Pathogen Of Citrus Fruit Research Articles

Bioinformatics-based identification of GH12 endoxyloglucanases in citrus-pathogenic Penicillium spp

Millions of tons of citrus peel waste are produced every year as a byproduct of the juice industry. Citrus peel is rich in pectin and xyloglucan, but while the pectin is extracted for use in the food industry, the xyloglucan is currently not valorized. To target hydrolytic degradation of citrus peel xyloglucan into oligosaccharides, we have used bioinformatics to identify three glycoside hydrolase 12 (GH12) endoxyloglucanases (EC 3.2.1.151) from the citrus fruit pathogens Penicillium italicum GL-Gan1 and Penicillium digitatum Pd1 and characterized them on xyloglucan obtained by alkaline extraction from citrus peel. The enzymes displayed pH-temperature optima of pH 4.6–5.3 and 35–37°C. PdGH12 from P. digitatum and PiGH12A from P. italicum share 84% sequence identity and displayed similar kinetics, although kcat was highest for PdGH12. In contrast, PiGH12B from P. italicum, which has the otherwise conserved Trp in subsite −4 replaced with a Tyr, displayed a 3 times higher KM and a 4 times lower kcat/KM than PiGH12A, but was the most thermostable enzyme of the three Penicillium-derived endoxyloglucanases. The benchmark enzyme AnGH12 from Aspergillus nidulans was more thermally stable and had a higher pH-temperature optimum than the enzymes from Penicillum spp. The difference in structure of the xyloglucan oligosaccharides extracted from citrus peel xyloglucan and tamarind xyloglucan by the new endoxyloglucanases was determined by LC-MS. The inclusion of citrus peel xyloglucan demonstrated that the endoxyloglucanases liberated fucosylated xyloglucan oligomers, implying that these enzymes have the potential to upgrade citrus peel residues to produce oligomers useful as intermediates or bioactive compounds.

A circular economy approach: A new formulation based on a lemon peel medium activated with lactobacilli for sustainable control of post-harvest fungal rots in fresh citrus fruit

The aim of the study was to develop an effective bio-formulation for preventing post-harvest fungal rots in fresh citrus fruit through a multi-step process that includes isolation and characterization of lactic acid bacteria (LABs) from citrus fruit peel, selection of LAB strains with high antifungal activity, chemical characterization of cell free supernatants (CFS), formulation of a lemon peel powdery-based medium (LM) activated with LAB fermentates, chemical characterization of fermented LM, and evaluation of the efficacy of the new bio-formulation against blue mold. Thirteen LABs were recovered from the rind of citrus fruit and identified by the peptide mass fingerprinting method. The antifungal activity of LABs, isolated from citrus fruit, as well as cell free supernatants (CFSs), obtained from their liquid cultures, was tested against diverse plant pathogenic fungi and oomycetes, using the dual-culture overlay and the diffusion agar assays, respectively. Two isolates designated as N3B2 and M2B2, both identified as Lactiplantibacillus plantarum, were selected among the others for their relevant antifungal activity. Both isolates showed a broad spectrum antagonistic activity comprising also major postharvest pathogens of citrus fruit, such as Penicillium digitatum and P. italicum, the causal agents of green and blue moulds, respectively, Alternaria alternata, Colletotrichum gloeosporioides, C. karsti, Phytophthora citrophthora and Ph. nicotianae. The N3B2 and M2B2 isolates were used as starter cultures to ferment a lemon peel powder medium enriched with a nutrient water solution (LM). The two formulations obtained by fermenting LM with the N3B2 and M2B2 isolates showed a strong in vitro inhibitory activity on the same broad range of pathogens used in the preliminary screening of LABs. Moreover, the two LM-based formulations reduced the severity of blue mold infection and inhibited the sporulation of P. italicum on artificially inoculated lemon fruit. The chemical analysis of LM-based bioformulations revealed their antifungal activity was very probably due to the LAB-derived acidic components, including lactic, acetic, DL-3-phenyllactic, 3-4-dihydroxyhydrocinnamic, salicylic acid vanillic acids. These innovative LM-based bioformulations activated with LAB-derived antifungal compounds will be exploited as edible and biodegradable fruit coatings to prolong the shelf-life and prevent post-harvest rots of fresh citrus fruit.

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.

Enhancing bioefficacy of Bacillus amyloliquefaciens SF14 with salicylic acid for the control of the postharvest citrus green mould

Penicillium digitatum is a major post-harvest pathogen of citrus fruit causing tremendous economic losses. In this study, salicylic acid (SA) and antagonistic bacteria Bacillus amyloliquefaciens (SF14) were tested in controlling P. digitatum (green mould) infections as an alternative to chemical controls. Three concentrations of SA (0.5, 1, and 2%) were tested for their antifungal activity in both in vitro and in vivo conditions alone and in combination with SF14. Fruit quality parameters including fruit firmness, weight loss, titratable acidity, and soluble solids content were evaluated. The impact of these biological treatments was also investigated by measuring the changes in organic and mineral properties of orange fruits using Fourier transform infrared spectroscopy (FT-IR). Enzymatic activity including phenylalanine ammonia-lyase (PAL), total polyphenols and flavonoids was also tested. In vitro assays showed that SF14 combined with 0.5% SA provided the highest inhibition of green mould growth reaching 72.98%. Inhibition of spore germination was also highest in the same treatment od reaching 63.40%. FTIR of fungal biomass confirmed an alteration of fungal well wall following biological treatments. Moreover, during in vivo assays, the combination of SF14 and 0.5% SA produced the lowest disease severity (5.43%) without considerably affecting the fruit quality. Further investigation of the effect of these treatments on the mineral composition of treated oranges showed that Ca, Fe, K, Mg, and Na concentrations were significantly affected. Changes in these elements may show a defence reaction induced by the biological treatments against the pathogen, which was also confirmed with the measurement of PAL activity, total polyphenols, and flavonoids. Interestingly, FTIR of citrus peel indicated significant changes in the content of bands denoting for phenolic and aromatic rings, suggesting an induced resistance against the pathogenic fungus. Overall, our findings suggested that the combination of both bacterial antagonist SF14 and 0.5% SA can control the green mould of citrus, and as such constitutes a safer alternative to chemicals during postharvest storage.

Treatment with Glyphosate Induces Tolerance of Citrus Pathogens to Glyphosate and Fungicides but Not to 1,8-Cineole.

During the postharvest period, citrus fruits are exposed to Penicillium italicum, Penicillium digitatum, and Geotrichum candidum. Pesticides such as imazalil (IMZ), thiabendazole (TBZ), orthophenylphenol (OPP), and guazatine (GUA) are commonly used as antifungals. Glyphosate (GP) is also used in citrus fields to eliminate weed growth. The sensitivity of fungal pathogens of citrus fruit to these pesticides and 1,8-cineole was evaluated, and the effect of GP on the development of cross-resistance to other chemicals was monitored over a period of 3 weeks. IMZ most effectively inhibited the mycelial growth and spore germination of P. digitatum and P. italicum, with minimum inhibitory concentrations (MICs) of 0.01 and 0.05 mg/mL, respectively, followed by 1,8-cineole, GP, and TBZ. 1,8-Cineole and GP more effectively inhibited the mycelial growth and spore germination of G. candidum, with minimum inhibitory concentrations (MICs) of 0.2 and 1.0 mg/mL, respectively, than OPP or GUA. For the spore germination assay, all substances tested showed a total inhibitory effect. Subculturing the fungal strains in culture media containing increasing concentrations of GP induced fungal tolerance to GP as well as to the fungicides. In soil, experiments confirmed that GP induced the tolerance of P. digitatum to TBZ and GP and the tolerance of P. italicum to IMZ, TBZ, and GP. However, no tolerance was recorded against 1,8-cineole. In conclusion, it can be said that 1,8-cineole may be recommended as an alternative to conventional fungicides. In addition, these results indicate that caution should be taken when using GP in citrus fields.

Antifungal features and properties of Pickering emulsion coating from alginate/lemongrass oil/cellulose nanofibers

SummaryThis study investigated the antifungal features of sodium alginate (Alg) 1% combined with lemongrass essential oil (LGO) 0.25%, 0.5% and 0.75% as a Pickering emulsion coating to control Penicillium digitatum and P. italicum, identified as citrus fruit pathogens. Cellulose nanofibers 0.24%, 42.55 ± 9.34 nm, were selected as a stabiliser. Confocal laser scanning microscopy and Fourier‐transform infrared spectroscopy were used to analyse the droplet size and morphology and chemical interaction of emulsified coating, respectively. The effective dosage of lemongrass oil (0.75%) may enhance the antifungal action confirmed with a series of in vitro tests on spore germination by 88.28% (P. digitatum) and 91.94% (P. italicum), germ tube elongation by 89.28% (P. digitatum) and 90.13% (P. italicum) and membrane integrity by 41.67% (P. digitatum) and 63% (P. italicum). Additionally, the incorporation of LGO Pickering emulsion improved the beneficial properties of Alg‐based coating film, including light transmission at UV and visible light wavelengths and hydrophobicity. Scanning electron microscopy was performed to evaluate the interior microstructure of the coating film. Pickering emulsion‐based coatings described in this study may have potential applications for active packaging, particularly for citrus fruit commodities.

Metabolomics Study at the Postharvest Conditions of Cold Storage and Fungicide (Imazalil Sulfate) Treatment in Navel Oranges and Clementine Mandarins.

Millions of citrus products are wasted every year due to postharvest fungal infections. To minimize fungal infections, packhouses utilize aqueous applications of fungicides to prevent infections that occur during harvest. The most prominent fungal pathogens of citrus fruit are commonly treated with imazalil sulfate (IMZ) due to its efficacy for controlling these pathogens at low cost and ease of handling. However, little is known on how it alters the tissues in the citrus fruit physiology. In this study, a nuclear magnetic resonance (NMR)-based metabolomics study is utilized to investigate the role of IMZ treatment in the juice, albedo, and flavedo tissues of two citrus commodities (navels and clementines). The experimental design consists of (a) fresh fruits at harvest, (b) raw fruits stored at 4 °C for 10 days, and (c) raw fruits treated with IMZ and stored at 4 °C for 10 days. Twenty-seven metabolites were identified, and several changes of metabolite composition due to either cold storage or IMZ treatment for both the spatial (albedo, flavedo, or juice) and temporal levels (days and storage) were found. The results show a notable difference between metabolomics profiles across the types and tissues, particularly significant changes on the albedo tissues of clementine. Furthermore, the pathways derived from the metabolomics profiles of the cold storage and the IMZ treatment are complementary to each other. Thus, the utility of metabolomics as a quality control tool in the citrus industry has the potential for broader applications to understand fruit growth and development.

Albedo- and Flavedo-Specific Transcriptome Profiling Related to Penicillium digitatum Infection in Citrus Fruit.

Penicillium digitatum is the main postharvest pathogen of citrus fruit. Although the inner fruit peel part (albedo) is less resistant than the outer part (flavedo) to P. digitatum, the global mechanisms involved in their different susceptibility remain unknown. Here, we examine transcriptome differences between both tissues at fruit harvest and in their early responses to infection. At harvest, not only was secondary metabolism, involving phenylpropanoids, waxes, and terpenoids, generally induced in flavedo vs. albedo, but also energy metabolism, transcription factors (TFs), and biotic stress-related hormones and proteins too. Flavedo-specific induced responses to infection might be regulated in part by ERF1 TF, and are related to structural plant cell wall reinforcement. Other induced responses may be related to H2O2, the synthesis of phenylpropanoids, and the stress-related proteins required to maintain basal defense responses against virulent pathogens, whereas P. digitatum represses some hydrolase-encoding genes that play different functions and auxin-responsive genes in this peel tissue. In infected albedo, the repression of transport and signal transduction prevail, as does the induction of not only the processes related to the synthesis of flavonoids, indole glucosinolates, cutin, and oxylipins, but also the specific genes that elicit plant immunity against pathogens.

Salicylic acid and Cinnamomum verum confer resistance against Penicillium rot by modulating the expression of defense linked genes in Citrus reticulata Blanco

Penicillium digitatum and Penicillium italicum are potentially important post-harvest pathogens of citrus fruit causing huge economic loss. In this study salicylic acid (SA) and Cinnamomum verum were tested to control the infection of P. digitatum (green mold) and P. italicum (blue mold) as an alternative to chemical control. In an in vitro assay methanolic extracts of five plants were tested for antifungal activity where C. verum exhibited the highest colony growth inhibition 74.6 and 76.4 % of green and blue mold respectively. Moreover, during In Planta assay the combination of C. verum and SA produced the lowest disease incidence (20 and 33.3%) and severity (13.1 and 6.1%) of green and blue mold respectively compared to stand-alone treatments without affecting the fruit quality considerably. Furthermore, an upsurge in the the activity of polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia lyase (PAL) encoding genes of citrus fruit was recorded during transcriptional profiling. The highest gene expression was recorded in fruit treated with C. verum and SA in combination compared to healthy control. Similar results were observed during quantification of corresponding gene products. This unveils the fact that the enhanced expression of defense-linked genes might be associated with the disease suppression. Conclusively, our findings indicate that C. verum and SA in combination can suppress green and blue mold of Citrus by modulating the expression of defense-linked genes. The combined use of plant extracts and resistance inducers is a safer alternate to chemicals to suppress green and blue mold during storage.

Coordinated activation of the metabolic pathways induced by LED blue light in citrus fruit

The biochemical changes induced by LED Blue Light (LBL) (450 nm) in Lane Late oranges were investigated. The selected quantum flux (60 µmol m−2 s−1, 2 days) was associated with resistance against Penicillium digitatum, the main postharvest pathogen of citrus fruit. A holistic overview was obtained by a comparative transcriptome profile analysis, which revealed that LBL favored energy metabolism and redirected metabolic pathways toward the synthesis of diverse primary and secondary metabolism products. LBL favored reactive oxygen species homeostasis and metabolic activities involving lipid metabolism, specifically the synthesis of pigments and oxylipins, and the metabolism of carbohydrates, amino acids and indol- and alkaloid-derivatives. LBL also repressed limonene catabolism and triggered phenylpropanoid derivatives-related changes, which increased content in total flavonoids. Transferring fruit from LBL to darkness favored those processes involving amino acids, different phenylpropanoid, alkaloid and terpenoid classes, and ferrochelatase activity.

EFE-Mediated Ethylene Synthesis Is the Major Pathway in the Citrus Postharvest Pathogen Penicillium digitatum during Fruit Infection.

Penicillium digitatum is the main fungal postharvest pathogen of citrus fruit under Mediterranean climate conditions. The role of ethylene in the P. digitatum–citrus fruit interaction is unclear and controversial. We analyzed the involvement of the 2-oxoglutarate-dependent ethylene-forming enzyme (EFE)-encoding gene (efeA) of P. digitatum on the pathogenicity of the fungus. The expression of P. digitatum efeA parallels ethylene production during growth on PDA medium, with maximum levels reached during sporulation. We generated ΔefeA knockout mutants in P. digitatum strain Pd1. These mutants showed no significant defect on mycelial growth or sporulation compared to the parental strain. However, the knockout mutants did not produce ethylene in vitro. Citrus pathogenicity assays showed no differences in virulence between the parental and ΔefeA knockout mutant strains, despite a lack of ethylene production by the knockout mutant throughout the infection process. This result suggests that ethylene plays no role in P. digitatum pathogenicity. Our results clearly show that EFE-mediated ethylene synthesis is the major ethylene synthesis pathway in the citrus postharvest pathogen P. digitatum during both in vitro growth on PDA medium and the infection process, and that this hormone is not necessary for establishing P. digitatum infection in citrus fruit. However, our results also indicate that ethylene produced by P. digitatum during sporulation on the fruit surface may influence the development of secondary fungal infections.

The mechanism involved in enhancing the biological control efficacy of Rhodotorula mucilaginosa with salicylic acid to postharvest green mold decay of oranges

The main postharvest pathogen of citrus fruit is Penicillium digitatum. The present study looked at the role of Rhodotorula mucilaginosa enhanced with 0.2 mM salicylic acid (SA) on pH, lignin content accumulation and growth dynamics as a resistance mechanism against P. digitatum in orange fruit. Our findings revealed that the increase in lignin content at 20 °C storage temperature resulted in the significant decrease (P ≤ 0.05) in the lesion diameter in fruit treated with R. mucilaginosa enhanced with or without 0.2 mM SA compared to the untreated fruit (control). In addition, the pH values of the fruit treated with R. mucilaginosa enhanced with or without 0.2 mM SA were 4.43 ± 0.07 and 4.15 ± 0.11, respectively, around the infection site compared to the untreated group. Moreover, the in vivo trial showed that the addition of 0.2 mM SA to the antagonist augmented its growth, and subdued substantially the hyphae and spore germination of P. digitatum in vitro. Also, lower levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) content were observed in fruit treated with the antagonist enhanced with or without 0.2 mM SA compared to the control. The results established that pH, lignin content accumulation and growth of the yeast played a significant role in the control of green mold decay of orange fruit.

The Myosin Motor Domain-Containing Chitin Synthases Are Involved in Cell Wall Integrity and Sensitivity to Antifungal Proteins in Penicillium digitatum.

Penicillium digitatum is the main postharvest pathogen of citrus fruit and is responsible for important economic losses in spite of the massive use of fungicides. The fungal cell wall (CW) and its specific component chitin are potential targets for the development of new antifungal molecules. Among these are the antifungal peptides and proteins that specifically interact with fungal CW. Chitin is synthesized by a complex family of chitin synthases (Chs), classified into up to eight classes within three divisions. Previously, we obtained and characterized a mutant of P. digitatum in the class VII gene (ΔchsVII), which contains a short myosin motor-like domain (MMD). In this report, we extend our previous studies to the characterization of mutants in chsII and in the gene coding for the other MMD-Chs (chsV), and study the role of chitin synthases in the sensitivity of P. digitatum to the self-antifungal protein AfpB, and to AfpA obtained from P. expansum. The ΔchsII mutant showed no significant phenotypic and virulence differences with the wild type strain, except in the production and morphology of the conidia. In contrast, mutants in chsV showed a more dramatic phenotype than the previous ΔchsVII, with reduced growth and conidial production, increased chitin content, changes in mycelial morphology and a decrease in virulence to citrus fruit. Mutants in chsVII were specifically more tolerant than the wild type to nikkomycin Z, an antifungal inhibitor of chitin biosynthesis. Treatment of P. digitatum with its own antifungal protein AfpB resulted in an overall reduction in the expression of the chitin synthase genes. The mutants corresponding to MMD chitin synthases exhibited differential sensitivity to the antifungal proteins AfpA and AfpB, ΔchsVII being more susceptible than its parental strain and ΔchsV being slightly more tolerant despite its reduced growth in liquid broth. Taking these results together, we conclude that the MMD-containing chitin synthases affect cell wall integrity and sensitivity to antifungal proteins in P. digitatum.

Functional and Pharmacological Analyses of the Role of Penicillium digitatum Proteases on Virulence.

Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. Previous results have shown that proteases is the largest enzyme family induced by P. digitatum during fruit infection. In the present work, we addressed the study of the role of P. digitatum’s proteases in virulence following two complementary approaches. In the first approach, we undertook the functional characterization of the P. digitatum prtT gene, which codes for a putative transcription factor previously shown to regulate extracellular proteases in other filamentous fungi. Deletion of prtT caused a significant loss in secreted protease activity during in vitro growth assays. However, there was no effect on virulence. Gene expression of the two major secreted acid proteases was barely affected in the ΔprtT deletant during infection of citrus fruit. Hence, no conclusion could be drawn on the role of these secreted acidic proteases on the virulence of P. digitatum. In the second approach, we studied the effect of different protease inhibitors and chelators on virulence. Co-inoculation of citrus fruit with P. digitatum conidia and a cocktail of protease inhibitors resulted in almost a complete absence of disease development. Analysis of individual inhibitors revealed that the metalloprotease inhibitor, 1,10-phenanthroline, was responsible for the observed effect. The application of metal ions reverted the protective effect caused by the metallopeptidase inhibitor. These results may set the basis for the development of new alternative treatments to combat this important postharvest pathogen.

Antifungal activity and mechanism of action of tannic acid against Penicillium digitatum

Penicillium digitatum is a destructive post-harvest pathogen of citrus fruit during storage and marketing. The aim of this study is to evaluate the activity of the natural polyphenolic compounds, tannins, on P. digitatum. Tannins acted against P. digitatum by inhibiting its mycelial growth and spore germination. In vivo tests showed that tannins significantly (P < 0.05) decrease the disease symptoms of P. digitatum in artificially inoculated citrus fruit. Storage tests also showed that tannins reduce the disease severity of green mold on citrus fruit by 70%. To determine the antifungal mechanism of tannins on P. digitatum, we evaluated the integrity of the cell wall and the permeability of the membrane. The disruption of cell wall and the plasma membrane caused leakage of the intracellular contents, such as sugars. These findings suggest that the cell wall of P. digitatum is the target for tannins and that they can potentially be used as an alternative to control citrus green mold.

Wound healing in citrus fruit is promoted by chitosan and Pichia membranaefaciens as a resistance mechanism against Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is the most important pathogen of citrus fruit and is a serious concern in rainy, humid regions and seasons. In this study, the effects of chitosan and Pichia membranaefaciens on anthracnose control, wound healing, and the cell wall changes in citrus fruit and the relationship between wound healing and cell wall composition were evaluated. We found that chitosan and P. membranaefaciens increased the wound healing process via wound tissue appearance changes, lignin accumulation, increases in cell wall compounds (pectin, cellulose) and reduced activities of cell wall enzymes - polygalacturonase (PG); pectin methylesterase (PME); cellulase (CEL) - to control anthracnose. During wound healing, acid-soluble pectin (ASP) and cellulose had strong positive correlations with lignin.

Temporal Occurrence and Niche Preferences of Phytophthora spp. Causing Brown Rot of Citrus in the Central Valley of California.

Brown rot of citrus fruit is caused by several species of Phytophthora and is currently of serious concern for the California citrus industry. Two species, Phytophthora syringae and P. hibernalis, are quarantine pathogens in China, a major export market for California citrus. To maintain trade and estimate the risk of exporting a quarantine pathogen, the distribution and frequency of Phytophthora spp. causing brown rot of orange in major growing areas of California was investigated. Symptomatic fruit were collected from navel (winter to late spring) and Valencia (late spring to summer) orange orchards from 2013 to 2015. Species identification of isolates was based on morphological characteristics, random amplified polymorphic DNA banding patterns, and sequencing of the internal transcribed spacer and the partial cox2/spacer/cox1 regions from axenic cultures, or directly on DNA from fruit tissue using a multiplex TaqMan quantitative polymerase chain reaction assay. In winter samplings, the incidence of P. syringae based on the number of fruit with Phytophthora spp. detection ranged from 73.6 to 96.1% for the two counties surveyed. The remaining isolates were identified as P. citrophthora. In late spring or summer, only P. citrophthora was recovered. P. hibernalis and P. nicotianae were not detected in any fruit with brown rot symptoms. These results indicate that P. syringae is currently an important brown rot pathogen of citrus fruit in California during the cooler seasons of the year. In winter 2016 and 2017, P. syringae was recovered by pear baiting at a high incidence from leaf litter and from a small number of rhizosphere soil or root samples but not from living leaves on the tree. In contrast, P. citrophthora was rarely found in leaf litter but was commonly detected in the rhizosphere. Thus, leaf litter is a major inoculum source for P. syringae and this species occupies a distinct ecological niche.

Effect of LED Blue Light on Penicillium digitatum and Penicillium italicum Strains.

Studies on the antimicrobial properties of light have considerably increased due in part to the development of resistance to actual control methods. This study investigates the potential of light-emitting diodes (LED) blue light for controlling Penicillium digitatum and Penicillium italicum. These fungi are the most devastating postharvest pathogens of citrus fruit and cause important losses due to contaminations and the development of resistant strains against fungicides. The effect of different periods and quantum fluxes, delaying light application on the growth and morphology of P. digitatum strains resistant and sensitive to fungicides, and P. italicum cultured at 20°C was examined. Results showed that blue light controls the growth of all strains and that its efficacy increases with the quantum flux. Spore germination was always avoided by exposing the cultures to high quantum flux (700 μmol m(-2) s(-1) ) for 18 h. Continuous light had an important impact on the fungus morphology and a fungicidal effect when applied at a lower quantum flux (120 μmol m(-2) s(-1) ) to a growing fungus. Sensitivity to light increased with mycelium age. Results show that blue light may be a tool for P. digitatum and P. italicum infection prevention during handling of citrus fruits.

Pathogenicity and Host Susceptibility of Penicillium spp. on Citrus.

Citrus fruit are exposed to numerous postharvest pathogens throughout the fresh produce supply chain. Well-known postharvest citrus fruit pathogens are Penicillium digitatum and P. italicum. Lesser-known pathogens include P. crustosum and P. expansum. This study examined pathogenicity and aggressiveness of Penicillium spp. present in fresh fruit supply chains on various Citrus spp. and cultivars. The impact of different inoculation methods and storage conditions on decay were also assessed. P. digitatum and P. italicum were the most aggressive Penicillium spp. on citrus but aggressiveness varied significantly over the evaluated citrus range. Decay and tissue-response lesions caused by P. crustosum were observed on 'Nules Clementine', 'Nova', 'Owari Satsuma', 'Delta Valencia', 'Cambria Navel', 'Eureka' seeded, and 'Star Ruby' for the first time. Likewise, these lesions caused by P. expansum were noted on Nules Clementine, Owari Satsuma, Delta Valencia, 'Midknight Valencia', and Eureka seeded for the first time. Tissue-response lesions affect fruit quality and some Penicillium spp. sporulated from the lesions, causing the inoculated species to complete their life cycle. New citrus-Penicillium spp. interactions were observed and the importance of monitoring inoculum loads of pathogens and nonhost pathogens were highlighted.

Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.

The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.