Postharvest Diseases Of Citrus Fruit Research Articles

Novel Essential Oil-Based Thiosemicarbazone Compounds as Potential Fungicides in Controlling Postharvest Diseases of Citrus Fruit

Novel Essential Oil-Based Thiosemicarbazone Compounds as Potential Fungicides in Controlling Postharvest Diseases of Citrus Fruit

Screening of pathogenicity-deficient Penicillium italicum mutants established by Agrobacterium tumefaciens-mediated transformation.

Blue mold, caused by Penicillium italicum, is one of the main postharvest diseases of citrus fruits during storage and marketing. The pathogenic mechanism remains largely unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of P. italicum PI5 was established via Agrobacterium tumefaciens-mediated transformation (ATMT). The system yielded 200-250 transformants per million conidia, and the transformants were genetically stable after five generations of successive subcultures on hygromycin-free media. 2700 transformants were obtained to generate a T-DNA insertion library of P. italicum. Only a few of the 200 randomly selected mutants exhibited significantly weakened virulence on citrus fruits, with two mutants displaying attenuated sporulation. The T-DNA in the two mutants existed as a single copy. Moreover, the mutant genes PiBla (PITC_048370) and PiFTF1 (PITC_077280) identified may be involved in conidia production by regulating expressions of the key regulatory components for conidiogenesis. These results demonstrated that the ATMT system is useful to obtain mutants of P. italicum for further investigation of the molecular mechanisms of pathogenicity and the obtained two pathogenesis-related genes might be novel loci associated with pathogenesis and conidia production.

Control of citrus blue and green molds by Actinomycin X2 and its possible antifungal mechanism

Citrus blue and green molds caused by Penicillium digitatum, P. italicum, and P. polonicum, are the major postharvest diseases of citrus fruit. In the present study, Actinomycin X2 (Act-X2), a naturally occurring antibiotic produced by Streptomyces species, was found to show excellent antifungal effect against these three pathogens with a minimum inhibitory concentration (MIC) value of 62.5 μg/mL for them all, which was better than the positive control thiophanate-methyl. Act-X2 significantly reduced the percentage of spore germination, and highly inhibited the mycelial growth of P. italicum, P. digitatum, and P. polonicum with EC50 values being 34.34, 13.76, and 37.48 μg/mL, respectively. In addition, Act-X2 greatly decreased the intracellular protein content while increasing the reactive oxygen species (ROS) level and superoxide anion (O2−) content in the mycelia of pathogens. In vivo test indicated that Act-X2 strongly inhibited the infection of navel oranges by these three Penicillium species, with an inhibition percentage of >50% for them all at the concentration of 10 MIC. Transcriptome analysis suggested that Act-X2 might highly influence the ribosomal functions of P. polonicum, which was supported as well by the molecular docking analysis of Act-X2 with some key functional proteins and RNAs of the ribosome. Furthermore, Act-X2 significantly reduced the decay percentage and improved the firmness, color, and sugar-acid ratio of navel oranges spray-inoculated with P. polonicum during the postharvest storage at 4 °C for 60 d.

The effect of pulcherriminic acid produced by Metschnikowia citriensis in controlling postharvest diseases of citrus fruits

The biocontrol effectiveness of Metschnikowia citriensis relies on its production of pulcherriminic acid (PA), which forms insoluble and stable pulcherrimin pigments by chelating iron ions, this inhibits pathogen growth by preventing their utilization of chelated Fe3+. In this study, ΔM. citriensis, which did not produce PA, was used as a control to examine changes in its biocontrol effectiveness by adding tryptophan to the medium. Tryptophan was shown to have no discernible impact on the growth and PA production of ΔM. citriensis; moreover, the PA synthesis-related genes PULs, Snf2, and leucyl-tRNA synthesis-related genes A3136 and A3022 were all down-regulated in ΔM. citriensis. The PA-free ΔM. citriensis eventually showed a much poorer inhibition zone against the pathogens in vitro, and a noticeably decreased control efficiency against postharvest diseases in citrus fruit. Tryptophan was added to the medium, which had no appreciable impact on inhibitory zone of ΔM. citriensis against pathogens in vitro, but enhanced its ability to control citrus postharvest diseases. Additionally, the control effects of culture broth of M. citriensis and ΔM. citriensis on postharvest diseases in citrus fruit were assessed. It was found that both culture broth of M. citriensis and ΔM. citriensis exhibited remarkable control effects against citrus postharvest diseases, with culture broth of M. citriensis which containing PA being more effective in controlling the disease. Last but not least, we extracted and dissolved pulcherrimin to obtain PA extracts, which were then injected to citrus fruits to assess the biocontrol effectiveness. The findings demonstrated that postharvest diseases of citrus fruit can be effectively controlled by PA extracts. This research suggested a new biological strategy for the management of citrus postharvest diseases.

The GRAS Salts of Na2SiO3 and EDTA-Na2 Control Citrus Postharvest Pathogens by Disrupting the Cell Membrane.

Sodium silicate (Na2SiO3) and ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) are inorganic salts classified as 'Generally Recognized as Safe' (GRAS) compounds with great advantages in controlling various pathogens of postharvest fruits and vegetables. Here, we determined the median effective concentration (EC50) of Na2SiO3 (0.06%, 0.05%, 0.07% and 0.08%) and EDTA-Na2 (0.11%, 0.08%, 0.5%, and 0.07%) against common pathogens affecting postharvest citrus fruit, including Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii, and Colletotrichum gloeosporioides. Na2SiO3 and EDTA-Na2 treatments at the EC50 decreased the spore germination rate, visibly disrupted the spore cell membrane integrity, and significantly increased the lipid droplets (LDs) of the four postharvest pathogens. Moreover, both treatments at EC50 significantly reduced the disease incidence of P. italicum (by 60% and 93.335, respectively) and G. citri-aurantii (by 50% and 76.67%, respectively) relative to the control. Furthermore, Na2SiO3 and EDTA-Na2 treatment resulted in dramatically lower disease severity of the four pathogens, while also demonstrating no significant change in citrus fruit quality compared with the control. Therefore, Na2SiO3 and EDTA-Na2 present a promising approach to control the postharvest diseases of citrus fruit.

Berberine as a promising natural compound to control Penicillium italicum causing blue mold of citrus fruit

The blue mold caused by Penicillium italicum is one of the most common diseases in citrus fruit. Berberine from Chinese herb has predominate bioactive effects. This study investigated the inhibitory effect of berberine on P. italicum and the underlying mechanisms. Results showed that berberine displayed excellent efficacy to inhibit mycelial growth and spore germination of P. italicum in vitro dose-dependently. Moreover, berberine damaged the plasma membrane integrity, indicated by increased relative conductivity but decreased contents of soluble protein and reducing sugar of P. italicum mycelia. Meanwhile, higher H2O2 content was also observed in berberine-treated P. italicum mycelia. Additionally, berberine suppressed the disease development in citrus fruit through increasing the enzymatic activities of polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and chitinase (CHI) as well as contents of total phenols and flavonoids in citrus peel. Furthermore, gene expression analysis suggested that genes encoding these defense-related enzymes were up-regulated by berberine. These results suggested that berberine could be expected as a new natural strategy to control postharvest diseases of citrus fruit.

Identification of Pathogenicity-Related Effector Proteins and the Role of Piwsc1 in the Virulence of Penicillium italicum on Citrus Fruits.

Blue mold caused by Penicillium italicum is one of the two major postharvest diseases of citrus fruits. The interactions of pathogens with their hosts are complicated, and virulence factors that mediate pathogenicity have not yet been identified. In present study, a prediction pipeline approach based on bioinformatics and transcriptomic data is designed to determine the effector proteins of P. italicum. Three hundred and seventy-five secreted proteins of P. italicum were identified, many of which (29.07%) were enzymes for carbohydrate utilization. Twenty-nine candidates were further analyzed and the expression patterns of 12 randomly selected candidate effector genes were monitored during the early stages of growth on PDA and infection of Navel oranges for validation. Functional analysis of a cell wall integrity-related gene Piwsc1, a core candidate, was performed by gene knockout. The deletion of Piwsc1 resulted in reduced virulence on citrus fruits, as presented by an approximate 57% reduction in the diameter of lesions. In addition, the mycelial growth rate, spore germination rate, and sporulation of ΔPiwsc1 decreased. The findings provide us with new insights to understand the pathogenesis of P. italicum and develop an effective and sustainable control method for blue mold.

A Combined Analysis of Transcriptome and Proteome Reveals the Inhibitory Mechanism of a Novel Oligosaccharide Ester against Penicillium italicum.

Blue mold caused by Penicillium italicum is one of the most serious postharvest diseases of citrus fruit. The aim of this study was to investigate the inhibitory effect of a novel oligosaccharide ester, 6-O-β-L-mannopyranosyl-3-O-(2-methylbutanoyl)-4-O-(8-methyldecanoyl)-2-O-(4-methyl-hexanoyl) trehalose (MTE-1), against P. italicum. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM), along with transcriptome and proteome analysis also, were conducted to illuminate the underlying mechanism. Results showed that MTE-1 significantly inhibited P. italicum growth in vitro in a dose-dependent manner. Moreover, MTE-1 suppressed the disease development of citrus fruit inoculated with P. italicum. Furthermore, ultrastructure observation, as well as transcriptome and proteome analysis, indicated that MTE-1 treatment damaged the cell wall and plasma membrane in spores and mycelia of P. italicum. In addition, MTE-1 regulated genes or proteins involved in primary metabolism, cell-wall metabolism, and pathogenicity. These results demonstrate that MTE-1 inhibited P. italicum by damaging cell walls and membranes and disrupting normal cellular metabolism. These findings contribute to the understanding of the possible molecular action of MTE-1. Finally, MTE-1 also provides a new natural strategy for controlling diseases in postharvest fruit.

A combination of cinnamaldehyde and citral greatly alleviates postharvest occurrence of sour rot in citrus fruits without compromising the fruit quality.

One of the most troublesome postharvest diseases of citrus fruits is sour rot, caused by Geotrichum citri-aurantii. Sour rot reduces the shelf life of the fruits leading to massive economic losses. This study investigated the potential for a combination of cinnamaldehyde and citral (CC; 1: 2, v/v) at reducing the incidence of sour rot postharvest and its possible effect on fruit quality. Our findings show that CC could totally inhibit germination of G. citri-aurantii spores, with the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) both being 0.80mL L-1. The combination (CC) acted against G. citri-aurantii by targeting the chitin content of the cell wall. Wax + CC (WCC; 1 × MFC) treatment also showed high efficiency in reducing the incidence of sour rot, which was 40% lower than in the control group by day 8 when all the fruits in the latter were rotten. Apart from vitamin c (Vc) content which was higher in the test group than in the control group, WCC treatment did not have any significant effect on the quality of the citrus fruits, the examined fruit quality parameters being weight loss rate, coloration index, firmness, pH, total soluble solid (TSS) content, Vc content, as well as solid acid ratio. These results indicate that the combination of cinnamaldehyde and citral (CC, 1: 2, v/v) can be used as a natural preservative to alleviate the progress of sour rot in citrus fruits postharvest.

Metschnikowia citriensis FL01 antagonize Geotrichum citri-aurantii in citrus fruit through key action of iron depletion

Metschnikowia citriensis FL01 has great potential for biocontrol applications for its excellent biocontrol efficacy on postharvest diseases of citrus fruit, and the iron depletion by pulcherriminic acid (PA) and then formation of insoluble pigment pulcherrimin had been speculated as an important action mechanism. To identify the genes involved in pulcherrimin synthesis and reutilization in M. citriensis FL01, we de novo assembled the genome of M. citriensis FL01 based on long-read PacBio sequencing. The final assembled genome consisted of 12 contigs with a genome size of 25.74 Mb, G + C content of 49.16% and 9310 protein-coding genes. The genome-wide BLAST of the PUL genes of M. pulcherrima APC 1.2 showed that the four PUL genes were clustered and located on Contig 4 of M. citriensis FL01. In order to further clarify the role of pulcherrimin pigment on biocontrol of M. citriensis FL01, CRISPR/cas9 technology was used to knock out PUL2 gene that was responsible for PA synthesis and the pigmentless mutants with stable phenotype were obtained. The mutant strains of M. citriensis FL01 lost the ability to produce pulcherrimin pigment, and simultaneously lost the ability to inhibit the growth of Geotrichum citri-aurantii in vitro. Moreover, the biocontrol efficacy of pigmentless mutant strains against sour rot was about 80% lower than that of wild-type M. citriensis FL01. These results directly proved that the iron depletion was an important mechanism of M. citriensis FL01.

Antofine inhibits postharvest green mold due to imazalil-resistant Penicillium digitatum strain Pdw03 by triggering oxidative burst.

The emergence of imazalil (IMZ) resistance in Penicillium digitatum has become a great threat for controlling citrus green mold. In this paper, we investigated the antifungal efficiency and mechanism of an alkaloid antofine against an IMZ-resistant P.digitatum strain Pdw03. Results showed that antofine exhibited a strong antifungal activity against the mycelial growth of strain Pdw03, with a minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of 1.56×10-3 and 1.25×10-2 g/L, respectively. In vivo application of antofine effectively delayed the disease progress and reduced the incidence of green mold in citrus fruit. The disease incidence of 10×MFC antofine-treated fruit after 6days of storage was only 11%±4%, which was significantly lower than that of the control (100%±0%). Antofine treatment altered mycelial morphology of strain Pdw03 without affecting the cell wall integrity. Although the ergosterol contents remained stable, a decrease in the total lipid content induced by lipid peroxidation was observed at 30min of exposure, indicating disruption of cell membrane permeability of strain Pdw03. In addition, the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) contents were also decreased at 60min of exposure. These results indicated that antofine inhibited the growth of strain Pdw03 by disrupting cell membrane permeability and impairing energy metabolism induced by oxidative burst. PRACTICAL APPLICATIONS: One of the most economically important postharvest diseases of citrus fruit is green mold caused by Penicillium digitatum. The pathogen is mainly controlled by using imazalil, but the prolonged and extensive application of this chemical fungicide has led to emergence of numerous IMZ-resistant strains among P.digitatum isolates. Consequently, new and safe strategies for controlling citrus green mold caused by IMZ-resistant P.digitatum strains are urgently needed. In this study, an alkaloid antofine effectively inhibited the growth of IMZ-resistant P.digitatum strain Pdw03 and significantly decreased green mold incidence in the affected citrus fruits. Antofine induced membrane lipid peroxidation of Pdw03 mycelia, resulting in damage to the cell membrane and impairment of energy metabolism. Antofine is therefore a potential antifungal agent for the control of green mold, which provide theoretical guidance for the food industry.

Protection of Citrus Fruits from Postharvest Infection with Penicillium digitatum and Degradation of Patulin by Biocontrol Yeast Clavispora lusitaniae 146.

Fungal rots are one of the main causes of large economic losses and deterioration in the quality and nutrient composition of fruits during the postharvest stage. The yeast Clavispora lusitaniae 146 has previously been shown to efficiently protect lemons from green mold caused by Penicillium digitatum. In this work, the effect of yeast concentration and exposure time on biocontrol efficiency was assessed; the protection of various citrus fruits against P. digitatum by C. lusitaniae 146 was evaluated; the ability of strain 146 to degrade mycotoxin patulin was tested; and the effect of the treatment on the sensory properties of fruits was determined. An efficient protection of lemons was achieved after minimum exposure to a relatively low yeast cell concentration. Apart from lemons, the yeast prevented green mold in grapefruits, mandarins, oranges, and tangerines, implying that it can be used as a broad-range biocontrol agent in citrus. The ability to degrade patulin indicated that strain 146 may be suitable for the control of further Penicillium species. Yeast treatment did not alter the sensory perception of the aroma of fruits. These results corroborate the potential of C. lusitaniae 146 for the control of postharvest diseases of citrus fruits and indicate its suitability for industrial-scale fruit processing.

Control of major citrus postharvest diseases by sulfur-containing food additives

Sodium metabisulfite (SMBS), potassium metabisulfite (PMBS), aluminum sulfate (AlS) and aluminum potassium sulfate (AlPS), common sulfur-containing salts used as food additives, were evaluated for their antifungal activity against Penicillium digitatum, Penicillium italicum and Geotrichum citri-aurantii, the most economically important pathogens causing postharvest diseases of citrus fruits. In vitro radial mycelial growth was measured on potato dextrose agar (PDA) Petri dishes amended with five different concentrations of the salts (10, 20, 30, 50, 100 mM) after 7 d of incubation at 25 °C. SMBS and PMBS at all concentrations, and AIS and AIPS above 20 mM, completely inhibited the growth of these fungi. The curative antifungal activity of the four salts to control citrus green (GM) and blue (BM) molds and sour rot (SR) was evaluated on ‘Valencia’ oranges artificially inoculated in rind wounds with P. digitatum, P. italicum and G. citri-aurantii, respectively. In vivo primary screenings showed no significant antifungal activity of AlS and AlPS to control the three diseases at any dose tested, but SMBS and PMBS reduced the incidence and severity of GM, BM and SR at various concentrations. Effective salts and concentrations were selected for in vivo dip treatments in small-scale trials. Dips at room temperature (20 °C) in SMBS and PMBS at 20 and 50 mM for 60 or 120 s significantly reduced the incidence and severity of GM and BM, with PMBS at 50 mM for 120 s the most effective treatment. Conversely, dips in SMBS and PMBS at 50 mM for 60 or 120 s did not reduce SR incidence and severity. SMBS and PMBS treatments are potentially new tools to be included in reduced-risk non-polluting strategies to control Penicillium diseases, but not SR, on citrus fruits.

Differential contribution of the two major polygalacturonases from Penicillium digitatum to virulence towards citrus fruit

The fungus Penicillium digitatum is the causal agent of the citrus green mould, the major postharvest diseases of citrus fruit. Lesions on the surface of infected fruits first appear as soft areas around the inoculation site, due to maceration of fruit. The macerating activity has been associated with pectinases secreted by the fungus during infection. In order to evaluate the contribution to virulence and macerating activity of the two major polygalacturonases (PGs) secreted by P. digitatum, we have obtained and characterized mutants lacking either pg1 or pg2, the genes encoding PG1 and PG2, respectively. Disease incidence of deletants in either gene was not different from that of the parental strain or ectopic transformants. However, disease progressed more slowly in deletants, especially in those lacking the pg2 gene. The lesions originated by the Δpg2 deletants were not as soft and the pH was not as acid as those originated by either the wild type strain or the ectopic transformants. Total PG activity in the macerated tissue was also lower in fruits infected with the Δpg2 deletants. Interestingly, the macerated tissue of oranges infected with Δpg2 deletants showed around 50% reduction in galacturonic acid content with respect to lesions caused by any other strain.

Effect of applying cinnamaldehyde incorporated in wax on green mould decay in citrus fruits.

Green mould caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Cinnamaldehyde (CA) is a food additive that has potential use in controlling postharvest disease of fruits and vegetables. In this study, the effectiveness of wax with CA (WCA) in controlling Ponkan (Citrus reticulata Blanco) green mould was investigated. The mycelial growth of P. digitatum was inhibited by CA in a dose-dependent manner. The minimum inhibitory concentration and minimum fungicidal concentration (MFC) were both 0.50 mL L-1 . In vivo tests demonstrated that WCA (1 × and 10 × MFC) applied to Ponkan fruits inoculated with P. digitatum could significantly decrease the incidence of green mould for up to 5 days. The WCA treatment increased the activities of catalase, superoxide dismutase, peroxidase, phenylalanine ammonia lyase, polyphenol oxidase, as well as the total phenols and flavonoids contents. Meanwhile, the treatment remarkably decreased the weight loss rate of fruits and maintained fruit quality. These results indicated that WCA treatment might induce defence responses against green mould in citrus fruit. Our findings suggest that WCA might be a promising approach in controlling green mould of citrus fruits. © 2017 Society of Chemical Industry.

Use of Food Additives to Control Postharvest Citrus Blue Mold Disease

The aim of this study was to find an alternative to the chemical fungicide currently used in the control of postharvest citrus diseases. The antifungal activity of 10 salt compounds, considered as common food additives was assayed in in vitro and in vivo trials against Penicillium italicum, causal agent of citrus blue mold. Among the 10 tested salt compounds, sodium carbonate, ammonium carbonate, copper sulfate, sodium EDTA and sodium metabisulfite completely inhibited mycelial growth of Penicillium italicum at 20 mM. Colony growth of P. italicum on pH adjusted medium was evaluated. Results indicate that P. italicum can grow on both acidic and alkaline pH, with the optimum growth occurred in the range of 4.0 and 8.0. Results of the in vivo trials with tested salt compounds indicate that sodium metabisulfite (100 and 200 mM), boric acid (400 mM), sodium salicylate and sodium sulfite (200, 300 and 400 mM) completely inhibited blue mold development on citrus fruit. Boric acid (400 mM) and sodium metabisulfite (100 mM) gave the best results as they completely inhibited the fungus development without damaging fruit rind. Such healthy products therefore may represent a sustainable alternative to the use of chemical fungicides for controlling postharvest diseases of citrus fruit.

Ag-zeolites as fungicidal material: Control of citrus green mold caused by Penicillium digitatum

One of the most economically important postharvest diseases of citrus fruit is green mold caused by the fungus Penicillium digitatum. In this investigation, the antifungal properties of silver FAU and silver LTA zeolites against P. digitatum either alone or incorporated into edible coatings were evaluated in citrus fruit. The Ag-zeolites were characterized by different techniques such as ICP, TPR, XRD and electronic microscopy. These techniques demonstrated that ion exchange was a suitable method for the incorporation of silver cations into zeolites without changing the structure of the matrix. It was observed that silver was present in the zeolite mainly as well dispersed Ag+ and that different silver species with different reduction properties were formed depending on the zeolite structure. It was found that the resulting materials had potential to control citrus green mold caused by P. digitatum. The results confirmed that incorporation of these zeolites into coatings provided an affordable and safe method to control decay of fresh oranges. The antifungal activity depended on the amount of silver contained in zeolites, but a high silver content induced phytotoxicity in the fruit surface. Nevertheless, Ag-zeolites with low silver content were also active without phytotoxic effects. The best results were obtained with the FAU zeolite, which had the largest pores and the highest Si/Al, showing the influence of the topology and composition of the zeolites on their antifungal properties.

Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral.

BackgroundGreen mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood.ResultsIn this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral.ConclusionOur present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2943-4) contains supplementary material, which is available to authorized users.

Novel mutations in CYP51B from Penicillium digitatum involved in prochloraz resistance.

Green mold caused by Penicillium digitatum is one of the most serious postharvest diseases of citrus fruit, and it is ubiquitous in all citrus growing regions in the world. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in the biological kingdom and a prime target of antifungal drugs. Mutations in CYP51s have been found to be correlated with resistance to azole fungicides in many fungal species. To investigate the mechanism of resistance to prochloraz (PRC) in P. digitatum, the PRC sensitivity was determined in vitro in this study to assess the sensitivity of 78 P. digitatum isolates collected in Hubei province. The results showed that 25 isolates were prochloraz-resistant (PRC-R), including six high-resistant (HR) strains, twelve medium-resistant (MR) and seven low-resistant (LR) strains. A sequence analysis showed no consistent point mutations of PdCYP51A in the PRC-R strains, but four substitutions of CYP51B were found, Q309H in LR strains, Y136H and Q309H in HR strains, and G459S and F506I in MR strains, which corresponded to the four sensitivity levels. Based on the sequence alignment analysis and homology modeling followed by the molecular docking of the PdCYP51B protein, the potential correlation between the mutations and PRC resistance is proposed.

Enhancement of biocontrol efficacy of Pichia membranaefaciens by hot water treatment in postharvest diseases of citrus fruit

Effects of hot water treatment (HWT; 53 °C for 2 min) and Pichia membranaefaciens, either alone or in combination, on controlling Penicillium italicum, Penicillium digitatum, and natural infection in citrus fruit were investigated. Results showed that the combined treatment significantly reduced disease incidence and lesion diameters of blue and green molds in artificially inoculated fruit whether the pathogens were inoculated after or before the treatment compared with the treatment of P. membranaefaciens or HWT alone. The combination of P. membranaefaciens with HWT was as effective as the fungicide treatment in natural infection trials. Application of HWT did not affect the growth of P. membranaefaciens in the wounds of citrus fruit at 20 °C or 4 °C. P. membranaefaciens combined with HWT effectively enhanced the phenylalanine ammonia-lyase, peroxidase, polyphenoloxidase, chitinase, and β-1,3-glucanase activities and stimulated the synthesis of phenolic compounds. These results suggest that the use of HWT is a useful approach to improve the efficacy of P. membranaefaciens in postharvest diseases of citrus fruit.