SntB Affects Growth to Regulate Infecting Potential in Penicillium italicum.

Penicillium italicum, a major postharvest pathogen, causes blue mold rot in citrus fruits through the deployment of various virulence factors. Recent studies highlight the role of the epigenetic reader, SntB, in modulating the pathogenicity of phytopathogenic fungi. Our research revealed that the deletion of the SntB gene in P. italicum led to significant phenotypic alterations, including delayed mycelial growth, reduced spore production, and decreased utilization of sucrose. Additionally, the mutant strain exhibited increased sensitivity to pH fluctuations and elevated iron and calcium ion stress, culminating in reduced virulence on Gannan Novel oranges. Ultrastructural analyses disclosed notable disruptions in cell membrane integrity, disorganization within the cellular matrix, and signs of autophagy. Transcriptomic data further indicated a pronounced upregulation of hydrolytic enzymes, oxidoreductases, and transport proteins, suggesting a heightened energy demand. The observed phenomena were consistent with a carbon starvation response potentially triggering apoptotic pathways, including iron-dependent cell death. These findings collectively underscored the pivotal role of SntB in maintaining the pathogenic traits of P. italicum, proposing that targeting PiSntB could offer a new avenue for controlling citrus fungal infections and subsequent fruit decay.

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.

The plasma membrane H+-ATPase is critical for cell growth and pathogenicity in Penicillium digitatum.

The plasma membrane H+-ATPase (PMA1) is a major cytosolic pH regulator and a potential candidate for antifungal drug discovery due to its fungal specificity and criticality. In this study, the function of Penicillum digitatum PMA1 was characterized through RNA interference (RNAi) and overexpression technology. The results showed that silencing the PMA1 gene reduces cell growth and pathogenicity, and increases susceptibility of P. digitatum to proton pump inhibitors (PPIs). Under scanning electron microscopy (SEM) and transmission electron microscopy (TEM) examination, cell morphology was significantly altered in the PMA1- silenced mutant (si57). When compared with wild type (WT) and the overexpressed mutant (oe9), the cell walls of the si57 mutant were thicker and their cell membrane damage manifested particularly at sites of polarized growth. Consistent with the morphological change on the cell wall, chitin and glucan content of the cell wall of si57 were significantly lower and accompanied with increased activities of chitinase and glucanase. The lower ergosterol content in the si57 mutant then increased cell membrane permeability, ultimately leading to leakage of cytoplasmic contents such as ions, reduced sugars and soluble proteins. Furthermore, significantly decreased activity of cell wall degrading enzymes of si57 during citrus fruit infections indicates a reduced pathogenicity in this mutant. We conclude that PMA1 in P. digitatum plays an important role in maintaining pathogenesis and PMA1 could be a candidate novel fungicidal drug discovery for citrus green mold. KEY POINTS: Silencing PMA1 gene decreased the growth and pathogenicity of P. digitatum. Silencing PMA1 gene damaged cell wall and cell membrane integrity of P. digitatum. PMA1 appears to be a suitable fungicidal target against citrus green mold.

Ergosterol depletion under bifonazole treatment induces cell membrane damage and triggers a ROS-mediated mitochondrial apoptosis in Penicillium expansum.

Penicillium expansum is the causal agent of blue mold in harvested fruits and vegetables during storage and distribution, causing serious economic loss. In this study we seek the action modes of bifonazole against this pathogen. Bifonazole exhibited strong antifungal activity against P. expansum by inhibiting ergosterol synthesis. The ergosterol depletion caused damage to the cell structure and especially cell membrane integrity as observed by SEM and TEM. With increased unsaturated fatty acids contents, the cell membrane viscosity decreases and can no longer effectively maintain the cytoplasm, which ultimately decreases extracellular conductivity, changes intracellular pH and ion homeostasis. Exposure of hyphal cells to bifonazole shows that mitochondrial respiration is inhibited and reactive oxygen species (ROS) levels-including H2O2 and malondialdehyde (MDA) - are significantly increased. The functional impairment of mitochondria and cell membrane eventually cause cell death through intrinsic apoptosis and necroptosis.

Active compound identification by screening 33 essential oil monomers against Botryosphaeria dothidea from postharvest kiwifruit and its potential action mode.

The antifungal activity of postharvest kiwifruit against the pathogen Botryosphaeria dothidea was evaluated for 33 essential oil monomers. The possible mechanism for the known active compounds were further assessed in this study. The results show all the EO components exhibit inhibitory effects on the pathogen to different degrees except for Farnesol. Carbon chain length and C2-C3 double bonds had a great effect on the antifungal activities of aldehydes. Of all of these, carvacrol had the strongest antifungal activity with EC50 of 12.58 µL/L and EC90 of 22.08 µL/L. Carvacrol also exhibits significant inhibitory effects on the pathogen, both in vivo and in vitro. Carvacrol evidently alters the hyphal morphology of B. dothidea and severely damages cell membrane and inhibits the formation of lipid components on the membrane. As cell membrane permeability increases, intracellular homeostasis including ion and biomacromolecules were destroyed by carvacrol. Furthermore, carvacrol appears to significantly inhibit mitochondrial activity and respiration rates, resulting in cell death of B. dothidea. Our results provide evidence that carvacrol could be a very useful compound for controlling postharvest rot soft in kiwifruit.

Physiological and iTRAQ-based proteomic analyses reveal the mechanism of pinocembrin against Penicillium italicum through targeting mitochondria.

The physiological and iTRAQ-based proteomic analyses were used to reveal the inhibitory roles of pinocembrin on mitochondria of P. italicum and its cell death mechanism. The results show that pinocembrin damages both mitochondrial structure and function. 167 and 807 differentially expressed proteins (DEPs) were detected in P. italicum mycelia after treatment with pinocembrin for 8 h and 24 h respectively, and the DEPs were significantly enriched in the oxidative phosphorylation (OXPHOS) pathway, especially for mitochondrial respiratory chain (MRC) complexes I and V. Furthermore, the expression levels of proteins related to programmed cell death (PCD) were significantly up-regulated in mycelia with Pinocembrin incubation for 24 h. Combined with the results of physio-chemical analysis, the data revealed that pinocembrin targeted MRC complexes I and V, to induce ATP depletion, enhance ROS accumulation, stimulate mitochondrial permeability transition pore (MPTP) opening, accelerate the loss of mitochondrial membrane potential (MMP) and promote cytochrome c release from mitochondria to the cytoplasm, which, as a result, effectively triggered three classical types of PCD pathways in mycelia of P. italicum.

The structure-antifungal activity relationship of 5,7-dihydroxyflavonoids against Penicillium italicum.

To evaluate the structure-activity relationship of 5,7-dihydroxyflavonoids against P. italicum, we tested the antifungal activity of 23 selected 5,7-dihydroxyflavonoids against spore germination of P. italicum, and the effects of hydroxyl group, hydrogenation, methylation and glycosylation on the antifungal activity are explored. C-4'-OH and C-3-OH are active groups for the 5,7-dihydroxyflavonoids against P. italicum. We find that hydrogenation of the C2/C3 bond decreases the antifungal activity of 5,7-dihydroxyflavonoids. Antifungal activity of 5,7-dihydroxyflavonoids against P. italicum was affected by the conjugation site of glycosylation and the class of sugar moiety. The correlation between antifungal activity and the inhibition of respiration of 5,7-dihydroxyflavonoids was further evaluated. We find no significant relationship among the IC50 of 5,7-dihydroxyflavonoids on spore germination and on respiration. Some 5,7-dihydroxyflavonoids even enhance the respiration of P. italicum. This indicate respiration is not the only target for 5,7-dihydroxyflavonoids against P. italicum.

Use of active extracts of poplar buds against Penicillium italicum and possible modes of action.

Antifungal components, from poplar buds active fraction (PBAF) against Penicillium italicum, the causal agent of blue mold in citrus fruits, were identified and possible action modes were investigated. Pinocembrin, chrysin and galangin were determined as active components in PBAF, using HPLC and HPLC-MS analysis. The antifungal activity is stable at temperatures ranging from 4 °C to 100 °C and pH levels ranging from 4 to 8. In the presence of PBAF, the hyphae become shriveled, wrinkled and the cell membrane became seriously disrupted. Further investigation on cell permeability, nucleic acid content and alkaline phosphatase suggest that the cell membrane might be the target. Mycelial oxygen consumption and the respiration-related enzymatic activity of succinate dehydrogenase, malate dehydrogenase and ATPase were all inhibited by PBAF. We propose that PBAF is a potentially useful alternative for blue mold control and may act against P. italicum by interfering with respiration and disrupting the cell membrane.

Effects of poplar buds as an alternative to propolis on postharvest diseases control of strawberry fruits.

BACKGROUND: Botrytis cinerea and Rhizopus stolonifer, two main postharvest pathogens, cause great loss of strawberry fruits. Here, the effects of poplar buds extracts, a main plant source for Chinese propolis, on disease control were investigated in vitro and in vivo. RESULTS: The HPLC profile of poplar buds ethanol extract (PBEE) was almost identical to that of propolis ethanol extract (PEE), with the active flavonoids identified as pinocembrin, chrysin and galangin. PBEE exhibited similar inhibitory activities on spore germination of both pathogens compared with PEE, and PBEE also strongly inhibited the mycelial growth of the pathogens. In vivo, PBEE could effectively reduce decay of strawberry fruits stored at 13 °C. Although the weight loss was slightly increased, the contents of total soluble solid, titritable acid, vitamin C and total anthocyanins were significantly higher in PBEE treated fruits than those of the control. CONCLUSION: PBEE had the similar antifungal activity with propolis and had great potential as an alternative to propolis to control strawberry fruits diseases.

[Accelerated senescence of fresh-cut Chinese water chestnut tissues in relation to hydrogen peroxide accumulation].

Accelerated senescence of fresh-cut Chinese water chestnut (CWC) tissues in relation to active oxygen species (AOS) metabolism was investigated. Fresh-cut CWC (2 mm thick) and intact CWC were stored at 4 degrees C in trays wrapped with plastic films. Changes in superoxide anion production rate, activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were monitored, while contents of hydrogen peroxide, ascorbic acid, MDA as well as electrolyte leakage were measured. Fresh-cutting of CWC induced activities of SOD, CAT and APX to a certain extent (Fig. 2B and Fig. 3), but simultaneously stimulated superoxide anion production markedly (Fig. 2A), enhanced hydrogen peroxide accumulation and accelerated loss in ascorbic acid (Figs. 4 and 5), which resulted in increased lipid peroxidation indicated by malondialdehyde (MDA) content and electrolyte leakage (Fig. 1). Statistics analysis indicated that there was a significantly positive correlation among hydrogen peroxide accumulation, MDA content and electrolyte leakage (Table 1). Histochemical detection with 3, 3'-diaminobenzidine further demonstrated that hydrogen peroxide accumulation increased in fresh-cut CWC during storage (Fig. 5). AOS production rate and activities of SOD, CAT and APX changed little while no obvious hydrogen peroxide accumulation was observed, in intact CWC during storage.