Osbeck Cv Research Articles

Excessive boron fertilization-induced toxicity is related to boron transport in field-grown pomelo trees.

Boron (B) is an essential micronutrient for plant growth and development; however, the process of B toxicity in citrus production is still poorly understood. We proposed a hypothesis that B toxicity in citrus trees is related to the characteristics of B transport from soil to leaf or fruit. For this, a field experiment was conducted for two treatments, control (B free or without B) and B fertilizer treatment (100 g Na2B4O7·10H2O plant-1), to investigate the effects on plant growth, nutrient uptake, fruit yield and quality, and B transport in 10-year-old pomelo trees [Citrus grandis (L.) Osbeck cv. Guanximiyou]. Our results showed that excess B fertilization directly led to B toxicity in pomelo trees by dramatically increasing soil total B and water-soluble B contents. B toxicity induced interveinal chlorosis in leaves and decreased leaf biomass and function, resulting in a decreased 45.3% fruit yield by reducing 30.6% fruit load and 21.4% single fruit weight. Also, B toxicity induced changes in mineral elements between leaf positions and fruit parts, in which the concentrations of B, potassium, and magnesium were increased while those of nitrogen and iron were decreased. Under B toxicity conditions, fruit quality parameters of total soluble solids (TSS), TSS/titratable acidity (TA), total soluble sugar, sucrose, pH, vitamin C, and total phenol contents decreased, which were regulated by the lower carbohydrate production in new leaves and the lower transport capacity in old leaves. Moreover, B toxicity significantly increased the transfer factor and bio-concentration factor of B in pomelo plants, with higher levels in leaf organs than in fruit organs. Taken together, excess B fertilization-induced B toxicity in pomelo trees, with induced growth inhibition and nutrient disorder, results in reduced fruit yield and quality, which are related to B transport from soil to organs. The findings of this study highlight the understanding of B toxicity in citrus plants and strengthen B management in pomelo production for high yield and high quality.

The underlying mechanisms by which boron mitigates copper toxicity in Citrus sinensis leaves revealed by integrated analysis of transcriptome, metabolome and physiology.

Both copper (Cu) excess and boron (B) deficiency are often observed in some citrus orchard soils. The molecular mechanisms by which B alleviates excessive Cu in citrus are poorly understood. Seedlings of sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) were treated with 0.5 (Cu0.5) or 350 (Cu350 or Cu excess) μM CuCl2 and 2.5 (B2.5) or 25 (B25) μM HBO3 for 24wk. Thereafter, this study examined the effects of Cu and B treatments on gene expression levels revealed by RNA-Seq, metabolite profiles revealed by a widely targeted metabolome, and related physiological parameters in leaves. Cu350 upregulated 564 genes and 170 metabolites, and downregulated 598 genes and 58 metabolites in leaves of 2.5μM B-treated seedlings (LB2.5), but it only upregulated 281 genes and 100 metabolites, and downregulated 136 genes and 40 metabolites in leaves of 25μM B-treated seedlings (LB25). Cu350 decreased the concentrations of sucrose and total soluble sugars and increased the concentrations of starch, glucose, fructose and total nonstructural carbohydrates in LB2.5, but it only increased the glucose concentration in LB25. Further analysis demonstrated that B addition reduced the oxidative damage and alterations in primary and secondary metabolisms caused by Cu350, and alleviated the impairment of Cu350 to photosynthesis and cell wall metabolism, thus improving leaf growth. LB2.5 exhibited some adaptive responses to Cu350 to meet the increasing need for the dissipation of excessive excitation energy (EEE) and the detoxification of reactive oxygen species (reactive aldehydes) and Cu. Cu350 increased photorespiration, xanthophyll cycle-dependent thermal dissipation, nonstructural carbohydrate accumulation, and secondary metabolite biosynthesis and abundances; and upregulated tryptophan metabolism and related metabolite abundances, some antioxidant-related gene expression, and some antioxidant abundances. Additionally, this study identified some metabolic pathways, metabolites and genes that might lead to Cu tolerance in leaves.

Adaptive Responses of Hormones to Nitrogen Deficiency in Citrus sinensis Leaves and Roots.

Some citrus orchards in China often experience nitrogen (N) deficiency. For the first time, targeted metabolomics was used to examine N-deficient effects on hormones in sweet orange (Citrus sinensis (L.) Osbeck cv. Xuegan) leaves and roots. The purpose was to validate the hypothesis that hormones play a role in N deficiency tolerance by regulating root/shoot dry weight ratio (R/S), root system architecture (RSA), and leaf and root senescence. N deficiency-induced decreases in gibberellins and indole-3-acetic acid (IAA) levels and increases in cis(+)-12-oxophytodienoic acid (OPDA) levels, ethylene production, and salicylic acid (SA) biosynthesis might contribute to reduced growth and accelerated senescence in leaves. The increased ethylene formation in N-deficient leaves might be caused by increased 1-aminocyclopropanecarboxylic acid and OPDA and decreased abscisic acid (ABA). N deficiency increased R/S, altered RSA, and delayed root senescence by lowering cytokinins, jasmonic acid, OPDA, and ABA levels and ethylene and SA biosynthesis, increasing 5-deoxystrigol levels, and maintaining IAA and gibberellin homeostasis. The unchanged IAA concentration in N-deficient roots involved increased leaf-to-root IAA transport. The different responses of leaf and root hormones to N deficiency might be involved in the regulation of R/S, RSA, and leaf and root senescence, thus improving N use efficiency, N remobilization efficiency, and the ability to acquire N, and hence conferring N deficiency tolerance.

Genomic basis of metabolome-mediated cultivar-specific flavor formation in juice sacs of the pomelo (Citrus grandis (L.) Osbeck) cultivars Shatian and Guanxi honey

Shatian pomelo (Citrus grandis (L.) Osbeck cv. Shatian) (ST) is one of the main cultivars of pomelo, and its flavor, aroma and mastication traits exhibit marked specificity compared to those of another major pomelo cultivar, Guanxi honey pomelo (Citrus grandis (L.) Osbeck cv. Guanxi honey) (BR). As parents, both cultivars generated many offspring cultivars with excellent quality. However, the limited study of pomelo flavor-specific metabolites and their metabolic pathways has hindered the understanding of pomelo flavor diversity. In this work, genome resequencing and metabolome analyses were performed to reveal the differences in cultivar-specific flavor metabolic pathways and cultivar-specificity formation molecular bases in taste flavor, aroma and mastication. A total of 397,056 and 427,885 SNP-InDels were found, with 53.67% and 54.76% of the SNP-InDels located in the gene intervals in ST and BR, respectively. 148 candidate genes distributed in carbohydrate, amino acid, flavonoid, lipid, terpene and lignin metabolism pathways were screened to uncover the molecular basis of flavor, aroma and mastication trait formation biological processes. Overall, revealing the origin of the cultivar-specific flavor of ST and BR may provide novel insight into the pomelo flavor formation mechanism and genetic resources for pomelo genetic breeding.

Oviposition by the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), on five citrus types in a laboratory

AbstractFrugivorous insects use visual, chemical and tactile cues to find a suitable host for oviposition. However, these cues can vary greatly among fruit cultivars and condition, changing their susceptibility to fruit fly oviposition. The aims of this study were to (1) determine the effects of ripeness stage and damage on oviposition propensity by sexually mature, mated female oriental fruit flies, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), on five citrus types (species and cultivars) under choice and no‐choice conditions and (2) describe the oviposition behaviour of B. dorsalis on ripe fruit of the same five citrus types that were either damaged or undamaged under no‐choice conditions. All tests were conducted in the laboratory. The citrus types tested were Citrus sinensis (L.) Osbeck cv Delta Valencia orange, Citrus sinensis (L.) Osbeck cv Glen Ora Late navel orange, Citrus limon (L) (Burm.f.) cv Eureka lemon, Citrus paradisi (Macfad.) cv Star Ruby grapefruit and Citrus reticulata (Blanco) cv Nadorcott mandarin. Peel physical properties and essential oil composition were determined for each citrus type and stage. Oviposition propensity of B. dorsalis was significantly greater on damaged citrus but was not correlated with fruit diameter, peel thickness, oil gland density or oil gland size. A total of 45 aromatic compounds were found to be significant between the five cultivars investigated, and 6 of 11 compounds were significantly associated with over‐ripe fruits. Bactrocera dorsalis spent a significantly greater proportion of time ovipositing in damaged citrus and showed higher aggression when oviposition occurred in undamaged citrus. These results suggest that the removal of damaged and fallen fruit is important for controlling this pest in citrus orchards.

First Report of Postharvest Fruit Rot on Gannan Navel Orange Caused by Diaporthe unshiuensis in Jiangxi Province, China.

The Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is one of the most widely planted citrus fruit cultivars in Ganzhou City, Jiangxi Province, China. A Gannan navel orange was harvested from an orchard in Yudu County, Ganzhou City, Jiangxi Province, China, in October 2022 (25.95N, 115.41E). Approximately 5% of the fruit rotted after being stored at room temperature for about two weeks.Infected fruits appear brown and rotted with slightly indented edges. Initially symptoms of infected fruits was small circular, light brown, which the rot expands, slightly water-stained halo circle with slightly indented edges. The surface of 10 infected fruits was sterilized with 75% ethanol, and the lesion edge was cut into 5-mm-diameter pieces, and the pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for five days. A total of eight morphologically similar isolates were obtained. PDA results showed dense white and fluffy aerial mycelia in the center of colonies with sparser edges. Two types of conidia were produced; the alpha conidia were hyaline, ellipsoidal or clavate, and aseptate, with 2 oil drops, 4.8 to 7.5 × 2.1 to 2.7 μm (n=30). The beta conidia were hyaline, aseptate, filiform, smooth, straight to sinuous, 16.9 to 27.5 × 1.3 to 1.6 μm (n=30). These isolates exhibit morphological characteristics similar to those of the Diaporthe genus. Genomic DNA of two representative isolates (JFRL-03-1130 and JFRL-03-1131) was extracted for further confirmation. The internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS3) genes were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b, respectively (Udayanga et al. 2015). These nucleotide sequences were deposited into the GenBank database with accession numbers OQ691637-OQ691638 (ITS), OQ701022-OQ701023 (TUB), OQ701016-OQ701017 (CAL), OQ701018-OQ701019 (TEF1-α) and OQ701020-OQ701021 (HIS3). The maximum likelihood analyses were performed for the combined ITS, TEF1-a, TUB, HIS3, and CAL data set using Phylosuite V1.2.2 (Zhang et al. 2020). The phylogenetic tree showed that the two isolates clustered with D. unshiuensis in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. unshiuensis based on morphological and molecular characteristics. To evaluate pathogenicity, a sterile scalpel was used to wound 10 surface-sterilized fruits, and a 5-mm-diameter mycelial plug of the isolate JFRL 03-1130, cultured on PDA at 25℃ for 7-days, was put on the wound. Another set of 10 fruits was similarly inoculated with sterile agar plugs as controls. The fruits were cultured at 25°C and 85% relative humidity, and the test was repeated twice. These fruits inoculated with D. unshiuensis showed similar rot symptoms after 10 days, while the control group remained symptomless. In order to prove Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as D. unshiuensis by molecular techniques, but never from the control fruits. Diaporthe unshiuensis has been reported as an endophyte associated with citrus and a pathogen that causes melanose disease in citrus (Chaisiri et al. 2020; Huang et al. 2015). However, to the best of our knowledge, this is the first reported case of D. unshiuensis causing postharvest fruit rot on Citrus sinensis. In the past, D. sojae has also been reported causing postharvest fruit brown rot disease on Citrus sinensis in China (Xiao, et al. 2023); Therefore, managers should pay more attention to postharvest fruit rot disease caused by Diaporthe species and implement storage strategies to control and reduce losses.

Impact of Integrated Nutrient Management on Growth, Yield and Quality Attributes of Sweet Orange (Citrus Sinensis L. Osbeck) CV. Phule Mosambi

Effects of integrated nutrient management (INM) approach on the growth, yield, and quality of sweet orange [Citrus sinensis (L.) Osbeck] cv. Phule Mosambi was investigated. Eleven treatments were imposed in Complete Randomized Design (CRD) for two seasons (2019 and 2020) and among the treatments, T11 [50% RDN + 20kg FYM/tree + 25% N from Castor Cake + 10ml Bio NPK Consortium + Micronutrient foliar spray (1% Grade IV)] recorded the maximum incremental plant height (35.28 cm), plant spread N-S (45.73 cm), plant spread E-W (45.32 cm) and canopy volume (0.061m3). Similarly treatment T11 significantly increased several yield parameters viz., the maximum fruit length (8.11cm), fruit diameter (7.96 cm), fruit weight (241.91 g), fruit volume (251.17 cc), no. of fruits per tree (255.17), yield (61.81 kg per tree and 17.18 t/ha) in pooled data. Amid quality traits, T11 stood significantly superior with respect to total sugar (6.99 %) content; it also showed higher sugar: acid ratio (14.27), ascorbic acid content (40.11 mg/100 ml pulp). Hence this particular treatment can be adopted preferably in sweet orange cultivation at middle Gujarat (India). Bangladesh J. Bot. 52(2): 345-355, 2023 (June)

Metabolomic and transcriptomic analyses reveal the effects of grafting on blood orange quality.

Blood orange (Citrus sinensis L.) is a valuable source of nutrition because it is enriched in anthocyanins and has high organoleptic properties. Grafting is commonly used in citriculture and has crucial effects on various phenotypes of the blood orange, including its coloration, phenology, and biotic and abiotic resistance. Still, the underlying genetics and regulatory mechanisms are largely unexplored. In this study, we investigated the phenotypic, metabolomic, and transcriptomic profiles at eight developmental stages of the lido blood orange cultivar (Citrus sinensis L. Osbeck cv. Lido) grafted onto two rootstocks. The Trifoliate orange rootstock provided the best fruit quality and flesh color for Lido blood orange. Comparative metabolomics suggested significant differences in accumulation patterns of metabolites and we identified 295 differentially accumulated metabolites. The major contributors were flavonoids, phenolic acids, lignans and coumarins, and terpenoids. Moreover, transcriptome profiling resulted in the identification of 4179 differentially expressed genes (DEGs), and 54 DEGs were associated with flavonoids and anthocyanins. Weighted gene co-expression network analysis identified major genes associated to 16 anthocyanins. Furthermore, seven transcription factors (C2H2, GANT, MYB-related, AP2/ERF, NAC, bZIP, and MYB) and five genes associated with anthocyanin synthesis pathway (CHS, F3H, UFGT, and ANS) were identified as key modulators of the anthocyanin content in lido blood orange. Overall, our results revealed the impact of rootstock on the global transcriptome and metabolome in relation to fruit quality in lido blood orange. The identified key genes and metabolites can be further utilized for the quality improvement of blood orange varieties.

P-Synephrine Indicates Internal Maturity of Citrus grandis (L.) Osbeck cv. Mato Peiyu-Reclaiming Functional Constituents from Nonedible Parts.

The processing of Citrus grandis Osbeck cv. Mato Peiyu (CGMP) fruits generates a considerable amount of waste, mainly the flavedo, albedo, and segment membrane; the generated waste yields severe environmental and economic challenges. In this study, we tried to reclaim some functional chemicals from the waste. Our data indicated that the essential oil content in the flavedo was 0.76-1.34%, with the major component being monoterpenes (93.75% in August, declining to 85.56% in November, including mainly limonene (87.08% to 81.12%) and others such as β-myrcene). p-Synephrine (mg/100 g dry weight) declined accordingly (flavedo, 10.40 to 2.00; albedo, 1.80 to 0.25; segment membrane, 0.3 in August, 0.2 in September, and none since October). Polyphenols (in μg/g) included gallic acid (70.32-110.25, 99.27-252.89, and 105.78-187.36, respectively); protocatechuic acid (65.32-204.94, 26.35-72.35, and 214.98-302.65, respectively), p-coumaric acid (30.63-169.13, 4.32-17.00, and 6.68-34.32, respectively), ferulic acid (12.36-39.36, 1.21-10.25, and 17.07-39.63, respectively), and chlorogenic acid (59.19-199.36, 33.08-108.57, and 65.32-150.14, respectively). Flavonoids (in μg/g) included naringin (flavedo, 89.32-283.19), quercetin (181.05-248.51), nobiletin (259.75-563.7), hesperidin, and diosmin. The phytosterol content (mg/100 g) was 12.50-44.00 in the flavedo. The total dietary fiber in the segment membrane was 57 g/100 g. The antioxidant activity against the DPPH• and ABTS+• free radicals was moderately high. In conclusion, the waste of CGMP fruits is worth reclaiming for essential oil, p-synephrine, polyphenolics, and dietary fiber. Notably, p-synephrine content (flavedo: <8 mg/100 g dry weight, albedo: <2.0, or segment membrane: <0.4 mg) can serve as a marker of the internal maturation of CGMP fruits.

Efficacy of an existing Ceratitis capitata (Diptera: Tephritidae) cold disinfestation treatment schedule for Ceratitis cosyra.

The efficacy of an existing cold disinfestation postharvest treatment targeting Ceratitis capitata (Wiedemann) in citrus was determined for the marula fly, Ceratitis cosyra (Walker). The cold tolerances of C. capitata and C. cosyra were first quantified in artificial diet at 3.5 °C at different exposure periods for up to 18 days. Ceratitis capitata was found to be more cold tolerant than C. cosyra. At 3.5 °C, the duration to achieve 99.9968% mortality was calculated to be 11.57 days for C. capitata and 9.10 days for C. cosyra. Under an existing C. capitata cold treatment schedule at 1 °C for 14 days, the conditions required for complete mortality of the third larval stage of C. cosyra in orange, Citrus sinensis (L.) Osbeck cv. Valencia, were then determined. No survivors of C. cosyra in oranges were recorded beyond 11 days of cold treatment at 1 °C. The efficacy of this C. capitata treatment for disinfestation of C. cosyra was thereafter confirmed in large scale trials in Valencia oranges. In the large-scale trial at the lowest mean temperature of 1.19 °C for 14 days, there were no survivors from a total of 85 490 treated C. cosyra third instars in oranges. Since C. capitata was shown to be more cold tolerant than C. cosyra and a large scale test demonstrated at least 99.9965% efficacy after 14 days at 1.19 °C, compared with the established effective C. capitata cold treatment of 14 days at 1.11 °C, cold disinfestation treatments for C. capitata should be at least equally effective against C. cosyra.

First Report of Brown Rot Caused by Phytophthora nicotianae on Navel Orange Fruit in China.

Navel orange (Citrus sinensis Osbeck cv. Newhall) is widely planted in southern China. From September to November 2021, severe outbreaks of Phytophthora brown rot were observed on navel orange fruit in three local orchards in Ganzhou City (28.80N, 115.53E), Jiangxi Province, China, with a disease incidence of 25 to 35%. Symptomatic fruit was mostly observed 1-m from the ground. Initial symptoms on infected fruit were circular, pale-brown to brown, water-soaked, slightly sunken lesions, covered with sparse white mycelia-like growth. As the disease progressed, the lesions turned dark brown and enlarged on the fruit surface. Three to four infected fruits were randomly collected from each orchard, placed in transparent plastic bags and immediately brought back to the laboratory for isolations. Infected fruits were surface-disinfested with 70% ethanol for 60 sec, and rinsed three times with sterile water. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 mm × 5 mm pieces, placed onto potato dextrose agar and incubated at 28°C for 5 days. Nine isolates were obtained. Colonies of three isolates (JFRL 03-16, 03-18, 03-19) in 10-day-old 20% V8 juice agar consisted of abundant, white, cottony aerial mycelia. Hyphal swellings and coenocytic mycelium were observed. Sporangia were ovoid, ellipsoid to spherical, papillate, and ranged in size from 17.2 to 60.1 µm × 15.8 to 48.6 µm (x ̅=46.2 ± 5.5 × 32.4 ± 4.8 µm, n=50). Chlamydospores were spherical, suborbicular, and ranged from 17.8 to 45.9 µm diam (x ̅=30.5 ± 3.5 µm, n=50). Oospores were not observed in pure cultures. These morphological characteristics were consistent with those of P. nicotianae (LaMondia et al. 2014). Genomic DNA was extracted from a representative isolate, JFRL 03-18, using the NuClean Plant Genomic DNA kit (CWBIO, China). The internal transcribed spacer (ITS) region, ras-related protein ypt1 (YPT), β-tubulin (TUB) gene were amplified by Polymerase Chain Reaction using primers ITS1/ITS4 (White et al. 1990), Yph1F/Yph2R (Schena et al. 2008), and TUBUF2/TUBUR1 (Kroon et al. 2004), respectively. All sequences were deposited in GenBank (Accession Nos. ON231777 for ITS, ON246910 for YPT, ON246908 for TUB). BLASTN homology search for these nucleotide sequences showed 100% identical to the ITS (MH341621), YPT (MK058408), TUB (MH760160) sequences of P. nicotianae. Sequences of twelve Phytophthora species and Pythium ostracodes were downloaded from GenBank. The phylogenetic tree of combined ITS, YPT, TUB sequences showed that the isolate JFRL 03-18 clustered with P. nicotianae. To complete Koch's postulates, zoospore suspensions were prepared from the cultures grown on 10-day-old V8 juice agar of isolates (JFRL 03-16, 03-18, 03-19). Pathogenicity tests were performed on healthy and surface-disinfested navel orange fruit. Nine fruits were gently wounded with a needle, inoculated with 10 µl zoospore suspension (104 zoospores/ml) of three isolates separately, and three fruit treated with sterilized water as controls. All fruit were incubated at 25℃ with 80% relative humidity and the test was repeated three times. After 7 days of incubation, the fruit inoculated with P. nicotianae showed similar brown rot symptoms and the control fruit remained symptomless. The pathogen was re-isolated from all inoculated fruits and confirmed as P. nicotianae by morphological and molecular analysis. Phytophthora nicotianae was previously reported on Hamlin sweet orange (Citrus sinensis (L.) Osbeck) fruit causing Phytophthora brown rot in Florida (Graham and Timmer 1995; Hao et al. 2018). To our knowledge, this is the first report of P. nicotianae causing Phytophthora brown rot of navel orange fruit in China. Based on the severity of this disease, local growers should develop and implement integrated disease management strategies for control.

First Report of Postharvest Fruit Brown Rot Disease on Navel Orange Caused by Diaporthe sojae in China.

In October 2020, a postharvest fruit brown rot symptom was observed on navel orange (Citrus sinensis Osbeck cv. Newhall) fruits in a local fruit market in Ganzhou, Jiangxi Province, China. The disease incidence increased up to 15% in 40 fruits with a 7-day-long storage at room temperature. The disease symptoms on the infected fruit were circular, light brown to brown, slightly sunken lesions, covered with whitish mycelium mass, and brown rot in the center. To isolate the causal organism, infected fruits were surface sterilized with 1% NaClO solution for 30 sec, and rinsed thrice with sterilized water. Symptomatic tissues at the margins were cut into 5-mm2 pieces, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. Thirteen morphologically similar single-spore fungal isolates were obtained from the isolation experiment. Fungal colonies were white, fluffy, cottony texture, reverse buff to light yellow, with black stromata at maturity. Alpha conidia were hyaline, aseptate, ellipsoid to clavate, tapering towards the ends, often biguttulate, and ranged in size from 6.8 to 9.8 µm × 2.7 to 4.5 µm (n=50). Beta conidia were hyaline, aseptate, smooth, straight to sinuous, and with size ranging from 12.1 to 21.3 µm × 0.9 to 2.2 µm (n=50). Morphological features were consistent with those of Diaporthe sojae (Dissanayake et al. 2015). For molecular identification, DNA was extracted from the representative isolate JFRL 03-13, the internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS) genes were amplified by using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b (Udayanga et al. 2015), respectively. The resulting sequences were deposited in GenBank (Accession Nos. OM281710 for ITS, OM289961 for TUB, OM289964 for CAL, OM289963 for TEF1-α, and OM289962 for HIS). BLAST analysis revealed that these sequences were 100% similar to the sequences of ITS (MN816426), TUB (MK941336), CAL (MN894375), TEF1-α (MN894447), HIS (MN894409) published for D. sojae. Phylogenetic analysis was conducted based on the concatenated sequences (ITS, TUB, CAL, TEF1-α, and HIS) by Maximum likelihood analysis (ML) and Bayesian inference (BI) using IQtree v.1.6.11 and MrBayes v.3.2.7 (Guo et al. 2020). The phylogenetic tree showed that the isolate clustered with D. sojae. To confirm pathogenicity, mature and healthy harvested fruits of navel orange (Citrus sinensis Osbeck cv. Newhall) were surface sterilized. Ten fruits were wounded by a sterile scalpel and put a 7-mm-diamter agar plug with 7-day-old mycelium of the isolate JFRL03-13 cultured on PDA at 25°C, noncolonized PDA plugs were used as the control. Inoculated fruits were incubated at 25℃ with 80% relative humidity. After 10 days, the similar symptoms were observed on the inoculated sites and spread on the surface of fruits, whereas the control remained symptomless. The pathogen was re-isolated from the lesions of inoculated fruits and confirmed as D. sojae via morphological and molecular analysis. The assays were repeated twice, fulfilling the Koch's postulates. Although D. sojae is known as the major causative agent of pod and stem blight, and has been reported as an endophyte in the twigs and leaves of citrus (Huang et al. 2015; Santos et al. 2011), but to our knowledge, this is the first report of postharvest fruits brown rot disease on navel orange caused by D. sojae in China. However, further investigation of the specific causes of this disease is necessary to help the local fruit farmers develop effective disease management strategies.

The Effects of Storage Temperature, Light Illumination, and Low-Temperature Plasma on Fruit Rot and Change in Quality of Postharvest Gannan Navel Oranges.

Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is an economically important fruit, but postharvest loss occurs easily during storage. In this study, the effects of different temperatures, light illuminations, and low-temperature plasma treatments on the water loss and quality of the Gannan navel orange were investigated. The fruit began to rot after 90 d of storage at 5 °C and 20-45 d at 26 °C. Navel oranges stored at 26 °C had 7.2-fold and 3.1-fold higher rates of water loss at the early and late storage stages, respectively, as compared with those stored at 5 °C. Storage at 5 °C decreased the contents of total soluble solids at the early storage stage and the contents of titratable acids at the late storage stage, whereas storage at 26 °C decreased the contents of total soluble solids at the late storage stage and the contents of titratable acids at the early storage stage, respectively. Application of low-temperature plasma produced by air ionization for 6 min, or continuous blue or red light illumination significantly inhibited water loss within 7 and 21 d of storage at 22 °C, respectively, but exhibited no significant effect on fruit quality. Furthermore, the low-temperature plasma treatment protected against fruit rot. Thus, treatment with low-temperature plasma followed by storage at a low temperature under continuous red or blue light illumination was of potential value as a green technology for preserving Gannan navel orange during storage.

Reducing Macronutrients and Increasing Micronutrient Fertilizers Are Key to Improving the Quality of Pomelo Citrus grandis (L.) Osbeck Cv. “Guanxi”

Due to long-term excessive fertilization, the fruit quality of the Guanxi pomelo (Citrus grandis) has been declining. The classification of fruit quality and its relationship with nutrients in soils and trees were studied to declaim the characteristics of nutrients in Guanxi pomelo orchards, ultimately guiding the fertilization for high-quality fruit production. By cluster analysis, 281 samples were grouped into four clusters. High-quality fruit (HF) showed a gourd shape with moderate weight size, high sweetness, edible rate (ER) up to 73%, and vitamin C content over 40 mg/100 g. Compared to sub-quality fruit (SF), common fruit (CF), and inferior fruit (IF), the content of magnesium (Mg) in the leaves of HF was 11.76, 11.76, and 18.75% higher, while the content of iron (Fe) was 6.45, 5.76 and 10.97% higher, respectively. Meanwhile, the contents of Zinc (Zn, 8.51, 6.44, and 11.22% higher than SF, CF, and IF, respectively) and Boron (B, 13.47, 13.83, and 25.40% higher than SF, CF, and IF, respectively) were also found to increase. However, the contents of Mn (35.34, 27.51, and 25.53% higher) and Cu (31.90, 31.99, and 5.64% higher) in IF were significantly higher than in HF, SF, and CF. Acid soils (4.24–4.40) with low OM content (23.00–26.71 g kg−1) led to an imbalance uptake of nutrients of citrus, ultimately resulting in poor quality. These results indicated that farmers should reduce the application of nitrogen (N), phosphorus (P), and K fertilizer and pesticides and increase micronutrient and organic fertilizer, which provides a theoretical basis for scientific fertilization to improve the fruit quality of Guanxi pomelo (Citrus grandis) of Pinghe County.

Characterization of copper-induced-release of exudates by Citrus sinensis roots and their possible roles in copper-tolerance

Copper (Cu) excess is often observed in old Citrus orchards. Little information is available on the characterization of Cu-induced-release of root exudates and their possible roles in plant Cu-tolerance. Using sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings as materials, we investigated the impacts of 0, 0.5, 25, 150, 350, 550, 1000, 2000 or 5000 μM CuCl2 (pH 4.8) on Cu uptake, root exudates [malate, citrate, total phenolics (TP), total soluble sugars (TSS) and total free amino acids (TFAA)], electrolyte leakage and malondialdehyde, and solution pH under hydroponic conditions; the time-course of root exudates and solution pH in response to Cu; and the impacts of protein synthesis and anion-channel inhibitors, and temperature on Cu-induced-secretion of root exudates and solution pH. About 70% of Cu was accumulated in 0 and 0.5 μM Cu-exposed roots, while over 97% of Cu was accumulated in ≥25 μM Cu-exposed roots. Without Cu, the seedlings could alkalize the solution pH from 4.8 to above 6.0. Cu-stimulated-secretion of root exudates elevated with the increment of Cu concentration from 0 to 1000 μM, then decreased or remained unchanged with the further increment of Cu concentration, while root electrolyte leakage and malondialdehyde (root-induced alkalization) increased (lessened) with the increment of Cu concentration from 0 to 5000 μM. Further analysis indicated that Cu-stimulated-secretion of root exudates was an energy-dependent process and could repressed by inhibitors, and that there was no discernible delay between the onset of exudate release and the addition of Cu. To conclude, both root-induced alkalization and Cu-stimulated-release of root exudates played a key role in sweet orange Cu-tolerance via increasing root Cu accumulation and reducing Cu uptake and phytotoxicity.

Vermiculite and shading in the formation of 'Rangpur' lime rootstock

‘Rangpur’ lime (Citrus limonia Osbeck cv. Cravo) is widely used as a rootstock for citrus seedlings, although factors such as luminosity and substrate composition are extremely important to obtain quality seedlings. The objective of this study was to analyze the formation of ‘Rangpur’ lime seedlings grown in different substrates and luminosities. A 3x4 factorial arrangement was used in an RBD with four replications of twelve plants. Three luminosities (full sun, 35%, and 70% shading) and four substrate: vermiculite proportions were tested (S1-1: 0, S2-2: 1, S3-1: 2, and S4-0: 1). The seeds were sown in 280 cm3 tubes and emergence was evaluated every two days for 30 days, and biometric assessments at 30, 60, 90, and 120 DAE by measuring the height, diameter, leaves, and mortality. Root length and shoot and root dry mass were measured at 30 and 120 days. Seeds in full sun showed high ESI values and a shorter time period, as well as high mortality at 120 DAE. Highly shaded environments cause etiolation and reduce biomass accumulation and the DQI. Seeds in S4 showed lower results for most variables. It is recommended to use 35% shading associated with the substrate proportions S1, S2, and S3.

Effect of nutrient feeding on controlling leaf chlorosis, yield and physico-chemical composition of sweet orange (Citrus sinensis L. Osbeck) cv. Mosambi

Effect of nutrient feeding on controlling leaf chlorosis, yield and physico-chemical composition of sweet orange (Citrus sinensis L. Osbeck) cv. Mosambi

Molecular and Physiological Responses of Citrus sinensis Leaves to Long-Term Low pH Revealed by RNA-Seq Integrated with Targeted Metabolomics.

Low pH-induced alterations in gene expression profiles and organic acids (OA) and free amino acid (FAA) abundances were investigated in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] leaves. We identified 503 downregulated and 349 upregulated genes in low pH-treated leaves. Further analysis indicated that low pH impaired light reaction and carbon fixation in photosynthetic organisms, thereby lowering photosynthesis in leaves. Low pH reduced carbon and carbohydrate metabolisms, OA biosynthesis and ATP production in leaves. Low pH downregulated the biosynthesis of nitrogen compounds, proteins, and FAAs in leaves, which might be conducive to maintaining energy homeostasis during ATP deprivation. Low pH-treated leaves displayed some adaptive responses to phosphate starvation, including phosphate recycling, lipid remodeling, and phosphate transport, thus enhancing leaf acid-tolerance. Low pH upregulated the expression of some reactive oxygen species (ROS) and aldehyde detoxifying enzyme (peroxidase and superoxidase) genes and the concentrations of some antioxidants (L-tryptophan, L-proline, nicotinic acid, pantothenic acid, and pyroglutamic acid), but it impaired the pentose phosphate pathway and VE and secondary metabolite biosynthesis and downregulated the expression of some ROS and aldehyde detoxifying enzyme (ascorbate peroxidase, aldo-keto reductase, and 2-alkenal reductase) genes and the concentrations of some antioxidants (pyridoxine and γ-aminobutyric acid), thus disturbing the balance between production and detoxification of ROS and aldehydes and causing oxidative damage to leaves.

Boron-mediated amelioration of copper-toxicity in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings involved reduced damage to roots and improved nutrition and water status

‘Xuegan’ (Citrus sinensis) seedlings were fertilized 6 times weekly for 24 weeks with 0.5 or 350 μM CuCl2 and 2.5, 10 or 25 μM H3BO3. Cu-toxicity increased Cu uptake per plant (UPP) and Cu concentrations in leaves, stems and roots, decreased water uptake and phosphorus, nitrogen, calcium, magnesium, potassium, sulfur, boron and iron UPP, and increased the ratios of magnesium, potassium, calcium and sulfur UPP to phosphorus UPP and the ratios of leaf magnesium, potassium and calcium concentrations to leaf phosphorus concentration. Many decaying and dead fibrous roots occurred in Cu-toxic seedlings. Cu-toxicity-induced alterations of these parameters and root damage decreased with the increase of boron supply. These results demonstrated that B supplementation lowered Cu uptake and its concentrations in leaves, stems and roots and subsequently alleviated Cu-toxicity-induced damage to root growth and function, thus improving plant nutrient (decreased Cu uptake and efficient maintenance of the other nutrient homeostasis and balance) and water status. Further analysis indicated that the improved nutrition and water status contributed to the boron-mediated amelioration of Cu-toxicity-induced inhibition of seedlings, decline of leaf pigments, large reduction of leaf CO2 assimilation and impairment of leaf photosynthetic electron transport chain revealed by greatly altered chlorophyll a fluorescence (OJIP) transients, reduced maximum quantum yield of primary photochemistry (Fv/Fm), quantum yield for electron transport (ETo/ABS) and total performance index (PIabs,total), and elevated dissipated energy per reaction center (DIo/RC). To conclude, our findings corroborate the hypothesis that B-mediated amelioration of Cu-toxicity involved reduced damage to roots and improved nutrient and water status. Principal component analysis showed that Cu-toxicity-induced changes of above physiological parameters generally decreased with the increase of B supply and that B supply-induced alterations of above physiological parameters was greater in 350 μM Cu-treated than in 0.5 μM Cu-treated seedlings. B and Cu had a significant interactive influence on C. sinensis seedlings.

Magnesium absorption, translocation, subcellular distribution and chemical forms in citrus seedlings.

Magnesium (Mg) is an essential macronutrient for plant growth and development; however, the adaptive mechanisms of Mg deficiency to underlying changes in Mg translocation, subcellular distribution and chemical forms in citrus plants are unknown. In this study, we conducted a sand culture experiment with 0 (Mg-deficiency) or 2 (Mg-sufficiency) mmoll-1Mg2+ treatments to investigate the responses underlying Mg adaptability, as well as the resulting growth and Mg transport features in citrus seedlings [Citrus sinensis (L.) Osbeck cv. 'Xuegan']. We found that Mg-deficiency significantly depressed biomass by 39% in the whole plant and by 66% in branch organs compared with Mg-sufficient conditions, which further resulted in a subsequent decrease in Mg concentration and accumulation with changes in its distribution in different organs and a reduction in root growth. Under Mg-sufficiency, >50% of Mg was sequestered in the soluble fraction and this was reduced by 30% under Mg-deficiency. Furthermore, >70% of Mg existed as inorganic (42%) and water-soluble (31%) forms with high mobility across treatments and organs. Under Mg-deficiency, the proportion of water-soluble Mg was reduced in leaf and increased in root, whereas the proportion of inorganic Mg increased in main stem leaves and decreased in branch leaves and root. However, under Mg-deficiency, the proportion of Mg forms with low mobility, including pectates and proteins, phosphates, oxalates and residues, was increased in leaf and root organs, with the exception of pectate and protein Mg, which was decreased in root. The Mg transfer factor showed that Mg-deficiency improved Mg transport from parent to branch organs, which was related to Mg subcellular distribution and chemical forms. Taken together, our study establishes a defined process to clarify the mechanisms of Mg absorption and translocation and reveals a possible strategy to effectively improve Mg mobility and availability in citrus plants.