Berberine hydrochloride delays citrus sour rot mainly by disrupting carbohydrate and energy metabolism of Geotrichum citri-aurantii spores.

Citrus sour rot is a common postharvest citrus disease caused by Geotrichum citri-aurantiiti, which has led to enormous economic losses, particularly during rainy seasons. In this study, we aimed to clarify the impact of berberine hydrochloride (BH), the hydrochloride form of an isoquinoline alkaloid, on the control efficiency of citrus sour rot and its antifungal mode against G. citri-aurantii. Results demonstrated that BH markedly impede the propagation of G. citri-aurantii by delaying the spores development from dormant stage into swollen and germinating stages, with the MIC and MFC value of 0.08 and 0.16 g L-1, respectively. When the artificially inoculated citrus fruit in control group were totally rotted, the disease incidence of BH-treated groups decreased by 35.00%-73.30%, which effectively delayed the disease progression and almost did not negatively affect fruit quality. SEM observation, CFW and PI staining images revealed that BH caused significant damage to both the cell membrane and cell wall of G. citri-aurantii spores, whereas only the cell membrane of the mycelium was affected. The impact of cell wall was related to the block of chitin and ß-1,3-glucan synthesis. Transcriptome results and further verification proved that 0.5 × MIC BH treatment affected the glycolysis pathway and TCA cycle mainly by inhibiting the production of acetyl-CoA and pyruvate. Subsequently, the activities of key enzymes declined, resulting in a further decrease in ATP levels, ultimately inhibiting the germination of spores. In conlusion, BH delays citrus sour rot mainly by disrupting carbohydrate and energy metabolism of G. citri-aurantii spores.

Starch-based antifungal edible coatings to control sour rot caused by Geotrichum citri-aurantii and maintain postharvest quality of 'Fino' lemon.

BACKGROUND: Two edible coating (EC) emulsions based on potato starch (F6 and F10) alone or formulated with sodium benzoate (SB, 2% w/w) (F6/SB and F10/SB) were evaluated to maintain postharvest quality of cold-stored 'Fino' lemons and control sour rot on lemons artificially inoculated with Geotrichum citri-aurantii. Previous research showed the potential of these ECs to improve the storability of 'Orri' mandarins and reduce citrus green and blue molds caused by Penicillum digitatum and Penicillium italicum, respectively. RESULTS: The coatings F6/SB and F10/SB significantly reduced sour rot incidence and severity compared to uncoated control samples on lemons incubated at 28 °C for 4 and 7 days. The F6/SB coating reduced weight loss and gas exchange compared to uncoated fruit after 2 and 4 weeks of storage at 12 °C plus a shelf life of 1 week at 20 °C, without adversely affecting the lemon physicochemical quality. CONCLUSION: Overall, the F6/SB coating formulation, composed of pregelatinized potato starch, glyceryl monostearate, glycerol, emulsifiers and SB, with a total solid content of 5.5%, showed the best results in reducing citrus sour rot and maintaining the postharvest quality of cold-stored 'Fino' lemons. Therefore, it showed potential as a new cost-effective postharvest treatment suitable to be included in integrated disease management programs for citrus international markets with zero tolerance to chemical residues. © 2021 Society of Chemical Industry.

Genome sequencing and transcriptome analysis of Geotrichum citri-aurantii on citrus reveal the potential pathogenic- and guazatine-resistance related genes.

Sour rot, caused by Geotrichum citri-aurantii, is a major postharvest disease of citrus，and it causes serious economic losses. In this study, a high-quality genome sequence of G. citri-aurantii was obtained by Single Molecule Real-Time Sequencing (SMRT). Approximately 5.43 Gb of clean data were obtained and a total of 27.94-Mb genomic sequence was mapped to 10 chromosome groups after high-through chromosome conformation capture (Hi-C) assembly. In addition, three polygalacturonase genes which were related to pathogenicity in G. citri-aurantii genome were discovered. And transcriptome data of guazatine-resistance had been analyzed, the results showed that the guazatine-resistance of G. citri-aurantii was related to two ATP-binding cassette (ABC) transporter family genes, six major facilitator superfamily (MFS) transporter family genes and two multidrug and toxic compound extrusion (MATE) transporter family genes. In summary, our research may provide novel insights into the effective control of this pathogen.

Purification of a killer toxin from Aureobasidium pullulans for the biocontrol of phytopathogens.

The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, ß-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including ß-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii.

Selective C-H dithiocarbamation of arenes and antifungal activity evaluation.

This paper discloses a transition metal-free selective C-H dithiocarbamation of drug skeletons using disulfiram (DSF) in the presence of KI/K2S2O8 in DMF/H2O. Drug skeletons, including 5-aminopyrazoles, indoles, pyrroloquinoline, and Julolidine, underwent C-H dithiocarbamation smoothly to afford a variety of drug-like molecules in moderate to good yields. It was found that the in situ formed 5-aminopyrazole iodide is the key intermediate for the dithiocarbamation. Bioassay results show that some of these N-heterocyclic dithiocarbamate derivatives exhibit good antifungal activity against Colletotrichum gloeosprioides and Fusarium oxysporum, F. proliferatum, Fusarium solani, Geotrichum candidum, Penicillium digitatum, Penicillium italicum, Phyricularia grisea.

Exploring the interaction between citrus flavonoids and phytopathogenic fungi through enzymatic activities.

Flavonoids are involved in citrus defense against phytopathogens. In this study, we applied in vitro biocatalysis assays using the flavanones glycosides hesperidin and naringin to explore the enzymatic activities involved in such interaction. The main enzymatic activity observed was the hydrolysis catalyzed by fungi naringinases and hesperidinases. Withing 7 days, the two citrus phytopathogenic fungi, Penicillium digitatum and Penicillium italicum, exhibited the highest hydrolyzing rate on the flavanones, reaching conversion values higher than 90%. In addition, Geothrichum citri-aurantii exhibited no enzymatic activity and Penicillium expansum only hydrolyzed hesperidin. In order to evaluate flavonoid biotransformation by the fungi in vivo, citrus fruits infected with P. digitatum were analyzed through molecular networking and Imaging Mass Spectrometry (IMS). In vivo assays revealed that citrus fruit in response to the infection is able to hydroxylate flavonoids, and novel flavonoid structures were associated to the citrus' defense. The data reported here present a new point of view in the relation between citrus flavonoids and phytopathogenic fungi and can be useful to understand the infection processes and host-pathogen interaction.

Thymol Encapsulated into HP-ß-Cyclodextrin as an Alternative to Synthetic Fungicides to Induce Lemon Resistance against Sour Rot Decay.

Consumers demand the use of eco-friendly fungicides to treat fruit and vegetables and governmental authorities have unauthorized the application of chemical antifungals for the efficient control of sour rot. In the present research, the microwave irradiation (MW) method was used to encapsulate thymol into 2-hydroxylpropyl-beta-cyclodextrin (HP-ß-CD) and the effect of these HP-ß-CD on controlling sour rot in citrus fruit, caused by Geotrichum citri-aurantii, was evaluated. Amounts of 25 and 50 mM of HP-ß-CD-thymol were used, and compared with propiconazole, to control the decay of inoculated lemon fruit. The treatments were performed in curative and preventive experiments. The incidence and severity of Geotrichum citri-aurantii in 25 and 50 mM HP-ß-CD-thymol-treated fruit were reduced in both experiments. The preventive 50 mM HP-ß-CD-thymol treatment showed the best effect, reducing the sour rot, respiration rate and fruit weight loss during storage at 20 °C. HP-ß-CD-thymol increased polyphenol concentration and the activity of antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) in lemon peel, and the highest effects were found with the 50-mM dose. In conclusion, the results show that the use of thymol encapsulated by MW into HP-ß-CD could be an effective and sustainable tool, a substitute to the synthetic fungicides, for G. citri-auriantii control in citrus fruit.

Inhibitory effects of glutaraldehyde on Geotrichum citri-aurantii and its possible mechanism.

AIMS: This study investigated the inhibitory effect of glutaraldehyde (GA) on sour rot in citrus fruit and the underlying antifungal mechanism on mycelial growth of the causative pathogen Geotrichum citri-aurantii. METHODS AND RESULTS: Glutaraldehyde exhibited a strong inhibitory effect on G. citri-aurantii, with a minimum inhibitory and fungicidal concentration (MFC) of 1·00 µl ml-1 . In addition, in vivo application of GA (1 × MFC and 5 × MFC) reduced the disease incidence of sour rot in citrus fruit by 60 and 80% respectively. Scanning electron microscopy results revealed that the morphology of G. citri-aurantii mycelia was greatly altered by GA treatment. Propidium iodide and Calcofluor White Staining revealed that the membrane permeability, rather than the cell wall integrity, of G. citri-aurantii mycelia was severely disrupted after the addition of GA. Massive accumulation of malonaldehyde and reactive oxygen species as well as an increase in lipoxygenase activity were observed. CONCLUSION: These results indicate that GA may inhibit the mycelia growth of G. citri-aurantii through a membrane damage mechanism induced by membrane peroxidation. SIGNIFICANCE AND IMPACT OF THE STUDY: Glutaraldehyde is expected to be a novel fungicide for controlling sour rot in citrus fruit.

Sequencing of FKS Hot Spot 1 from Saprochaete capitata To Search for a Relationship to Reduced Echinocandin Susceptibility.

Saprochaete capitata, formerly known as Geotrichum capitatum, is an emerging fungal pathogen with low susceptibility to echinocandins. Here, we report the nucleotide sequence of the S. capitata hot spot 1 region of the FKS gene (FKS HS1), which codifies for the catalytic subunit of ß-1,3-d-glucan synthase, the target of echinocandins. For that purpose, we first designed degenerated oligonucleotide primers derived from conserved flanking regions of the FKS1 HS1 segment of 12 different fungal species. Interestingly, analysis of the translated FKS HS1 sequences of 12 isolates of S. capitata revealed that all of them exhibited the same F-to-L substitution in a position that is highly related to reduced echinocandin susceptibility.

Fungal Infection Caused by Geotrichum capitatum in a Severe Aplastic Anemia Patient: a Case Report and Review of the Literature.

BACKGROUND: Invasive fungal infections often occur in immunocompromised patients. Here, we report an infection case caused by Geotrichum capitatum in a severe aplastic anemia patient. METHODS: Identification of the pathogenic bacteria was done by sequencing and mass spectrometric analysis. RESULTS: The fungal infection was isolated from blood cultures. The pathogenic bacteria were identified as Geotrichum capitatum. The infection was primarily cured by voriconazole and caspofungin monotherapy. However, the effect was not obvious. Then a combination of liposomal amphotericin B and caspofungin was used. Body temperature of the patient decreased, and clinical symptoms improved. CONCLUSIONS: Sequencing and mass spectrometric analysis could have a role for Geotrichum capitatum diagnosis. Curative effect of using a single antifungal drug was unsatisfactory. Using liposome amphotericin B combined with caspofungin might obtain certain curative effect. Early diagnosis and appropriate combined therapy were necessary to improve the outcome of patients with Geotrichum capitatum infection.

Antifungal mechanism of sodium dehydroacetate against Geotrichum citri-aurantii.

This study investigated the potential anti-fungal mechanisms of sodium dehydroacetate (SD) against Geotrichum citri-aurantii. The results showed that the cell wall integrity of G. citri-aurantii was not affected, whereas the membrane permeability of G. citri-aurantii mycelia was visibly altered by SD. Dramatic morphological changes of the mycelia, such as loss of cytoplasm, plasmolysis, and dissolution of intracellular substances, were observed by scanning electron microscopy and transmission electron microscopy analyses, indicating that the mycelium is severely damaged by the SD treatment. Furthermore, SD apparently induced a decrease in the intracellular ATP content before 30 min of exposure. An increase in the activity of the Na+/K+-ATPase was also observed, indicating that Na+ ions might enter the cell and thus disturb the energy supply. Taken together, this study's findings suggest that the anti-fungal activity of SD against G. citri-aurantii can be attributed to the disruption of cell membrane permeability and energy metabolism.

Use of Cistus aqueous extracts as botanical fungicides in the control of Citrus sour rot.

In this work, we investigated the in vitro and in vivo antifungal activity of aqueous extracts obtained from eight Cistus plant species against the development of Geotrichum citri-aurantii, the causal agent of citrus sour rot. The results demonstrate the in vitro effectiveness of all tested Cistus species aqueous extracts against G. citri-aurantii, the inhibition of mycelial growth ranged between 80 and 100%. Furthermore, Cistus aqueous extracts totally inhibited germination of G. citri-aurantii arthrospores at a concentration of 5 mg/mL. Among the plant species tested, C. laurifolius, C. salviifolius, C. monspeliensis, C. ladanifer and C. populifolius displayed the best fungistatic activity since the minimum inhibitory concentration (MIC) was <0.625 mg/mL. Under in vivo conditions, incidence of sour rot was lowered to 8.33% when fruits were treated with aqueous extracts of C. populifolius and C. creticus compared to 100% in the control. Disease severity was lowered to 3.74, 4.47 and 5.13% when fruits were treated with C. creticus, C. ladanifer and C. populifolius aqueous extracts, respectively. Using such these biopesticides in a replacement for synthetic fungicides or in combination with other established disease management practices could help control citrus postharvest decay in a more sustainable and eco-friendly way.

Invasive infections due to Saprochaete and Geotrichum species: Report of 23 cases from the FungiScope Registry.

Saprochaete and Geotrichum spp. are rare emerging fungi causing invasive fungal diseases in immunosuppressed patients and scarce evidence is available for treatment decisions. Among 505 cases of rare IFD from the FungiScope™ registry, we identified 23 cases of invasive infections caused by these fungi reported from 10 countries over a 12-year period. All cases were adults and previous chemotherapy with associated neutropenia was the most common co-morbidity. Fungaemia was confirmed in 14 (61%) cases and deep organ involvement included lungs, liver, spleen, central nervous system and kidneys. Fungi were S. capitata (n=14), S. clavata (n=5), G. candidum (n=2) and Geotrichum spp. (n=2). Susceptibility was tested in 16 (70%) isolates. All S. capitata and S. clavata isolates with the exception of one S. capitata (MIC 4 mg/L) isolate had MICs>32 mg/L for caspofungin. For micafungin and anidulafungin, MICs varied between 0.25 and >32 mg/L. One case was diagnosed postmortem, 22 patients received targeted treatment, with voriconazole as the most frequent first line drug. Overall mortality was 65% (n=15). Initial echinocandin treatment was associated with worse outcome at day 30 when compared to treatment with other antifungals (amphotericin B ± flucytosine, voriconazole, fluconazole and itraconazole) (P=.036). Echinocandins are not an option for these infections.

Syntheses of Novel 4-Substituted N-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamide Derivatives with Potential Antifungal Activity.

Candidiasis represent a serious threat for patients with altered immune responses. Therefore, we have undertaken the synthesis of compounds comprising a pyridine-3-sulfonamide scaffold and known antifungally active 1,2,4-triazole substituents. Thus a series of novel 4-substituted N-(5-amino-1H-1,2,4-triazol-3-yl)pyridine-3-sulfonamides have been synthesized by multistep reactions starting from 4-chloropyridine-3-sulfonamide via N'-cyano-N-[(4-substitutedpyridin-3-yl)sulfonyl]carbamimidothioates which were further converted with hydrazine hydrate to the corresponding 1,2,4-triazole derivatives 26-36. The final compounds were evaluated for antifungal activity against strains of the genera Candida, Geotrichum, Rhodotorula, and Saccharomycess isolated from patients with mycosis. Many of them show greater efficacy than fluconazole, mostly towards Candida albicans and Rhodotorula mucilaginosa species, with MIC values ≤ 25 µg/mL. A docking study of the most active compounds 26, 34 and 35 was performed showing the potential mode of binding to Candida albicans lanosterol 14α-demethylase. Also in vitro cytotoxicity of selected compounds have been evaluated on the NCI-60 cell line panel.

A cluster of Geotrichum clavatum (Saprochaete clavata) infection in haematological patients: a first Italian report and review of literature.

Invasive fungal infections, usually Aspergillus and Candida, represent a major cause of morbidity and mortality in patients with malignant haematological diseases, but in the last years rare fungal infections have more frequently been reported. Here, we report the clinical history of three patients affected with haematological malignancies who developed an infection caused by Geotrichum (G.) clavatum. Two out of three patients were affected by acute myeloid leukaemia (AML), and one by mantle cell lymphoma (MCL). All patients received cytarabine-based chemotherapeutic regimens and developed G. clavatum infection within 3 weeks from therapy initiation. In all cases, G. clavatum was isolated from central venous catheter and peripheral blood cultures. In vitro susceptibility test confirmed an intrinsic resistance to echinocandins and, in all cases, visceral localisations (spleen, liver and lung) were documented by total body computed tomography (CT) scan. A prolonged antifungal therapy with high doses liposomal amphotericin-B was necessary to obtain fever resolution. Only the patient with MCL died while the other two AML recovered, and one of them after received an allogeneic stem cell transplantation. We consecutively reviewed all published cases of infection caused by G. clavatum. Our experience and literature review indicate that G. clavatum can cause invasive infection in haematological patients, mainly in those with acute leukaemia.

Antifungal properties of organic extracts of eight Cistus L. species against postharvest citrus sour rot.

UNLABELLED: The effectiveness of methanol and chloroform extracts of eight Cistaceae species to control citrus sour rot decay, caused by Geotrichum citri-aurantii, was investigated in both in vitro and in vivo conditions. Methanol extracts of these plant species exhibited more interesting activity against G. citri-aurantii, in both in vitro and in vivo conditions, compared with chloroforme extracts. Under in vitro trials, obtained results showed that methanol extracts of all tested plants revealed a highest significant antifungal activity with inhibition zones that ranged between 12·33 and 16·33 mm in diameter. All tested methanol extracts totally inhibited spore germination when tested at 10 mg ml(-1) . Incidence of sour rot was significantly lowered to 11·11% when fruits were treated with Cistus populifolius and Cistus ladanifer methanol extracts compared with 100% in the control. The disease severity was lowered to 5·19% and 6·04% when fruits were treated with the same methanol extracts respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The methanol Cistus extracts had sufficient antifungal activities in vitro and in vivo against G. citri-aurantii to consider its use in the citrus industry after it has been tested under production and natural infection conditions. Such natural products therefore represent a viable alternative approaches for sour rot postharvest management of citrus.

Ellagitannins from Raspberry (Rubus idaeus L.) Fruit as Natural Inhibitors of Geotrichum candidum.

The paper presents the chemical characteristics of ellagitannins isolated from raspberry (Rubus idaeus L.) fruit and their in vitro and in situ antifungal activity against Geotrichum candidum LOCK 0511. The study investigated a complex preparation containing various raspberry ellagitannins at a concentration of 86% w/w, as well as pure lambertianin C and sanguiin H-6. The ellagitannin preparation was obtained by extracting raspberry press cake and purifying the extract using Amberlite XAD resin, while individual compounds were isolated by means of preparative HPLC. The complex preparation was analyzed for the content of ellagitannins, anthocyanins, and flavan-3-ols using HPLC and LC-MS. The antifungal activity of the complex ellagitannin preparation and the isolated ellagitannins was determined for the strain Geotrichum candidum. The MIC and MFC values (10.0 mg/mL and 30.0 mg/mL, respectively) were found to be the same for lambertianin C, sanguiin H-6, and the complex ellagitannin preparation. The fungistatic activity of the studied ellagitannin preparation at a concentration of 10 mg/mL, as determined by the poisoned medium method, was 65.2% following 6 day incubation of Geotrichum candidum, with the linear growth rate of only 16.2 mm/day. The corresponding parameters for the control sample were 0% and 56 mm/day, respectively. The study demonstrated both in vitro and in situ antifungal activity of raspberry ellagitannins against Geotrichum candidum.

Mode of action of metabolites from Bacillus sp. strain IBA 33 on Geotrichum citri-aurantii arthroconidia.

Geotrichum citri-aurantii is a postharvest phytopathogenic fungus of lemons. We studied the mode of action of antifungal metabolites from Bacillus sp. strain IBA 33 on arthroconidia of G. citri-aurantii. These metabolites are lipopeptides belonging to the iturin family. Membrane permeabilization of G. citri-aurantii was analyzed and mitochondrial respiratory rate was evaluated. Disturbance of the plasma membrane promotes the leakage of many cellular components into the surrounding media, and mitochondrial membrane disorganization promotes the inhibition of the respiratory rate. Our findings provide insights into the ability of lipopeptides to suppress plant fungal pathogens and their possible agronomical applications.

Exploring the Efficacy of Using Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus, Clay Minerals, and Walnut Nutshells for Mycotoxin Remediation.

The aim of this study was to evaluate the effectiveness of nine different biological compounds to reduce mycotoxins concentrations. The hypothesis of this study was that a static in vitro gastrointestinal tract model, as an initial screening tool, can be used to simulate the efficacy of Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus yeast cell walls and their polysaccharides, red and white clay minerals, and walnuts nutshells claiming to detoxify AFB1, ZEA, DON, and T-2 toxin mycotoxins. Mycotoxin concentrations were analyzed using high-performance liquid chromatography (HPLC) with fluorescent (FLD) and ultraviolet detectors (UV). The greatest effects on reducing mycotoxin concentrations were determined as follows: for AFB1, inserted G. fermentans cell wall polysaccharides and walnut nutshells; for ZEA, inserted R. rubra and G. fermentans cell walls and red clay minerals; for DON, R. rubra cell wall polysaccharides and red clay minerals; and for T-2 toxin, R. rubra cell walls, K. marxianus, and G. fermentans cell wall polysaccharides and walnut nutshells. The present study indicated that selected mycotoxin-detoxifying biological compounds can be used to decrease mycotoxin concentrations.

Antifungal efficiency and mechanisms of ethyl ferulate against postharvest pathogens.

Postharvest loss caused by a range of pathogens necessitates exploring novel antifungal compounds that are safe and efficient in managing the pathogens. This study evaluated the antifungal activity of ethyl ferulate (EF) and explored its mechanisms of action against Alternaria alternata, Aspergillus niger, Botrytis cinerea, Penicillium expansum, Penicillium digitatum, Geotrichum candidum and evaluated its potential to inhibit postharvest decay. The results demonstrated that EF exerts potent antifungal activity against a wide board of postharvest pathogens. Results also revealed that its antifungal mechanism is multifaceted: EF may be involved in binding to and disturbing the integrity of the fungal plasma membrane, causing leakage of intracellular content and losing normal morphology and ultrastructure. EF also induced oxidative stress in the pathogen, causing membrane lipid peroxidation and malondialdehyde accumulation. EF inhibited the critical gene expression of the pathogen, affecting its metabolic regulation, antioxidant metabolism, and cell wall degrading enzymes. EF exhibited antifungal inhibitory activity when applied directly into peel wounds or after incorporation with chitosan coating. Due to its wide board and efficient antifungal activity, EF has the potential to provide a promising alternative to manage postharvest decay.