agriculture

Article Paenibacillus brasilensis YS-1: A Potential Biocontrol Agent to Retard Tangerine Senescence

Chuying Chen 1 , Chunpeng Wan 1,* , Juanhua Guo 1,2 and Jinyin Chen 1,3,*

1 Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits and Vegetables, College of Agronomy, Jiangxi Agricultural University, 330045, ; [email protected] (C.C.); [email protected] (J.G.) 2 Bureau of Agriculture and Rural Affairs in Lianxi Area, 332000, China 3 College of Materials and Chemical Engineering, University, Pingxiang 337055, China * Correspondence: [email protected] (C.W.); [email protected] (J.C.); Tel.: +86-791-83813185 (C.W. & J.C.)  Received: 15 June 2020; Accepted: 28 July 2020; Published: 5 August 2020 

Abstract: The Xinyu tangerine (Citrus reticulata Blanco) is a non-climacteric fruit that is widely cultivated and consumed in China but highly susceptible to fungal infections. Antagonistic microorganisms can control postharvest diseases and extend the storage life of citrus fruits. However, little work has been done to investigate the effects of applying Paenibacillus brasilensis YS-1 by immersion to enhance the cold storability of Xinyu tangerines. Fruits were soaked with P. brasilensis YS-1 fermented filtrates for 10 min and in sterile water as the control. The decay incidence, weight loss, nutrient content, respiration rate, malondialdehyde (MDA) content, and defensive enzymes activities in citrus fruit were measured during cold storage at 5 0.5 C. The results showed that ± ◦ P. brasilensis YS-1 treatment significantly reduced postharvest decay and effectively maintained the nutritional quality compared to the control under cold storage. The weight loss, respiration rate, and MDA content were lower in P. brasilensis YS-1-treated fruits than the control fruits, indicating that P. brasilensis YS-1 treatment increased the activities of superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), and phenylalnine ammonia-lyase (PAL). According to the results, a postharvest application of P. brasilensis YS-1 can control the postharvest decay and maintain fruit quality, as well as increase the defensive enzyme activity, so as to achieve the purpose of retarding postharvest senescence in citrus fruit.

Keywords: Paenibacillus brasilensis YS-1; Citrus reticulata Blanco; postharvest senescence; storability

1. Introduction Citrus reticulata Blanco is a widely cultivated fruit that is consumed in China because it has rich juicy contents, a delicious taste, it is easy to peel, has fewer dregs, a higher yield, and abundant nutrients, such as vitamin C, flavonoids, anthocyanins, and various other antioxidants [1–3]. Among mandarin varieties, Xinyu tangerines (Citrus reticulata Blanco) belong to the loose-skinned citrus group and are listed in the national geographic indication for the protection of products in China due to its equitable color, high juice content, fresh flavor, and promotion of human health [2]. However, it is a perishable citrus fruit, with a short postharvest storage life of 30 d at room temperature [4]. The majority of fresh mandarins ripen in early October to late November, and with their tender peel, postharvest diseases (green mold, blue mold, sour rot, and stem-end rot) caused by fungal pathogens such as Penicillium digitatum Sacc., P. italicum Wehmer, Geotrichum citri-aurantii E.E. Butler, and Diaporthe citri (Faw.) Wolf contribute to significant and heavy economic losses during harvest, storage, transportation, and even marketing [5–9]. A pathogenic fungal infection is an important factor that affects the

Agriculture 2020, 10, 330; doi:10.3390/agriculture10080330 www.mdpi.com/journal/agriculture Agriculture 2020, 10, 330 2 of 13 nutritional value and storage life of harvested citrus fruits. Therefore, it is a matter of great urgency to reduce the postharvest fungal rots of mandarins and other horticultural fruits. Traditionally, the postharvest fungal decay of citrus fruits is chiefly controlled by a variety of synthetic fungicides including imazalil, pyrimethanil, thiabendazole, and polyhexamethylene guanidine, which have been commonly used to ensure a stable supply of fruit at markets to cope with an ever-increasing demand [10,11]. However, the widespread use of synthetic fungicides has become a global health concern due to their potential undesirable risk to human health, resistant strains, and environmental contamination [12,13]. Keeping in view these environmental and health issues, controlling postharvest fungal diseases using various biologically degradable compounds sourced from antagonist microorganisms shows a great potential as an alternative to synthetic preservatives to enhance the storage life of citrus fruit. In recent decades, the use of antagonistic microorganisms and/or their secondary metabolites has been reported to have great potential as a promising alternative to synthetic fungicides for controlling postharvest diseases in citrus and several other horticultural crops [12,14]. Many antagonistic microorganisms such as Aureobasidium pullulans strain ACBL-77 [15], Bacillus amyloliquefaciens BUZ-14 [16], B. subtilis ABS-S14 [17], Kloeckera apiculata 34-9 [18], lactic acid bacteria [19], Pseudomonas fluorescens ZX [20], Rhodosporidium paludigenum Fell and Tallman [21], and Yarrowia lipolytica W29 [22] have been reported and considered as promising biological control agents (BCAs) for the postharvest disease control of citrus fruit. Biocontrol bacteria Paenibacillus brasilensis YS-1 is a soil-born Gram-positive bacteria and phylogenetically resembles P. polymyxa [23,24], while P. brasilensis displays a strong antimicrobial activity against various phytopathogenic fungi in vitro, including Penicillium italicum Wehmer which frequently causes blue mold disease in citrus fruit [23,25]. Additionally, P. brasilensis has been reported to exhibit antagonist activities against many human pathogenic fungal strains, such as Cryptococcus spp., Candida albicans sorotype B, Fonsecaea pedrosoi (Brumpt), Histoplasma capsulatum var. duboisii and Fusarium moniliforme Sheldon LGM-2 [26], and it has high security in terms of posing no threat to humans and the environment, and is generally recognized as safe (GRAS) [24]. Previously, our lab successfully isolated a biocontrol bacterium P. brasilensis YS-1 from the soil of a kumquat (Fortunella japonica Swingle) root, which was identified by 16S rDNA sequencing combined with the Biolog microbial identification system [23]. One of main antifungal compounds was identified as cytosine, which was analyzed by mass spectrometry and nuclear magnetic resonance (1H-NMR, 13C-NMR, and 2D-NMR) [27]. However, the effects of postharvest treatments with P. brasilensis YS-1 on the postharvest storability and fruit quality of Xinyu tangerines have not been evaluated yet. In the current study, the effects of P. brasilensis YS-1 dipping treatment on the fruit quality, senescence-associated material contents, and defensive enzymes activities in harvested Xinyu tangerines were investigated. Furthermore, the study also focused on developing a safe and effective BCA to enhance postharvest storability and prolong the storage life of Xinyu tangerines.

2. Materials and Methods

2.1. Fruit and Bacterial Strain Healthy Xinyu tangerines (Pengjia No. 39) were picked at commercial maturity from an orchard in Yushui of Xinyu City, China, and were promptly transported to the laboratory within 3 h. The fruits were sorted based on the uniformity of size and color, and the defected fruits with any mechanical wounds or disease were removed. The antagonistic bacterium P. brasilensis YS-1 was isolated from the soil of a kumquat (Fortunella japonica Swingle) root, and identified by 16S rDNA sequencing [23]. This strain was maintained on potato-dextrose agar (PDA) and embedded in 30% glycerol at –80 ◦C for conservation. Paenibacillus brasilensis YS-1 fermented filtrates: P. brasilensis YS-1 was cultured in Luria-Bertani liquid medium (10 g/L of tryptone, 3 g/L of beef extract, 20 g/L of glucose, 5 g/L of NaCl) at 27 ◦C for Agriculture 2020, 10, 330 3 of 13

24 h with 180 r/min. Then, 2.0% of the fermented filtrate was migrated to liquid fermentation medium (60 g/L of soluble starch, 10 g/L of yeast extract, 6 g/L of NaCl, 2 g/L of MgSO , 2 g/L of K HPO 3H O) 4 2 4· 2 at 27 C for 48 h with 180 r/min, and centrifuged (8000 g, 20 min; 5804R, Eppendorf, Hamburg, ◦ × Germany). The supernatant was collected and heated for 20 min in a boiling water bath, and then centrifuged to obtain YS-1 fermented filtrates.

2.2. Sample Treatments A total of 1000 selected fruits were washed with sterile water and divided into two groups (500 fruits per group). The P. brasilensis YS-1 group was dipped in a fermented filtrate suspension of P. brasilensis YS-1 for 10 min, and the control group was immersed in sterile water for 10 min. After being air dried at room temperature, all fruits were individually film packaged and stored at 5 0.5 C with a relative humidity (RH) of 80–90%. During 60 d of cold storage, the P. brasilensis ± ◦ YS-1-treated and control fruits were evaluated for fruit decay and sampled every 10 d to analyze the physicochemical indicators.

2.3. Evaluation of Fruit Decay and Weight Loss The percentage of fruit decay incidence and weight loss were measured according to the method described by Chen et al. [28] with certain modifications. The same 100 Xinyu tangerines were taken out to evaluate the fruit decay at intervals of 10 d during cold storage at 5 ◦C. Fruits with any indication of fungal rot were defined as decayed fruits. The fruit weight was measured every 10 d by an AX224ZH digital weighing balance ( 0.0001 g, Ohaus Co., Ltd., Parsippany, NJ, USA) and compared with the ± harvested weight.

2.4. Analysis of the Total Soluble Solids, Total Sugar, Titratable Acidity, and Vitamin C Contents in Fruit Pulp Ten grams of juice was extracted from 10 fruits in each replicate and was centrifuged at 8000 g × for 15 min. The supernatant was collected to determine the contents of total soluble solids (TSS), total sugar, titratable acidity (TA), and vitamin C (VC) following the procedures defined by Chen et al. [2]. The TSS content (%) was determined using a RA-250WE Brix-meter (Atago, Tokyo, Japan). The total sugar content was assayed using the anthrone colorimetric method. Both the TA and VC contents in the supernatant were determined by a titration with 0.1 MNaOH (pH 8.0) and 2, 6-dichlorophenol indophenols, respectively. The results are expressed as a percentage (%) of the citric acid on a fresh 1 weight basis and milligrams of ascorbic acid per 100 g of juice (mg 100 g− ). Each analysis was carried out in three replicates with 10 fruits per replicate.

2.5. Determination of the Respiration Rate and Malondialdehyde Content The fruit respiration rate of 10 fruits from the P. brasilensis YS-1-treated and control groups was determined according to the method described by Chen et al. [2,28]. The respiration rate was measured by a GHX-3051H fruit and vegetable breathing apparatus (Jingmi Scientific LLC., Shanghai, China), from the CO production and the results are expressed as mg/(kg h). 2 · The malondialdehyde (MDA) content in the P. brasilensis YS-1-treated and control groups was determined according to the thiobarbituric acid (TBA) method described by Mahunu et al. [14] with some minor modifications. The pericarp tissues of 10 fruits were ground in a MM 400 grinder (Retsch GmbH., Arzberg, Germany), and 3.0 g of powder was homogenized in 15 mL of 10% (w/v) TBA. After centrifugation (12,000 g at 4 C, 20 min), 2 mL of the supernatant was mixed with 2 mL of × ◦ 0.67% (w/v) TBA, and immersed in a boiling water bath for 30 min. Then, it was cooled and centrifuged at 8000 g (5804R, Eppendorf) for 10 min. Finally, the absorbance of the solution was measured at × three wavelengths (450, 532, and 600 nm) by a M5 Multiscan Spectrum microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). The result of the MDA content is expressed as mmol/g frozen weight (FW). Agriculture 2020, 10, 330 4 of 13

2.6. Assay of the Defensive Enzyme Activity Each sample (2.0 g) derived from the pericarp tissues of 10 fruits was homogenized with various ice-cold extraction buffers to obtain extracts for assaying the following defensive enzymes: 8 mL of a 50Agriculture mM ice-cold 2020, 10 phosphate, x FOR PEER buREVIEWffer (pH 7.8) containing 1 mM ethylene diamine tetraacetic acid (EDTA),4 of 13 and 2% (w/v) polyvinyl pyrrolidone (PVP) for superoxide dismutase (SOD, EC 1.15.1.1); 8 mL of a 100100 mMmM ice-coldice-cold acetateacetate bubufferffer (pH(pH 5.5)5.5) containingcontaining 1 mM polyethylene glycol (PEG), 4% ( w//v)) PVP, andand 1%1% ( w(w/v/)v Triton) Triton X-100 X-100 for peroxidasefor peroxidase (POD, (POD, EC 1.11.1.7) EC 1.11.1.7) and polyphenol and polyphenol oxidase oxidase (PPO, EC (PPO, 1.10.3.1), EC and1.10.3.1), 5 mL andof a 5 50 mL mM of a ice-cold 50 mM Tris-HClice-cold Tris-HCl buffer (pH buffer 8.8) (pH containing 8.8) containing 15 mM β15-mercaptoethanol, mM β-mercaptoethanol, 5 mM VC,5 mM 5 mM VC, EDTA, 5 mM 1 mMEDTA, phenylmethylsulfonyl 1 mM phenylmethyl fluoridesulfonyl (PMSF), fluoride and (PMSF), 0.15% (w and/v) PVP 0.15% for phenylalanine(w/v) PVP for ammonia-lyasephenylalanine ammonia-lyase (PAL, EC 4.3.1.5). (PAL, After EC centrifugation4.3.1.5). After centrifugation (12,000 g at 4 (12,000×C, 30 min), g at 4 the °C, supernatants 30 min), the × ◦ weresupernatants collected were and usedcollected as crude and used enzyme as crude extracts enzyme for the extracts assays. for the assays. TheThe SODSOD activityactivity waswas assessedassessed usingusing nitrobluenitroblue tetrazoliumtetrazolium (NBT)(NBT) asas previouslypreviously describeddescribed byby WanWan etet al.al. [[29].29]. OneOne unitunit ofof enzymeenzyme activityactivity waswas defineddefined asas aa 50%50% inhibition ofof NBT photoreduction. BothBoth thethe PODPOD andand PPOPPO activityactivity waswas determineddetermined accordingaccording toto thethe methodsmethods ofof guaiacolguaiacol oxidationoxidation atat 470470 nmnm and and catechol catechol oxidation oxidation at 420at 420 nm, nm, respectively respectively [14,30 [14,30].]. One unitOne of unit enzyme of enzyme activity activity was defined was asdefined an increment as an increment of 0.01 per of minute. 0.01 per The minute. PAL activity The PAL was activity determined was determined by using a PAL by using assay a kit PAL (Nanjing assay Jianchengkit (Nanjing Bioengineering Jiancheng Bioengineering Inst., China) measuring Inst., China) the measuring absorbance the at 290absorbance nm using at the 290 M5 nm microplate using the readerM5 microplate (Molecular reader Devices (Molecular Corp., Sunnyvale,Devices Corp., CA, Sunnyvale, USA). CA, USA).

2.7.2.7. Statistical Analysis AllAll datadata fromfrom thethe physicochemicalphysicochemical experimentsexperiments areare displayeddisplayed asas thethe meanmean ± standard error (SE). ± TheThe SPSSSPSS softwaresoftware versionversion 17.017.0 (SPSS(SPSS Inc.,Inc., Chicago,Chicago, IL,IL, USA)USA) waswas usedused toto determinedetermine thethe didifferencesfferences withwith thethe independentindependent samplessamplest t-test-test ((pp< < 0.05)0.05) forfor eacheach storagestorage time.time.

3.3. Results

3.1.3.1. Changes in the Decay Incidence andand WeightWeight LossLoss ofof CitrusCitrus FruitsFruits afterafter P.P.brasilensis brasilensisYS-1 YS-1 Treatment Treatment AA postharvestpostharvest treatmenttreatment withwith P.P. brasilensisbrasilensis YS-1YS-1 significantlysignificantly preventedprevented fungalfungal infectionsinfections andand reducedreduced fruit decay during during cold cold stor storage.age. Citrus Citrus fruits fruits treated treated with with P. brasilensisP. brasilensis YS-1YS-1 became became infected infected with withfungal fungal pathogens pathogens at 40 d at of 40 storage, d of storage, while whilevisible visible signs of signs fruit of rot fruit were rot found were on found fruits on in fruits the control in the controlgroup at group 30 d of at cold 30 d storage of cold (Figure storage 1). (Figure At the1 end). At of the cold end storage of cold (60 storage d), the (60percentage d), the percentageof decay of the of decaycontrol of group the control sharply group rose sharply to 7.33%, rose with to 7.33%, a significa with ant significant 2.20-fold 2.20-foldincrease increaseover fruits over treated fruits with treated P. withbrasilensisP. brasilensis YS-1. YS-1.

Control a 8 P. brasilensis YS-1

6 a

b 4 a b

Decay rate (%) Decayrate 2 b

0 30 40 50 60 Storage time (d) Figure 1. Effect of P. brasilensis YS-1 treatment on the decay rate of citrus fruits stored at 5 0.5 C for Figure 1. Effect of P. brasilensis YS-1 treatment on the decay rate of citrus fruits stored at 5 ± 0.5± °C◦ for 60 60 d. Vertical bars represent the mean SE (n = 3). Letters indicate the statistical differences according d. Vertical bars represent the mean ± SE± (n = 3). Letters indicate the statistical differences according to the toindependent the independent samples samples t-test (tp-test < 0.05) (p < on0.05) each on storage each storage day. day. The percentage of weight loss increased with the extension of cold storage time (Figure2). The percentage of weight loss increased with the extension of cold storage time (Figure 2). Compared Compared with the non-treated control citrus fruits, a lower amplification of weight loss was observed with the non-treated control citrus fruits, a lower amplification of weight loss was observed in the P. brasilensis YS-1-treated fruits during cold storage, and a significant difference between the weight loss of P. brasilensis YS-1-treated and control groups was observed after 30 d of cold storage.

Agriculture 2020, 10, 330 5 of 13 in the P. brasilensis YS-1-treated fruits during cold storage, and a significant difference between the Agriculture 2020, 10, x FOR PEER REVIEW 5 of 13 weight loss of P. brasilensis YS-1-treated and control groups was observed after 30 d of cold storage.

4 Control P. brasilensis YS-1 a b 3 a a b

2 a b b

Weight loss (%) loss Weight 1

0 10 20 30 40 50 60 Storage time (d) Figure 2. Effect of P. brasilensis YS-1 treatment on the weight loss of citrus fruits stored at 5 0.5 C for Figure 2. Effect of P. brasilensis YS-1 treatment on the weight loss of citrus fruits stored at± 5 ± ◦0.5 °C 60 d. Vertical bars represent the mean SE (n = 10). Letters indicate statistical differences according to for 60 d. Vertical bars represent the mean± ± SE (n = 10). Letters indicate statistical differences according an independent samples t-test (p < 0.05) on each storage day. to an independent samples t-test (p < 0.05) on each storage day. 3.2. Changes in the Postharvest Quality Attributes in Citrus Fruits after P. brasilensis YS-1 Treatment 3.2. Changes in the Postharvest Quality Attributes in Citrus Fruits after P. brasilensis YS-1 Treatment The contents of the TSS and total sugar in the P. brasilensis YS-1-treated and control groups exhibitedThe acontents quick increase of the TSS at 30 and d and total 20 d,sugar respectively, in the P. and brasilensis then gradually YS-1-treated reduced and to control the end groups of the coldexhibited storage a quick period increase (Table 1at). 30 The d and independent 20 d, respecti samplesvely, andt-test then indicated gradually that reduced the contents to the ofend the of TSS the andcold total storage sugar period in the (TableP. brasilensis 1). TheYS-1-treated independent group samples were t-test significantly indicated ( pthat< 0.05) the contents higher than of the that TSS of theand control total sugar fruits in during the P. brasilensis the last 40 YS-1-treated days of storage. group were significantly (p < 0.05) higher than that of the control fruits during the last 40 days of storage. Table 1. Variation in the total soluble solids (TSS), total sugar, titratable acidity (TA), and vitamin C (VC)Table contents 1. Variation of citrus in the fruit total under soluble cold solids storage (TSS), in relation total sugar, to P. brasilensis titratable YS-1acidity treatment. (TA), and vitamin C (VC) contents of citrus fruit under cold storage in relation to P. brasilensis YS-1 treatment. 1 Storage Time (d) Treatment TSS Content (%) Total Sugar Content (%) TA Content (%) VC Content (mg 100 g− ) Storage Control TSS Content Total Sugar TA Content VC Content 0 Treatment 12.03 0.058 11.15 0.182 0.66 0.012 21.42 0.272 Time (d) P. brasilensis YS-1 ± (%) Content± (%) ± (%) (mg± 100g−1) Control 12.23 0.058a 11.88 0.124a 0.63 0.010a 22.73 0.368b 10 ± ± ± ± P. brasilensisControlYS-1 12.07 0.115a 12.05 0.148a 0.61 0.012a 23.78 0.513a 0 12.03± ± 0.058 11.15± ± 0.182 ±0.66 ± 0.012 21.42± ± 0.272 P. brasilensisControl YS-1 12.80 0.000a 12.44 0.251 0.60 0.012a 23.95 0.488b 20 ± ± ± ± P. brasilensisControlYS-1 12.63 12.230.058b ± 0.058a 12.33 11.880.058 ± 0.124a 0.58 0.630.006b ± 0.010a 25.81 22.730.423a ± 0.368b 10 ± ± ± ± Control 12.43 0.115b 12.18 0.113b 0.51 0.012b 21.40 0.440b 30 P. brasilensis YS-1 12.07± ± 0.115a 12.05± ± 0.148a ± 0.61 ± 0.012a 23.78± ± 0.513a P. brasilensis YS-1 13.10 0.000a 12.89 0.128a 0.55 0.012a 24.68 0.525a Control 12.80± ± 0.000a 12.44± ± 0.251 ± 0.60 ± 0.012a 23.95± ± 0.488b 20 Control 12.13 0.058b 11.62 0.273b 0.45 0.015b 19.24 0.263b 40 ± ± ± ± P.P. brasilensis YS-1 YS-1 12.53 12.630.058a ± 0.058b 12.53 12.330.058a ± 0.058 0.51 0.580.010a ± 0.006b 23.59 25.810.557a ± 0.423a ± ± ± ± ControlControl 11.70 12.430.100b ± 0.115b 10.75 12.180.176b ± 0.113b 0.37 0.510.017b ± 0.012b 18.38 21.400.488b ± 0.440b 3050 ± ± ± ± P. brasilensis YS-1 12.10 0.100a 12.10 0.100a 0.44 0.017a 21.07 0.164a P. brasilensis YS-1 13.10± ± 0.000a 12.89± ± 0.128a ± 0.55 ± 0.012a 24.68± ± 0.525a Control 11.57 0.058b 10.15 0.224b 0.34 0.015b 15.92 0.225b 60 Control 12.13± ± 0.058b 11.62± ± 0.273b 0.45± ± 0.015b 19.24± ± 0.263b 40 P. brasilensis YS-1 11.90 0.000a 11.90 0.000a 0.40 0.008a 19.49 0.327a P. brasilensis YS-1 12.53± ± 0.058a 12.53± ± 0.058a ± 0.51 ± 0.010a 23.59± ± 0.557a Control 11.70 ± 0.100b 10.75 ± 0.176b 0.37 ± 0.017b 18.38 ± 0.488b 50 The TA contentP. brasilensis in the P.YS-1 brasilensis 12.10YS-1-treated ± 0.100a and12.10 control± 0.100a groups 0.44 dropped ± 0.017a gradually 21.07 ± 0.164a with a prolonged storage timeControl (Table 1). It was 11.57 found ± 0.058b that a slower 10.15 reduction± 0.224b of 0.34 the TA± 0.015b content 15.92 was observed± 0.225b 60 in the P. brasilensisP. brasilensisYS-1-treated YS-1 group 11.90 compared± 0.000a to 11.90 the ± non-treated 0.000a 0.40 controls. ± 0.008aFurther 19.49 statistical± 0.327a comparisons indicated that the TA content in the P. brasilensis YS-1-treated group was notably (p < 0.05) higherThe than TA in content the control in the fruits P. brasilensis from 20 d YS-1-treated to 60 d. and control groups dropped gradually with a prolongedTable1 storage illustrates time that (Table the VC 1). contentIt was found in the thatP. brasilensis a slower reductionYS-1-treated of the and TA control content groups was increasedobserved slightlyin the P. and brasilensis reached theYS-1-treated highest level group at 20compared d, and then to droppedthe non-treated quickly controls. as storage Further time progressed, statistical whilecomparisons the P. brasilensis indicatedYS-1-treated that the TA groupcontent presented in the P. abrasilensis significantly YS-1-treated (p < 0.05) group lower VCwas loss notably than ( thep < control0.05) higher group than during in the the control whole fruits storage from time. 20 d to 60 d. Table 1 illustrates that the VC content in the P. brasilensis YS-1-treated and control groups increased slightly and reached the highest level at 20 d, and then dropped quickly as storage time progressed, while the P. brasilensis YS-1-treated group presented a significantly (p < 0.05) lower VC loss than the control group during the whole storage time.

3.3. Change in the Respiration Rate of Citrus Fruits after P. brasilensis YS-1 Treatment

Agriculture 2020, 10, x FOR PEER REVIEW 6 of 13 Agriculture 2020, 10, 330 6 of 13 Fruit respiration is a vital factor for evaluating the postharvest quality and storability of citrus fruit Agriculture 2020, 10, x FOR PEER REVIEW 6 of 13 3.3.and Change other horticultural in the Respiration products; Rate of th Citruse higher Fruits the after fruit P. brasilensisrespiration, YS-1 the Treatmentmore nutrients are consumed and theFruit lower respiration the storability is a vital of factorharvested for evaluating fruit. After th fallinge postharvest sharply quality in the first and 30 storability d of cold of storage, citrus fruit the Fruit respiration is a vital factor for evaluating the postharvest quality and storability of citrus respirationand other horticulturalrate of the control products; group th showede higher a noticeabthe fruitle respiration, increase from the 30 more d to thenutrients end of arestorage, consumed while fruit and other horticultural products; the higher the fruit respiration, the more nutrients are consumed theand P. the brasilensis lower the YS-1-treated storability of fruit harvested exhibited fruit. a Afterslight falling upward sharply trend in (Figure the first 3). 30 Further d of cold comparisons storage, the and the lower the storability of harvested fruit. After falling sharply in the first 30 d of cold storage, demonstratedrespiration rate a of significant the control difference group showed between a noticeab the respirationle increase rate from of 30 the d toP. thebrasilensis end of storage, YS-1-treated while the respiration rate of the control group showed a noticeable increase from 30 d to the end of storage, andthe P.control brasilensis groups YS-1-treated after 20 d offruit cold exhibited storage. a slight upward trend (Figure 3). Further comparisons while the P.brasilensis YS-1-treated fruit exhibited a slight upward trend (Figure3). Further comparisons demonstrated a significant difference between the respiration rate of the P. brasilensis YS-1-treated demonstrated a significant difference between the respiration rate of the P. brasilensis YS-1-treated and and control groups after 2040 d of cold storage. control groups after 20 d of cold storage. Control P. brasilensis YS-1 )

-1 35

h -1 40 30 Control P. brasilensis YS-1 )

-1 35 a

25 h a -1

30 a a 20 a a 25 b b 15 b a b a b a

Respirationrate (mg kg 20 10 a b b 15 0 102030405060b b Storage btime (d) Respirationrate (mg kg 10 Figure 3. Effect of P. brasilensis0 YS-1 102030405060 treatment on the respiration rate of citrus fruits stored at 5 ± 0.5 °C for 60 d. Vertical bars represent the meanStorage ± SE time (n (d) = 3). Letters indicate statistical differences Figureaccording 3. E fftoect an of independentP. brasilensis samplesYS-1 treatment t-test (p on < the0.05) respiration on each storage rate of citrusday. fruits stored at 5 0.5 C Figure 3. Effect of P. brasilensis YS-1 treatment on the respiration rate of citrus fruits stored ±at 5 ±◦ 0.5 for°C 60 for d. 60 Vertical d. Vertical bars represent bars repres theent mean the SEmean (n =±3). SE Letters (n = 3). indicate Letters statistical indicate distatisticalfferences accordingdifferences 3.4. Change in the MDA Content of Citrus Fruits± after P. brasilensis YS-1 Treatment toaccording an independent to an independent samples t-test samples (p < 0.05) t-test on (p each < 0.05) storage on each day. storage day. 3.4. ChangeThe MDA in the content MDA Contentincreased of Citrusgradually Fruits regardless after P. brasilensis of treatment YS-1 during Treatment the complete cold storage period.3.4. Change Postharvest in the MDA treatment Content of of P. Citrus brasilensis Fruits YS-1 after delayedP. brasilensis the increase YS-1 Treatment in MDA content. At the end of storage,The MDA the contentMDA content increased in the gradually P. brasilensis regardless YS-1-treated of treatment fruits during was about the complete19.4% lower cold than storage that The MDA content increasedP. brasilensisgradually regardless of treatment during the complete cold storage period.in control Postharvest fruit (Figure treatment 4). During of the last 50YS-1 d of delayed cold storage the increase (except in for MDA day content.20), a slower At the increase end of storage,period. thePostharvest MDA content treatment in the ofP. P. brasilensis brasilensisYS-1-treated YS-1 delayed fruits the increase was about in 19.4%MDA lowercontent. than At thatthe end in inof thestorage, MDA the content MDA contentwas observed in the P.in brasilensis the P. brasilensis YS-1-treated YS-1-treated fruits was fruit about compared 19.4% lower to the than control that controlgroup. fruit (Figure4). During the last 50 d of cold storage (except for day 20), a slower increase in the MDAin control content fruit was (Figure observed 4). During in the P. the brasilensis last 50 dYS-1-treated of cold storage fruit (except compared for today the 20), control a slower group. increase in the MDA content was observed in the P. brasilensis YS-1-treated fruit compared to the control

group. 3.5 Control a P. brasilensis YS-1

)

-1 3.0 a 3.5 b Control a a 2.5 P. brasilensis YS-1 b )

-1 3.0 a b a b 2.0 b a 2.5 b a b 1.5

MDA content (mmol g (mmol content MDA 2.0 b 1.0 0 102030405060 1.5 Storage time (d) MDAcontent (mmol g Figure 4. Effect of P. brasilensis1.0 YS-1 treatment on the malondialdehyde (MDA) content of citrus fruits Figure 4. Effect of P. brasilensis YS-10 treatment 102030405060 on the malondialdehyde (MDA) content of citrus fruits stored at 5 0.5 C for 60 d. Vertical bars representStorage the time mean (d) SE (n = 3). Letters indicate statistical stored at 5± ± 0.5◦ °C for 60 d. Vertical bars represent the mean± ± SE (n = 3). Letters indicate statistical differences according to an independent samples t-test (p < 0.05) on each storage day. differences according to an independent samples t-test (p < 0.05) on each storage day. Figure 4. Effect of P. brasilensis YS-1 treatment on the malondialdehyde (MDA) content of citrus fruits 3.5. Changestored inat the5 ± Defensive0.5 °C for Enzyme60 d. Vertical Activity bars of represen Citrus Fruitt the mean after P.± SE brasilensis (n = 3). Letters YS-1 Treatment indicate statistical 3.5. Change in the Defensive Enzyme Activity of Citrus Fruit after P. brasilensis YS-1 Treatment Thedifferences SOD activity according in theto an control independent fruits samples increased t-test during (p < 0.05) the on first each 20 storage d of cold day. storage and then droppedThe sharply SOD activity in the remainingin the control storage fruits time increased (Figure5 duringA). The theP. brasilensisfirst 20 d YS-1of cold treatment storage peaked and then at 3.5. Change in the Defensive Enzyme Activity of Citrus Fruit after P. brasilensis YS-1 Treatment daydropped 30, with sharply the peak in the of remaining SOD activity storage in the timeP. brasilensis (Figure 5A).YS-1-treated The P. brasilensis fruit being YS-1 significantlytreatment peaked higher at (pday< 0.05) 30,The with thanSOD the that activity peak of theof in SOD control the activitycontrol fruits. infruits Duringthe P.increased brasilensis the last during 40 YS-1-treated d of the cold first storage, fruit 20 d being of there cold significantly was storage a significant and higher then didroppedfference sharply in the SOD in the activity remaining between storage the twotime groups.(Figure 5A). The P. brasilensis YS-1 treatment peaked at day 30, with the peak of SOD activity in the P. brasilensis YS-1-treated fruit being significantly higher

Agriculture 2020, 10, x FOR PEER REVIEW 7 of 13

(p < 0.05) than that of the control fruits. During the last 40 d of cold storage, there was a significant difference in the SOD activity between the two groups. The POD activity in the P. brasilensis YS-1-treated and control groups increased rapidly during the first 30 d of cold storage, followed by a decline during the subsequent storage period (Figure 5B). Compared with the control group, further comparisons indicated that a higher value of POD activity was observed in the P. brasilensis YS-1-treated fruits from 20 d to 50 d of cold storage. The PPO activity in the P. brasilensis YS-1-treated and control fruits showed a similar tendency, rising gradually during the first 40 d of cold storage, then dropping sharply over the following days (Figure 5C). However, after 30 d of storage, the PPO activity in the P. brasilensis YS-1-treated fruits was higher than that of the control fruits. The change in the PAL activity exhibited a similar trend to the POD activity during the whole storage period, in the control and P. brasilensis YS-1-treated groups, with PAL activity initially increasing, then peaking at 30 d, after which the activity decreased gradually, and the treatment of P. Agriculturebrasilensis2020 YS-1, 10 maintained, 330 a higher PAL activity compared with the control group after 20 d of storage7 of 13 (Figure 5D).

25 40 A a Control B Control P. brasilensis YS-1 a P. brasilensis YS-1 20 a 35 ) )

-1 b b a -1 a

a 30 15 b

a b b a 25 a 10 b

SOD activity (U g (U activity SOD b g (U activity POD b 20 b 5 0 102030405060 0 102030405060 Storage time (d) Storage time (d)

0.50 Control 1200 Control C P. brasilensis YS-1 D a a P. brasilensis YS-1 0.45 a ) ) a a -1 -1 1000 a 0.40 a b 0.35 b a a 800 b b b 0.30 b 0.25 b 600 PPO activity(U g PAL activityPAL (U g b 0.20 b 400 0 102030405060 0 102030405060 Storage time (d) Storage time (d) Figure 5. Effects of P. brasilensis YS-1 treatment on SOD (A), POD (B), PPO (C) and PAL (D) activities ofFigure citrus 5. fruits Effects stored of P. atbrasilensis 5 0.5 YS-1C for treatment 60 days. Verticalon SOD bars(A), POD represent (B), PPO the mean(C) and SEPAL (n (=D3).) activities Letters ± ◦ ± indicateof citrus statisticalfruits stored differences at 5 ± 0.5 according °C for 60 to days. an independent Vertical bars samples representt-test the (p < mean0.05) on± SE each (n storage= 3). Letters day. indicate statistical differences according to an independent samples t-test (p < 0.05) on each storage Theday. POD activity in the P. brasilensis YS-1-treated and control groups increased rapidly during the first 30 d of cold storage, followed by a decline during the subsequent storage period (Figure5B). Compared3.6. Pearson with Correlation the control of Citrus group, Fruits further after comparisonsP. brasilensis YS-1 indicated Treatment that a higher value of POD activity was observedA correlation-based in the P. brasilensis approachYS-1-treated using the Pearson fruits from coefficient 20 d to 50was d ofchosen cold storage.to evaluate the positive and negativeThe PPO relationships activity in the betweenP. brasilensis the decayYS-1-treated incidence and and control defensive fruits enzyme showed (SOD, a similar POD, tendency, PPO and risingPAL) activity, gradually VC during content, the respiration first 40 d of rate cold and storage, MDA then content dropping and enzyme sharply activity over the for following P. brasilensis days (FigureYS-1-treated5C). However, and control after citrus 30 d offruits storage, during the cold PPO storage activity at in 5 the ± 0.5P. brasilensis°C for 60 YS-1-treatedd. Significant fruits positive was highercorrelations than that(in red) of the and control negative fruits. correlations (in blue) are displayed in Figure 6. The changenumerical in thevalue PAL and activity color intensity exhibited are a similarproportional trend to the Pearson POD activity correlation during coefficients. the whole storageThe correlation period, in coefficient the control indicates and P.brasilensis that theYS-1-treated defensive enzymes, groups, with respiration, PAL activity and initiallyMDA involved increasing, in thenreactive peaking oxygen at 30 species d, after which(ROS), the energy, activity and decreased memb gradually,rane lipid and metabolism the treatment in of citrusP. brasilensis fruits,YS-1 are maintaineddifferentially a higherregulated PAL by activity postha comparedrvest treatments with the and control cold groupstorage, after as previously 20 d of storage demonstrated (Figure5D). in other horticultural crops, such as kiwifruits [31], pears [30], and broccoli [32]. 3.6. Pearson Correlation of Citrus Fruits after P. brasilensis YS-1 Treatment A correlation-based approach using the Pearson coefficient was chosen to evaluate the positive and negative relationships between the decay incidence and defensive enzyme (SOD, POD, PPO and PAL) activity, VC content, respiration rate and MDA content and enzyme activity for P. brasilensis YS-1-treated and control citrus fruits during cold storage at 5 0.5 C for 60 d. Significant positive ± ◦ correlations (in red) and negative correlations (in blue) are displayed in Figure6. The numerical value and color intensity are proportional to the Pearson correlation coefficients. The correlation coefficient indicates that the defensive enzymes, respiration, and MDA involved in reactive oxygen species (ROS), energy, and membrane lipid metabolism in citrus fruits, are differentially regulated by postharvest treatments and cold storage, as previously demonstrated in other horticultural crops, such as kiwifruits [31], pears [30], and broccoli [32]. Agriculture 2020, 10, x FOR PEER REVIEW 8 of 13

The SOD activity was positively correlated with the POD activity (r = 0.879; p < 0.01) and the VC content (r = 0.861; p < 0.01). This result indicates that SOD, POD, and VC are the main important antioxidant enzymes or compounds responsible for scavenging excess ROS and reducing oxidative damage, and together with PPO and PAL make up a defensive system to improve fruit disease Agricultureresistance.2020 Furthermore,, 10, 330 the SOD activity was negatively correlated with the respiration rate8 of(r 13 = −0.553; p < 0.05), MDA content (r = −0.438; p < 0.05) and decay incidence (r = −0.734; p < 0.01).

FigureFigure 6. CorrelationCorrelation matrix matrix based based on Pearson’s on Pearson’s correlati correlationon coefficient coeffi betweencient between the defensive the defensive enzyme enzyme(superoxide (superoxide dismutase dismutase (SOD), peroxidase (SOD), peroxidase (POD), po (POD),lyphenol polyphenol oxidase (PPO), oxidase and (PPO), phenylalnine and phenylalnine ammonia- ammonia-lyaselyase (PAL)) activity, (PAL)) activity, respiration respiration rate (RR), rate(RR), MDA MDA content, content, and and decay decay incidence incidence (DI). PositivePositive correlationscorrelations areare displayeddisplayed inin redred andand negative negative correlations correlationsin in blue. blue. NumericalNumerical valuesvalues andand thethe colorcolor intensityintensity are are proportional proportional to to the the correlation correlation coe coefficient.fficient.

TheThe SODresults activity show wasa significant positively positive correlated correlation with the PODbetween activity the MDA (r = 0.879; contentp < and0.01) decay and theincidence VC content (r = 0.912; (r = 0.861;p < 0.01),p < highlighting0.01). This that result membrane indicates li thatpid SOD,peroxidation POD, and is responsible VC are the for main the importantaccumulation antioxidant of MDA, enzymes which decreases or compounds fruit diseas responsiblee resistance for scavenging and leads to excess the occurrence ROS and reducing of decay oxidativerot. damage, and together with PPO and PAL make up a defensive system to improve fruit disease resistance. Furthermore, the SOD activity was negatively correlated with the respiration rate (r = 0.553; p < 0.05), MDA content (r = 0.438; p < 0.05) and decay incidence (r = 0.734; p < 0.01). 4. Discussion− − − The results show a significant positive correlation between the MDA content and decay incidence (r = 0.912;The potentialp < 0.01), of highlighting P. brasilensis that YS-1 membrane as a biocontrol lipid peroxidation agent for controlling is responsible citrus for postharvest the accumulation disease ofhas MDA, been whichwell reported decreases [25,33]. fruit diseaseHowever, resistance the current and study leads tois unique the occurrence as it deals of decaywith P. rot. brasilensis YS- 1 application for enhancing the postharvest preservation of citrus fruits. The results of the current 4.study Discussion indicate that P. brasilensis YS-1 could significantly reduce fruit decay incidence, maintain a high nutritional quality, suppress fruit respiration and MDA accumulation, while increasing the activities The potential of P. brasilensis YS-1 as a biocontrol agent for controlling citrus postharvest disease of defense-associated enzymes (SOD, POD, PPO, and PAL) in citrus fruit. has been well reported [25,33]. However, the current study is unique as it deals with P. brasilensis Fruit decay incidence and weight loss are two important indicators in evaluating the postharvest YS-1 application for enhancing the postharvest preservation of citrus fruits. The results of the current storability of harvested fruits [17,34]. Generally, the decay incidence and weight loss of harvested study indicate that P. brasilensis YS-1 could significantly reduce fruit decay incidence, maintain a high fruits increase with prolonged storage periods due to the attenuation of disease resistance and nutritional quality, suppress fruit respiration and MDA accumulation, while increasing the activities of continuous fruit respiration. The data obtained in the current study show that a 2.0% fermented defense-associated enzymes (SOD, POD, PPO, and PAL) in citrus fruit. filtrate of P. brasilensis YS-1 efficiently reduced the decay incidence and weight loss in citrus fruit, Fruit decay incidence and weight loss are two important indicators in evaluating the postharvest mimicking earlier studies. Lai et al. [35] and Wu et al. [36] demonstrated that a postharvest treatment storability of harvested fruits [17,34]. Generally, the decay incidence and weight loss of harvested fruits with antagonistic microorganisms (Photorhabdus luminescens (Enterobacteriaceae) Hb1029 and increase with prolonged storage periods due to the attenuation of disease resistance and continuous Bacillus amyloliquefaciens subsp. LY-1) developed biofilm formations on the surface of litchi fruits since fruit respiration. The data obtained in the current study show that a 2.0% fermented filtrate of they have a latent effect in suppressing fungal infections and fruit respiration. Furthermore, both in P.brasilensis YS-1 efficiently reduced the decay incidence and weight loss in citrus fruit, mimicking earlier vitro and in vivo tests showed that P. brasilensis YS-1 prominently inhibited the spore germination of studies. Lai et al. [35] and Wu et al. [36] demonstrated that a postharvest treatment with antagonistic P. italicum to induce blue mold development on Xinyu tangerines in our previous study [23,33]. Less microorganisms (Photorhabdus luminescens (Enterobacteriaceae) Hb1029 and Bacillus amyloliquefaciens decay incidence was observed in P. brasilensis YS-1-treated citrus fruits, which might be linked to its subsp. LY-1) developed biofilm formations on the surface of litchi fruits since they have a latent effect in suppressing fungal infections and fruit respiration. Furthermore, both in vitro and in vivo tests showed that P. brasilensis YS-1 prominently inhibited the spore germination of P. italicum to induce blue mold development on Xinyu tangerines in our previous study [23,33]. Less decay incidence was observed in P. brasilensis YS-1-treated citrus fruits, which might be linked to its antimicrobial potential. Agriculture 2020, 10, 330 9 of 13

Therefore, given that its biocontrol capacity to reduce decay incidence and weight loss is directly linked with its antimicrobial and biofilm-forming ability on the surface of citrus fruits, it should be suggested that P. brasilensis YS-1 should be considered as a promising biocontrol agent to control postharvest diseases and prolong the storage-life of harvested citrus fruit. TSS, total sugar, TA, and VC are considered as important indicators to evaluate the nutritional quality, texture, and flavor of harvested citrus fruits [2]. The TSS is one of the best quality parameters to appraise the texture and nutritional value of fresh horticultural products [29,37]. Total sugar comprises multiple monosaccharides, including glucose, fructose, maltose, sucrose, and certain hydrolysable starches [38]. The TA of sweet oranges, mandarins, tangerines, ponkans, and pummelos are generally interpreted as the percentage of citric acid present in citrus fruits, which are a major source of citric acid [38]. Fresh citrus fruit is the best source of vitamin C (VC), which has an antioxidant capacity and nutritional benefits for human health [37,39]. Additionally, VC is commonly used as a quality parameter for estimating the nutritional value of horticultural products [28,34]. In the current study, P. brasilensis YS-1-treated citrus fruits had higher TSS, total sugar, TA, and VC contents after 30 d of storage, indicating that the treatment of P. brasilensis YS-1 could potentially reduce nutrient degradation in the later storage period (Table1). Similar results were previously reported by Habiba and colleagues [ 40] who noted that Kinnow mandarin (Citrus reticulata Blanco) treated with two epiphytic yeasts (HAB-31 and HAB-53) had higher contents of TSS, TA, and VC than the control fruit. Lai and co-workers [35] reported that the change in TSS, TA, and trehalose contents were decreased by treating litchi fruit with a suspension of P. luminescens Hb1029 (1.0 108 CFU mL 1). A possible explanation for these results × − may be that P. brasilensis YS-1 formed a biofilm to suppress the vital activities of fruits, such as reducing the fruit respiration rate, reducing water evaporation and delaying nutrient degradation. These results reveal that the treatment of P. brasilensis YS-1 could significantly delay the degradation of TSS, TA, and VC, and subsequently lead to maintaining a higher nutritional quality in the pulp of harvested citrus fruits. Fruit respiration is another important parameter that affects the deterioration of nutrients and limits the storage life of harvested citrus fruit [28,37]. It is linked with deleterious changes in organic acids and soluble carbohydrates. In our current study, the increased respiration rate was significantly slowed down by the treatment of P. brasilensis YS-1 compared with the non-treated citrus fruit (Figure3), corresponding with the lower reduction of TA and TSS content (Table1). The lower decay incidence (Figure1), weight loss (Figure2), and MDA accumulation (Figure4), indicate that the potential of P. brasilensis YS-1 to delay postharvest fruit senescence can be linked with the inhibition of the respiration rate. The rate of fruit respiration increased in the control group, suggesting that vigorous respiration and oxidative stress probably lead to a high decay incidence in citrus fruit. Additionally, the increase in the respiration rate during storage life can be due to the loss of organic acids, especially citric acid [38]. Similarly, several studies reported the same phenomenon for pears [41], plums [39] and table grapes [42]. These findings collectively demonstrate that a postharvest treatment of P. brasilensis YS-1 down-regulated fruit respiration and delayed the deterioration of the nutritional quality of citrus fruits under cold storage. Membrane damage or deterioration is a peculiar physiological feature of plant senescence, and this process is chiefly accompanied by an intensified membrane lipid peroxidation with the accumulation of MDA, a final product of cell oxidative damage [14,35,42]. In harvested citrus fruit, the MDA content gradually increased as fruit ripening and senescence progressed during postharvest storage. However, the postharvest application of P. brasilensis YS-1 significantly suppressed the accumulation of the MDA content in citrus fruits (Figure4). The result clearly showed that the lower the disease resistance, the higher the decay incidence. This indicates that a treatment with P. brasilensis YS-1 could delay the process of membrane lipid peroxidation and hence reduce oxidative cellular damage and fruit decay, and delay postharvest senescence in citrus fruit. This is consistent with studies showing that treatment with antagonistic microorganisms such as P. luminescens Hb1029, Bacillus subtilis, B. amyloliquefaciens L-1 Agriculture 2020, 10, 330 10 of 13 and R. paludigenum, could reduce the membrane lipid peroxidation in sweet oranges [34], litchis [35], pears [43] and jujubes [44] during postharvest storage. Both SOD and POD play crucial roles in scavenging reactive oxygen species (ROS) and reducing membrane lipid peroxidation, and hence protecting fruits from oxidative stress [31,34]. In addition, polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) are the main defense enzymes in plant resistance physiology. PPO is a critical enzyme that participates in the oxidation of phenolic compounds, and PAL remains the key enzyme involved in the phenylpropanoid pathway. Therefore, PPO and PALactivity could be used as important indicators to assess disease resistance processes in plant tissues [17,22]. It is also reported that an enhanced disease resistance in harvested citrus fruits and other horticultural products is closely related to increased levels of SOD, POD, PPO, and PAL [17,34,41,42]. Herein, the results demonstrated that postharvest treatments of P. brasilensis YS-1 significantly increased the activities of SOD, POD, PPO, and PAL in citrus fruits (Figure5). Based on these findings, it is demonstrated that the treatment of P. brasilensis YS-1 could significantly enhance and maintain a higher activity of defensive enzymes, such as SOD, POD, PPO and PAL in citrus peel, and suppressed fruit respiration and the accumulation of MDA content, resulting in a lower decay incidence and retarding postharvest fruit senescence of Xinyu tangerines. Similarly, other botanical preservatives, as alternatives to synthetic fungicides, have been applied to improve SOD and POD activity in Xinyu tangerines [2]. Wang and co-workers [34] also found that the co-fermentation of probiotics and Chinese herbs, as a novel liquid fermentation compound (LFC), induced the elevation of SOD, PPO, PAL, and POD activities in citrus fruits. Meanwhile, an interesting finding in the current study is that postharvest treatment with P. brasilensis YS-1 could markedly delay the oxidation of VC (ascorbic acid, AsA) and maintain its high level in harvested citrus fruits (Table1). As an important nutrient, VC has potent antioxidant capacity to eliminate ROS and reduce oxidative cellular damage in citrus fruit in postharvest storage, implying that a higher VC content could delay the process of citrus fruit senescence and ultimately prolong its storage life [28,39]. Similar results were reported for different fruits (litchis, strawberries, pears, apples, and jujubes) treated with other antagonistic microorganisms, such as P. luminescens Hb1029 [35], B. amyloliquefaciens LY-1 [36], Lactobacillus plantarum [45], B. amyloliquefaciens L-1 [46], Sporidiobolus pararoseus Y16 [47], and R. paludigenum [44]. Hence, the results in our current study indicate that the treatment of P. brasilensis YS-1 can effectively delay postharvest senescence and prolong the storage life of citrus fruits. This is probably due to the high levels of defensive enzymes and VC content in the P. brasilensis YS-1-treated fruits during the middle and late stage of the storage period at 5 ◦C. This may be due to a higher ROS scavenging capacity, contributing to the prevention of oxidative damage that begins during fruit senescence due to membrane lipid peroxidation.

5. Conclusions The results led us to conclude that the use of P. brasilensis YS-1 retarded the postharvest senescence of citrus fruit. Moreover, P. brasilensis YS-1-treated fruits showed a lower decay incidence, weight loss, respiration rate and MDA content, and postponed the degradation of TSS, total sugar, TA, and VC. This contributed to maintaining higher SOD, POD, PPO, and PAL activities. Taking all the data together, P. brasilensis YS-1 could be considered as a potential biocontrol agent for the postharvest preservation of citrus fruit. However, the applicability and commercialized production of P. brasilensis YS-1 need to be investigated.

Author Contributions: Conceptualization, J.C.; methodology, C.C. and J.G.; validation, C.C., C.W., J.G. and J.C.; formal analysis, C.C.; data curation, J.G.; writing—original draft preparation, C.C.; writing—review and editing, C.W.; visualization, C.C.; supervision, C.W.; project administration, J.C.; funding acquisition, J.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Foundation of China (grant no. 31760598), Jiangxi 2011 Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables (grant no. JXGS-03) and the Modern Agricultural Citrus Industrial Technology System of Jiangxi, China (grant no. JXARS-07). Conflicts of Interest: The authors declare no conflict of interest. Agriculture 2020, 10, 330 11 of 13

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