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

2020 ◽  
Vol 6 (3) ◽  
pp. 175
Author(s):  
Ana-Rosa Ballester ◽  
Luis González-Candelas
Keyword(s):  
Ethylene Production ◽  
Fungal Infections ◽  
Citrus Fruit ◽  
Infection Process ◽  
Penicillium Digitatum ◽  
Ethylene Synthesis ◽  
Knockout Mutant ◽  
Major Pathway ◽  
Knockout Mutants

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. digitatumefeA 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.

scholarly journals Developing Penicillium digitatum Management Strategies on Post-Harvest Citrus Fruits with Metabolic Components and Colonization of Bacillus subtilis L1-21

2022 ◽  
Vol 8 (1) ◽  
pp. 80
Author(s):  
Yongmei Li ◽  
Mengyuan Xia ◽  
Pengbo He ◽  
Qiaoming Yang ◽  
Yixin Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Culture Filtrate ◽  
Management Strategies ◽  
Citrus Fruit ◽  
Inhibition Zone ◽  
Penicillium Digitatum ◽  
Antifungal Compounds ◽  
Fruit Peel ◽  
Post Harvest

Citrus is among the most important plants in the fruit industry severely infected with pathogens. Citrus green mold caused by Penicillium digitatum is one of the most devastating diseases during post-harvest stages of citrus fruit. In this study, a potential endophyte Bacillus subtilis L1-21, isolated from healthy citrus plants, was assessed for its biocontrol activity against the pathogen P. digitatum. Based on an in vitro crosstalk assay, we suggested that B. subtilis L1-21 inhibits the pathogen with an inhibition zone of 3.51 ± 0.08 cm. Biocontrol efficacy was highest for the fermented culture filtrate of B. subtilis L1-21. Additionally, using GC-MS analysis, 13 compounds were detected in the extract of this endophyte. The culture filtrate in Landy medium could enlarge and deform pathogen spores and prevent them from developing into normal mycelium. Accordingly, the Landy culture filtrate of B. subtilis L1-21 was stable in the temperature range of 4–90 °C and pH of 3–11. Further, MALDI-TOF-MS for B. subtilis L1-21 detected surfactin, fengycin, bacillaene and bacilysin as potential antifungal compounds. GFP-tagged B. subtilis L1-21 easily colonized in citrus fruit peel and pulp, suggesting its role in eliminating the fungal pathogen. Altogether, it is highly expected that the production of antifungal compounds, and the colonization potential of B. subtilis L1-21 are required against the post-harvest P. digitatum pathogen on citrus fruit.

scholarly journals A Novel Secreted Cysteine-Rich Anionic (Sca) Protein from the Citrus Postharvest Pathogen Penicillium digitatum Enhances Virulence and Modulates the Activity of the Antifungal Protein B (AfpB)

2020 ◽  
Vol 6 (4) ◽  
pp. 203
Author(s):  
Sandra Garrigues ◽  
Jose F. Marcos ◽  
Paloma Manzanares ◽  
Mónica Gandía
Keyword(s):  
Citrus Fruit ◽  
Penicillium Digitatum ◽  
Antifungal Protein ◽  
Penicillium Expansum ◽  
Wild Type ◽  
Phenotypic Differences ◽  
Antifungal Proteins ◽  
Ascomycete Fungi ◽  
In Vitro Antifungal Activity

Antifungal proteins (AFPs) from ascomycete fungi could help the development of antimycotics. However, little is known about their biological role or functional interactions with other fungal biomolecules. We previously reported that AfpB from the postharvest pathogen Penicillium digitatum cannot be detected in the parental fungus yet is abundantly produced biotechnologically. While aiming to detect AfpB, we identified a conserved and novel small Secreted Cysteine-rich Anionic (Sca) protein, encoded by the gene PDIG_23520 from P. digitatum CECT 20796. The sca gene is expressed during culture and early during citrus fruit infection. Both null mutant (Δsca) and Sca overproducer (Scaop) strains show no phenotypic differences from the wild type. Sca is not antimicrobial but potentiates P. digitatum growth when added in high amounts and enhances the in vitro antifungal activity of AfpB. The Scaop strain shows increased incidence of infection in citrus fruit, similar to the addition of purified Sca to the wild-type inoculum. Sca compensates and overcomes the protective effect of AfpB and the antifungal protein PeAfpA from the apple pathogen Penicillium expansum in fruit inoculations. Our study shows that Sca is a novel protein that enhances the growth and virulence of its parental fungus and modulates the activity of AFPs.

scholarly journals The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Natacha S. Ogando ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Yvonne van der Meer ◽  
Peter J. Bredenbeek ◽  
Clara C. Posthuma ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Antiviral Drug ◽  
Genome Replication ◽  
Large Set ◽  
Knockout Mutant ◽  
Additional Function ◽  
Knockout Mutants ◽  
Mrna Capping

ABSTRACT Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless—as happened occasionally—reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity. IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

scholarly journals Clinical Candida albicans Vaginal Isolates and a Laboratory Strain Show Divergent Behaviors during Macrophage Interactions

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Franziska Gerwien ◽  
Christine Dunker ◽  
Philipp Brandt ◽  
Enrico Garbe ◽  
Ilse D. Jacobsen ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Infections ◽  
Laboratory Strain ◽  
Infection Process ◽  
Vulvovaginal Candidiasis ◽  
Host Immune System ◽  
Host Pathogen Interactions ◽  
Content Type ◽  
Host Pathogen

ABSTRACT Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans, the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced β-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient. IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays.

scholarly journals Amelioration of Salt Tolerance in Soybean (Glycine Max. L) by Plant-Growth Promoting Endophytic Bacteria Produce 1-Aminocyclopropane-1-Carboxylase Deaminase

2018 ◽  
Vol 22 (2) ◽  
pp. 81 ◽  
Author(s):  
Rumella Simarmata ◽  
Ngadiman Ngadiman ◽  
Saifur Rohman ◽  
Partomuan Simanjuntak
Keyword(s):  
Salinity Stress ◽  
Ethylene Production ◽  
Endophytic Bacteria ◽  
Dry Weight ◽  
Crop Productivity ◽  
Germination Percentage ◽  
Stress Conditions ◽  
Ethylene Synthesis ◽  
Soybean Seedlings

     Salinity is a major abiotic stress that can induce ethylene synthesis beyond the normal limits as plants response to stress and hence reduces crop productivity. The 1-aminocyclopropane-1-carboxylase deaminase (ACCD)-producing bacteria can reduce excessive ethylene synthesis by taking ACC (ethylene precursor) as a nitrogen source. This study showed the possibility of using endophytic bacteria in order to reduce the undesirable effects of salinity. Strain Pseudomonas putida PIR3C and Roultella terrigena PCM8 exhibited promising performance for promoting the growth of plant under salinity stress conditions. The results showed that bacterial inoculation was effective even in the presence of higher salinity levels. Strain P. putida PIR3C was the most efficient strain compared to the other strains and significantly increased shoot length, root length, dry weight, germination percentage, and reduced stem diameter. The role of ACCD in reducing ethylene production under salinity stress conditions was also studied by measuring the evolution of ethylene in vitro by soybean seeds treated with some ACCD bacterial strain. The maximum ethylene lowering capacity was observed in R. terrigena PCM8, the strain reduced ethylene production from 622.81 nmol.g-1(control) to 352.78 nmol.g-1 (43% reduction). The production of α-ketobutyrate, chlorophyll content and germination percentage from P. putida PIR3C was higher than other strains. The results suggested that strain P. putida PIR3C and R. terrigena PCM8 can be employed for salinity tolerance in soybean seedlings and may have better prospects for an amelioration of stress condition.

scholarly journals Effects of Peptide Thanatin on the Growth and Transcriptome of Penicillium digitatum

2020 ◽  
Vol 11 ◽  
Author(s):  
Guirong Feng ◽  
Xindan Li ◽  
Wenjun Wang ◽  
Lili Deng ◽  
Kaifang Zeng
Keyword(s):  
Ribosome Biogenesis ◽  
Molecular Mechanisms ◽  
High Efficiency ◽  
Citrus Fruit ◽  
Underlying Mechanism ◽  
Treated Group ◽  
Penicillium Digitatum ◽  
Major Facilitator Superfamily

Penicillium digitatum is the most damaging pathogen provoking green mold in citrus fruit during storage, and there is an urgent need for novel antifungal agents with high efficiency. The aim of this study was to investigate the antifungal effects of peptide thanatin against P. digitatum and the molecular mechanisms. Results showed that peptide thanatin had a prominent inhibitory effect on P. digitatum by in vitro and in vivo test. A total of 938 genes, including 556 downregulated and 382 upregulated genes, were differentially expressed, as revealed by RNA-seq of whole P. digitatum genomes analysis with or without thanatin treatment. The downregulated genes mainly encoded RNA polymerase, ribosome biogenesis, amino acid metabolism, and major facilitator superfamily. The genes associated with heat shock proteins and antioxidative systems were widely expressed in thanatin-treated group. DNA, RNA, and the protein content of P. digitatum were significantly decreased after thanatin treatment. In conclusion, thanatin could inhibit the growth of P. digitatum, and the underlying mechanism might be the genetic information processing and stress response were affected. The research will provide more precise and directional clues to explore the inhibitory mechanism of thanatin on growth of P. digitatum.

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

2019 ◽  
Author(s):  
Ana-Rosa Ballester ◽  
Mario López-Pérez ◽  
Beatriz de la Fuente ◽  
Luis González-Candelas
Keyword(s):  
Protease Inhibitors ◽  
Functional Characterization ◽  
Citrus Fruit ◽  
Significant Loss ◽  
Penicillium Digitatum ◽  
Extracellular Proteases ◽  
Climate Conditions ◽  
Virulence Gene Expression

Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. In the present work, we have addressed the study of the role of P. digitatum’s proteases in virulence following two complementary approaches. In a first approach, we have undertaken the functional characterization of the P. digitatum prtT gene, which codes for a 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 a second approach, we have studied the effect of different protease inhibitors and chelators in 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.

Synergistic Antifungal Activity of Sodium Hypochlorite, Hydrogen Peroxide, and Cupric Sulfate against Penicillium digitatum

2009 ◽  
Vol 72 (8) ◽  
pp. 1660-1665 ◽  
Author(s):  
LUCIANA CERIONI ◽  
VIVIANA ANDREA RAPISARDA ◽  
MIRNA HILAL ◽  
FERNANDO EDUARDO PRADO ◽  
LUISA RODRÍGUEZ-MONTELONGO
Keyword(s):  
Hydrogen Peroxide ◽  
Antifungal Activity ◽  
Sodium Hypochlorite ◽  
Citrus Fruit ◽  
Causal Agent ◽  
Penicillium Digitatum ◽  
Sequential Treatment ◽  
Cupric Sulfate ◽  
In Vitro Treatment

Oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2) are widely used in food sanitization because of their antimicrobial effects. We applied these compounds and metals to analyze their antifungal activity against Penicillium digitatum, the causal agent of citrus green mold. The MICs were 300 ppm for NaClO and 300 mM for H2O2 when these compounds were individually applied for 2 min to conidia suspensions. To minimize the concentration of these compounds, we developed and standardized a sequential treatment for conidia that resulted in loss of viability on growth plates and loss of infectivity on lemons. The in vitro treatment consists of preincubation with 10 ppm of NaClO followed by incubation with 100 mM H2O2 and 6 mM CuSO4 (cupric sulfate). The combination of NaClO and H2O2 in the presence of CuSO4 produces a synergistic effect (fractional inhibitory concentration index of 0.36). The sequential treatment applied in situ on lemon peel 24 h after the fruit was inoculated with conidia produced a significant delay in the fungal infection. The in vitro treatment was effective on both imazalil-sensitive and imazalil-resistant strains of P. digitatum and Geotrichum candidum, the causal agent of citrus sour rot. However, this treatment inhibited 90% of mycelial growth for Penicillium italicum (citrus blue mold). These results indicate that sequential treatment may be useful for postharvest control of citrus fruit diseases.

scholarly journals Functionalization of the Chalcone Scaffold for the Discovery of Novel Lead Compounds Targeting Fungal Infections

Molecules ◽  
2019 ◽  
Vol 24 (2) ◽  
pp. 372 ◽  
Author(s):  
Francesca Bonvicini ◽  
Giovanna Gentilomi ◽  
Francesca Bressan ◽  
Silvia Gobbi ◽  
Angela Rampa ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Infection Process ◽  
New Drugs ◽  
Added Value ◽  
Yeast Growth ◽  
Candida Spp ◽  
Lead Compounds

The occurrence of invasive fungal infections represents a substantial threat to human health that is particularly serious in immunocompromised patients. The limited number of antifungal agents, devoid of unwanted toxic effects, has resulted in an increased demand for new drugs. Herein, the chalcone framework was functionalized to develop new antifungal agents able to interfere with cell growth and with the infection process. Thus, a small library of chalcone-based analogues was evaluated in vitro against C. albicans ATCC 10231 and a number of compounds strongly inhibited yeast growth at non-cytotoxic concentrations. Among these, 5 and 7 interfered with the expression of two key virulence factors in C. albicans pathogenesis, namely, hyphae and biofilm formation, while 28 emerged as a potent and broad spectrum antifungal agent, enabling the inhibition of the tested Candida spp. and non-Candida species. Indeed, these compounds combine two modes of action by selectively interfering with growth and, as an added value, weakening microbial virulence. Overall, these compounds could be regarded as promising antifungal candidates worthy of deeper investigation. They also provide a chemical platform through which to perform an optimization process, addressed at improving potency and correcting liabilities.

scholarly journals A viral protein promotes host SAMS1 activity and ethylene production for the benefit of virus infection

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Shanshan Zhao ◽  
Wei Hong ◽  
Jianguo Wu ◽  
Yu Wang ◽  
Shaoyi Ji ◽  
...  
Keyword(s):  
Ethylene Production ◽  
Host Plants ◽  
Ethylene Biosynthesis ◽  
Rice Dwarf Virus ◽  
Ethylene Synthesis ◽  
Ethylene Signaling ◽  
Ethylene Precursor ◽  
Knockout Mutants ◽  
Biotic Stress Response ◽  
Host Antiviral Response

Ethylene plays critical roles in plant development and biotic stress response, but the mechanism of ethylene in host antiviral response remains unclear. Here, we report that Rice dwarf virus (RDV) triggers ethylene production by stimulating the activity of S-adenosyl-L-methionine synthetase (SAMS), a key component of the ethylene synthesis pathway, resulting in elevated susceptibility to RDV. RDV-encoded Pns11 protein specifically interacted with OsSAMS1 to enhance its enzymatic activity, leading to higher ethylene levels in both RDV-infected and Pns11-overexpressing rice. Consistent with a counter-defense role for ethylene, Pns11-overexpressing rice, as well as those overexpressing OsSAMS1, were substantially more susceptible to RDV infection, and a similar effect was observed in rice plants treated with an ethylene precursor. Conversely, OsSAMS1-knockout mutants, as well as an osein2 mutant defective in ethylene signaling, resisted RDV infection more robustly. Our findings uncover a novel mechanism which RDV manipulates ethylene biosynthesis in the host plants to achieve efficient infection.

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