Inhibition of Aspergillus flavus growth and aflatoxin B1 production by natamycin

Aspergillus flavus causes huge crop losses, reduces crop quality and has adverse effects on human and animal health. A large amount of food contaminated with aflatoxin can greatly increase the risk of liver cancer. Therefore, prevention and control of aflatoxin production have aroused attention of research in various countries. Natamycin extracted from Streptomyces spp. has been widely used in production practice due to its good specificity and safety. Here, we found that natamycin could significantly inhibit fungal growth, conidia germination, ergosterol and AFB1 production by A. flavus in a dose-dependent manner. Scanning electron microscope analysis indicated that the number of conidia was decreased, the outer wall of conidia was destroyed, and the mycelia were shrivelled and tangled by natamycin. RNA-Seq data indicated that natamycin inhibited fungal growth and conidia development of A. flavus by significantly down-regulating some genes involved in ergosterol biosynthesis, such as Erg13, HMG1 and HMG2. It inhibited conidia germination by significantly down-regulating some genes related to conidia development, such as FluG and VosA. After natamycin exposure, the decreased ratio of aflS/aflR caused by the down-regulation of all the structural genes, which subsequently resulted in the suppression of AFB1 production. In conclusion, this study served to reveal the inhibitory mechanisms of natamycin on fungal growth and AFB1 biosynthesis in A. flavus and to provide solid evidence for its application in controlling AFB1 contamination.

Biological Control of Celery Powdery Mildew Disease Caused by Erysiphe heraclei DC In Vitro and In Vivo Conditions

The present study aimed to investigate the potentiality of certain biocontrol agents, namely Bacillus subtilis, B. pumilus, B. megaterium, Pseudomonas fluorescens, Serratia marcescens, Trichoderma album, T. harzianum and T. viride, as well as the synthetic fungicide difenoconazole to control celery powdery mildew caused by Erysiphe heraclei DC, in vitro (against conidia germination and germ tube length of E. heraclei) and in vivo (against disease severity and AUDPC). In vitro, it was found that the antifungal activity of the tested biocontrol agents significantly reduced the germination percentage of the conidia and germ tube length of the pathogen. The reduction in conidia germination ranged between 88.2% and 59.6% as a result of the treatment with B. subtilis and T. album, respectively compared with 97.1% by the synthetic fungicide difenoconazole. Moreover, the fungicide achieved the highest reduction in germ tube length (92.5%) followed by B. megaterium (82.0%), while T. album was the least effective (62.8%). Spraying celery plants with the tested biocontrol agents in the greenhouse significantly reduced powdery mildew severity, as well as the area under the disease progress curve (AUDPC), after 7, 14, 21 and 28 days of application. In this regard, B. subtilis was the most efficient followed by B. pumilus, S. marcescens and B. megaterium, with 80.1, 74.4, 73.2 and 70.5% reductions in disease severity, respectively. In AUDPC, reductions of those microorganisms were 285.3, 380.9, 396.7 and 431.8, respectively, compared to 1539.1 in the control treatment. On the other hand, the fungicide difenoconazole achieved maximum efficacy in reducing disease severity (84.7%) and lowest AUDPC (219.3) compared to the other treatments. In the field, all the applied biocontrol agents showed high efficiency in suppressing powdery mildew on celery plants, with a significant improvement in growth and yield characteristics. In addition, they caused an increase in the concentration of leaf pigments, and the activities of defense-related enzymes such as peroxidase (PO) and polyphenol oxidase (PPO) and total phenol content (TPC). In conclusion, the results showed the possibility of using tested biocontrol agents as eco-friendly alternatives to protect celery plants against powdery mildew.

Alternative Control of Phragmidium rubi-idaei Infecting Two Rubus Species

Organic berry plantations have been gaining popularity among farmers during recent years. Even so, farmers experience serious challenges in disease control management, which is a concern in organic farming. Phragmidiumrubi-idaei (DC) P. Karst is the pathogen responsible for blackberry and raspberry rust disease, one of the most present and active diseases in plantations. The antifungal certified products found on the organic farming market offer the opportunity for an efficient control strategy over plant pathogens in fruit shrub plantations. In this study, 5 natural based products—namely Altosan, Mimox, Canelys, Zitron, and Zeolite—were tested for their fungistatic effect over P. rubi-idaei. The experiments were carried out under laboratory conditions, performing observations over the impact of organic products, used at different concentration levels, on rust conidia germination. Moreover, field experiments were conducted in order to evaluate the efficiency of different treatments for rust control on raspberry (‘Polka’, ‘Veten’ and ‘Heritage’) and blackberry (‘Thorn Free’, ‘Chester’ and ‘Loch Ness’) varieties. Data analysis based on ANOVA tests showed significant differences between the tested variants and the control sample at p < 0.001. Furthermore, LSD test confirmed differences between all substances tested (p < 0.005). The natural products Canelys (formulated with cinnamon) and Zytron (based on citrus extract) have proven the highest inhibitory capacity for conidia germination during in vitro tests registering values of 80.42% and 78.34%, respectively. The same high inhibitory rates against rust pathogen were kept also in the field tests using the same two natural-based products mentioned earlier. In addition, outcomes from this study demonstrated that Zeolite is not recommended for raspberry or blackberry rust control.

Variation in UV-mediated damage recovery among Pseudoidium neolycopersici isolates: possible mechanisms.

The cryptochrome/photolyase family (CPF) consists of a diversified class of flavoproteins that are evolutionarily related. Although their domain architecture is highly conserved, they perform entirely different physiological functions. Previous studies have confirmed the presence of a functional photolyase in Pseudoidium neolycopersici, which repairs UV-C-induced DNA damage by using near UV-A/blue light as an energy source. Similar doses of UV-C treatment followed by incubation with dark or blue light was tested on conidia germination of five isolates of P. neolycopersici collected from different regions of Norway and the Netherlands. The results showed variations in the effect of UV on germination and germination recovery under blue light incubation after UV treatment. Evolutionary studies confirmed that P. neolycopersici photolyase is highly conserved among different isolates of P. neolycopersici and among different species. All CPF members have a core domain consisting of an identical cofactor Flavin adenine dinucleotide (FAD), and an additional photoantenna chromophore. An amino acid sequence analysis demonstrated that CPF members have highly conserved C-terminals compared with their N-terminals, as FAD binds in the C-terminal region. When compared with other CPF members, varying lengths of N- and C-terminals were noticed in P. neolycopersici photolyase and putative cryptochrome, respectively. Further research using comparative genomics targeting pyrimidine base composition, the role of regulatory elements including promotor architecture, and the characterization of optical properties of native photolyase among isolates may help to explain the differences in the biological responses of conidia germination of P. neolycopersici treated with UV.

CgNPG1 as a Novel Pathogenic Gene of Colletotrichum gloeosporioides From Hevea brasiliensis in Mycelial Growth, Conidiation, and the Invasive Structures Development

The rubber tree (Hevea brasiliensis) is a tropical perennial crop for the primary source of natural rubber. Colletotrichum gloeosporioides from Hevea brasiliensis (C. gloeosporioides Hb) and Colletotrichum acutatum from Hevea brasiliensis (C. acutatum Hb) are the causal agents of rubber tree anthracnose and lead to serious loss of natural rubber production. Inoculation tests showed that C. gloeosporioides Hb possessed higher pathogenicity than C. acutatum Hb to the rubber tree. Genomic analysis revealed that an unknown gene, named CgNPG1 (a Novel Pathogenic Gene 1), was presented in the genome of C. gloeosporioides Hb but not identified in C. acutatum Hb. CgNPG1 was predicted to encode a small secretory protein without any conserved domain. To investigate the functions of CgNPG1 in C. gloeosporioides Hb and in C. acutatum Hb, the gene deletion and overexpression mutants were generated. The phenotype analysis showed that deletion of CgNPG1 led to changed conidia morphology, decreased mycelial growth, conidiation, conidia germination rate, appressorium formation rate, and pathogenicity of C. gloeosporioides Hb to the rubber tree. Meanwhile, heterogeneous expression of CgNPG1 in C. acutatum Hb significantly changed the conidia morphology and improved the mycelial growth rate, conidiation, conidia germination rate, appressorium formation rate, and the pathogenicity of C. acutatum Hb to the rubber tree. Consistently, CgNPG1 increased the expression level of CaCRZ1 and CaCMK1 in C. acutatum Hb. These data suggested that CgNPG1 contributed to mycelial growth, conidiation, the development of invasive structures, and the pathogenicity of Colletotrichum to the rubber tree, which might be related to the modulation of CaCRZ1 and mitogen-activated protein kinase CMK1. Our results provided new insight into CgNPG1 in regulating growth and pathogenicity of the Colletotrichum spp.

Synergistic antimicrobial effect of chitosan polymers and oligomers

This study evaluated the efficacy of the combined application of well-characterized chitosan polymer (degree of acetylation DA = 10%, degree of polymerization DPn = 90, dispersity ÐDP = 2.8) and oligomers (paCOS) (DP = 2-17) on conidia germination and mycelial growth of Fusarium graminearum, the major causal agent of Fusarium Head Blight in wheat. The polymer alone showed a higher inhibitory effect than the paCOS mixture alone, with half-maximal inhibitory concentrations (IC50) of less than 50 µg mL⁻¹ and more than 100 µg mL⁻¹, respectively. Using time-lapse microscopy, we also showed that paCOS did not affect conidia germination at 50 μg mL⁻¹, while chitosan polymer at the same concentration led to a delay in germination and in elongation of germ tubes. Scanning electron microscopy was used to observe the chitosan-induced changes in hyphal morphology. Surprisingly, the combination of chitosan polymer and paCOS led to strong synergistic effects in inhibiting conidia germination and fungal growth, as quantified by both the Abbot and Wadley equation. To our knowledge, this is the first report on a synergistic effect of a combination of chitosan polymers and oligomers, also highlighting for the first time the importance of ÐDP when studying structure-function relationships of functional biopolymers such as chitosan. The consequences of this finding for the improvement of chitosan-based antimicrobial or plant protective products are discussed. Given the economic importance of F. graminearum, this study suggests that the combination of chitosan polymer and oligomers can be used to support an efficient, sustainable plant protection strategy.

Novel Metabolites Identified From Bacillus Safensis and Their Antifungal Property Against Alternaria Alternata

Plant growth promoting rhizobacteria offer an effective and eco-sustainable solution to protect crops against phytopathogens. In the present study, Bacillus safensis STJP (NAIMCC-B-02323) from the rhizospheric soil of Stevia rebaudiana showed strong biocontrol activity against phytopathogen, Alternaria alternata. B. safensis STJP produced antifungal volatile organic compounds (AVOC). In the presence of AVOC, there was no conidia germination, mycelium growth was inhibited, and hyphae ruptured as observed by scanning electron microscopy (SEM). When mycelium of the fungus from bacterial treated plate was transferred into fresh potato dextrose agar plate, A. alternata could not grow. Extracted AVOC from B. safensis STJP were identified by thin-layer chromatography (TLC), fourier-transform-infrared spectroscopy (FT-IR) and gas-chromatography-mass spectrometry (GC-MS). In total 25 antifungal metabolites were identified by GC-MS analysis having alcohol, alkane, phenol, alkyl halide and aromatic compounds. Five compounds (phenol, 2,4-bis (1,1-dimethylethyl)-, 3-hexadecanol, pyrrolo(1,2-a)pyrazine-1,4-dione, 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo(1,2-a:1',2'-d)pyrazine and hexadecanoic acid) completely inhibited the mycelium growth, controlling spore formation and conidia germination of A. alternata. This study concluded that AVOC producing B. safensis can be used as a green-fungicide against A. alternata. Bacterial metabolites could pave the way for the development of next generation biopesticide. This can be a reliable technology to enhance the quality and reliability of biopesticides.

In vitro and in vivo antifungal activity of Cuminum cyminum essential oil against Aspergillus aculeatus causing bunch rot of postharvest grapes

Bunch rot in grapes is an aggressive disease and needs to be controlled during the postharvest period. We investigate the antifungal potential of Zanthoxylum bungeanum Maxim., Zanthoxylum rhetsa, Cuminum cyminum, Coriandrum sativum, and Zingiber montanum (J. Koenig) Link ex A. Dietr. essential oils against Aspergillus aculeatus that cause bunch rot disease on postharvest grapes. C. cyminum essential oil exhibited stronger significantly inhibition percentage of 95.08% than other treatments in in vitro assay. Cumin aldehyde (33.94%) and α-terpinen-7-al (32.20%) were identified as major volatile compounds in C. cyminum oil. Antifungal potential of C. cyminum oil was then tested in conidia germination and in vitro tests compared to cumin aldehyde and α-terpinen-7-al. Their EC50 values against the conidial germination were also estimated. Significant reduction of conidia germination was also detected in C. cyminum essential oil and cumin aldehyde at a concentration of 1,000 and 100 μg/mL, respectively. EC50 values of the C. cyminum essential oil, cumin aldehyde, and α-terpinen-7-al were 67.28 μg/mL, 9.31 μg/mL, and 13.23 μg/mL, respectively. In vivo assay, the decrease of the disease severity (0.69%) and incidence (1.48%) percentage of A. aculeatus on grape berries treated at 1,000 μg/mL of C. cyminum essential oil was significantly greater than that obtained from other treatments after 10 days incubation. In addition, grape berries treated with C. cyminum essential oil decreased weight loss and retained fruit firmness. The changing of total soluble solids, total phenolic content, and antioxidant activity are also delayed in treated fruits. Therefore, essential oil of C. cyminum may be applied as a biological antifungal agent to control A. aculeatus in postharvest grapes without any negative effects on its quality.

Protective Efficacy of Lectin-Fc(IgG) Fusion Proteins In Vitro and in a Pulmonary Aspergillosis In Vivo Model

Aspergillosis cases by Aspergillus fumigatus have increased, along with fungal resistance to antifungals, urging the development of new therapies. Passive immunization targeting common fungal antigens, such as chitin and β-glucans, are promising and would eliminate the need of species-level diagnosis, thereby expediting the therapeutic intervention. However, these polysaccharides are poorly immunogenic. To overcome this drawback, we developed the lectin-Fc(IgG) fusion proteins, Dectin1-Fc(IgG2a), Dectin1-Fc(IgG2b) and wheat germ agglutinin (WGA)-Fc(IgG2a), based on their affinity to β-1,3-glucan and chitooligomers, respectively. The WGA-Fc(IgG2a) previously demonstrated antifungal activity against Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. In the present work, we evaluated the antifungal properties of these lectin-Fc(s) against A. fumigatus. Lectin-Fc(IgG)(s) bound in a dose-dependent manner to germinating conidia and this binding increased upon conidia germination. Both lectin-Fc(IgG)(s) displayed in vitro antifungal effects, such as inhibition of conidia germination, a reduced length of germ tubes and a diminished biofilm formation. Lectin-Fc(IgG)(s) also enhanced complement deposition on conidia and macrophage effector functions, such as increased phagocytosis and killing of fungi. Finally, administration of the Dectin-1-Fc(IgG2b) and WGA-Fc(IgG2a) protected mice infected with A. fumigatus, with a 20% survival and a doubled life-span of the infected mice, which was correlated to a fungal burden reduction in lungs and brains of treated animals. These results confirm the potential of lectin-Fc(IgGs)(s) as a broad-spectrum antifungal therapeutic.

Evaluation of Yeast Potential in the Biological Control of Colletotrichum orbiculare in Cucumber

This work aimed to evaluate the effect of the yeasts Candida albicans, Pichia guilliermondii, Rhodotorula glutinis, Saccharomyces cerevisiae and Zygoascus hellenicus in the control of anthracnose in cucumbers. The influence of volatile and non-volatile compounds on mycelial growth and the effect of cell suspension and culture filtrate on conidial germination were evaluated. In a greenhouse, yeasts were tested on cucumber cotyledons, one cotyledon being treated and the other not; afterwards both cotyledons were challenged with C. orbiculare. The severity of the disease in both cotyledons was assessed by determining biological control and resistance induction. The production of volatile compounds from the yeasts R. glutinis and C. albicans reduced the pathogen growth by 90.7 and 90.0%, respectively. The production of non-volatile compounds stimulated the pathogen development. Conidia germination was affected when exposed to cell suspension of all tested isolates, ranging from 43 to 75%. For the culture filtrate from Z. hellenicus, it reduced the conidia germination by 11.4%. In the in vivo test, on the treated cotyledon, the yeasts Z. hellenicus, R. glutinis and S. cerevisiae reduced the anthracnose severity by 52.5, 50.0 and 42.5%, respectively.