Hydrogen sulfide enhances kiwifruit resistance to soft rot by regulating jasmonic acid signaling pathway.

As the third active gas signal molecule in plants, hydrogen sulfide (H2S) plays important roles in physiological metabolisms and biological process of fruits and vegetables during postharvest storage. In the present study, the effects of H2S on enhancing resistance against soft rot caused by Botryosphaeria dothidea and the involvement of jasmonic acid (JA) signaling pathway in kiwifruit during the storage were investigated. The results showed that 20 µL L-1 H2S fumigation restrained the disease incidence of B. dothidea-inoculated kiwifruit during storage, and delayed the decrease of firmness and the increase of soluble solids (SSC) content. H2S treatment increased the transcription levels of genes related to JA biosynthesis (AcLOX3, AcAOS, AcAOC2, and AcOPR) and signaling pathway (AcCOI1, AcJAZ5, AcMYC2, and AcERF1), as well as the JA accumulation. Meanwhile, H2S promoted the expression of defense-related genes (AcPPO, AcSOD, AcGLU, AcCHI, AcAPX, and AcCAT). Correlation analysis revealed that JA content was positively correlated with the expression levels of JA biosynthesis and defense-related genes. Overall, the results indicated that H2S could promote the increase of endogenous JA content and expression of defense-related genes by regulating the transcription levels of JA pathway-related genes, which contributed to the inhibition on the soft rot occurrence of kiwifruit.

Bioaccumulation mediated by water solubility leads to differences in the acute toxicity of organophosphorus insecticides to zebrafish (Danio rerio).

The use of some organophosphate insecticides is restricted or even banned in paddy fields due to their high toxicity to aquatic organisms. The aim of this study is to elucidate the main pathways and target organs of organophosphate insecticide toxicity to fish exposed via different routes by integrating histopathological and biochemical techniques. Using malathion as the model drug, when the dosage is 20-60 mg/L, the toxicity of whole body and head immersion drugs to zebrafish is much higher than that of trunk immersion drugs. A dose of 21.06-190.44 mg/kg of malathion feed was fed to adult zebrafish. Although the dosage was already high, no obvious toxicity was observed. Therefore, we believe that the drug mainly enters the fish body through the gills. When exposed to a drug solution of 20 mg/L and 60 mg/L, the fish showed significant neurological behavioral abnormalities, and the pathological damage to key organs and brain tissue was the most severe, showing obvious vacuolization and the highest residual amount (8.72-47.78 mg/L). The activity of acetylcholinesterase was the most inhibited (54.69-74.68%). Therefore, brain tissue is the key toxic target organ of malathion in fish. In addition, we compared the bioaccumulation effects of different water-soluble organophosphorus insecticides in fish and their toxic effects. We found that the higher the water solubility of organophosphorus insecticides, the lower their toxicity to fish.

Gum arabic coating alleviates chilling injury of cold-stored peach by regulating reactive oxygen species, phenolic, and sugar metabolism.

In this study, gum arabic (GA) coating was employed to mitigate chilling injury in peach fruit, and it was observed that 10% GA coating exhibited the most favorable effect. GA coating significantly inhibited the decline of AsA content and enhanced antioxidant enzyme activity in peach fruit, thereby enhancing reactive oxygen species (ROS) scavenging rate while reducing its accumulation. Simultaneously, GA coating inhibited the activity of oxidative degradation enzymes for phenolics and enhanced synthase activity, thus maintaining higher levels of total phenolics and flavonoids in fruits. Additionally, compared to the control fruit, GA-coated fruits demonstrated higher concentrations of sucrose and sorbitol, accompanied more robust activity of sucrose synthase and sucrose phosphate synthase, as well as reduced activity of acid invertase and neutral invertase. Our study demonstrates that GA coating can effectively enhance the cold resistance of peach fruit by regulating ROS, phenolics, and sugar metabolism, maintaining high levels of phenolics and sucrose while enhancing antioxidant activity.

Red light induces salicylic acid accumulation by activating CaHY5 to enhance pepper resistance against Phytophthora capsici.

Pepper (Capsicum annuum L.) is frequently challenged by various pathogens, among which Phytophthora capsici is the most devastating to pepper production. Red light signal acts as a positive induction of plant resistance against multiple pathogens. However, little is known about how the red light signal affects pepper resistance to P. capsici infection (PCI). Here, we report that red light regulates salicylic acid (SA) accumulation by activating elongated hypocotyl5 (CaHY5), a basic leucine zipper (bZIP) transcription factor, thereby decreasing pepper susceptibility to PCI. Exogenous SA treatment reduced pepper susceptibility to PCI, while silencing of CaPHYB (a red light photoreceptor) increased its susceptibility. PCI significantly induced CaHY5 expression, and silencing of CaHY5 reduced SA accumulation, accompanied by decreases in the expression levels of phenylalanine ammonia-lyase 3 (CaPAL3), CaPAL7, pathogenesis-related 1 (CaPR1), and CaPR1L, which finally resulted in higher susceptibility of pepper to PCI. Moreover, CaHY5 was found to activate the expression of CaPAL3 and CaPAL7, which are essential for SA biosynthesis, by directly binding to their promoters. Further analysis revealed that exogenous SA treatment could restore the resistance of CaHY5-silenced pepper plants to PCI. Collectively, this study reveals a critical mechanism through which red light induces SA accumulation by regulating CaHY5-mediated CaPAL3 and CaPAL7 expression, leading to enhanced resistance to PCI. Moreover, red light-induced CaHY5 regulates pepper resistance to PCI, which may have implications for PCI control in protected vegetable production.

First Report of Vacuiphoma oculihominis Causing Fruit Rot on Navel Orange (Citrus sinensis) in China.

Newhall navel orange [Citrus sinensis (L.) Osbeck] is an economically important agricultural product in China. In February 2022, a rare lesion symptom was observed on Newhall navel oranges that were harvested from an orchard Ganzhou city, Jiangxi province, China (25.53° N, 114.79° E) and stored for 90 days (18±2℃, 80 to 90% RH) at the Jiangxi Key Laboratory for Postharvest Technology and Non-destructive Testing of Fruits and Vegetables (28.68° N, 115.85° E). Approximately 2% (15/750) of the oranges exhibited symptoms, with normal appearance but ink-black flesh and juice, yellowish lesions on edges of the symptoms, and no unusual odor. To isolate the pathogen, three 5 × 5 mm pieces of symptomatic tissue from a diseased orange were disinfected in 75% ethanol for 30 s, rinsed three times with sterile water, and inoculated on potato dextrose agar (PDA) at 25±1℃ and a 12:12 h photoperiod for 7 days. A pure isolate named ND-hsp was obtained. The colony was light yellow center with pale edge on the top and brown on the bottom. Conidia and pycnidia were observed on PDA medium after 2 months. Conidia were long oval, no septa, 2.9 × 3.4 µm (n = 50), and pycnidia were solitary, 39.4 × 43.9 µm (n = 20), with one or no orifice, brown to dark brown. The morphological characteristics of ND-hsp strain on PDA, oatmeal agar and malt extract agar were similar to those of the Didymellaceae (Aveskamp et al. 2010). Ulteriorly, the genomic DNA of the ND-hsp isolate was extracted from its mycelia using a fungal genomic DNA extraction kit (Solarbio, Beijing, China) for subsequent phylogenetic analyses. Four primer sets, LR0R (Rehner and Samuels 1994) /LR7 (Vilgalys and Hester 1990), V9G (Hoog and Gerrits 1998) /ITS4 (White et al. 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/RPB2-7cR (Liu et al. 1999) were used to amplify the corresponding DNA fragments of large subunit ribosomal RNA (LSU), internal transcribed spacer region (ITS), beta-tubulin gene (TUB2) and RNA polymerase Ⅱ second largest subunit (RPB2), respectively. The obtained sequences were assigned GenBank accession numbers and showed 99 to 100% identity with their counterparts of Vacuiphoma oculihominis UTHSC DI16-308. A phylogenetic tree was constructed in MEGA 7.0 using the concatenated sequences, placing the isolate within the V. oculihominis clade by 100% bootstrap support. Pathogenicity experiments were performed in triplicate. Ten Newhall navel oranges were surface sterilized with 75% ethanol and inoculated with 15µL of a spore suspension (2×106 spores/ml) into a 3 mm-diameter wound on the equator. The control group received sterile water instead of the spore suspension. Treated and control oranges were incubated at 25±1 ℃ and about 90% relative humidity for 20 days. All oranges were cut longitudinally or transversely through the inoculated wound and examined internally. The oranges inoculated with ND-hsp exhibited ink-black flesh and juice symptoms consistent with the initial oranges. The control oranges remained asymptomatic. Under the Koch's rule, V. oculihominis was reisolated from diseased oranges and kept in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. GenBank database analysis confirms that V. oculihominis has been found in human eye secretions and decayed trees. This is the first report of V. oculihominis as a pathogen on navel oranges in China. Our findings contribute to understanding of citrus fruit pathogens.

First Report of Colletotrichum fructicola Causing Anthracnose on Paeonia lactiflora in China.

Paeonia lactiflora Pall is a traditional famous flower with long cultivated history in China, and has important medical and ornamental functions (Duan et al. 2022). In the middle of June 2022, anthracnose disease was observed nearly 25% (n=90) on P. lactiflora in Poyang County, Shangrao City, Jiangxi Province (29.00° N, 116.67° E) (Figure 1 E). The symptoms of the disease were small, round, light brown spots then grew bigger to round or irregular dark brown lesions (5 to 7 mm diameter) progressively on the leaves with disease spread (Figure 1 A). Subsequently, necrotic tissue was formed in the center and caused fade and wilt on the leaves ultimately, which reduced the medicinal and aesthetic value severely. Small pieces of diseased tissue (5 × 5 mm) were cut from the diseased junction, disinfected with 75% ethanol for 30 to 45 seconds, then 1% NaClO for 1 to 2 minutes, rinsed three times with sterile water. To identify the pathogen, tissues were placed on PDA and incubated for 3 days at 28°C. Single spore isolates were cultured on PDA, the colonies of one representative strain (SY4) were originally white with a lot of aerial mycelium after 5 to 7 days at 28°C in the incubator. The center of the colony turned greyish-white, released tiny orange-yellow particles (conidia) (Figure 1 F and 1 G), which were single, colorless, elongated ovals with rounded ends and measured 11.29 to 23.24 × 3.94 to 5.60 µm (av=15.89 µm × 4.74 µm, n=50) (Figure 1 H and 1 I). The isolate SY4 was identified to Colletotrichum fructicola based on morphological characteristics (Yang et al. 2021; Li et al. 2022b). For further molecular identification, the rDNA-ITS, actin gene (ACT), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), chitin synthase (CHS) and calmodulin gene (CAL) genes were amplified and sequenced with primers of ITS1/ITS4 (Gardes et al. 1993), ACT-512F/ACT-783R, GDF/GDR (Templeton et al. 1992), CHS-79F/CHS-345R (Carbone et al. 1999) and CL1C/ CL2C (Weir et al. 2012) respectively. The accession numbers in GenBank were OP523977 (ITS-rDNA), OP547618 (ACT), OP605733 (GAPDH), OP605732 (CHS), and OP605731 (CAL). The BLAST analysis revealed that these sequences were identical more than 99% with those of C. fructicola (GenBank accession Nos. MZ437948.1, MN525803.1, MN525860.1, MZ13360.1 and ON188684.1) (Figure 2). To confirm pathogenicity, the leaves were cleaned with 75% ethanol, rinsed with sterile water. After the leaf surface was dried naturally, 20 leaves were pricked at two symmetrical places on either side of the main veins of the leaf with a sterilized inoculum needle (2.0 mm in diameter), half of the wounded leaves were inoculated with 20 µL spore suspension (1.0 × 106 spores/mL) (Figure 1 C and 1 D), while the other half were inoculated with sterile water as controls (Figure 1 B). Inoculated leaves were grown for 5 days in an incubator at 28 °C and above 90% relative humidity, repeated three times. The results demonstrated that the wounded leaves with C. fructicola showed the same signs of wilting with the original disease leaves, while control leaves remained healthy. The same fungus was reisolated from the diseased leaves which confirmed with Koch's postulates. The same fungus was re-isolated from the diseased leaves while it was not isolated from control leaves, confirmed with Koch's postulates. In China, it had been reported that C. fructicola caused anthracnose on Persea americana (Li et al. 2022a) and Myrica rubra (Li et al. 2022b). To the best of our knowledge, this is the first report of anthracnose on P. lactiflora caused by C. fructicola in China. The results will help to develop effective control strategies for anthracnose on P. lactiflora.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Wisteria sinensis in China.

Wisteria (Wisteria sinensis) is a well-known ornamental plant for environmental protection in the garden, which also has a high value for medicinal use. In December 2021, leaf spots were observed on W. sinensis plants growing on the campus of Jiangxi Agricultural University in Nanchang, Jiangxi Province (28.45° N, 115.49° E), with a incidence rate of 40% plants were infested (n = 100 investigated plants). Initially leaf spots were small and pale brown (Approx. 2 mm in diameter), which gradually expanded into round or irregular dark brown spots as disease progressed, and lesions developed greyish-white necrotic tissues in the center at later stages, eventually causing the leaves to rot. To isolate the pathogen, tissues (5 × 5 mm) at the margin of lesions were cut from ten symptomatic leaves, surface disinfected with 75% ethanol for 30 s followed by 2% sodium chloride (NaClO) for 1 min, rinsed three times with sterile distilled water, and the dried tissues were cultured on potato dextrose agar (PDA) at 28 ± 1℃ in darkness for 3 days. After culture purification, five isolates were obtained and the representative single spore isolate (ZTTJ1) was used for subsequent identification tests. After 10 days of incubation on PDA medium, colonies had dense aerial mycelium with a gray center and dark gray-green mycelium outward, with orange-red conidial masses distributed in a ring on the surface. The underside of the colonies was light gray to dark gray. Conidia were cylindrical, with ends obtuse-rounded, 11.83 to 20.74 × 3.34 to 5.33 µm (av=16.11 µm × 4.26 µm, n = 50) in size. These morphological characteristics were consistent with Colletotrichum gloeosporioides (Shi et al, 2019). Six conserved regions of isolate (ZTTJ1), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), ß-tublin (TUB), actin (ACT), and chitin synthase 1 (CHS1) gene regions were amplified using ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Li et al, 2018), Bt2a/Bt2b (Prihastuti et al, 2009), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999) primers, respectively. Using BLAST, ITS, GAPDH, CAL, TUB, ACT, and CHS1 gene sequences (GenBank Accession No. OP703312, OP713773, OP713775, OP713776, OP713772, OP713774, respectively) were over 99% identical to C. gloeosporioides (GenBank Accession No. MK967281, MH594288, MT449307, MN624110, MN107239 and MN908602, respectively). A maximum likelihood (ML) phylogenetic analysis based on ITS-ACT-GAPDH-CHS1-CAL-TUB2 sequences using MEGA7.0, placed isolate (ZTTJ1) within C. gloeosporioides. To complete Koch's postulates, 10 µL spore suspension (1.0 × 106 conidia/mL) of ZTTJ1 (7-day-old culture on PDA medium) was dropped onto 10 leaves wounded with a sterilized needle and 10 non-wounded leaves, respectively. Ten wounded leaves were inoculated with sterile water as controls. All leaves were incubated at 28 ± 1 °C and 90 % relative humidity (12 h/12 h light/dark). After 7 days, all wounded leaves inoculated with C. gloeosporioides developed symptoms as previously observed, while the control and non-wounded leaves remained healthy. The fungus re-isolated from the inoculated leaves were identified as C. gloeosporioides by morphological and molecular identification; the pathogen causing disease in W. sinensis was determined to be C. gloeosporioides. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. sinensis in China. This work has identified the pathogenic species of the disease, which helps to take targeted measures to control its spread, providing a basis for the prevention and treatment of the disease.

First Report of Black Spot Disease Caused by Alternaria alternata on Persimmon Fruit in China.

Sweet persimmon is native to Japan and valued for its fruit, which are high in sugar and vitamins. In October 2021, symptoms were observed on persimmon (Diospyros kaki L. cv. Yangfeng) fruits in cold storage room in Suiping county, Henan Province (32.59 °N, 15 113.37 °E). Initially, small circular dark-brown spots were visible on the fruit rind, turning into irregular sunken dark areas, and eventually rotting 15% of 200 fruits after four weeks of cold storage (10°C, 95% relative humidity). To isolate the causal agent, 10 fruits of symptomatic tissues (4 mm2) were surface-sterilized in 2% sodium hypochlorite (NaOCl) for 1 minute, washed three times in sterile distilled water, then aseptically transferred to potato dextrose agar (PDA) and incubated for 7 days at 25°C. Fungal colonies were isolated from plant tissue, and on three colonies of similar morphology, single-spore isolation was performed. On PDA, the isolates produced circular colonies of fluffy aerial mycelia, gray-brown in the center with gray-white margins. Conidia were dark brown, obclavate or pyriform, with 0 to 3 longitudinal septa and 1 to 5 transverse septa, and a size range of 19.2 - 35.1 × 7.9 - 14.6 µm (n=100). Conidiophores were olivaceous, septate, straight, or bent, with a length of 18 - 60 × 1 - 3 µm (n=100). These morphological characteristics identify the isolates as Alternaria alternata (Simmons. 2007). Genomic DNA was extracted from a representative isolate YX and re-isolated strain Re-YX by cetyltrimethylammonium bromide (CTAB). The primers of ITS1/4, Alt-F/R, GPD-F/R, EF1/2, EPG-F/R (Chen et al. 2022), RPB2-5F/7cR (Liu et al. 1999), and H3-1a/1b (Lousie et al. 1995) were used to amplify the partial internal transcribed spacer (ITS) region, Alternaria major allergen (Alt a1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF), endo-polygalacturonase (endoPG), RNA polymerase second largest subunit (RPB2) and Histone 3 (His3), respectively. GenBank accession No of ITS, Alt a1, GAPDH, TEF, endoPG, RPB2, His3 were ON182066, ON160008 to ON160013 for YX and OP559163, OP575313 to OP575318 for Re-YX respectively. Sequence data of Alternaria spp. were downloaded from GenBank and the BLAST analysis showed 99%-100% homology between various A. alternata strains (ITS: MT498268; Alt a1: MF381763; GAPDH: KY814638; TEF: MW981281; endoPG: KJ146866; RPB2: MN649031; His3: MH824346). A phylogenetic analysis based on ITS, Alt a1, GAPDH, TEF, and RPB2 sequences using MEGA7 (Molecular Evolutionary Genetics Analysis) revealed that the isolate YX and Re-YX were clustered in A. alternata clade (Demers M. 2022). For the pathogenicity test, seven-day-old cultures were used to create spores suspensions (5.0 × 105 spores/mL) of each of the three isolates. Ten µL aliquots from each isolate were inoculated onto ten needle-wounded persimmon fruits; ten additional fruits were inoculated with water only to serve as controls. The pathogenicity test was three replications. Fruits were deposited in a climate box at 25°C, 95% relative humidity. Seven days post-inoculation, the wounded fruit treated with spore suspensions displayed black spot symptoms similar to the symptoms on the original fruit. There were no symptoms on the control fruits. The strain Re-YX was re-isolated from the symptomatic tissue of inoculated fruits and its identity confirmed using the morphological and molecular methods previously mentioned, fulfilling Koch's postulates. The persimmon fruit rot caused by A. alternata had been reported in Turkey and Spain (Kurt et al., 2010, Palou et al., 2012). According to our knowledge, this is the first report of black spot disease on persimmon fruits caused by A. alternata in China. The disease could infect persimmon fruits during cold storage, so more control methods should be developed to prevent postharvest disease of persimmon in the future.

Alleviatory effects of salicylic acid on postharvest softening and cell wall degradation of 'Jinshayou' pummelo (Citrus maxima Merr.): A comparative physiological and transcriptomic analysis.

The regulatory mechanisms underlying the salicylic acid (SA)-mediated inhibition of senescence in pummelo fruit, the largest known citrus variety, remain unclear. Herein, postharvest 0.3% SA treatment was demonstrated to delay postharvest 'Jinshayou' pummelo senescence, as evidenced by the inhibitions in firmness loss, electrolyte leakage increase, and color change. Using comparative transcriptomic data, a total of 4367, 3769, and 1659 DEGs were identified between CK0 and CK60, CK0 and SA60, and CK60 and SA60, respectively. Further GO analysis revealed that DEGs were mainly implicated in the processes of cell wall modification and phenylpropanoid pathway during fruit senescence. More importantly, postharvest exogenous 0.3% SA treatment was observed to inhibit CWDEs activities and their encoding gene expression, retain higher protopectin, cellulose, and hemicelluloses contents, as well as reduce WSP content, thus maintaining cell wall structure. These findings collectively indicated that postharvest SA treatment was a green and useful preservative for alleviating fruit senescence and prolonging the storage life of harvested 'Jiashayou' pummelo fruit.

First Report of Fruit Rot Caused by Fusarium graminearum on Ponkan (Citrus reticulata Blanco cv. Ponkan) in China.

Ponkan (Citrus reticulata Blanco cv. Ponkan) is a Chinese citrus species with tasty fruit. In November 2021, an unknown postharvest disease of Ponkan fruit caused nearly 15% losses of 2000 fruits in Nanchang, Jiangxi Province (28.68° N, 115.85° E). The initial fruit's surface necrosis was brown (Xu et al. 2022) (Figure 1A). Disease spots spread to the entire fruit, white or grey hyphae appeared, and the fruit rotted. Twenty diseased fruits were surface-disinfested with 2% sodium hypochlorite and 75% ethanol, then rinsed with sterile distilled water to isolate the pathogen. Diseased tissue sections (5 × 3 mm) were incubated on potato dextrose agar (PDA) for 7 days at 25°C. Twelve of 15 monoconidial isolates have similar morphology. On PDA, the isolates produced copious white aerial mycelia. After 5-7 days on straw juice medium, two types of conidia appeared (Rice straw 60 g, Agar 20 g, distilled water 1000 mL) (Figure 1E-I). Macroconidia were abundant, falcate, slender, and slightly curved with 0-8 septa, mostly 4-5 septa (average 41.70 × 3.81 m, n=100) (Figure 1J). Microconidia were globose, oval, or piriform with 0-1 septa, 2.72 to 8.57 × 2.53 to 7.47 m (average 5.49 × 4.52 m, n=50) (Figure 1L), and chlamydospores were not observed. Conidial and colony morphology identified 12 monoconidial isolates as Fusarium graminearum (Fisher et al., 1982; Yulfo-Soto et al., 2021). Genomic DNA was extracted from three isolates using a DNA Extraction Kit (Yeasen, Shanghai, China). The ITS1/4 region combined with partial gene fragments of translation elongation factor-1 alpha (TEF-1α, primer TEF1/2, O'Donnell et al. 1998), RNA polymerase second largest subunit (RPB2, primer fRPB2-5F/7cR, Liu et al. 1999) and ß-tubulin (ß-tub, primer Bt2a/2b, Li et al. 2013) from the isolates were amplified and sequenced. The three tested isolates showed identical gene sequences. Sequences amplified from one representative isolate (PG16) have been submitted to GenBank. BLAST searches revealed that ITS (OM019317), TEF-1α (OM048103), RPB2 (ON364348), and ß-tub (OM048104) had 99 to 100% identity compared with F. graminearum (MH591453.1, KX087136.1, MF662636.1, and MZ078952.1, respectively) in GenBank. The phylogenetic analysis combined ITS - TEF-1α - RPB2 (O'Donnell et al. 2015) concatenated sequences using MEGA7.0 (Mao et al. 2021) showed the isolate was clustered with the F. graminearum clade with 100% bootstrap support (Figure 2). The isolate PG16 was used for pathogenicity tests. Ponkan fruits were surface-disinfested with 75% ethanol and rinsed with sterile distilled water three times. Then, 30 punctured wound fruits (2-mm-diameter, 2-mm-depth) with a sterile needle and 30 unwounded fruits were inoculated with conidial suspension (10 µL, 3.0 × 105 conidia/mL). while the control fruits were inoculated with 10 µL sterile distilled water. All fruits were incubated at 25°C and 90% relative humidity. Two days later, all wounded fruits inoculated with conidial suspension showed disease spots, similar symptoms to the original rotten fruits (Figure 1D). Control and conidial-inoculated unwounded fruits were healthy (Figure 1B-C). The Pathogenicity test was repeated twice, and similar symptoms were observed. Morphologically and molecularly, the re-isolated fungus matched the inoculated isolate. First report of F. graminearum causing Ponkan fruit rot in China. As Ponkan is an important citrus crop with high economic value in China, identification of the causing agent, F. graminearum, for fruit rot allows the development of control measures to manage this disease.

First Report of Leaf Spot Disease on Chamaedorea elegans Caused by Fusarium oxysporum in China.

Chamaedorea elegans, native to Mexico and Guatemala, is a commonly planted indoor and small-scale garden ornamental due to its stately appearance, tolerance of low light levels, and its ability to improve air quality (El-Khateeb et al. 2010). In December 2021, an unknow leaf-spot disease was observed on C. elegans in Ganzhou City of Jiangxi Province, China (25.83 °N, 114.93 °E). The symptoms were small brown spots on the leaves, gradually expanded into irregular dark brown spots with necrotic tissue forming in the center of the lesions (Figure 2 A-1 and A-2). To isolate the pathogen, the diseased leaves were surface sterilized in 75% ethanol for 30 s. Small pieces of tissue (5 × 5 mm) were taken from the margin between diseased and healthy tissue, disinfected 1% NaClO for 45 s, washed three times in sterile water, and then placed on PDA at 25 ± 1°C for 5 days. Later, five isolates were purified from single spores and each of the five isolates has the same properties as described below. The isolates had abundant pale purple flocculent hyphae with purple pigmentation (Figure 2 C-1 and C-2). Macroconidia were falciform, straight or slightly curved, 1-2 septate, 11.75 to 22.99 × 3.06 to 4.44 µm (µ=16.08 µm × 3.37 µm, n=50) (Figure 2 D-1). Microconidia were oval or elliptical, a septate, 4.03 to 9.19 × 1.92 to 3.73 µm (µ=5.88 µm × 2.66 µm, n=50) (Figure 2 D-2). Chlamydospores formed singly or in pairs, and were terminal or intercalary in hyphae (Figure 2 D-3). Based on morphological characteristics, the fungus was preliminarily identified as a Fusarium sp. (Leslie et al. 2006). To confirm the identification, primers ITS1/ITS4 (White et al. 1990), RPB2-5f2/RPB2-7cr (O'Donnell et al. 2010; Liu et al. 1999) and TEF 1-αF/TEF 1-αR (O'Donnell et al. 2000) were used to amplify and sequence apportion of the ITS, RPB2 and TEF (Table 1). The sequences (Genebank accession number: OM780148, OM782679, OM782680) shared 100% idnetity with Fusarium oxysporum (Genebank accession number: MH866024.1, MH484930.1, MH485021.1). The maximum likelihood (ML) phylogenetic analysis of the concantenated ITS, RPB2 and TEF sequences was performed in MEGA7.0. (Sudhir et al. 2016), assigning the isoaltes to the F. oxysporum species complex (Figure 1). To confirm the pathogenicity, nine pots of healthy 3-year-old C. elegans plants were inoculated in the greenhouse (12 h light/12 h dark cycle, RH 90 %, three for wounded inoculation, three for nonwounded inoculation and three for control). Fifty disinfected leaves were wounded with sterile needles and fifty remained unwounded. The wounded (Figure 2 B-1 and B-2) and unwounded leaves were inoculated with a 10 µL spore suspension (1.0 × 106 conidia/ml) which was taken from each of the five isolates cultured for 7 days. Fifty leaves were mock-inoculated with sterile water (Figure 2 B-3 and B-4). After incubation for 7 days, the wounded leaves inoculated with the spore suspension had similar symptoms to the original diseased leaves, while the unwounded leaves and the control leaves did not develop symptoms. The experiment was repeated three times and the pathogens was reisolated from wound-inoculated leaves with the same morphological characteristics to the original pathogens, and identified as F. oxysporum by morphological and molecular analysis, completing Koch's postulates. F. oxysporum, a pathogen with a broad spectrum of hosts, ranks 5th among the top 10 fungal plant pathogens (Amjad et al. 2018.) and has been reported to Carpinus betulus, Citrullus lanatus, Pinus pinea (Mao et al. 2021; Muhammad et al. 2021; Monther et al. 2021). To our knowledge, this is the first report of leaf spot disease on C. elegans caused by F. oxysporum in China. C. elegans is an important ornamental plant in China with high economic value, so the disease has the potential to be a threat to its cultivation industry.

Amelioration of Chilling Injury by Fucoidan in Cold-Stored Cucumber via Membrane Lipid Metabolism Regulation.

Cucumber fruit is very sensitive to chilling injury, which rapidly depreciates their commodity value. Herein, the effect of fucoidan treatment on cucumber under cold stress were investigated. Fucoidan treatment of cold-stored cucumber alleviated the occurrence of chilling injury, delayed weight loss, lowered electrolyte leakage and respiration rate, and retarded malondialdehyde accumulation. Different from the control fruit, fucoidan treated fruit showed a high level of fatty acid unsaturated content, fatty acid unsaturation, and unsaturation index and increased ω-FDAS activity, along with upregulated expression levels of CsSAD and CsFAD genes. Fucoidan reduced the phosphatidic acid content and membrane lipid peroxidation, lowered the phospholipase D (PLD) and lipoxygenase (LOX) activity, and downregulated the expression levels of CsPLD and CsLOX genes. Collectively, fucoidan treatment maintained the integrity of cell membrane in cold-stress cucumbers. The results provide a new prospect for the development of fucoidan as a preservative agent in the low-temperature postharvest storage of cucumbers.

Comparative transcriptomic and metabolomic analyses reveal the delaying effect of naringin on postharvest decay in citrus fruit.

Introduction: Naringin exhibits antioxidant capacity and can partially inhibit pathogens in many horticultural products, such as citrus fruit; however, the effects of naringin on the storage quality and mechanisms that regulate senescence in citrus fruit have not been comprehensively analyzed. Methods and results: In this study, exogenous naringin treatment was found to significantly delay citrus fruit disease, decreasing the H2O2 content, increasing the antioxidant capacity and maintaining the quality of the fruit. Metabolomic analysis of citrus peel indicated the vast majority (325) of metabolites belonging to flavonoids. Moreover, the auraptene, butin, naringenin, and luteolin derivative levels within the phenylpropanoid pathway were significantly higher in the naringin-treated fruit than in the control fruit. Transcriptomic analysis also revealed that twelve genes in the phenylpropanoid and flavonoid biosynthesis pathways were significantly upregulated. Further analysis with a co-expression network revealed significant correlation between these differential genes and metabolites. Additionally, MYC and WRKY, screened from the MAPK signaling pathway, may contribute to naringin-induced disease resistance. Conclusion: In conclusion, naringin treatment can efficiently delay decay and maintain the quality of citrus fruit, mainly by promoting metabolites accumulation, and upregulating differentially expressed genes in phenylpropanoid and flavonoid biosynthesis pathway. This study provides a better understanding of the regulatory mechanisms through which naringin delays citrus fruit decay and maintains fruit quality.

Exogenous melatonin treatment affects ascorbic acid metabolism in postharvest 'Jinyan' kiwifruit.

Ascorbic acid (AsA) is an important nutritious substance in fruits, and it also can maintain the biological activity of fruits during storage. This research investigated the effect of exogenous melatonin (MT) on AsA metabolism in postharvest kiwifruit. Our results indicated that exogenous MT delayed the decrease of fruit firmness and titratable acid (TA), inhibited the increase of soluble solids content (SSC), reduced the respiration rate and ethylene production, and maintained a higher AsA content in kiwifruit during storage. The high expression of L-galactose pathway key genes in the early storage and regeneration genes in the later storage maintained the AsA content in postharvest kiwifruit. MT treatment enhanced the expression levels of AsA biosynthesis (AcGME2, AcGalDH, and AcGalLDH) and regeneration (AcGR, AcDHAR, and AcMDHAR1) genes. Meanwhile, the expression of the degradation gene AcAO was inhibited in MT-treated kiwifruits.

Hyperspectral dimension reduction and navel orange surface disease defect classification using independent component analysis-genetic algorithm.

Canker is a common disease of navel oranges that is visible before harvest, and penicilliosis is a common disease occurring after harvest and storage. In this research, the typical fruit surface, canker spots, penicillium spore, and hypha of navel oranges were, respectively, identified by hyperspectral imaging. First, the light intensity on the edge of samples in hyperspectral images was improved by spherical correction. Then, independent component images and weight coefficients were obtained using independent component analysis. This approach, combined with use of a genetic algorithm, was used to select six characteristic wavelengths. The method achieved dimension reduction of hyperspectral data, and the testing time was reduced from 46.21 to 1.26 s for a self-developed online detection system. Finally, a deep learning neural network model was established, and the four kinds of surface pixels were identified accurately.

First Report of Postharvest Fruit Rot on Citrus reticulata Blanco Caused by Fusarium concentricum in China.

Nanfeng tangerine (Citrus reticulata Blanco) is highly regarded for its nutritional and economic value. In January 2022, an unknown fruit rot was observed on Nanfeng tangerine fruits harvested from Nanfeng County (27.22 °N, 116.53 °E), Fuzhou City, Jiangxi Province after 70 days in storage (25 °C, 90% relative humidity). The disease mostly started from the pedicel or a wound. Symptoms initiated with dark brown lesions that rapidly expanded between the fruit center and pulp capsule causing total fruit rot. The surface of symptomatic fruit was sterilized with 75% ethanol for 30 s and 2% NaClO for 30 s. Small diseased tissue pieces (2 mm2) between diseased and healthy tissues were placed on potato dextrose agar (PDA) and put in an incubator (25 ± 1 °C) for 3 days. The representative isolate NFMJ-1 was subcultured onto PDA using single-spore purification. Colonies on PDA were light yellow to white, with abundant flocculent aerial hyphae. Microconidia were oval, obovoid to allantoid, 0 septate, occasionally 1 septate, 4.07 to 17.53 × 1.69 to 3.56 µm (average=7.40 µm × 2.55 µm, n=50). Macroconidia were slender, with a beaked apical cell and a foot-shaped basal cell, 3 to 5 septate, 22.99 to 81.12 × 2.34 to 3.81 µm (average=45.04 µm × 3.12 µm, n=50). According to morphological characteristics, the isolate was tentatively identified as Fusarium sp. (Leslie and Summerell 2006). To confirm the identification, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), RNA polymerase II second largest subunit (RPB2), beta-tubulin gene (TUB2), and calmodulin gene (CaM) sequences were amplified with primers ITS1/ITS4 (Gardes et al. 1993), TEF1/TEF2 (O'Donnell et al. 2010), RPB2-5f2/RPB2-7cr (Liu et al. 1999), Bt2a/Bt2b (Glass and Donaldson 1995), and CL1C/CL2C (Weir et al. 2012), respectively. The obtained sequences (ON184033, ON212051, ON212052, ON212053, ON212054) showed homology with F. concentricum ITS (MW016417.1; 514/514 bp), TEF (MK609902.1; 667/667 bp), RPB2 (LC631461.1; 941/972 bp), TUB2 (MT942588.1; 331/337 bp), and CaM (MK609916.1; 558/597 bp). A phylogenetic analysis of concatenated ITS-RPB2-TEF sequences was performed by MEGA7.0 with the maximum likelihood and Kimura 2-parameter model, revealing that the isolate was placed in the F. concentricum clade. To confirm pathogenicity, 36 healthy tangerine fruits were surface sterilized with 75% alcohol, then 18 disinfected fruits were wounded with sterile needles and 18 remained unwounded. Half of the wounded and un-wounded fruits were inoculated with 10 µL of a conidial suspension (1.0 × 106 conidia/ml) of isolate NFMJ-1 cultured for 7 days on PDA. Half of the wounded and un-wounded fruits were mock-inoculated with sterile water as controls. After incubation in an incubator (25 ± 1°C, 90% relative humidity) for 7 days, the wounded fruits inoculated with F. concentricum showed similar symptoms to the original diseased fruits, while the mock-inoculated fruits were asymptomatic. The pathogenicity test was repeated three times. The pathogen was re-isolated from the wound-inoculated fruits and identified as F. concentricum by morphological and molecular analysis, completing Koch's postulates. F. concentricum has been reported as a pathogen of Podocarpus macrophyllus (Dong et al. 2022), Capsicum annuum (Wang et al. 2013) and Zea mays (Du et al. 2020) in China. This is the first report of fruit rot caused by F. concentricum on Citrus reticulata in China. Appropriate prevention and control measure of the pathogen need to be developed to preserve marketability of this economically important citrus fruit.

An Antifungal Role of Hydrogen Sulfide on Botryosphaeria Dothidea and Amino Acid Metabolism Involved in Disease Resistance Induced in Postharvest Kiwifruit.

Botryosphaeria dothidea is a major pathogen responsible for postharvest kiwifruit soft rot. This study aimed to determine the influence of hydrogen sulfide (H2S) on postharvest resistance to kiwifruit soft rot and the antifungal role of H2S against B. dothidea. The results indicated that H2S (20 µl L-1) restricted the lesion area following inoculation with B. dothidea. H2S enhanced the production of shikimic acid, tyrosine, tryptophan, and phenylalanine while also increasing the total phenols, flavonoids, and lignin. H2S upregulated the expression of AcDHQS, AcSDH, AcSK, AcPAL, AcCAD, and AcCHS. Additionally, sodium hydrosulfide (NaHS)-released H2S inhibited mycelial growth. NaHS concentrations of 20 and 40 mmol L-1 significantly decreased the mycelial weight and malondialdehyde content (MDA) content while increasing cell membrane conductivity and membrane leakage. The results indicate that H2S induces resistance in kiwifruit via a microbicidal role and amino acid metabolism involved in postharvest kiwifruit disease resistance.

First report of Fusarium tricinctum causing fruit rot on navel orange (Citrus sinensis (L.) Osbeck) in China.

Citrus sinensis (L.) Osbeck is popular with consumers for its delicious taste. In December 2020, a rot symptom causing about 15% losses of a total of 450 fruits was observed on 'Newhall' navel oranges after 70 d storage (20℃, 85%-90% RH) at Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables (28.68° N, 115.85° E). The fruits were harvested from an orchard in Ganzhou City, Jiangxi Province, China (25.53° N, 114.79° E). Incipiently, the pedicles of infected fruits were brown, the peels became softened and showed yellowish-brown lesions which, gradually expanded and had white hyphae (Fig. S1A). To isolate the pathogen, the surface of diseased fruits was disinfected with NaClO (2%) for 2 min and ethanol (75%) for 0.5 min, then washed with sterile water three times. Tissues (5 × 5 mm) around the lesion were incubated on potato dextrose agar (PDA) at 28 ± 1℃ (L: D=12: 12) for 5 days. Five cultures with similar morphology were obtained and colonies initially produced white aerial hyphae and became khaki then turned pink on PDA (Fig. S1F, G, H). Abundant microconidia, macroconidia and rare chlamydospores were observed after 10 days on PDA and no glucose PDA media (Zhang et al. 2020). Macroconidia were falciform and curved to lunate, 2-4 septa, 29.38 × 3.75 µm in size (n=50) (Fig. S1K, Fig. S3). Microconidia were oval, napiform or pyriform, 0-1 septa, 12.00 × 3.43 µm in size (n=50) (Fig. S1L1 to L4, Fig. S3). Chlamydospores were found in hyphae, ellipsoidal or orbicular (Fig. S1I-1 to I-2, J-1 to J-2). The morphological features of five isolates were similar to Fusarium (Leslie and Summerell 2006). Genomic DNA of five isolates was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), ITS1/ITS4, EF1Ha/EF2Tb and fRPB2-5F/fRPB2-7cR primers were used to amplify the internal transcribed spacer region (ITS), and the transcriptional elongation factor-1 alpha (TEF-1α), and RNA polymerase II (RPB2) gene sequences (White et al. 1990; Carbone and Kohn 1999; Liu et al. 1999). The ITS, TEF-1α and RPB2 sequences of five isolates were deposited in GenBank and showed 99-100% identity with corresponding sequences from F. tricinctum (Table S1). A phylogenetic tree was constructed with ITS-TEF-1α-RPB2 concatenated sequences in MEGA7.0 (Li et al. 2021) and all five isolates were placed in F. tricinctum clade with 100% bootstrap support (Fig. S2). To confirm pathogenicity, ten healthy C. sinensis fruits were surface-sterilized with 75% ethanol and inoculated with 10 µL spore suspension (1.0 × 106 spore/mL) including five wounded (with sterilized needle) and five unwounded (Fig. S1B to E). Control fruits were inoculated with 10 µL sterile water. All fruits were incubated at 28 ± 1℃, 90% RH for 7 days. The experiment was conducted three times. The lesion diameter of inoculated wounded fruits was 21.01 ± 2.52 mm and showed similar symptoms to original rotten fruits. However, the control and unwounded fruits remained healthy. To fulfill Koch's postulates, F. tricinctum was re-isolated from the inoculated fruits and deposited in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. To our knowledge, F. tricinctum has been reported on apple tree and kiwi plant in China (Zhang et al. 2021; Ma et al. 2022), but this is the first report of F. tricinctum causing fruit rot on navel orange in China. This finding provides important information for preventing postharvest disease of citrus.

Lignin Biosynthesis Pathway and Redox Balance Act Synergistically in Conferring Resistance against Penicillium italicum Infection in 7-Demethoxytylophorine-Treated Navel Orange.

7-Demethoxytylophorine (DEM), a natural water-soluble phenanthroindolizidine alkaloid, has a great potential for in vitro suppression of Penicillium italicum growth. In the present study, we investigated the ability of DEM to confer resistance against P. italicum in harvested "Newhall" navel orange and the underlying mechanism. Results from the in vivo experiment showed that DEM treatment delayed blue mold development. The water-soaked lesion diameter in 40 mg L-1 DEM-treated fruit was 35.2% lower than that in the control after 96 h. Moreover, the decrease in peel firmness loss and increase in electrolyte leakage, superoxide anion (O2•-) production, and malondialdehyde (MDA) content were significantly inhibited by DEM treatment. Hydrogen peroxide (H2O2) burst in DEM-treated fruit at the early stage of P. italicum infection contributed to the conferred resistance by increasing the activities of lignin biosynthesis-related enzymes, along with the expressions of their encoding genes, resulting in lignin accumulation. The DEM-treated fruit maintained an elevated antioxidant capacity, as evidenced by high levels of ascorbic acid and glutathione content, and enhanced or upregulated the activities and gene expression levels of APX, GR, MDHAR, DHAR, GPX, and GST, thereby maintaining ROS homeostasis and reducing postharvest blue mold. Collectively, the results in the present study revealed a control mechanism in which DEM treatment conferred the resistance against P. italicum infection in harvested "Newhall" navel orange fruit by activating lignin biosynthesis and maintaining the redox balance.

Antofine Triggers the Resistance Against Penicillium italicum in Ponkan Fruit by Driving AsA-GSH Cycle and ROS-Scavenging System.

Postharvest fungal infection can accelerate the quality deterioration of Ponkan fruit and reduce its commodity value. Penicillium italicum is the causal pathogen of blue mold in harvested citrus fruits, not only causing huge fungal decay but also leading to quality deterioration. In our preliminary study, antofine (ATF) was found to have a great potential for significant in vitro suppression of P. italicum growth. However, the regulatory mechanism underpinning ATF-triggered resistance against P. italicum in citrus fruit remains unclear. Here, the protective effects of ATF treatment on blue mold development in harvested Ponkan fruit involving the enhancement of ROS-scavenging system were investigated. Results showed that ATF treatment delayed blue mold development and peel firmness loss. Moreover, the increase of electrolyte leakage, O2 •- production, and malonyldialdehyde accumulation was significantly inhibited by ATF treatment. The ATF-treated Ponkan fruit maintained an elevated antioxidant capacity, as evidenced by inducted the increase in glutathione (GSH) content, delayed the declines of ascorbic acid (AsA) content and GSH/oxidized GSH ratio, and enhanced the activities of superoxide dismutase, catalase, peroxidase, and six key AsA-GSH cycle-related enzymes, along with their encoding gene expressions, thereby maintaining ROS homeostasis and reducing postharvest blue mold in harvested Ponkan fruit. Collectively, the current study revealed a control mechanism based on ATF-triggered resistance and maintenance of a higher redox state by driving AsA-GSH cycle and ROS-scavenging system in P. italicum-infected Ponkan fruit.