Fruit Wounds Research Articles

Application of Lactobacillus spp. for the biological management of green mold (Penicillium digitatum) on sweet orange fruit under in vitro and storehouse conditions

ABSTRACT This study aimed to investigate the use of probiotic lactic acid bacteria including Lactobacillus brevis, L. plantarum, L. helveticus and L. delbrueckii from dairy products against Penicillium digitatum (green mold) on sweet orange fruit. Antifungal activity of Lactobacillus spp. was first assayed using bacterial cells through the overlay method against 105 spore/mL suspension of P. digitatum. Also, P. digitatum growth inhibition was measured in the PDA medium containing 15 and 30% cell-free supernatant (CFS) of Lactobacillus spp. Antifungal activity of Lactobacillus spp. was evaluated under storehouse conditions by treating wounded fruits with 108/mL cell suspension (CS) and 30% CFS of Lactobacillus spp., which then infected with 105/mL spore suspension of P. digitatum. The CS and CFS of L. plantarum had the best antifungal activity under both in vitro and in vivo conditions followed by L. helveticus, L. delbrueckii and L. brevis. L. plantarum. The 15 and 30% L. plantarum CFS were able to inhibit the growth of P. digitatum by 30.31%, 76.82% and 97.6% respectively, under in vitro conditions. Also, L. plantarum CS and 30% CFS reduced the growth of the P. digitatum on the fruit by 92.77 and 98.9% respectively, under storehouse conditions. The total content of all sugars of treated fruits with Lactobacillus spp., CS and CFS showed significant difference compared to untreated fruits. Therefore, the present study highlights the successful application of biological fungicides based on secondary compounds of Lactobacillus spp. due to greater stability of metabolites than bacterial cells in the environment.

Influence of Fruit Wounding on Subsequent Monilinia laxa Infection of Nectarines

Despite the fact that brown rot is the most economically important disease in stone fruits, the relationship between mechanical stress and infection has never been explored. Thus, to explore this connection, we carried out four experiments linking nectarine wounding and M. laxa infection. First, we evaluated a possible systemic reaction of the fruit to wounding that could impact the M. laxa infection. Afterward, we study the impact of the fruit in the environment on M. laxa colonies grown in vitro. Subsequently, we tested the disease susceptibility of fruits inoculated with M. laxa placed in the same environment as wounded fruits. Finally, in the fourth experiment, the effect of wounding on the subsequent fruit infections was evaluated at three fruit stages. As a result, we observed that there was no evidence of a fruit systemic reaction to wounding and M. laxa infection. In the study related to the impact of the fruit in the environment of M. laxa, the findings suggest that M. laxa “perceived” the fruit’s presence, resulting in accelerated in vitro growth. Moreover, the presence of wounded fruits in the box increased the susceptibility to brown rot. Inoculated fruits showed a delayed and reduced infection 7 h after being wounded, during the second stage of fruit development (15–19 weeks after full bloom). Moreover, a red reaction associated with inoculation was detected immediately after the fruits were wounded. Nine phenolic compounds exclusively related to the red areas were extracted, while six other compounds were present in higher proportions. These compounds may be actively involved in plant–pathogen interactions and the activation of metabolic pathways involved in nectarine susceptibility/resistance to M. laxa.

Chitosan and chitooligosaccharide regulated reactive oxygen species homeostasis at wounds of pear fruit during healing

Both chitosan (CTS) and chitooligosaccharide (COS) can promote fruit healing. However, whether the two chemicals regulate reactive oxygen species (ROS) homeostasis during wound healing of pear fruit remains unknown. In this study, the wounded pear fruit (Pyrus bretschneideri cv. Dongguo) was treated with a 1 g L−1 CTS and COS. We found CTS and COS treatments increased NADPH oxidase and superoxide dismutase activities, and promoted O2.- and H2O2 production at wounds. CTS and COS also enhanced the activities of catalase, peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, and elevated the levels of ascorbic acid and glutathione. In addition, the two chemicals improved antioxidant capacity in vitro and maintained cell membrane integrity at fruit wounds during healing. Taken together, CTS and COS can regulate ROS homeostasis at wounds of pear fruit during healing by scavenging excessive H2O2 and improving antioxidant capacity. Overall, the COS demonstrated superior performance over the CTS.

First Report of Gliocephalotrichum simplex Causing Fruit Rot of Choerospondias axillaris in China

First Report of <i>Gliocephalotrichum simplex</i> Causing Fruit Rot of <i>Choerospondias axillaris</i> in China

Bacillus cereus B8W8 an effective bacterial antagonist against major postharvest fungal pathogens of fruit

The antifungal activity of Bacillus cereus (B8W8) was investigated against major post-harvest fungal pathogens affecting citrus and apples fruits namely Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii, Penicillium expansum, Botrytis cinerea, Monilinia laxa and Monilinia fructigena. The in vitro dual culture of B8W8 and fungal pathogens showed high inhibition rates of the mycelial growth of Monilinia laxa and Penicillium digitatum (71.4% and 72.5% respectively). Subsequently, the volatile compounds (VOCs) have a high antagonistic effect against Monilinia laxa (86.4%) and Monilinia fructigena (73.4%). In addition, the bacterial-free cell filtrate of B8W8 had a significantly important effect on the inhibition of spore’s germination and mycelial growth of Penicillium expansum, Botrytis cinerea, Penicillium italicum and Geotrichum citri-aurantii. The in vivo bioassays revealed a significant impact of B8W8 on fruit decays either by reducing the disease severity of causative agents or by completely preventing their occurrence, particularly brown rot of apple caused by M. laxa and green mold of citrus caused by P. digitatum (DS = 0%). However, the severity of sour rot of citrus caused by G. citri-aurantii was only reduced by 50%. The semi-commercial trials showed a high potential of B. cereus B8W8 to reduce the incidence of gray mold of apple and green mold of citrus to very low values during at storage. In addition, the biological treatment with B8W8 was shown generally more effective than imazalil, the chemical treatment reference. Furthermore, the rapid colonization of fruit wounds by B8W8 could promote its antagonistic ability. Moreover, biochemical tests showed the ability of B8W8 to produce protease, amylase, and hydrocyanic acid (HCN). Interestingly, the molecular analysis demonstrated the ability of this bacterium to produce the fengycin. The secretion of this lipopeptide and lytic enzymes is more likely the major mechanism of action involved in the biocontrol activity of this bacterium. Results indicated that quality parameters of fruit, when challenged post-harvest pathogens and treated with B8W8, were mostly very close to those of the untreated control, suggesting that B8W8 did not affect the fruit quality. Accordingly, it was concluded that B. cereus strain B8W8 could be an effective biological agent to control the main fungal pathogens of apples and citrus fruits during post-harvest period.

First report of Preharvest Decay caused by Colletotrichum chrysophilum on apples in Italy (South Tyrol).

Apple (Malus × domestica Borkh.) is economically the most important fruit crop in South Tyrol (Italy). At the end of the growing season 2020, necrotic lesions and chlorosis developed on leaves and premature leaf dropping was observed on the cultivars Gala, Granny Smith and Cripps Pink(cov)/Rosy Glow(cov) in the Etsch/Adige valley. After the appearance of these symptoms, small circular brownish spots were observed on above 90 % of apples in the respective orchards. Fungal isolates were obtained from symptomatic apples by culturing small portions of fruit flesh from the lesion margin on potato dextrose agar (PDA) at 25°C in the dark. The colonies showed a white to peachy color on the upper surface and were greyish on the reverse side. Conidia were cylindrical, predominantly rounded and averaged 16.39 ± 1.37 µm and 5.75 ± 0.81 µm (n = 33), consistent morphological characteristics as described recently in Fuentes-Aragón et al. (2021). A multi-locus sequence analysis according to Astolfi et al. (2022) and Weir et al. (2022) was conducted based on the internal transcribed spacer (ITS) region and on fragments of actin (ACT), DNA-(apurinic or apyrimidinic site) lyase (APN2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), beta-tubulin (TUB2), and intergenic spacer and partial mating type (Mat1-2) genes. MegaBlast analysis revealed 100 % identity to the reference genome of C. chrysophilum for ITS (NR_160821), for ACT (KX093982), APN2 (KX094018), CAL (KX094063), GAPDH (KX094183), 99.29 % for TUB2 (KX094285), 99.85 % for Mat1-2 (KX094325) and 99.87 % for GS (KX094204). Sequences were deposited in GenBank (ITS: OK485032; ACT: OK539650; APN2: ON624100; CAL: ON624104; GAPDH: OK539654; GS: ON624108; TUB2: OK539658; Mat1-2: ON645973). Pathogenicity assays were performed on wounded and unwounded apples of cvs. Gala and Rosy Glow(cov). Apples of both cvs. were wounded with a sterile needle. For each variant, 8 fruits were inoculated with 10 µL spore suspension (1 × 106 spores mL-1) in 0.05 % Tween®20 or with 10 µL 0.05 % Tween®20 as control. The apples were put into plastic boxes containing moist tissue and incubated at 25°C, 100 % relative humidity and 12 h photoperiod. First lesions appeared on inoculated wounded fruits after 5 days, whereas unwounded and control fruits remained asymptomatic. After 15 days, symptoms could be observed only on inoculated wounded apples with spore suspension: on 95 % of individual wounds of Rosy Glow(cov) and on 77.5 % of Gala. Koch's postulates were fulfilled by re-isolating the fungus and by identifying the re-isolates as C. chrysophilum (ITS: OK485033-OK485035; ACT: OK539651-OK539653; APN2: ON624101-ON624103; CAL: ON624105-ON624107; GAPDH: OK539655-OK539657; GS: ON624109; TUB2: OK539659-OK539661; Mat1-2: ON645974-ON645976). To date, only one study confirmed C. chrysophilum as causal agent of apple bitter rot in Europe (Cabrefiga et al. 2022). Recently, Astolfi et al. (2022) reclassified C. chrysophilum as the main causal agent of GLS on apples in Southern Brazil and Uruguay. The authors stressed that C. chrysophilum might also be the potential agent of GLS in Europe (Astolfi et al. 2022). This confirms the observations made in 2020 in South Tyrol, where massive leaf spots preceded the symptoms on fruit. To the best of our knowledge, this is the first report of a preharvest decay on apples caused by C. chrysophilum in Italy (South Tyrol).

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.

Combining the biocontrol agent Meyerozyma guilliermondii with UV-C treatment to manage postharvest gray mold on kiwifruit

Biological and physical controls as eco-friendly methods are being pursued actively to manage postharvest diseases. Postharvest gray mold is one of the major causes of significant economic losses for kiwifruit industry. Effects of the biocontrol yeast Meyerozyma guilliermondii and UV-C to control gray mold and natural infection on kiwifruit were determined in this study. M. guilliermondii or UV-C as an individual treatment inhibited gray mold and natural infection development. The combined treatment of M. guilliermondii and UV-C provided greater control efficacy than each individual treatment. The growth of M. guilliermondii in fruit wounds was not negatively impacted by UV-C. The analysis at the molecular and physiological levels showed that M. guilliermondii and UV-C alone or in combination induced defensive gene expression and total phenolics in kiwifruit. The disease resistance response induced by UV-C may partially contribute to enhancement in the biocontrol effectiveness of M. guilliermondii on kiwifruit. Taken together, these findings suggested that the hurdle technology combining biocontrol and physical controls may represent a promising method to manage gray mold on kiwifruit.

Sodium silicate accelerates the deposition of lignin and silicon by activating the biosynthesis of lignin monolignols and increasing the relative silicon content in muskmelon wounds

Silicate is the main constituent of soil and is widely distributed throughout the Earth's. Sodium silicate (SS) is a common water-soluble silicate. Our previous research found that SS could prevent water evaporation and pathogen infection by promoting the deposition of suberin polyphenols on muskmelon fruit wounds, bur few reports are available on the effect of SS on the deposition of lignin and silicon on muskmelon wounds. In this study, SS treatment enhanced the activities of enzymes related to lignin synthesis, increased ρ-coumaryl, coniferyl, and sinapyl alcohols, and lignin levels, increased peroxidase activity and H2O2 content, and accelerated the deposition of lignin on wounds. In addition, SS treatment increased the relative silicon content to increase the rate of silicon deposition, which altered the morphological layer of cells compared to the control. These results suggest that SS accelerated lignin deposition on muskmelon wounds by activating lignin biosynthesis. SS also improved silicon deposition by increasing relative silicon content of the wounds. Since SS promotes lignin and silicon deposition at muskmelon wounds, the chemical can be used for wound healing of muskmelon fruit.

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.

Biocontrol activity of Bacillus halotolerans strain Pl7 against Botryosphaeria dothidea causing apple postharvest decay and potential mechanisms.

Apple ring rot, one of the most common apple postharvest diseases during storage, is caused by Botryosphaeria dothidea. Fungicide application is the most widely used method to control this disease, but the increasing environmental and food safety concerns greatly limit their use. The present study aimed to examine the biocontrol activity and underlying action mechanism of Bacillus halotolerans strain Pl7 against B. dothidea. The results revealed that B. halotolerans strain Pl7 exhibited strong inhibitory activity against B. dothidea by 69% in vitro. The culture filtrate of strain Pl7 possessed cellulase, β-1, 3-glucanase, protease activity and mediated the antifungal activity against B. dothidea. Further analysis demonstrated that culture filtrate of strain Pl7 could cause cell membrane permeabilization of B. dothidea. Apple fruit suffering from ring rot induced by a carbendazim (CBZ)-sensitive or -resistant B. dothidea isolate was much suppressed after being treated with strain Pl7, maintaining postharvest quality. The ability of strain Pl7 to swiftly colonize and thrive in apple fruit wounds was demonstrated by a re-isolation assay. Additional transcriptome studies of untreated and treated apple fruit with strain Pl7 revealed that strain Pl7 mostly changed the expression of genes functioning in plant secondary metabolite biosynthesis and plant-pathogen interaction. In light of these outcomes, the underlying antagonistic mechanism was investigated, and B. halotolerans strain Pl7 was identified as a promsing microbial biocontrol agent against apple postharvest decay.

Phenylalanine Promotes Biofilm Formation of Meyerozyma caribbica to Improve Biocontrol Efficacy against Jujube Black Spot Rot.

During storage and transportation after harvest, the jujube fruit is susceptible to black spot rot, which is caused by Alternaria alternata. The present study aimed to evaluate the effectiveness of the yeast Meyerozyma caribbica in controlling A. alternata in postharvest jujube fruits, and to explore the biofilm formation mechanism. The results showed that M. caribbica treatment significantly reduced the A. alternata decay in jujube fruits. M. caribbica could rapidly colonize jujube fruit wounds, adhering tightly to hyphae of A. alternata, and accompanied by the production of extracellular secretions. In in vitro experiments, we identified that M. caribbica adhered to polystyrene plates, indicating a strong biofilm-forming ability. Furthermore, we demonstrated that M. caribbica can secrete phenylethanol, a quorum sensing molecule which can affect biofilm development. Phenylalanine (a precursor substance for phenylethanol synthesis) enhanced the secretion of phenylethanol and promoted the formation of M. caribbica biofilms. Meanwhile, phenylalanine enhanced the biological control performance of M. caribbica against jujube black spot rot. Our study provided new insights that enhance the biological control performance of antagonistic yeast.

BTH-induced joint regulation of wound healing at the wounds of apple fruit by JA and its downstream transcription factors

Jasmonic acids (JAs) are important injury signaling molecules, which participate in the process of wound healing in plants. However, how JA and its downstream transcription factors involve in wound healing in apple fruit mediated by BTH has not been reported yet. In the present study, BTH treatment up-regulated gene expression of MdLOX3.1, MdAOS1, MdAOC, and MdOPR3, promoting JA synthesis at fruit wounds. Moreover, BTH up-regulated the gene expression of MdMYC2, MdGAIPB, and MdMYB108 transcription factors and increased MdPAL1, Md4CL2, MdCOMT1, and MdCAD6 expression. In addition, BTH facilitated the synthesis of phenylpropanoid metabolism products and accelerated suberin polyphenolics deposition at the wounds, which effectively reduced fruit weight loss and lesion diameter of apple fruit inoculated with Penicillium expansum during healing. It is suggested that BTH induced wound healing in apple fruit by the stimulating JA and its downstream transcription factors, and phenylpropanoid metabolism.

Caffeic acid increases the fitness of Candida oleophila to the microenvironment of kiwifruit and its biocontrol performance against postharvest decay fungi

Reactive oxygen species (ROS) in harvested fruit wound tissues result in an oxidative microenvironment. The pretreatment of some antagonistic yeasts with stress-ameliorating compounds has been shown to enhance tolerance to subsequent exposure to oxidative stress and thus improves biocontrol performance. The effect of caffeic acid (CA) on ROS in Candida oleophila was investigated after administration to kiwifruit wounds. CA treatment enhanced the tolerance of C. oleophila to the microenvironment of kiwifruit wounds. CA-treated yeast recovered from the microenvironment had lower ROS, less mitochondrial impairment and cellular oxidative damage, relative to non-CA (NCA) treated yeast. CA-treated yeast also exhibited improved biocontrol efficacy on kiwifruit against the decay fungi, Botrytis cinerea and Penicillium expansum, and more rapid growth in fruit wounds, than NCA-treated yeast. The antioxidant gene expression of C. oleophila was elicited by the CA treatment. In conclusion, the activation of antioxidant response induced by the by CA treatment of C. oleophila contributed to enhanced tolerance to oxidative stress and improved biocontrol efficacy against postharvest decay fungi.

Tryptophan enhances biocontrol efficacy of Metschnikowia citriensis FL01 against postharvest fungal diseases of citrus fruit by increasing pulcherriminic acid production

The aim of this work was to study the capability and mechanism of enhancing the yield of pulcherriminic acid (PA) produced by Metschnikowia citriensis FL01 with the help of tryptophan for the control of postharvest diseases on citrus caused by Penicillium italicum, Geotrichum citri-aurantii and Penicillium digitatum. The adding of 10 mmol/L tryptophan to the growth medium resulted in the widest pulcherrimin pigment zone produced by M. citriensis FL01. The adding of tryptophan to the growth medium upregulated A3136 and A3022 gene expression (responsible for leucyl-tRNA biosynthesis from leucine), downregulated A1350 gene expression (responsible for the biosynthesis of leucine to branched-chain fatty acids), and decreased the content of intracellular leucine in M. citriensis FL01, speculating that the addition of tryptophan in the growth medium induced leucine conversion toward leucyl-tRNA in M. citriensis FL01. Moreover, the adding of tryptophan to the growth medium upregulated PULs (responsible PA biosynthesis) and Snf2 (transcriptional regulator) gene expression and promoted intracellular, extracellular or total PA production by M. citriensis FL01 in liquid medium. In addition, the addition of tryptophan in the growth medium showed no effect on the growth of M. citriensis FL01 itself in liquid medium, while the population dynamics in citrus fruit wounds of M. citriensis FL01 with the addition of tryptophan in the growth medium were increased compared with those of M. citriensis FL01. What's more, M. citriensis FL01 with the addition of tryptophan in the growth medium completely inhibited the growth of pathogens in vitro. The disease incidences and lesion diameters of blue mold, sour rot and green mold on citrus fruit were lower in group which treated with M. citriensis FL01 containing tryptophan in the growth medium than that treated with M. citriensis FL01 alone. Overall, the postharvest biocontrol of citrus with M. citriensis FL01 containing 10 mmol/L tryptophan in the growth medium is a promising approach to protect these fruits from blue mold, sour rot and green mold.

Transcription factor AacmrA mediated melanin synthesis regulates the growth, appressorium formation, stress response and pathogenicity of pear fungal Alternaria alternata.

CmrA, as transcription factor for regulating DHN-melanin synthesis, controls melanin synthesis gene expression, and also regulate growth, development, stress response and virulence of plant fungi. However, little is known about the roles of CmrA on infection structure formation, penetration and pathogenicity of pear fungal Alternaria alternata. Here, we identified cmrA gene in A.alternata and assigned as AacmrA, sequence alignment and phylogenetic analysis revealed that AacmrA is highly conserved among fungi and encoded protein contain two Cys2His2 zinc finger motifs and one Zn(II)2Cys6 zinc cluster protein motif. ΔAacmrA severely decreased melanin production and DHN melanin synthesis related genes expression. Deletion of AacmrA impaired the morphology of spore and hyphae. Spore germination and appressorium formation induced by hydrophobicity surfaces and fruit wax significantly decreased in ΔAacmrA mutant. ΔAacmrA mutants were more sensitive than the wild type to osmotic stress and cell wall inhibitors, especially more sensitive to oxidative stress. In addition, lesion diameter of pear fruit wound inoculated with the ΔAacmrA mutant was reduced by 40.8% compared with the wild type 12d after inoculation. All findings of this study suggested that AacmrA is required for melanin biosynthesis, infection structure formation, and pathogenicity in A.alternata.

Penicillium growth inhibition, fruit decay reduction, and polymethoxyflavones and scoparone induction in satsuma mandarin irradiated with ultraviolet-A light-emitting diodes

In this study, we investigated the effects of ultraviolet (UV)-A light-emitting diodes (LEDs) on the inhibition of fungal spore germination, fruit decay, and accumulation of secondary metabolites, including phytoalexins, in satsuma mandarin. We examined the growth and development of blue (Penicillium italicum) and green (Penicillium digitatum) molds exposed to UV-A LED irradiation in vitro and found that UV-A LEDs with an emission peak of 375 nm and light intensity of 9.4 W m−2 significantly reduced the growth of both these fungi. In addition, wounded fruits inoculated with P. digitatum were irradiated with UV-A LEDs for 6 days, and we monitored the subsequent changes in green mold symptoms. The results indicated that low-intensity UV-A LED treatment significantly reduced green mold symptoms in fruit, being the effect most pronounced inhibitory on Penicillium sporulation. Furthermore, wounded fruits subjected to UV-A LED treatment were found to have significantly higher contents of the coumarin compound scoparone than fruits maintained in the dark. Contrastingly, scoparone was barely detectable in non-wounded fruits, irrespective of UV-A LED irradiation. The contents of nobiletin, tangeretin, and hesperidin were likewise found to be higher in wounded fruits treated with UV-A LED than in either non-wounded or non-irradiated fruits. These results indicate that treatment with low-intensity UV-A LED irradiation is sufficient to reduce mold symptom development, and accordingly could represent a promising safe approach to the control of post-harvest decay in citrus fruits. On the basis of our observations, we speculate that the biosynthesis of scoparone, polymethoxyflavones, and flavanones could be induced when the peel is injured and irradiated with UV-A contributing to a reduction in fruit decay.

Post-harvest chitosan treatment suppresses oxidative stress by regulating reactive oxygen species metabolism in wounded apples.

Mechanical wound on fruit triggers the formation of reactive oxygen species (ROS) that weaken cell walls, resulting in post-harvest losses. This mechanism can be controlled by using fruit preservatives to stimulate fruit antioxidant enzyme activities for the detoxification of ROS. Chitosan is a safe and environmentally friendly preservative that modulates ROS in whole fruits and plant cells, but the effects of chitosan on the ROS metabolism of mechanically wounded apples during storage are unknown. Our study focused on exploring the effects of post-harvest chitosan treatment on ROS production, cell membrane integrity, and enzymatic and non-enzymatic antioxidant systems at fruit wounds during storage. Apple fruits (cv. Fuji) were artificially wounded, treated with 2.5% (w/v) chitosan, and stored at room temperature (21–25°C, RH = 81–85%) for 7 days. Non-wounded apples were used as healthy controls. The results showed that chitosan treatment stimulated the activities of NADPH oxidase and superoxide dismutase and increased the formation of superoxide anions and hydrogen peroxide in fruit wounds. However, malondialdehyde, lipoxygenase, and membrane permeability, which are direct biomarkers to evaluate lipid peroxidation and membrane integrity, were significantly decreased in the wounded fruits after chitosan treatment compared to the wounded control fruits. Antioxidant enzymes, such as peroxidase and catalase activities, were induced by chitosan at fruit wounds. In addition, ascorbate-glutathione cycle-related enzymes; ascorbate peroxide, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase and the content of substrates, mainly ascorbic acid, dehydroascorbate, reduced glutathione, and glutathione, were increased at fruit wounds by chitosan compared to the wounded control fruits. Our results show that wounding stimulated the production of ROS or oxidative stress. However, treatment with chitosan triggered antioxidant systems to scavenge ROS and prevent loss of fruit membrane integrity. Therefore, chitosan promises to be a favorable preservative in inducing tolerance to stress and maintaining fruit quality.

First Report of Stemphylium eturmiunum Causing Leaf Spot and Black Spot on Apple in Zhaotong, Yunnan, China.

Apple is the largest fruit tree crop in the world, and China is the largest apple-producing County in the world. Zhaotong, Yunnan Province is a typical cold and mountainous apple-producing area in China. However, apple production is threatened by diseases during the entire growing season, and among them, apple leaf spot and fruit black spot are severe. In previous reports, the main pathogen causing apple leaf spot and fruit black spot was Alternaria sp. (Lior, et al, 2017), while different pathogens were identified. In the current study, seven red Fuji apple fruit with typical black spot samples were collected randomly in Dongda company orchard, Sujiayuan town, Zhaotong, Yunnan on March 25, 2021. The spots on the surface of these apples appear rounded, the diseased parts turn brown or black in colour and the flesh became soften and rotten. The tissues of fruit epidermis at the edge between diseased and healthy parts were cut, soaked in 75% alcohol for 30 s, washed with sterile water three times, and air-dried. Five pieces of tissue were placed on PDA medium amended with rifampicin (50 mg/ml) and incubated in the dark at 25 ℃ for 3-5 days. After colonies grew, mycelial clumps were picked out from the edges of the colonies, transferred to new PDA plates, and incubated at 25 ℃ for 6 days. The diameter of the colonies reached up to 5.7 cm. A representative isolate was retained for further work and was named P6-3-1. The hyphae were white and dense at an early stage, the culture medium on the underside became yellow and the middle parts of the colonies were darker. With maturity, hyphae were clumped, became red with other colors interspersed, and the medium became dark red. Light brown spores were produced, with more vertical septa and fewer transverse septa. Two to three transverse septa were generally observed with obvious constriction at the transverse septa. Average spore size was 22.83 µm ± 2.04 µm × 14.58 µm ± 1.97 µm. DNA was extracted from mycelium, purified and amplified with two pairs of primers, ITS1/ITS4 (White et al. 1990) and gpdF/gpdR (Marcos P. S. Câmara, et al. 2002). The PCR products were sequenced and deposited in GenBank (accession NO.OK560128 and OK627661 ). The similarity of ITS sequences between the isolate and MH843733 (Stemphylium eturmiunum strain ST14) was 100%, and that of gpd sequences between the isolate and MH843728 (Stemphylium eturmiunum strain ST20) was 100%. The maximum parsimony method of Mega7.0 was used and demonstrated that the studied isolate converged to the same branch as Stemphylium eturmiunum. Koch's postulates was applied to identify the pathogenicity of this isolate. A disc of P6-3-1-culture on PDA (5 mm in diameter) was placed on apple leaves and fruit wounds. Sterile PDA was used as a control. All plants were kept in a growth chamber at 25-30 ℃. Four days after inoculation, the disease spot was observed on the inoculated sites and fruit, and with the extension of incubation time, the diseased spots continue to grow, and the leaf spots were not limited by the veins. The pathogen was re-isolated from the inoculated leaves and fruit, satisfying Koch's postulates. This pathogen can also cause postharvest rot of sweet cherry (Alice Spadoni, et al, 2020), postharvest rot on tomato (Prencipe Simona, et al, 2021), etc. This is the first report that Stemphylium eturmiunum can cause apple leaf spot and fruit black spot in Yunnan province, China. The apple black spot caused by Stemphylium eturmiunum was accurately identified. By distinguishing between the two similar diseases mentioned above, resistance to the host and management practices can be accrued based on the characteristics of the pathogen, its epidemiological pattern and the choice of an effective chemical fungicide.

Sodium silicate treatment promotes suberin poly phenolic and silicon deposition, and enhances hardness and brittleness at muskmelon wounds

Silicon can enhance plant resistance to biotic stress by directly inhibiting pathogens, forming physical barriers, and inducing the synthesis of defense compounds. However, no study is available on silicon on fruit wound healing. Here, we found that sodium silicate enhanced the activities of phenylalanine ammonia-lyase, 4-coumarate coenzyme A ligase, and cinnamate-4-hydroxylase, and elevated levels of cinnamic, ρ-coumaric, caffeic, ferulic, sinapic acids, and total phenols at fruit wounds. Sodium silicate accelerated the deposition of suberin poly phenolic and silicon at wounds, increased the hardness and brittleness and reduced their resilience of the healing tissues, which slowed weight loss by wounded fruit, and disease index in fruit subjected to Trichothecium roseum. Taken together, sodium silicate may promote wound healing in muskmelons by activating phenylpropanoid pathway, accelerating suberin poly phenolic and silicon deposition at wounds, and increasing the hardness and brittleness of healing tissues.