Inhibition Of Spore Germination Research Articles

Cell-free culture filtrate of Trichoderma longibrachiatum AD-1 as alternative approach to control Fusarium solani and induce defense response Phaseolus vulgaris L. plants

The present study was conducted to evaluate the antifungal activities of cell-free culture filtrate of Trichoderma isolated from rhizospheric soil of common bean (Phaseolus vulgaris L.) plants against growth and spore germination of Fusarium solani. The results noted that the cell-free culture filtrate of Trichoderma at different concentrations of isolated Trichoderma longibrachiatum AD-1 significantly inhibited mycelial growth of F. solani with growth reduction ranging from 28.8 to 97.3% and inhibited spore germination up to 96%. Compared to control, soil treated with cell-free culture filtrate of T. longibrachiatum AD-1 exhibited a significant reduction in the frequency of root rot disease and boosted the percentage of surviving plants and vegetative growth. To get insight into the biocontrol mechanism, we employed Real-Time Quantitative Reverse Transcription PCR (RT-qPCR), which revealed that the cell-free culture filtrate of Trichoderma in common bean (Phaseolus vulgaris L.) showed significant upregulation of defense-related genes PR1, PR2, PR3, and PR4. This study revealed that the cell-free culture filtrate of Trichoderma could be promising to develop an alternative natural agent to control root rot diseases caused by Fusarium spp.

Polymethoxylated flavonoids (PMFs)-loaded citral nanoemulsion controls green mold in citrus by damaging the cell membrane of Penicillium digitatum

Citrus is susceptible to Penicillium digitatum (P. digitatum) infection in post-harvest storage, resulting in enormous economic losses. This study aimed to investigate the antifungal activity and potential mechanism of the combination of Polymethoxylated flavones (PMFs) and citral (two natural antifungal components derived from citrus) against P. digitatum in vitro and citrus fruit. The results show that PMFs can enhance the antifungal activity of citral nanoemulsion, and PMFs-loaded citral nanoemulsion (PCT) has significant antifungal activity in a concentration-dependent manner. PCT can evidently inhibit spore germination and mycelial growth in vitro, and effectively control the growth of green mold on postharvest citrus fruit. Furthermore, PCT treatment resulted in the alteration of mycelia morphology, accumulation of reactive oxygen species, and membrane lipid peroxidation. These changes can disrupt the normal structure and function of the cell membrane, as evidenced by the reduction of total lipid and ergosterol content in the mycelia and the stronger red fluorescence of the cells emitted after PI staining. Based on the above results, we infer that PCT has a strong inhibitory effect on P. digitatum, and its potential mechanism is related to the destruction of the cell membrane. Therefore, PCT can be considered as a botanical fungicide for citrus preservation.

Calcium lactate efficiently induces production of bacillomycin D in Bacillus subtilisNS‐174 and inhibition of spore germination of Aspergillus flavus in rice

SummaryThe present study provided a new alternative for efficiently producing lipopeptides by calcium lactate and expanded potential applications of lipopeptides for ensuring food safety. The results indicated that the addition of calcium lactate significantly improved bacillomycin D production. When fed 150 mL of fermentation medium with 6.5 g/L calcium lactate at the batch fermentation time of 102 h, the maximum production of C14 bacillomycin D (C14BD), C15 bacillomycin D (C15BD), C16 bacillomycin D (C16BD), C17 bacillomycin D (C17BD) and total C14‐17 bacillomycin D (TBD) were 298.96, 345.43, 149.56, 216.6 and 1010.55 mg/L at 162 h, respectively. In addition, calcium lactate effectively induced biosynthesis of bacillomycin D through upregulating 2‐ketobutyric acid in the valine, leucine and isoleucine biosynthesis (VLIB) pathway that was closely related to threonine dehydratase and threonine and increasing expression of bacillomycin D synthase genes. Moreover, bacillomycin D could remarkably inhibit spore germination of Aspergillus flavus in rice.

Isolation, identification, and evaluation of the biocontrol potential of aBacillus velezensisstrain against tobacco root rot caused byFusarium oxysporum

Tobacco (Nicotiana tabacum) is an economically important crop. Root rot caused by Fusarium oxysporum has become a damaging disease in N. tabacum crops grown in Henan province of China. Therefore, the objectives of this study were to screen bacterial isolates against F. oxysporum from rhizosphere soils of tobacco growing areas and to evaluate their antifungal activities, biocontrol effects, and effects on plant growth. Nineteen strains with antifungal inhibition effects of >60% against F. oxysporum were obtained using the method of flat confrontation; the strain Ba-0321 was the strongest, with an antifungal effect of 75%. Moreover, this strain had broad spectrum antimicrobial activity to eight additional tobacco pathogens. The strain was identified as Bacillus velezensis by morphology and the 16S rDNA sequence. The B. velezensis strain Ba-0321 had strong UV resistance as well as tolerance to high temperatures and low nutrition. The bacteria inhibited spore germination and mycelial growth of F. oxysporum under in vitro co-culture conditions. In vivo assays demonstrated that the Ba-0321 strain significantly reduced the pathogenicity of F. oxysporum, resulting in a control effect on tobacco root rot of 81.00%. Simultaneously, the bacteria significantly promoted root development and the growth of tobacco plants. Our results confirmed that the B. velezensis strain Ba-0321 has a strong antifungal effect and stress resistance that enable it to be used as a biological control agent for tobacco root rot caused by F. oxysporum. Tobacco root rot caused by F. oxysporum has become a damaging disease in China. The B. velezensis strain Ba-0321 has promising application value for controlling tobacco root rot diseases, and it could provide a new biocontrol agent against root rot caused by F. oxysporum in other plant species.

Synthesis of Biogenic Hematite Nanocubes as Recyclable Dark Fenton-like Catalysts at Neutral pH and Plant Growth Applications of Degraded Waste Water.

The goal of this study is to fabricate bioinspired metal oxide nanocubes from lemon peel extract in an environmentally friendly manner and evaluate its impact on environmental remediation. In neutral pH, the degradation kinetics of methylene blue dye (MB) in the aqueous phase was investigated using iron oxide nanoparticles as a catalyst. The obtained results revealed that under optimum conditions, synthesized Fe2O3 nanoparticles (IONPs) offered ultrafast dark Fenton-like reaction to degrade MB. The size, morphological structures, and stability were confirmed through dynamic light scattering, field emission scanning electron microscopy, X-ray diffraction, and ζ potential analysis. The overall environmental impact of the process was assessed by growing wheat plants with treated wastewater and evaluating their biochemical attributes. Antibacterial activity was investigated against Gram-positive (Bacillus megaterium, Bacillus subtilis) and Gram-negative (Escherichia coli, Salmonella typhimurium) aerobics and Gram-positive cocci (Staphylococcus aureus). The antifungal activity was measured against Fusarium solani by spore germination inhibition and zone inhibition of fungal pathogens for different nanocube concentrations.

Antifungal Activity and Possible Mechanism of Bacillus amyloliquefaciens FX2 Against the Postharvest Apple Ring Rot Pathogen.

Botryosphaeria dothidea-induced apple ring rot is one of the most serious postharvest diseases in apple production. In our preliminary work, we isolated a bacterial strain (FX2) from an infested apple orchard. Here, we confirmed the strong antifungal activity of FX2 on B. dothidea. Through phylogenetic analysis and morphological observations, we identified FX2 as a Bacillus amyloliquefaciens strain. We also found that 10% cell-free supernatant (CFS) of FX2 significantly affected mycelial growth and morphology and almost completely inhibited spore germination and germ tube elongation in B. dothidea. Furthermore, 10% CFS damaged the cell ultrastructure, resulting in a remarkable increase in cellular leakage in B. dothidea mycelia. Thus, CFS has the potential to effectively reduce in vivo B. dothidea infection, reduced lesion diameters to 64.7% compared with the control group, and reduced disease incidence by 15%. Finally, ultrafiltration, desalting chromatography, and anion exchange chromatography showed that the antifungal constituents in CFS are composed mainly of antifungal proteins. We further characterized these potential antifungal proteins via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Herein, we provide novel insights into the antifungal mechanisms of B. amyloliquefaciens FX2, and we highlight its potential as a novel biocontrol agent for controlling postharvest apple ring rot.

The G protein subunit α1, CaGα1, mediates ethylene sensing of mango anthracnose pathogen Colletotrichum asianum to regulate fungal development and virulence and mediates surface sensing for spore germination.

Mango is an important tropic fruit, but its production is highly restricted by anthracnose diseases. Mango anthracnose development is related to the fruit-ripening hormone ethylene, but how the pathogen senses ethylene and affects the infection remains largely unknown. In this study, mango pathogen Colletotrichum asianum strain TYC-2 was shown to sense ethylene to enhance spore germination, appressorium formation and virulence. Upon further analysis of ethylene sensing signaling, three histidine kinase genes (CaHKs) and a G-protein gene (CaGα1) were functionally characterized. Ethylene upregulated the expression of the three CaHKs but had no influence on CaGα1 expression. No function in ethylene sensing was identified for the three CaHKs. Ethylene enhanced spore germination and multiple appressorium formation of the wild-type TYC-2 but not CaGα1 mutants. TYC-2 has extremely low germination in water, where self-inhibition may play a role in ethylene sensing via CaGα1 signaling. Self-inhibitors extracted from TYC-2 inhibited spore germination of TYC-2 and CaGα1 mutants, but ethylene could not rescue the inhibition, indicating that the self-inhibition was not mediated by CaGα1 and had no interactions with ethylene. Interestingly, spore germination of CaGα1 mutants was significantly enhanced in water on hydrophobic but not hydrophilic surfaces, suggesting that CaGα1 is involved in surface sensing. In the pathogenicity assay, CaGα1 mutants showed less virulence with delayed germination and little appressorium formation at early infection on mango leaves and fruit. Transcriptome and qRT-PCR analyses identified several pathogenicity-related genes regulated by ethylene, indicating that ethylene may regulate TYC-2 virulence partially by regulating the expression of these genes.

English

Fusarium oxysporum f.sp. elaeidis (Foe) is a fungus that attacks oil palm and affects its production. This study aimed at characterizing phenolic compounds and evaluating their spatiotemporal evolution in two cultivars (sensitive and tolerant to fusariosis). For this, total phenols were quantified during the latency period [0 to 28 Days After Inoculation (DAI)] of Foe. Thereafter, phenolic extracts from plant organs (roots and pseudobulbs) were used for in vitro inhibition tests of Foe spore germination. Lastly, Gas Chromatography coupled to Mass Spectrophotometry (GC-MS) was used to detect polyphenols. These results revealed that phenolic reaction in roots was earlier (1st DAI or T0) in the tolerant cultivar than in the sensitive (8th DAI). However, it occurred simultaneously in the pseudobulbs (3rd DAI) in both cultivars. Spore Germination Inhibition Rate (SGIR) of Foe reached 100% in the tolerant cultivarb against the sensitive cultivar in which a maximum rate of 49, 64% was recorded. GC-MS revealed that in roots, para-hydroxybenzoic acid is a phytoanticipin while meta-hydroxybenzoic acid is a phytoalexin. However, in pseudobulbs, these two polyphenols are phytoalexins. These results will allow exploring the biosynthetic pathway of phenolic acids in order to improve the phenolic response of disease-sensitive but productive hybrids. Key words: Fusarium oxysporum f.sp. elaeidis, inhibition, polyphenols, GC-MS, meta-hydroxybenzoic acid, para-hydroxybenzoic acid.

The ester derivatives of ferulic acid exhibit strong inhibitory effect on the growth of Alternaria alternata in vitro and in vivo

Alternaria alternata is the principal pathogenic fungus causing postharvest black spot disease of Korla pear, leading to enormous economic losses. This study aimed to explore the antifungal activity of ferulic acid (FA) and its two simple esterified derivatives, methyl ferulate (MF) and ethyl ferulate (EF), against A. alternata in vitro and in vivo, and to reveal the underlying inhibitory mechanisms. The results showed that MF and EF exhibited stronger inhibitory effects than FA on mycelial growth, spore germination and germ tube elongation of A. alternata in vitro, and on reducing the development of black spot disease in Korla pear fruit. Scanning electron microscopy images indicated that the surface morphology of A. alternata hyphae treated with FA, MF, and EF presented irreversible deformation, and transmission electron microscopy observation further demonstrated that MF and EF caused more serious damage to the cell membranes, cytoplasm, and organelles of A. alternata than FA, which impeded the normal growth of A. alternata. Notably, EF was slightly better than MF in inhibiting spore germination and damaging cell ultrastructure of A. alternata. In addition, propidium iodide staining suggested that FA, MF, and EF treatments damaged the plasma membrane integrity of A. alternata spores. At the same conditions, MF and EF treatments induced more leakage of intercellular electrolytes, nucleic acids and proteins of A. alternata than FA, indicating the increase of cell membrane permeability. Taken together, all these results evidenced that MF and EF exerted stronger antifungal activity than FA against A. alternata in vitro and in vivo through membrane damage mechanism, therefore they can be used as promising green substitutes to control postharvest black spot disease on Korla pear fruit.

Transcriptomic analysis shows the antifungal mechanism of honokiol against Aspergillus flavus

Plant-derived substances as antifungal agents have received considerable attention in recent years to reduce the use of chemical fungicides in food preservatives. In this study, honokiol, a type of phenolic compound in Magnolia officinalis, was found to inhibit spore germination and mycelia growth of Aspergillus flavus at 100 μg/mL. In addition, a pathogenicity assay showed that honokiol had potent antifungal activity against A. flavus in corn flour by suppressing conidia production. Fluorescence staining, transmission electron microscopy and biochemical assays were performed to explore its possible inhibition mechanisms against A. flavus. The results showed that the destructive effect of honokiol on A. flavus appeared to be related to increased plasma membrane permeability, the inhibition of ATPase activity, mitochondrial dysfunction and the accumulation of reactive oxygen species. Furthermore, a transcriptomic analysis showed that honokiol treatment resulted in 1578 different expressed genes. Gen Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that some genes of A. flavus related to spore development, integrity of the cell wall and membrane, oxidative stress and energy metabolism were significantly downregulated. In addition, RNA-seq results were validated by quantitative real-time polymerase chain reactions. Our finding enhanced the understanding of the antifungal activity of honokiol and underlying mechanisms of action at the molecular level, supporting honokiol as a potential agent in preventing contamination by A. flavus.

Improved Bioherbicidal Efficacy of Bipolaris eleusines through Herbicide Addition on Weed Control in Paddy Rice.

Bipolaris eleusines was mixed with herbicides to improve the control of barnyardgrass (Echinochloa crus-galli), a noxious weed in rice fields. The compatibility of B. eleusines with herbicides was evaluated for toxic effects on spore germination and mycelium growth in vitro tests, and varied effects were observed with different chemical products. Briefly, 25 g/L penoxsulam OD plus 10% bensulfuron-methyl WP were much more compatible with B. eleusines, and there was no inhibition of spore germination but the promotion of mycelium growth of B. eleusines at all treatment rates. Under greenhouse conditions, the coefficient of the specificity of B. eleusines conidial agent was determined as 3.91, closer to the herbicidal control of 2.89, showing it is highly specific between rice and barnyardgrass. Field experiments in 2011 and 2012 showed that B. eleusines conidial agent displayed good activity on barnyardgrass, monochoria [Monochoria vaginalis (Burm.f.) Presl. Ex Kunth.], and small-flower umbrella sedge (Cyperus difformis L.) and had no negative impact on the rice plant. It also reduced the loss of rice yield when compared with the non-treated control and could make this pathogen a conidial agent for commercial bioherbicidal development in the future.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6h) and a 1.46-fold upregulation during the infection hyphae formation period (8h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

Activity and cell toxicology of fluazinam on Fusarium graminearum

Fusarium graminearum is an important plant pathogen and the causal agent of Fusarium head blight (FHB). At present, the principal method of controlling FHB is through fungicides. Fluazinam is an agent with strong broad-spectrum antifungal activity and has been used to control many diseases. However, there are no reported uses of fluazinam for controlling FHB. This study reports the activity and cell toxicology mechanisms of fluazinam on the filamentous fungus F. graminearum and its effect on fungal growth and development. The activity of fluazinam was tested for 95 wild-type field strains of F. graminearum. The EC50 values (the 50% effective concentration) of fluazinam for inhibition of mycelial growth and spore germination ranged from 0.037 μg/ml to 0.179 μg/ml and from 0.039 μg/ml to 0.506 μg/ml, respectively. The fluazinam sensitivity of these strains varied in 4.9 and 13.0 folds, implying that the target of the fungicide remained unchanged. After treatment with 0.3 μg/ml (≈EC90) fluazinam, the production of conidia was reduced, and the cell wall and cell membrane had shrunked; the cell nucleus and septum morphology, cell membrane permeability, and sexual development were not affected. When treated with 0.1 μg/ml (≈EC50) or 0.3 μg/ml fluazinam, the mycelial respiration and deoxynivalenol (DON) synthesis of F. graminearum were decreased. Confocal images showed that the formation of toxisomes was disturbed after fluazinam treatment, suggesting that fluazinam reduces DON synthesis by inhibiting toxisome formation. Infection of wheat coleoptiles revealed that fluazinam had a strong protective activity against F. graminearum. At 250 μg/ml fluazinam the control efficacy of protective treatments reached 100% and controlled strains resistant to carbendazim. These results contribute to the understanding of the mode of action of fluazinam and its application.

Application of Pinhole Plasma Jet Activated Water against Escherichia coli, Colletotrichum gloeosporioides, and Decontamination of Pesticide Residues on Chili (Capsicum annuum L.).

Plasma activated water (PAW) generated from pinhole plasma jet using gas mixtures of argon (Ar) and 2% oxygen (O2) was evaluated for pesticide degradation and microorganism decontamination (i.e., Escherichia coli and Colletotrichum gloeosporioides) in chili (Capsicum annuum L.). A flow rate of 10 L/min produced the highest concentration of hydrogen peroxide (H2O2) at 369 mg/L. Results showed that PAW treatment for 30 min and 60 min effectively degrades carbendazim and chlorpyrifos by about 57% and 54% in solution, respectively. In chili, carbendazim and chlorpyrifos were also decreased, to a major extent, by 80% and 65% after PAW treatment for 30 min and 60 min, respectively. E. coli populations were reduced by 1.18 Log CFU/mL and 2.8 Log CFU/g with PAW treatment for 60 min in suspension and chili, respectively. Moreover, 100% of inhibition of fungal spore germination was achieved with PAW treatment. Additionally, PAW treatment demonstrated significantly higher efficiency (p < 0.05) in controlling Anthracnose in chili by about 83% compared to other treatments.

Penicillium growth inhibition, fruit decay reduction, and polymethoxyflavones and scoparone induction in satsuma mandarin irradiated with ultraviolet-A light-emitting diodes

In this study, we investigated the effects of ultraviolet (UV)-A light-emitting diodes (LEDs) on the inhibition of fungal spore germination, fruit decay, and accumulation of secondary metabolites, including phytoalexins, in satsuma mandarin. We examined the growth and development of blue (Penicillium italicum) and green (Penicillium digitatum) molds exposed to UV-A LED irradiation in vitro and found that UV-A LEDs with an emission peak of 375 nm and light intensity of 9.4 W m−2 significantly reduced the growth of both these fungi. In addition, wounded fruits inoculated with P. digitatum were irradiated with UV-A LEDs for 6 days, and we monitored the subsequent changes in green mold symptoms. The results indicated that low-intensity UV-A LED treatment significantly reduced green mold symptoms in fruit, being the effect most pronounced inhibitory on Penicillium sporulation. Furthermore, wounded fruits subjected to UV-A LED treatment were found to have significantly higher contents of the coumarin compound scoparone than fruits maintained in the dark. Contrastingly, scoparone was barely detectable in non-wounded fruits, irrespective of UV-A LED irradiation. The contents of nobiletin, tangeretin, and hesperidin were likewise found to be higher in wounded fruits treated with UV-A LED than in either non-wounded or non-irradiated fruits. These results indicate that treatment with low-intensity UV-A LED irradiation is sufficient to reduce mold symptom development, and accordingly could represent a promising safe approach to the control of post-harvest decay in citrus fruits. On the basis of our observations, we speculate that the biosynthesis of scoparone, polymethoxyflavones, and flavanones could be induced when the peel is injured and irradiated with UV-A contributing to a reduction in fruit decay.

Combination of Selenium and Methyl Jasmonate Controls Postharvest Tomato Gray Mold by Damaging the Membrane System

Tomato, which is regarded as an important worldwide crop, is susceptible to gray mold caused by Botrytis cinerea. Selenium and methyl jasmonate can act as antifungal agents against pathogenic infections. To clarify the effect of selenium and methyl jasmonate on the fungal pathogen, the spore germination and mycelial growth of B. cinerea were investigated in vitro using the growth rate method. Additionally, the electrical conductivity, soluble protein content, malondialdehyde content and oxalic acid secretion of B. cinerea mycelium were also determined to further explore the antifungal mechanism of selenium and methyl jasmonate. The results showed that selenium application significantly increased cell membrane permeability and malondialdehyde content, and methyl jasmonate treatment decreased the soluble protein content in mycelium of B. cinerea. Furthermore, supplementation of the medium with both selenium and methyl jasmonate effectively inhibited spore germination and colony growth of B. cinerea by compromising membrane integrity, and significantly reduced soluble protein content and the oxalic acid secretion of hypha. The resulting incidence of postharvest tomato gray mold with the combination of selenium and methyl jasmonate was 34.7%, which was approximately half of that of the control. To sum up, the combined use of selenium and methyl jasmonate inhibited the normal physiological activity and pathogenicity of B. cinerea, which suggests that selenium and methyl jasmonate have the potential for controlling gray mold disease caused by B. cinerea in postharvest fruits and vegetables. These findings may offer a promising and eco-friendly strategy to control gray mold disease in postharvest fruits and vegetables.

Content and spatial distribution of dairy-related Clostridium spores in Grana Padano cheese during the ripening period

This study investigated the stress factors that affect the content and the spatial distribution of Clostridium spores in Grana Padano (GP) wheels and in GP samples purchased from the market. The cheeses analyzed were produced adding lysozyme to prevent late blowing defects (LBD). The highest NaCl amount was detected in the external part (5.2 ± 0.6%) while a lower level was observed in the core (2.8 ± 0.3%). The clostridial community differed in cheeses with different ripening times along with pH and salt changes that occur slowly over time. The spore level in the core (0.24 ± 0.19 and 0.16 ± 0.14 MPN/g after 11 and 20 months of ripening) was significantly lower than in the peripheral zones (0.48 ± 0.26 and 0.45 ± 0.31 MPN/g). Moreover, in the wheels aged 11 months, the presence of C. tyrobutyricum was higher in the areas with the lowest salt content, while after 20 months of ripening when a homogeneous concentration of salt and pH within the wheel occurred, C. sporogenes was the prevalent species. Our results showed that in the GP wheels changes in the presence of clostridia responsible for LBD still occur until the salt content in the center of cheeses is higher than threshold NaCl needed to inhibit spore germination.

Synergistic Activity of Rhamnolipid Biosurfactant and Nanoparticles Synthesized Using Fungal Origin Chitosan Against Phytopathogens.

Phytopathogens pose severe implications in the quantity and quality of food production by instigating several diseases. Biocontrol strategies comprising the application of biomaterials have offered endless opportunities for sustainable agriculture. We explored multifarious potentials of rhamnolipid-BS (RH-BS: commercial), fungal chitosan (FCH), and FCH-derived nanoparticles (FCHNPs). The high-quality FCH was extracted from Cunninghamella echinulata NCIM 691 followed by the synthesis of FCHNPs. Both, FCH and FCHNPs were characterized by UV-visible spectroscopy, DLS, zeta potential, FTIR, SEM, and Nanoparticle Tracking Analysis (NTA). The commercial chitosan (CH) and synthesized chitosan nanoparticles (CHNPs) were used along with test compounds (FCH and FCHNPs). SEM analysis revealed the spherical shape of the nanomaterials (CHNPs and FCHNPs). NTA provided high-resolution visual validation of particle size distribution for CHNPs (256.33 ± 18.80 nm) and FCHNPs (144.33 ± 10.20 nm). The antibacterial and antifungal assays conducted for RH-BS, FCH, and FCHNPs were supportive to propose their efficacies against phytopathogens. The lower MIC of RH-BS (256 μg/ml) was observed than that of FCH and FCHNPs (>1,024 μg/ml) against Xanthomonas campestris NCIM 5028, whereas a combination study of RH-BS with FCHNPs showed a reduction in MIC up to 128 and 4 μg/ml, respectively, indicating their synergistic activity. The other combination of RH-BS with FCH resulted in an additive effect reducing MIC up to 128 and 256 μg/ml, respectively. Microdilution plate assay conducted for three test compounds demonstrated inhibition of fungi, FI: Fusarium moniliforme ITCC 191, FII: Fusarium moniliforme ITCC 4432, and FIII: Fusarium graminearum ITCC 5334 (at 0.015% and 0.020% concentration). Furthermore, potency of test compounds performed through the in vitro model (poisoned food technique) displayed dose-dependent (0.005%, 0.010%, 0.015%, and 0.020% w/v) antifungal activity. Moreover, RH-BS and FCHNPs inhibited spore germination (61–90%) of the same fungi. Our efforts toward utilizing the combination of RH-BS with FCHNPs are significant to develop eco-friendly, low cytotoxic formulations in future.

Amphotericin B and 5-flucytosine as fungicides against Penicillium italicum for citrus fruit rot

The expansion of the global population and improved living standards has created an increasing demand for fresh fruit. The combination of agricultural and medical development for new fungicides is one of the most effective approaches for keeping fruit fresh. Both amphotericin B and 5-flucytosine are quite safe and have a wide spectrum of antifungal activity. For the purpose of investigating their potential applications for preserving fruit freshness, we evaluated their antifungal efficiencies against Penicillium italicum in vitro and in vivo. The results showed that amphotericin B and 5-flucytosine significantly inhibited spore germination and the mycelial growth of P. italicum. The 5-flucytosine showed much better bioactivity than the well-known agricultural fungicide prochloraz, both in vitro and in vivo. Furthermore, crystal violet staining, propidium iodide dyeing, ultrastructural observation and the ergosterol content indicated that amphotericin B and 5-flucytosine destroyed the cellular ultrastructure and membrane integrity. In addition, nucleic acids (OD260) and proteins (OD280) escaped from P. italicum cells in a dose-dependent manner. The results of this study indicated that amphotericin B and 5-flucytosine have good potential as antifungal preservatives for postharvest citrus fruit due to their excellent antifungal effects, and that the primary mode of action was the destruction of the membrane integrity and permeability of P. italicum.

Efficacy of Debaryomyce hansenii in the biocontrol for postharvest soft rot of strawberry and investigation of the physiological mechanisms involved

The postharvest disease of strawberries caused by Rhizopus stolonifer caused enormous financial losses. This study investigates the efficacy of antagonistic yeast Debaryomyce hansenii in the biocontrol of postharvest soft rot of strawberries and the physiological mechanisms involved. The results showed that D. hansenii significantly reduced the incidence of postharvest soft rot of strawberry, reduced the natural decay incidence of postharvest strawberry, and had no adverse effects on quality parameters of strawberries. In vitro experiments showed that D. hansenii reduced the spore germination rate and germ tube length of R. stolonifer. D. hansenii can also stably colonize the surface and wounds of strawberries at either 4 °C or 20 °C. Meanwhile, the activities of antioxidant-related enzymes superoxide dismutase, peroxidase, catalase, ascorbate oxidase and resistance-related enzymes polyphenol oxidase and phenylalanine ammonia-lyase were significantly higher in D. hansenii-treated strawberries than in control. Meanwhile, the contents of resistance-related substances including total phenols, flavonoids and anthocyanins were also increased in D. hansenii-treated strawberries. This study suggested that D. hansenii has the potential to control postharvest soft rot of strawberry by the mechanism involving inhibition of spore germination and germ tube length of R. stolonifer and induction of increased activities of defense-related enzymes and substances in strawberry.