and proanthocyanin B2. Cyanidin-3-glucoside and cyanidin-3-rutinoside were two anthocyanidins conferring a red color on litchi pericarp. Although no significant variations for pericarp epicatechin, proanthocyanin A2, proanthocyanin B2, ferulic acid, and cyanidin-3-glucoside concentrations were observed among treatments and fruit types, sulfur spray significantly boosted the accumulation of cyanidin-3-rutinoside (p < 0.05), effectively promoting fruit pigmentation in litchi. Meanwhile, flavonols, rutin in particular, are co-pigments that can enhance the color manifestation of anthocyanidins [30,31]. Therefore, the considerably increased rutin by sulfur spray was conducive to promoting the color display of cyanidin-3-rutinoside in litchi pericarp as well. Although similar levels of quercetin-3-glucoside and kaempferol-3-glucoside were determined for various treatments, significantly higher levels of these phenolic compounds were detected in the DPF compared to the normal fruit (p < 0.05).

3.4. Activities of Antioxidant Enzymes

The symptoms of Mn phytotoxicity differ with plant species. However, dark speckle is the typical symptom of Mn toxicity, which is due to the buildup of oxidized Mn [32,33], or oxidized phenols [34,35], and or the combination of both oxidized Mn and phenols [36]. The regulation of antioxidase activity is a key mechanism for plants to resist Mn stress. SOD can eliminate superoxide anion radical and act as a crucial enzyme in the frontier of plant tolerance to oxidative injury linked with excess Mn [37]. POD, PPO, and lactase can catalyze Mn oxidation and the oxidation/polymerization of phenols to form dark materials in plants under Mn stress [38,39,40]. As shown in Figure 3a, the sprays of both ascorbic acid and sulfur significantly enhanced pericarp SOD activity (p < 0.05), while the adjuvant spray insignificantly promoted it, as compared to the control. Meanwhile, significantly higher pericarp SOD activity was observed in the normal fruit than in the DPF (p < 0.01). The significantly boosted SOD activities by the sprays of ascorbic and sulfur reflected the contribution of SOD to resist oxidative stress caused by excess Mn in the pericarp. In contrast to the control, the three sprays, sulfur spray in particular, significantly promoted pericarp PPO activity (p < 0.05) (Figure 3b). Generally, higher pericarp PPO activity was observed in the DPF than in the normal fruit, with the exception of an opposite trend for both types of fruits treated with the adjuvant. Spraying ascorbic acid significantly promoted pericarp POD activity, but sulfur particle spray significantly inhibited it (p < 0.05), and the adjuvant spray insignificantly depressed it (Figure 3c). The three sprays did not affect POD activities in both types of fruits. The three sprays significantly enhanced pericarp laccase activities (p < 0.05), except for an insignificant increase by the adjuvant spray, as compared to the control (Figure 3d). Laccase activities in the DPF were markedly lower than those in the normal fruit (p < 0.01). The above suggests that SOD contributed to Mn tolerance in litchi and POD, superior to PPO and laccase, was involved in Mn resistance as well. The promoted activity of SOD and depressed activity of POD partially explain the significantly lower DPD morbidity of the sulfur-sprayed litchi fruit.

3.5. Sugar Components in Litchi Pericarp

Anthocyanidin glycosylation is essential for anthocyanin synthesis, and sugars act as the glycosyl donor. Sucrose, glucose, galactose, and fructose were detectable sugar components in litchi pericarp (

Table 3. The contents of sugar components in litchi pericarp as influenced by treatment and fruit type.

Item	Sucrose (g kg−1)	Glucose (g kg−1)	Galactose (g kg−1)	Fructose (g kg−1)
Treatment
Control	3.92 ± 1.52 b	3.91 ± 1.00 ab	8.76 ± 1.08 a	4.22 ± 1.83 a
Ascorbic acid	4.34 ± 0.88 b	3.08 ± 1.07 b	8.09 ± 0.87 a	4.33 ± 1.40 a
Sulfur	5.53 ± 0.97 a	3.89 ± 0.81 ab	8.15 ± 0.79 a	5.38 ± 1.08 a
Adjuvant	4.85 ± 1.50 ab	4.22 ± 0.99 a	8.78 ± 0.79 a	4.93 ± 1.65 a
Fruit type
Normal fruit	4.88 ± 1.36 a	3.86 ± 1.16 a	8.35 ± 0.81 a	5.02 ± 1.62 a
DPF	4.43 ± 1.34 a	3.69 ± 0.89 a	8.54 ± 1.02 a	4.41 ± 1.41 a
Treatment × Fruit type	p > 0.05	p > 0.05	p > 0.05	p > 0.05

Note: The lowercase letters affiliated with data in each column denote significance at 0.05 level.

). The spray of ascorbic acid reduced pericarp glucose, while the sulfur spray did not affect it. The three sprays, sulfur spray in particular, enhanced the accumulation of sucrose and fructose and provided more glycosyl donors for anthocyanidin glycosylation, thereby facilitating the synthesis/accumulation of anthocyanin. Galactose is a major component of non-cellulosic neutral sugar, hemicellulose and pectin in plant cell walls; thus, the slightly elevated galactose might be unrelated to anthocyanin synthesis in the DPF.

3.6. Sugar and Organic Acid Components in Litchi Pulp

To evaluate the effect of the three sprays on fruit quality, sugar and organic acid components in litchi pulp were examined. Sucrose, glucose, galactose, and fructose were detectable in litchi pulp as well (

Table 4. The contents of sugar components in litchi pulp as influenced by treatment and fruit type.

Item	Sucrose (g kg−1)	Glucose (g kg−1)	Galactose (g kg−1)	Fructose (g kg−1)
Treatment
Control	31.7 ± 7.5 a	40.6 ± 4.0 a	2.6 ± 0.4 b	43.0 ± 4.2 a
Ascorbic acid	29.7 ± 7.1 a	37.8 ± 3.6 a	3.5 ± 0.8 a	40.0 ± 3.8 a
Sulfur	29.7 ± 7.9 a	37.2 ± 2.0 a	3.0 ± 0.4 ab	40.1 ± 2.0 a
Adjuvant	31.3 ± 9.0 a	40.5 ± 5.0 a	2.6 ± 0.5 b	43.0 ± 4.7 a
Fruit type
Normal fruit	29.3 ± 6.0 a	39.4 ± 4.1 a	2.8 ± 0.5 a	42.1 ± 4.0 a
DPF	31.9 ± 9.0 a	38.7 ± 3.9 a	3.0 ± 0.8 a	41.0 ± 4.0 a
Treatment × Fruit type	p > 0.05	p > 0.05	p > 0.05	p > 0.05

Note: The lowercase letters affiliated with data in each column denote significance at 0.05 level.

). Generally, the sprays of ascorbic acid, sulfur, and adjuvant did not affect sugar components, with the exception of increased galactose levels by the sprays of ascorbic acid and sulfur, as compared to the control. The four sugar components stayed at similar levels in both fruit types.

Ten organic acids were determined, and tartaric acid, malic acid, and quinic acid were the top three organic acids in litchi pulp (

Table 5. The contents of organic acids in litchi pulp as influenced by treatment and fruit type.

Item	Quinic Acid (g kg−1)	Malic Acid (g kg−1)	Tartaric Acid (g kg−1)	Oxalic Acid (mg kg−1)	Shikimic Acid (mg kg−1)	Ketoglutaric Acid (mg kg−1)	Citric Acid (mg kg−1)	Fumaric Acid (mg kg−1)	Ascorbic Acid (mg kg−1)	Acetic Acid (mg kg−1)
Treatment
Control	2.80 ± 0.47 a	3.57 ± 0.92 b	3.81 ± 0.93 a	286.9 ± 47.7 B	155.6 ± 37.1 a	96.0 ± 14.1 B	293.9 ± 37.1 a	13.3 ± 3.6 a	556.1 ± 213.6 a	729.4 ± 265.8 b
Ascorbic acid	2.83 ± 0.54 a	2.73 ± 0.53 b	3.27 ± 0.52 a	362.4 ± 49.6 A	145.1 ± 40.5 ab	88.7 ± 14.0 B	277.0 ± 28.3 ab	11.5 ± 4.5 ab	608.2 ± 92.8 a	892.0 ± 187.2 ab
Sulfur	2.70 ± 0.50 a	3.54 ± 1.21 b	3.46 ± 0.60 a	340.6 ± 45.5 A	135.2 ± 21.7 ab	88.1 ± 20.1 B	269.0 ± 30.4 ab	13.3 ± 3.2 a	531.5 ± 99.8 a	747.6 ± 99.5 b
Adjuvant	2.66 ± 0.26 a	4.74 ± 0.90 a	3.59 ± 0.55 a	349.1 ± 19.7 A	121.9 ± 16.9 b	131.8 ± 15.4 A	260.4 ± 22.6 b	9.6 ± 2.6 b	555.0 ± 119.4 a	977.4 ± 176.9 a
Fruit type
Normal fruit	2.76 ± 0.41 a	3.49 ± 1.31 a	3.55 ± 0.72 a	338.9 ± 52.9 a	142.3 ± 33.6 a	100.7 ± 22.9 a	272.0 ± 31.7 a	11.9 ± 3.8 a	566.0 ± 146.1 a	826.7 ± 219.4 a
DPF	2.73 ± 0.48 a	3.85 ± 0.95 a	3.51 ± 0.64 a	330.6 ± 48.0 a	136.6 ± 31.1 a	101.6 ± 25.4 a	277.1 ± 31.2 a	11.8 ± 3.7 a	559.3 ± 132.2 a	846.5 ± 208.6 a
Treatment × Fruit type	p > 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05	p > 0.05

Note: The lowercase or uppercase letters affiliated with data in each column denote significance at 0.05 or 0.01 level, respectively.

). Although significant differences were observed for most of the organic acids except for quinic acid, tartaric acid, and ascorbic acid among treatments, these differences did not show an obvious trend among the three sprays. Tartaric acid is the greatest contributor to the sourness of litchi flesh [41], indicating the taste of litchi pulp associated with organic acids might not be affected by the three sprays. Additionally, there were no significant variations in organic acids between the normal fruit and the DPF.

3.7. Soil Properties

The application of an alkaline soil conditioner had a vital effect on soil attributes, as shown in

Table 6. Variations of soil properties as influenced by application of soil conditioner.

Soil Attribute	Before the Use of Soil Conditioner	After the Use of Soil Conditioner (at Fruit Harvest)
pH	4.54 ± 0.02 B	5.37 ± 0.10 A
Organic matter (g kg−1)	18.4 ± 0.1 B	28.5 ± 0.4 A
Alkali-hydrolyzed N (mg kg−1)	207 ± 1 A	156 ± 7 B
Available P (mg kg−1)	17.3 ± 0.4 a	17.9 ± 0.7 a
Available K (mg kg−1)	622 ± 23 a	585 ± 53 a
Available Fe (mg kg−1)	15.0 ± 1.0 a	13.7 ± 3.7 a
Available Mn (mg kg−1)	657 ± 26 A	150 ± 24 B
Available Ca (mg kg−1)	1583 ± 100 B	2665 ± 79 A

Note: The lowercase or uppercase letters affiliated with data in each row denote significance at 0.05 or 0.01 level, respectively.

. Prior to the experiment, soil pH was low to 4.54 ± 0.02 and significantly increased to 5.37 ± 0.10 when the litchi was harvested (p < 0.01). Soil organic matter, available Ca and Mg were significantly increased by the amendment of the soil conditioner due to the fact that this soil conditioner contained organic matter, Ca and Mg (p < 0.01). Moreover, the increased soil pH was conducive to promoting soil organic matter in acid soil as well [42]. Soil alkali-hydrolyzed N was significantly reduced by the soil conditioner broadcast owing to the increased ammonia loss caused by the alkalinity of the soil conditioner. Specifically, the initial soil available Mn was 657 ± 26 mg kg⁻¹ before the use of soil conditioner and markedly reduced to 150 ± 24 mg kg⁻¹ at fruit harvest (p < 0.01).

4. Discussion

Soil available Mn of 63 litchi orchards in Guangdong, southern China, varies in the range of 0.8~139.3 mg kg⁻¹, with a mean of 24.3 mg kg⁻¹ [43]. In this work, soil available Mn in the experimental orchard was extraordinarily high (657 ± 26 mg kg⁻¹), well explaining the successive and serious DPD in this orchard every year during the last ten years. Additionally, according to the weather report issued by the government of Yingjiang county (https://www.tianqi24.com/yingjiang/history.html, accessed on 25 August 2023), the rainy days between 1 May and 31 August from 2011 to 2022 were 115, 104, 113, 104, 101, 106, 101, 117, 98, 111, 109, and 97 d in Yingjiang county (the amount of rainfall was lacking), when the litchi fruit in this orchard was swelling. Therefore, the substantial rainfall further increased the soil available Mn during the litchi fruit growth, being another contributor to the continuous and serious DPD in this orchard. Totally, the combination of soil conditioner broadcast and foliar sulfur spray efficiently alleviated DPD in this study, which could be ascribed to the three processes.

4.1. The Role of Soil Conditioner

Land application of alkaline soil conditioner substantially decreased the availability of soil Mn (Table 6), due to the increased soil pH [6,18]. It has been demonstrated that prior liming in soil leads to lower available Mn and retards the reduction of Mn oxides during succedent waterlogging. Moreover, after drainage, available Mn²⁺ is rapidly re-oxidized in limed soil [44]. In contrast to the previous high DPD morbidity (70~85%) during the last ten years, the application of an alkaline soil conditioner might be the primary contributor to the markedly reduced DPD in 2022 in this study.

4.2. The Effect of Sulfur Spray

Foliar sulfur spray significantly depressed Mn accumulation in the litchi pericarp compared to the control (Table 1). The inhibited Mn uptake and upward translocation by sulfur addition, regardless of elemental sulfur, sulfur particles or S-containing compounds, have been observed in several plants grown under Mn-stress conditions as well [45,46,47]. However, the genetic and molecular mechanisms of this effect are still not well explained to date. S is taken up by plant roots as sulfate via sulfate transporters [48,49], but the oxidation of sulfur particles in plant tissue is not revealed yet. Therefore, the physiological and biochemical pathways that sulfur regulates Mn accumulation need to be further investigated. Meanwhile, pericarp Mg and Ca were increased by sulfur spray, which facilitated the accumulation of sugars, thereby promoting the supply of glycosyl donors for anthocyanin synthesis. On the other hand, the higher pericarp Mg and Ca can enhance the synthesis of anthocyanidin via up-regulating gene expression and promoting activities of enzymes related to anthocyanidin synthesis in litchi [50], leading to improved fruit pigmentation in the present study.

S is an ingredient of a range of defense components, such as glutathione, phytochelatins, glucosinolates, and vitamins in plants [51,52]. S plays a crucial role in Mn tolerance by counteracting oxidative injury and enhancing the antioxidant defense system [25]. The significantly increased SOD activity and the decreased POD activity suggest that the strongly regulated antioxidant system also contributed to DPD alleviation in this work. In addition, moderate S application can mitigate Mn toxicity by segregating excess Mn into vacuoles and chelating metals within vacuoles via up-regulating genes involved in glutathione and phytochelatin synthesis [46,53].

4.3. The Influence of Ascorbic Acid Spray

It is documented that external supply of moderate ascorbic acid (≤50 mg L⁻¹) mediates Mn toxicity in V. natans via antioxidant defence system, cytomembrane stability, soluble material, and photosynthetic pigment, but high dose of ascorbic acid (≥100 mg L⁻¹) exacerbates Mn toxicity, although the pathway by which excess ascorbic acid inversely acts as a stress factor remains vague [54]. The extra addition of ascorbic acid at 1 mM alleviates Mn stress in hydroponic non-heading Chinese cabbage [55], and foliar spray of 5–10 μM ascorbic acid mitigates Mn toxicity in cowpea [56]. Further, transgenic technology has been adopted to increase foliar ascorbate levels as a strategy to adapt to multiple abiotic stresses, including Mn toxicity in rice [57]. Therefore, the above investigations suggest that the proper utilization of ascorbic acid is a promising approach to alleviating Mn stress in plants. We suppose that the insignificantly reduced pericarp Mn and DPD morbidity by ascorbic acid spray might be linked with its use dose in the present study. However, the optimum dose of ascorbic acid for DPD prevention in litchi is required to be further examined in field conditions.

4.4. The Impact of Adjuvant Spray

Although the adjuvant spray had an influence on the accumulation of some mineral elements, phenolic compounds and sugar components, as well as the activities of some antioxidases in litchi pericarp, it did not affect the occurrence of DPD as supported by the similar DPD morbidity with the control in the present study (Figure 2). It highly suggests that the adjuvant used in this work was reasonable and did not alter the role of sulfur spray.

The current study demonstrates that the joint use of soil conditioner and foliar toxinicide effectively alleviated Mn toxicity in litchi. In fact, mitigation of Mn phytotoxicity can be undertaken from three strategies: (1) reduction of soil available Mn by liming or applying soil conditioner; (2) depression of Mn uptake by plant roots and concomitant upward translocation from root to shoot (canopy) via the use of antagonists for Mn; (3) screening of suitable foliar toxinicides with proper doses for a specific plant species. However, the integrated approaches need to be examined in field conditions.

5. Conclusions

Field experiment demonstrates that the broadcast of an alkaline soil conditioner before flower differentiation and following foliar sulfur spray during fruit development efficiently prevents the occurrence of DPD in litchi through the reduction of soil acidity and Mn bioavailability, the inhibition of Mn accumulation, and the regulation of antioxidase activity in the pericarp. The foliar sulfur spray does not impact fruit quality and improves fruit pigmentation. We conclude that the combination of soil conditioner broadcast and foliar sulfur spray is a promising approach to control DPD in litchi, even when subject to continuous and severe DPD.