Effects of Soil Aeration and Fertilization Practices on Alleviating Iron Deﬁciency Chlorosis in “Huangguan” Pears Grafted Onto Quince a in Calcareous Soils

horticulturae

Article Effects of Soil Aeration and Fertilization Practices on Alleviating Iron Deﬁciency Chlorosis in “Huangguan” Pears Grafted onto Quince A in Calcareous Soils

Yanyan Zhao 1,2, Haigang Li 1, Mingde Sun 2, Zhenxu Liang 2, Futong Yu 3, Fei Li 1,* and Songzhong Liu 2,*

1 Inner Mongolia Key Laboratory of Soil Quality and Nutrient Resource, College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, Hohhot 010018, China; [email protected] (Y.Z.); [email protected] (H.L.) 2 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Beijing Engineering Research Center for Deciduous Fruit Trees, Beijing Academy of Forestry and Pomology Sciences, Beijing 100093, China; [email protected] (M.S.); [email protected] (Z.L.) 3 College of Agricultural Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China; [email protected] * Correspondence: author: [email protected] (F.L.); [email protected] (S.L.)

Abstract: In North China, the high-quality pear cultivar “Huangguan” (Pyrus bretschneideri Rehd. cv), which is grafted onto dwarf quince A (Cydonia oblonga Mill. cv) rootstock and grown in calcareous soil, experiences severe iron (Fe) deficiency; this deficiency greatly constrains tree growth as well as fruit yield and quality. Therefore, we evaluated the effects of six practices for alleviating chlorosis caused by Fe deficiency in “Huangguan” grafted onto quince A (HG-QA). The practices included ridging Citation: Zhao, Y.; Li, H.; Sun, M.; with landscape fabric mulching as a control, flattening with landscape fabric mulching (FM), ridging Liang, Z.; Yu, F.; Li, F.; Liu, S. Effects without landscape fabric mulching (R), flattening without landscape fabric mulching (F), Fe fertilizer of Soil Aeration and Fertilization application in soil (SFe), foliar Fe application (FFe), and manure application (M). The results showed Practices on Alleviating Iron that the leaf Fe concentration increased by 356% under FFe, compared to that under the control, but Deficiency Chlorosis in “Huangguan” the practice failed to alleviate Fe deficiency chlorosis. In contrast, an increase in leaf Fe concentration Pears Grafted onto Quince A in Calcareous Soils. Horticulturae 2021, 7, and chlorosis alleviation were observed under F. F alleviated chlorosis mainly by increasing the root 172. https://doi.org/10.3390/ ferric-chelate reductase activity. These results indicate that Fe uptake and utilization in leaves are horticulturae7070172 independent biochemical processes and soil aeration improvement have positive effect on increasing Fe uptake. M improved both the soil active Fe concentration and leaf Fe utilization. Thus, manure Academic Editor: Esmaeil Fallahi application should be the first choice for alleviating Fe deficiency chlorosis in HG-QA grown in calcareous soils. Combining manure application with other practices that increase Fe uptake would Received: 1 June 2021 likely be an effective way to address the problem of Fe deficiency chlorosis. Accepted: 24 June 2021 Published: 2 July 2021 Keywords: pears; Fe deficiency; fertilization; chlorosis alleviation; calcareous soil

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Iron (Fe) is a necessary micronutrient for plant growth and development and plays an important role in intracellular metabolism [1,2], such as electron transfer and is required for many enzymes to complete biological functions. Fe deficiency chlorosis is a worldwide nutritional disorder in crop plants as well as fruit trees. It reduces or even arrests the Copyright: © 2021 by the authors. growth of fruit trees and consequently decreases fruit yield and quality. Licensee MDPI, Basel, Switzerland. Fe deficiency chlorosis often occurs when plants grow in calcareous soils, even though This article is an open access article the total Fe concentration in such soils is high [3]. A previous study showed that this distributed under the terms and conditions of the Creative Commons occurred due to the high pH and high bicarbonate concentration in the soil, which reduced Attribution (CC BY) license (https:// Fe acquisition in plants by decreasing Fe bioavailability in soil and inducing Fe precipitation creativecommons.org/licenses/by/ in the apoplast of root cells, respectively [4,5]. Moreover, inactive Fe tends to accumulate 4.0/). in chlorotic leaves in calcareous soils [6–8]. This indicates that Fe precipitation also occurs

Horticulturae 2021, 7, 172. https://doi.org/10.3390/horticulturae7070172 https://www.mdpi.com/journal/horticulturae Horticulturae 2021, 7, 172 2 of 14

in the leaf veins and apoplast as a result of high apoplast pH or low leaf ferric-chelate reductase (FCR) activity [9]. China has been the world’s largest pear producer since 1977 [10]. Dwarf culture is a new and highly efficient intensive cultivation mode used in pear production because of its high yields, labor savings, and simple management practices, and it is being widely adopted in the EU and USA [11]. However, quince, one of the popular dwarf rootstocks, has low Fe utilization efficiency, which often leads to Fe deficiency in the grafted pear trees [12]. In North China, pears grafted onto quince show severe interveinal chlorosis in younger leaves when grown in calcareous soil, which greatly restricts the expansion of dwarf pear culture in this region. To alleviate Fe deficiency chlorosis in fruit trees, several direct Fe replenishment strategies have been developed, such as fertilizer application to soil [13,14] and foliar spraying [15]. However, the efficiency of these strategies is variable and depends on the experimental conditions and plant species [15–17]. Foliar sprays of Fe(III)-DTPA and FeSO4 temporarily increase the Fe level in pear leaves [18]. The application of Fe-EDDHA chelate increases Fe uptake by roots and its distribution to leaves in lemon trees and alleviates the chlorosis [13]. Moreover, these chelates are expensive and inevitably increase production costs [18]. Many studies have also shown that some practices can indirectly promote Fe uptake by plants [19,20]. Conventional tillage (soil plowing at 20 cm) significantly increases Fe accumulation in peanuts by 51.2% compared with that under no tillage [21]. Short, high- frequency irrigation periods alleviate leaf chlorosis in apple and pear trees in calcareous soils with a caliche layer [22]. Ridging with mulching is a routine practice in fruit cultivation [20,23,24]. It is presumed possible to change soil Fe bioavailability by modifying soil aeration. Farmers often apply manure to build soil fertility in fruit orchards. Application of manure changes most issues such as texture, microorganisms, and organic matter content that effects Fe uptakes. Meanwhile, many micronutrients contained in the manure, including Fe, are input into the soil at the same time. In some studies, the soil bioavailable Fe content and the Fe concentration in pear leaves increased after manure application [19,25]. These low-cost practices may be optimal ways to alleviate chlorosis in fruit trees grown in calcareous soils. In this study, we compared the effects of six practices on alleviating Fe deficiency chlorosis in pear, including flattening with mulching, ridging without mulching, flattening without mulching, Fe fertilizer application in soil, foliar Fe application, and manure application (ridging with mulching was used as a control), with the goal of identifying a low-cost, high-efficiency practice for dwarf pear cultivation in North China.

2. Materials and Methods 2.1. Plant Materials and Experimental Conditions This study was conducted in Beijing, North China (40◦1107.0000 N, 116◦29042.1700 E), at an altitude of 40 m above sea level. This area experiences a continental monsoon climate. The annual precipitation and average temperature are 550 mm and 10 ◦C, respectively. Most of the annual precipitation occurs during a few large rainfall events from July to September. The soil in the experimental orchard was a silt loam with clay, silt, and sand proportions of 5.40%, 64.7%, and 29.9%, respectively. Three-year-old trees of the pear cultivar “Huangguan” (Pyrus bretschneideri Rehd. cv) grafted onto quince A (Cydonia oblonga Mill. cv) with Hardy as the interstock (abbreviated as HG-QA) were used in this study. The trees were trimmed to slender spindles and grown at high density (spacing of 1.0 m within rows and 3.5 m between rows, 2857 trees ha−1). To ensure high quality and quantity in the pears, ridging with landscape fabric mulching was implemented throughout the whole orchard before the treatments were established. The ridges along the tree rows were 30 cm high and 140 cm wide. Under these conditions, HG-QA leaves exhibited obvious interveinal chlorosis (Figure1A). A foliar Fe fertilizer was Horticulturae 2021, 7, x FOR PEER REVIEW 3 of 14

established. The ridges along the tree rows were 30 cm high and 140 cm wide. Under these Horticulturae 2021, 7, x FOR PEER REVIEW 3 of 14 conditions, HG-QA leaves exhibited obvious interveinal chlorosis (Figure 1A). A foliar Fe fertilizer was sprayed onto the chlorotic leaves, and the leaves turned partially green, which confirmed that the chlorosis in these leaves was caused by Fe deficiency (Figure 1B). Horticulturae 2021, 7, 172 established. The ridges along the tree rows were 30 cm high and 140 cm wide. Under3 of 14 these conditions, HG-QA leaves exhibited obvious interveinal chlorosis (Figure 1A). A foliar Fe fertilizer was sprayed onto the chlorotic leaves, and the leaves turned partially green, which confirmed that the chlorosis in these leaves was caused by Fe deficiency (Figure sprayed onto the chlorotic leaves, and the leaves turned partially green, which conﬁrmed 1B).that the chlorosis in these leaves was caused by Fe deﬁciency (Figure1B).

Figure 1. Leaves’ color of “Huangguan” pear grafted onto quince A grown in pear research base of Beijing with calcareous soil (A) and the green dots in pear leaves caused by a foliar Fe application (B).

2.2. Experimental Implementation Seven experimental practices were implemented in mid-April 2019 (Figure 2): (1) Figure 1. Leaves’ color of “Huangguan” pear grafted onto quince A grown in pear research base of Beijing with calcareous soil (A) and Figure 1. Leaves’ color of “Huangguan”ridging with pear landscape grafted onto fabric quince mulching A grown (con introl), pear (2)research flattening base ofwith Beijing landscape with calcar- fabric the green dots in pear leaves caused by a foliar Fe application (B). eous soil (A) and the green dotsmulching in pear leaves(FM), caused(3) ridging by a withoutfoliar Fe landscapeapplication fabric(B). mulching (R), (4) flattening without landscape2.2. Experimental fabric mulching Implementation (F), (5) manure applied to the control conditions (M), (6) soil 2.2.Fe Experimental fertilizer applied Implementation to the control conditions (SFe), and (7) foliar Fe applied to the control Seven experimental practices were implemented in mid-April 2019 (Figure2): (1) ridg- conditionsingSeven with landscapeexperimental (FFe). The fabric manure practices mulching and (control),Fewere fertilizer implem (2) flatteningapplicationented in with mid-Aprilpractices landscape were 2019 fabric as (Figure mulchingfollows: 2): for (1) ridgingthe(FM), M treatment,with (3) ridging landscape two without ditches fabric landscape (40 mulching cm fabricdeep, mulching40(con cmtrol), long, (R), (2) and (4) flattening flattening30 cm wide) with without along landscape landscape the rows fabric on mulchingbothfabric sides mulching (FM), of trees (3) (F), ridging(30 (5) cm manure away without appliedfrom landscape the to tr theee) control werefabric dug, conditions mulching and then (M), (R), sheep (6)(4) soil flattening manure Fe fertilizer (0.01% without landscapeFe,applied 40% organic tofabric thecontrol mulching carbon, conditions pH (F), 8.14) (5) (SFe), manuremixed and with (7)applied foliar soil Fe(sheepto appliedthe controlmanure-soil to the conditions control ratio conditions of (M), 1:3) (6) was soil placed into each ditch; for the SFe treatment, two ditches (10 cm deep, 40 cm long, and 30 Fe fertilizer(FFe). The applied manure to and the Fe control fertilizer conditions application (SFe), practices and were(7) foliar as follows: Fe applied for the to M the treat- control cmment, wide) two were ditches dug (40 in cm the deep, same 40 location cm long, as and described 30 cm wide) previously, along the an rowsd 1.0 on g both Fe as sides polysac- of conditions (FFe). The manure and Fe fertilizer application practices were as follows: for charidetrees (30 chelate cm away Fe fromsolution the tree)(pH were5.5, 0.5 dug, g Fe and L− then1) was sheep poured manure into (0.01% each ditch Fe, 40% every organic fifteen the M treatment, two ditches (40 cm deep, 40 cm long, and 30 cm wide) along the rows on days;carbon, for pHthe 8.14)FFe treatment, mixed with 25 soil mg (sheep Fe L−1 manure-soilmannitol chelate ratio ofFe 1:3) was was sprayed placed on into both each sides bothofditch; the sides leaves for of the trees every SFe (30 treatment, ten cm days. away two from ditches the (10 tree) cm deep,were 40dug, cm and long, then and sheep 30 cm wide)manure were (0.01% Fe, dug40%There in organic the were same carbon, location5 replicates pH as described 8.14)of each mixed previously,practice, with wi andsoilth 1.01(sheep tree g Fe per asmanure-soil polysaccharidereplicate. In ratio mid-July, chelate of 1:3) Fe soil was placedsolution into (pHeach 5.5, ditch; 0.5 g for Fe Lthe−1 )SFe was treatment, poured into two each ditches ditch every (10 cm fifteen deep, days; 40 cm for long, the FFe and 30 and plant samples were− collected. cm treatment,wide) were 25 mgdug Fe in L the1 mannitol same location chelate Feas wasdescribed sprayed previously, on both sides an ofd the1.0 leavesg Fe as every polysac- charideten days. chelate Fe solution (pH 5.5, 0.5 g Fe L−1) was poured into each ditch every fifteen days; for the FFe treatment, 25 mg Fe L−1 mannitol chelate Fe was sprayed on both sides of the leaves every ten days. There were 5 replicates of each practice, with 1 tree per replicate. In mid-July, soil and plant samples were collected.

Figure 2. Schematic diagram of the practices. Control: ridging with landscape fabric mulching and without other treatments; FM: ﬂattening with landscape fabric mulching, R: ridging without landscape fabric mulching; F: ﬂattening without landscape fabric mulching; M: Manure application; SFe: Fe application in soil; FFe: Foliar Fe application.

There were 5 replicates of each practice, with 1 tree per replicate. In mid-July, soil and plant samples were collected.

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2.3. Leaf Soil Plant Analysis Development (SPAD) Value A SPAD−502 (Minolta, Osaka, Japan) was used to determine the SPAD of fully expanded leaves (the fourth leaves from the tips of the new shoots). Then, the measured leaves were harvested and used for the Fe concentration determination.

2.4. Rhizosphere pH, Soil Active Fe Concentration, and Soil Moisture A gasoline-powered soil sampler was used to sample soils near the root zone (unfertil- ized area) at a depth of 0−40 cm. After removing the loosely adhering soil around the roots, the tightly adhering soils were collected as the rhizosphere soil. Aliquots of rhizosphere soil were used for pH measurements (water:soil ratio of 1:1) with a pH meter (FE20K PLUS PH, Mettler-Toledo, Switzerland). Soil active Fe measurements were performed by extraction with DTPA and determination by plasma emission spectrometry (ICP-OES, OPTIMA 3300 DV, Perkins-Elmer Inc., Waltham, Massachusetts, USA) at 248.3 nm band and with the error not to exceed 1% deﬂection on the absorbance scale [26]. The rest of the soil in each sample was weighed immediately and then dried to a constant weight.

Soil moisture (%) = (fresh weight − dry weight)/dry weight × 100% (1)

2.5. Root FCR Activity Fresh root samples were used to determine the FCR activity according to the modiﬁed procedure of Zheng et al. [27]. The capillary roots were gently cut off and immediately washed with deionized water and dipped in the reaction solution (0.5 mM CaSO4, 0.1 mM MES, 0.1 mM BPDS, 100 µM Fe(III)-EDTA; pH 5.5) for 30 min. A rootless reaction solution was run simultaneously as the blank control. The absorbance of the Fe(II)-BPDS complex was measured at 535 nm with a UV–Vis spectrophotometer (UV1900PC). A standard curve was used to calculate the activity of root FCR.

2.6. Leaf Fe Concentration The leaves harvested from the trees were washed three times with deionized water and then dried in a forced-draft oven at 105 ◦C for the ﬁrst 2 h and then to constant weight at 85 ◦C. The dried samples were ground into powder and digested at 220 ◦C for 30 min in the presence of 13 M HNO3 and 8.8 M H2O2. The Fe concentrations of the leaves were measured with a plasma emission spectrometer (ICP-OES, OPTIMA 3300 DV, Perkins- Elmer Inc., Waltham, Massachusetts, USA) at 248.3 nm band and with the error not to exceed 1% deﬂection on the absorbance scale [26].

2.7. Statistical Data Analysis The experiments were set up in a completely randomized design with 5 replicates. The factor levels are control, FM, R, F, M, SFe, and FFe. Data were obtained from soil (active Fe concentration and soil moisture), leaves (SPAD, leaf Fe concentration and the ratio of SPAD to the leaf Fe concentration), and roots (root FCR activity and rhizosphere pH). Three independent one-way MANOVA (multivariate analysis of ANOVA) models were used in the data, one for “soil” variables, one for “root” variables and one for “leaf” variables. Before analysis, Shapiro–Wilk test and Levene’s test were used to check the hypoth- esis of the normality of the residuals and variance homogeneity along the factor levels, respectively. The Shapiro–Wilk test showed that the residuals were normally distributed (p > 0.05). The Levene’s test showed that the variances of active Fe concentration, soil moisture, the ratio of SPAD to the leaf Fe concentration and rhizosphere pH were all homogeneous (p > 0.05), and the variances of SPAD, leaf Fe concentration and root FCR activity were heterogeneous (p ≤ 0.05). Tukey’s test (p < 0.05) was used as a post hoc test method to calculate the signiﬁcant differences among treatment means with the SPSS 20.0 program (Inc., Chicago, IL, USA) when variances were homogeneous, and Games–Howell test (p < 0.05) was used when variances were heterogeneous. Finally, Benjamini–Hochberg false Horticulturae 2021, 7, x FOR PEER REVIEW 5 of 14

moisture, the ratio of SPAD to the leaf Fe concentration and rhizosphere pH were all homogeneous (p > 0.05), and the variances of SPAD, leaf Fe concentration and root FCR activity were heterogeneous (p ≤ 0.05). Tukey’s test (p < 0.05) was used as a post hoc test method to calculate the significant differences among treatment means with the SPSS 20.0 program (Inc., Chicago, IL, USA) when variances were homogeneous, and Games–Howell test (p < 0.05) was used when variances were heterogeneous. Finally, Benjamini–Hochberg false discovery rate (FDR) method was used to correct for multiple testing, and the FDR p value was considered statistically significant only if it was less than 0.05. Horticulturae 2021, 7, 172 5 of 14 3. Results

3.1. Leaf SPAD Values discovery rate (FDR) method was used to correct for multiple testing, and the FDR p value The analysis resultswas consideredshowed statisticallythat the differ significantences only of if it“leaf” was less variables than 0.05. under different treatments were statistically3. Results significant (Wilks’ Lambda = 0.002, F = 30.689, p < 0.001). The results of between-subjects3.1. Leaf effects SPAD Values showed that the differences of SPAD among different treatments were statisticallyThe analysissignificant results (p showed < 0.001). that the differences of “leaf” variables under different treatments were statistically significant (Wilks’ Lambda = 0.002, F = 30.689, p < 0.001). The The leaf SPAD valuesresults and of between-subjects post hoc test effects resu showedlts are thatshown the differences in Figure of SPAD3. The among leaf SPAD different values ranged from 11.7treatments to 29.7 were among statistically the significant different (p < treatments. 0.001). The leaf SPAD values under F and M were 167%The and leaf 165% SPAD valueshigher and than post that hoc test under results the are showncontrol, in Figurerespectively.3. The leaf SPADThe values ranged from 11.7 to 29.7 among the different treatments. The leaf SPAD values lowest SPAD value underunder R F andwas M 11.7, were 167%which and was 165% only higher 65.7% than that of that under under the control, the respectively.control. The The leaf SPAD values underlowest FM SPAD and value FFe under were R the was same 11.7, which as that was onlyunder 65.7% the of thatcontrol. under the control. The leaf SPAD values under FM and FFe were the same as that under the control.

Figure 3. The soil plant analysis development (SPAD) values of HG-QA leaves grown in different practices. HG-QA: Figure“Huangguan” 3. The pearsoil grafted plant ontoanalysis quince A.development Control: ridging (SPAD) with landscape values fabric of HG-QA mulching andleaves without grown other treatments;in different practices.FM: flattening HG-QA: with landscape “Huangguan” fabric mulching, pear R: grafted ridging withoutonto quince landscape A. fabric Control: mulching; ridging F: flattening with without landscape landscape fabric mulchingfabric mulching; and without M: Manure other application; treatments; SFe: Fe applicationFM: flatteni in soil;ng FFe: with Foliar landscape Fe application. fabric The mulching, different letters R: meanridging withoutstatistical landscape differences betweenfabric treatments,mulchingp;< F: 0.05 flattening (Benjamini–Hochberg without correction).landscape fabric mulching; M: Manure application; SFe: Fe application in soil; FFe: Foliar Fe application. The different letters mean statisti- 3.2. Soil Active Fe Concentrations and Soil Moisture cal differences between treatments, p < 0.05 (Benjamini–Hochberg correction). The analysis results showed that the differences of “soil” variables under different treatments were statistically significant (Wilks’ Lambda = 0.013, F = 34.305, p < 0.001). 3.2. Soil Active Fe ConcenThetrations results of and between-subjects Soil Moisture effects showed that the differences of soil active Fe concentrations and soil moisture among different treatments were statistically significant The analysis results(p < 0.001).showed that the differences of “soil” variables under different treatments were statisticallyThe soilsignificant active Fe concentrations (Wilks’ Lambda and post = hoc 0.013, test results F = 34.305, are shown p < in 0.001). Figure4A. The The results of between-subjectssoil active effe Fects concentrations showed that were the similar differences among the FM, of soil R, F, andactive FFe treatmentsFe concentra- and the control at approximately 8.00 mg kg−1. The soil active Fe concentrations under M and SFe tions and soil moisturewere among 11.31 mgdifferent kg−1 and tr 13.39eatments mg kg− were1, respectively; statistically they were significant not significantly (p < 0.001). different The soil active Fefrom concentrations each other but were and significantly post hoc higher test thanresults those are under shown FM and in the Figure control. 4A. The soil active Fe concentrations were similar among the FM, R, F, and FFe treatments and the control at approximately 8.00 mg kg−1. The soil active Fe concentrations under M

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and SFe were 11.31 mg kg−1 and 13.39 mg kg−1, respectively; they were not significantly different from each other but were significantly higher than those under FM and the control. The soil moisture data and post hoc test results are shown in Figure 4B. The soil moisture levels under R and F were 14.0% and 13.5%, respectively, which were significantly lower than that under the control (19.1%). The lowest soil moisture level was observed Horticulturae 2021, 7, 172 under F. The soil moisture levels under FM, M, SFe, and FFe were the same6 of 14 as that under the control and ranged from 19.73 to 20.29%.

Figure 4. Active Fe concentration (A) and moisture of soil (B) in 0−40 cm depth of HG-QA grown in different practices. Figure 4. ActiveHG-QA: Fe concentration “Huangguan” pear (A grafted) and ontomoisture quince A.of Control:soil (B ridging) in 0− with40 cm landscape depth fabric of HG-QA mulching grown and without in different other practices. HG-QA: “Huangguan”treatments; FM: pear flattening grafted with onto landscape quince fabric A. mulching, Control: R: ridging ridging without with landscape landscape fabric fabric mulching; mulching F: flattening and without other treatments; FM:without flattening landscape with fabric landscape mulching; M: fabric Manure mulching, application; R: SFe: ridging Fe application without in soil; landscape FFe: Foliar fabric Fe application. mulching; The F: flattening different letters mean statistical differences between treatments, p < 0.05 (Benjamini–Hochberg correction). without landscape fabric mulching; M: Manure application; SFe: Fe application in soil; FFe: Foliar Fe application. The different letters mean statistical differencesThe soilbetween moisture treatments, data andpost p < 0.05 hoc test(Benjamini–Hochberg results are shown in Figurecorrection).4B. The soil moisture levels under R and F were 14.0% and 13.5%, respectively, which were significantly lower than that under the control (19.1%). The lowest soil moisture level was observed 3.3. Rootunder FCR F. TheActivities soil moisture and Rhizosphere levels under FM, pH M, SFe, and FFe were the same as that under Thethe analysis control and results ranged fromshowed 19.73 tothat 20.29%. the differences of “root” variables under different treatments were statistically significant (Wilks’ Lambda = 0.189, F = 5.858, p < 0.001). The results of between-subjects effects showed that the differences of root FCR activity among

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different treatments were statistically significant (p < 0.001) and as well as the rhizosphere pH3.3. (p Root = 0.001). FCR Activities and Rhizosphere pH TheThe root analysis FCR resultsactivities showed and post that thehoc differences test results of are “root” shown variables in Figure under 5A. different The root FCRtreatments activity were under statistically F was 0.31 significant Fe µg min (Wilks’−1 g−1 Lambda FW, which = 0.189, was F much = 5.858, higherp < 0.001). than Thethose underresults R, of M, between-subjects SFe, and the control effects (by showed 163%–207 that the%). differences No significant of root difference FCR activity in root among FCR activitydifferent was treatments found among were statisticallyR, SFe, and significantthe control. (p The< 0.001) root andFCR as activity well as under the rhizosphere M was 0.15 FepH µg (p min= 0.001).−1 g−1 FW, which was significantly lower than those under F and FM but not significantlyThe root different FCR activities from that and under post hocthe testcontrol. results The are root shown FCR inactivity Figure under5A. The FM root (0.24 −1 −1 FeFCR µg activitymin−1 g− under1 FW) and F was FFe 0.31 (0.18 Fe µFeg minµg ming−1 gFW,−1 FW) which showed was muchno significant higher than differences those tounder that under R, M, the SFe, control. and the control (by 163–207%). No significant difference in root FCR activityThe rhizosphere was found among pH data R, and SFe, post and hoc the control.test results The are root shown FCR in activity Figure under 5B. The M wasrhizo- 0.15 Fe µg min−1 g−1 FW, which was significantly lower than those under F and FM but sphere pH under SFe was 7.65, which was approximately 0.30 units lower than those un- not significantly different from that under the control. The root FCR activity under FM der FM and F. The rhizosphere pH values under the FM, F, SFe, M, FFe, and control treat- (0.24 Fe µg min−1 g−1 FW) and FFe (0.18 Fe µg min−1 g−1 FW) showed no significant mentsdifferences ranged to from that under7.78 to the 8.03 control. but were not significantly different from each other.

Figure 5. Ferric-chelate reductase (FCR) activity of roots (A) and rhizosphere pH (B) of HG-QA grown in different practices. FigureHG-QA: 5. Ferric-chelate “Huangguan” reductase pear grafted (FCR) onto activity quince of A. roots Control: (A) and ridging rhizosphere with landscape pH (B) fabricof HG-QA mulching grown and in without different other practices. HG-QA: “Huangguan” pear grafted onto quince A. Control: ridging with landscape fabric mulching and without treatments; FM: ﬂattening with landscape fabric mulching, R: ridging without landscape fabric mulching; F: ﬂattening other treatments; FM: flattening with landscape fabric mulching, R: ridging without landscape fabric mulching; F: flatten- without landscape fabric mulching; M: Manure application; SFe: Fe application in soil; FFe: Foliar Fe application. The ing without landscape fabric mulching; M: Manure application; SFe: Fe application in soil; FFe: Foliar Fe application. The different letters mean statistical differences between treatments, p < 0.05 (Benjamini–Hochberg correction). different letters mean statistical differences between treatments, p < 0.05 (Benjamini–Hochberg correction).

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3.4. Leaf Fe Concentrations and the Ratio of SPAD to the Leaf Fe Concentration (RSFe) The rhizosphere pH data and post hoc test results are shown in Figure5B. The The results of between-subjects effects showed that the differences of leaf Fe concen- rhizosphere pH under SFe was 7.65, which was approximately 0.30 units lower than those trations and RSFe among different treatments were statistically significant (p < 0.001). under FM and F. The rhizosphere pH values under the FM, F, SFe, M, FFe, and control The leaf Fe concentrations and post hoc test results are shown in Figure 6A. The leaf treatments ranged from 7.78 to 8.03 but were not significantly different from each other. Fe concentrations under F and SFe were similar and were more than 1.2-fold higher than −1 −1 −1 those3.4. Leaf under Fe Concentrations FM (129.2 mg andkg the), M Ratio (124.5 of SPADmg kg to) theand Leaf the Fecontrol Concentration (100.4 mg (RSFe) kg ). The leaf Fe concentrations under the FM, R, M, and SFe treatments were not significantly different The results of between-subjects effects showed that the differences of leaf Fe concen- from that under the control. In addition, the leaf Fe concentration under FFe was 357.2 mg trations and RSFe among different treatments were statistically significant (p < 0.001). kg−1, which was nearly 2-fold higher than those under FM, R, F, M, and SFe, and 3-fold The leaf Fe concentrations and post hoc test results are shown in Figure6A. The leaf higher than that under the control. Fe concentrations under F and SFe were similar and were more than 1.2-fold higher than The values and post hoc test results of RSFe are shown in Figure 6B. The RSFe values those under FM (129.2 mg kg−1), M (124.5 mg kg−1) and the control (100.4 mg kg−1). −1 −1 underThe leaf R (0.099 Fe concentrations mg kg) and under FFe (0.0549 the FM, mg R, M,kg) and were SFe significantly treatments lower were notthan significantly that under −1 thedifferent control from (0.181 that mg under kg), the while control. the RSFe In addition, values theunder leaf F, Fe FM, concentration SFe, and the under control FFe were was −1 not357.2 significantly mg kg−1, which different. was However, nearly 2-fold the higherRSFe under than thosethe M under group FM, (0.236 R, F, mg M, andkg) SFe,was 1.3- and fold3-fold higher higher than than that that under under the the control control. group.

Figure 6. Leaf Fe concentration (A) and RSFe (B) of HG-QA grown in different practices. RSFe: the ratio of SPAD to Figure 6. Leaf Fe concentration (A) and RSFe (B) of HG-QA grown in different practices. RSFe: the ratio of SPAD to the leafthe Fe leaf concentration. Fe concentration. HG-QA: HG-QA: “Huangguan “Huangguan”” pear grafted pear grafted onto quince onto A. quince Control: A. Control:ridging with ridging landscape with landscape fabric mulching fabric andmulching without and other without treatments; other treatments; FM: flatteni FM:ng ﬂatteningwith landscape with landscape fabric mulching, fabric mulching, R: ridging R: without ridging withoutlandscape landscape fabric mulch- fabric ing;mulching; F: flattening F: ﬂattening without without landscape landscape fabric fabric mulching; mulching; M: Manure M: Manure application; application; SFe: SFe: FeFe application application in in soil; soil; FFe: FFe: Foliar Foliar Fe Fe application. The different letters mean statistical differences between treatments, p < 0.05 (Benjamini–Hochberg correction).

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The values and post hoc test results of RSFe are shown in Figure6B. The RSFe values under R (0.099 mg−1 kg) and FFe (0.0549 mg−1 kg) were signiﬁcantly lower than that under the control (0.181 mg−1 kg), while the RSFe values under F, FM, SFe, and the control were not signiﬁcantly different. However, the RSFe under the M group (0.236 mg−1 kg) was 1.3-fold higher than that under the control group.

4. Discussion Many practices can increase the Fe concentration of leaves by directly providing Fe through fertilization, increasing the Fe absorption ability of roots or mobilizing Fe in leaves [18,19,25]. We tested six practices and found that only F and FFe increased the Fe concentration of pear leaves (Figure6A). Leaf chlorosis was alleviated by only some of the tested practices, including F and M (Figure3). We provide further discussion of these results below.

4.1. Improving the Leaf Fe Concentration The low concentration of soil active Fe is the factor that most limits root Fe absorption in strategy I plant species grown in calcareous soils [28]. Soil organic matter is an important source of chelates and other soluble organic complexes and therefore helps to dissolve and mobilize Fe [29]. Manure application replenishes Fe-mobilizing chelates/complexes in soils [30,31] and thereby increases the concentration of soil active Fe (Figure4A). However, high leaf Fe concentrations were not observed under the M treatment, indicating that the iron absorption process was not improved. Low soil pH facilitates Fe(III) reduction to Fe(II) in soil solution, and Fe(II) can be absorbed by roots directly [7,32]. Fe-deficient plants often acidify the rhizosphere to enhance Fe(III) reduction [33]. However, rhizosphere acidification was observed only under the SFe treatment; leaves under this treatment also exhibited the mildest chlorosis. The active substances such as amino acids in SFe fertilizer can increase root activity [34], which further enhances proton secretion. Thus, the active adaptation of pear trees to Fe deficiency was not the only reason for the observed rhizosphere acidification; the application of acidic fertilizer in SFe also decreased the soil pH, which will contribute to chlorosis alleviation. These evens should be a concern, even though the chlorosis alleviation effect was not significant. In addition to rhizosphere acidification, an increase in root FCR activity is another strategy for enhancing Fe(III) reduction in the rhizosphere of Fe-deficient plants [35–38]. Root FCR activity in strategy I plants was significantly inhibited by a high bicarbonate concentration in soil, which was attributed to the high soil moisture in calcareous soils [39–41], and these conditions caused Fe accumulation in the root apoplast [5,42,43]. Maintaining soil moisture at an optimal level decreases the soil bicarbonate concentration by increasing gas exchange in rhizosphere soil [44,45], which eliminates the inhibition of root FCR activity by high bicarbonate concentrations. Under the F treatment, soil evaporation was assumed to be higher than that under other practices without ridges and film mulching, and the higher soil evaporation resulted in lower soil moisture. High root FCR activity was responsible for the high leaf Fe concentration under this treatment. However, confusing results were observed under the FM and R treatments, possibly because the impact of the individual treatment on the interaction of soil moisture and root FCR activity was not as easy to detect as the impact of both treatments combined.

4.2. Fe Activation in Leaves An “Fe chlorosis paradox”, in which the Fe concentration in Fe-deficient leaves is similar to or even higher than that in Fe-sufficient leaves, often occurs in the field [46,47]. Not all Fe in leaves is involved in biological processes because a large amount of Fe in leaves deposits into apoplasts as inactive Fe. The higher proportion of inactive Fe in chlorotic leaves than in non-chlorotic leaves causes this paradox [48]. Thus, activating leaf Fe is an effective strategy for alleviating chlorosis in calcareous soils [7,9]. Horticulturae 2021, 7, 172 10 of 14

The high Fe concentrations in the HG-QA leaves under the F treatment successfully alleviated the chlorosis, indicating that the Fe absorbed into leaves was not deposited in the apoplasts as inactive Fe in these trees. However, more Fe were inactivated in leaves under R treatment (Figure6B). High bicarbonate concentrations are proposed to be a main reason for Fe inactivation in leaf apoplasts [4,9,49]. Under ridging and mulching practices, the bicarbonate concentration is promoted by the high levels of soil moisture incorporated with the carbonate supply in calcareous soil [44]. This is likely the reason of the inhibition of leaf Fe activation occurred under R than under F. In previous studies, foliar Fe spraying increased leaf chlorophyll content [15,18,50]. However, we did not find any significant improvement in the SPAD values (Figure3) of HG-QA under FFe, although the leaf Fe concentration was 3.56-fold smaller than the control. This result indicated that the Fe added to the leaves by spraying may have been deposited in the apoplast as inactive Fe. However, recently, the addition of fullerenol to Fe(II)-sulfate sprays has been shown to increase leaf active Fe and successfully suppress Fe deficiency symptoms by increasing the stability of the applied Fe(II) [51]; increasing Fe(II) stability is important for enhancing the potential of foliar Fe fertilization. Manure application not only increased soil Fe bioavailability, but also activated Fe in the leaves of HG-QA (Figure6B). However, the mechanism of Fe activation in leaves under manure application is still not clear. Fe inactivation in leaves in calcareous soils is probably related to the high pH of the leaf apoplast. Manure application may have acidified the apoplast and thus led to Fe activation. After combining our data and analyses, we proposed a model illustrating how the different practices alleviated Fe deficiency in HG-QA grown in calcareous soils (Figure7). The practices of F and FFe both increased the leaf Fe concentration. However, Fe chlorosis was alleviated only under the F and M treatments. More of the Fe in the apoplast may have been inactivated under R and FFe than under the other treatments, which would have resulted in the continuation of leaf chlorosis. The increase in root FCR activity under F and in soil active Fe under SFe and the activation of leaf Fe under M all contributed to alleviating leaf chlorosis. However, ridging with mulching is considered to be an effective and adaptable field management method to improve productivity in fruit cultivation [20,23,24,52], which restricts the feasibility of F for alleviating Fe deficiency. However, it reminds us of the importance of soil aeration management on improving Fe nutrition. In addition, manure application can improve leaf Fe utilization and reduce the cost of pear production because it is less expensive than other Fe fertilizers. Therefore, manure is more appropriate for combating Fe deficiency in HG-QA trees grown in calcareous soil. However, manure can only alleviate Fe deficiency in HG-QA trees and does not provide a complete solution. Measures that can address the fundamental reasons for Fe deficiency chlorosis in HG-QA trees grown in calcareous soil need to be explored further. Horticulturae 2021, 7, 172 11 of 14 Horticulturae 2021, 7, x FOR PEER REVIEW 11 of 14

FigureFigure 7. ModelModel of how differentdifferent practicespractices alleviate alleviate Fe Fe deficiency deficiency in in pear pear trees trees in in calcareous calcareous soil. soil. The The black black arrow arrow represents represents the direction of the process. The steps inside the dotted ovals represent a complete process. The diamonds with the direction of the process. The steps inside the dotted ovals represent a complete process. The diamonds with different different numbers represent the different practices. The practices shown within a dotted oval will affect the corresponding numbers represent the different practices. The practices shown within a dotted oval will affect the corresponding process. process. The size of the diamond represents the magnitude of the impact of the corresponding practice. Green diamonds representThe size of positive the diamond effects, represents and red diamon the magnitudeds represent of the negative impact effects. of the correspondingFM: flattening practice.with landscape Green diamondsfabric mulching, represent R: ridgingpositive without effects, landscape and red diamonds fabric mulching; represent F: negativeflattening effects. without FM: landscape flattening fabric with mulching; landscape M: fabric Manure mulching, application; R: ridging SFe: Fewithout application landscape in soil; fabric FFe: mulching;Foliar Fe application. F: flattening without landscape fabric mulching; M: Manure application; SFe: Fe application in soil; FFe: Foliar Fe application. 5. Conclusions 5. ConclusionsPractice F aimed at improving soil aeration, and it increased the Fe concentration in HG-QAPractice leaves F by aimed decreasing at improving soil moisture, soil aeration, indicating and the it increasedimportance the of Fesoil concentration aeration improvement.in HG-QA leavesThe application by decreasing of Fe soilfertilizers moisture, did indicatingnot increase, the and importance could even of soildecrease, aeration Fe utilizationimprovement. in leaves. The application This result of indicated Fe fertilizers that didFe uptake not increase, and utilization and could in even leaves decrease, were independentFe utilization biochemical in leaves. This processes. result indicated However, that only Fe uptakemanure and application utilization had in leavespositive were ef- fectsindependent on both soil biochemical Fe increase processes. and leaf However, Fe utilization only manureeven though application the leaf had Fe positiveconcentration effects wason both not increased. soil Fe increase This may and leafhave Fe been utilization due to evenchelate though exudation the leaf to Femobilize concentration soil Fe and was thenot acidification increased. This of the may leaf have apoplast been to due acti tovate chelate leaf Fe. exudation Thus, manu to mobilizere application soil Fe was and the the firstacidification choice for of alleviating the leaf apoplast Fe deficiency to activate chlorosis leaf Fe. in Thus, HG-QA manure trees application grown in was calcareous the first soils.choice Combining for alleviating various Fe practices deficiency that chlorosis increase in Fe HG-QA uptake treesis likely grown to achieve in calcareous greater soils.suc- cessCombining in addressing various this practices problem. that increase Fe uptake is likely to achieve greater success in addressing this problem. Author Contributions: Conceptualization, H.L.; Data curation, Y.Z.; Formal analysis, Z.L. and F.L.; FundingAuthor Contributions:acquisition, S.L.;Conceptualization, Investigation, Y.Z. H.L.; and DataM.S.; curation,Methodology, Y.Z.; Formal H.L.; Project analysis, administration, Z.L. and F.L.; M.S.;Funding Resources, acquisition, S.L.; Supervision, S.L.; Investigation, F.Y. and Y.Z. F.L.; and Validation, M.S.; Methodology, Z.L.; Visualization, H.L.; Project S.L. administration,and F.L.; Writ- ing—originalM.S.; Resources, draft, S.L.; Y.Z.; Supervision, Writing—review F.Y. and F.L.; and Validation, editing, H.L. Z.L.; and Visualization, F.Y. All authors S.L. and have F.L.; read Writing— and agreedoriginal to draft, the published Y.Z.; Writing—review version of the and manuscript. editing, H.L. and F.Y. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by Science and Technology Innovation Capacity Building Program of Beijing Academy of Agriculture and Forestry Sciences (KJCX20200114 and KJCX20200602), and National Key R&D Program of China (2020YFD1000202).

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Funding: This study was funded by Science and Technology Innovation Capacity Building Program of Beijing Academy of Agriculture and Forestry Sciences (KJCX20200114 and KJCX20200602), and National Key R&D Program of China (2020YFD1000202). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Raw data are available on request. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations Fe Iron SPAD Soil plant analysis development FCR Ferric-chelate reductase FDR False discovery rate HG-QA “Huangguan” grafted onto quince A with Hardy as an interstock FM Flattening with landscape fabric mulching R Ridging without landscape fabric mulching F Flattening without landscape fabric mulching M Manure application SFe Fe application in soil FFe Foliar Fe application RSFe The ratio of SPAD to the leaf Fe concentration

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