Antifungal Activity of Ageritin, a Ribotoxin-like Protein from Cyclocybe aegerita Edible Mushroom, against Phytopathogenic Fungi.

Ageritin from poplar mushrooms is a specific endonuclease that hydrolyzes a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA, thereby blocking protein synthesis. Considering the possible biotechnological use of this enzyme, here we report its antifungal activity against virulent fungi affecting crops of economic interest. Our results show that ageritin (200 µg/plug; ~13.5 nmole) inhibits the growth of Botrytis cinerea (57%), Colletotrichum truncatum (42%), and Alternaria alternata (57%), when tested on potato dextrose agar plates. At the same time, no effect was observed against Trichoderma harzianum (a fungus promoting beneficial effects in plants). To verify whether the antifungal action of ageritin against B. cinerea and T. harzianum was due to ribosome damage, we tested ageritin in vitro on partially isolated B. cinerea and T. harzianum ribosomes. Interestingly, ageritin was able to release the Endo's fragment from both tested fungal ribosomes. We therefore decided to test the antifungal effect of ageritin on B. cinerea and T. harzianum using a different growth condition (liquid medium). Differently from the result in solid medium, ageritin can inhibit both B. cinerea and T. harzianum fungal growth in liquid medium in a concentration-dependent manner up to 35.7% and 38.7%, respectively, at the highest concentration tested (~200 µg/mL; 12 µM), and the analysis of RNA isolated from ageritin-treated cells revealed the presence of Endo's fragment, highlighting its ability to cross the fungal cell wall and reach the ribosomes. Overall, these data highlight that the efficacy of antifungal treatment to prevent or treat a potential fungal disease may depend not only on the fungal species but also on the conditions of toxin application.

Alternaria alternata Isolated from Infected Pears (Pyrus communis) in Italy Produces Non-Host Toxins and Hydrolytic Enzymes as Infection Mechanisms and Exhibits Competitive Exclusion against Botrytis cinerea in Co-Infected Host Fruits.

Alternaria alternata is one of the most devastating phytopathogenic fungi. This microorganism causes black spots in many fruits and vegetables worldwide, generating significant post-harvest losses. In this study, an A. alternata strain, isolated from infected pears (Pyrus communis) harvested in Italy, was characterized by focusing on its pathogenicity mechanisms and competitive exclusion in the presence of another pathogen, Botrytis cinerea. In in vitro assays, the fungus produces strong enzymatic activities such as amylase, xylanase, and cellulase, potentially involved during the infection. Moreover, it secretes four different toxins purified and identified as altertoxin I, alteichin, alternariol, and alternariol 4-methyl ether. Only alteichin generated necrotic lesions on host-variety pears, while all the compounds showed moderate to slight necrotic activity on non-host pears and other non-host fruit (lemon, Citrus limon), indicating they are non-host toxins. Interestingly, A. alternata has shown competitive exclusion to the competitor fungus Botrytis cinerea when co-inoculated in host and non-host pear fruits, inhibiting its growth by 70 and 65%, respectively, a result not observed in a preliminary characterization in a dual culture assay. Alteichin and alternariol 4-methyl ether tested against B. cinerea had the best inhibition activity, suggesting that the synergism of these toxins and enzymatic activities of A. alternata are probably involved in the competitive exclusion dynamics in host and non-host pear fruits.

Sporulation efficiency and spore quality in a human intestinal isolate of Bacillus cereus.

Bacteria classified as Bacillus cereus sensu stricto cause two different type of gastrointestinal diseases associated with food poisoning. Outbreaks of this opportunistic pathogen are generally due to the resistance of its spores to heat, pH and desiccation that makes hard their complete inactivation from food products. B. cereus is commonly isolated from a variety of environments, including intestinal samples of infected and healthy people. We report the genomic and physiological characterization of MV19, a human intestinal strain closely related (ANI value of 98.81%) to the reference strain B. cereus ATCC 14579. MV19 cells were able to grow in a range of temperatures between 20 and 44 °C. At the optimal temperature the sporulation process was rapidly induced and mature spores efficiently released, however these appeared structurally and morphologically defective. At the sub-optimal growth temperature of 25 °C sporulation was slow and less efficient but a high total number of fully functional spores was produced. These results indicate that the reduced rapidity and efficiency of sporulation at 25 °C are compensated by a high quality and quantity of released spores, suggesting the relevance of different performances at different growth conditions for the adaptation of this bacterium to diverse environmental niches.

Use of halotolerant Bacillus amyloliquefaciens RHF6 as a bio-based strategy for alleviating salinity stress in Lotus japonicus cv Gifu.

Halotolerant (HT) bacteria are a group of microorganisms able to thrive in environments with relatively high salt concentrations. HT-microorganisms with plant growth-promoting (PGP) characteristics have been proposed to increase plant tolerance in salty soil. Here, we evaluated the PGP properties at increasing NaCl concentrations of HT-Bacillus strains, previously shown to have beneficial effects under physiological conditions. Most of the isolated showed indole acetic acid and ammonia production and were able to solubilize phosphate and suppress the proliferation of the phytopathogenic fungus Macrophomina phaseolina 2013-1 at high salt concentrations. One of the selected strains, Bacillus amyloliquefaciens RHF6, which retained its beneficial properties up to 400 mM NaCl in vitro, was tested on the legume model plant Lotus japonicus cv Gifu under salt stress. The inoculation with RHF6 significantly improved the survival of plants under high salinity conditions, as reflected in seedling root and shoot growth and total fresh weight (increased by 40%) when compared with non-inoculated plants. The ability of RHF6 to induce a plant antioxidant response, secrete the osmoprotectant proline and reduce ethylene level via the enzymatic ACC deaminase activity indicated this strain as a potentially helpful PGPB for the treatment of degraded soils.

Truncatenolide, a Bioactive Disubstituted Nonenolide Produced by Colletotrichum truncatum, the Causal Agent of Anthracnose of Soybean in Argentina: Fungal Antagonism and SAR Studies.

A bioactive disubstituted nonenolide, named truncatenolide, was produced by Colletotrichum truncatum, which was collected from infected tissues of soybean showing anthracnose symptoms in Argentina. This is a devastating disease that drastically reduces the yield of soybean production in the world. The fungus also produced a new trisubstituted oct-2-en-4-one, named truncatenone, and the well-known tyrosol and N-acetyltyramine. Truncatenolide and truncatenone were characterized by spectroscopic (essentially one-dimensional (1D) and two-dimensional (2D) 1H and 13C NMR and HR ESIMS) and chemical methods as (5E,7R,10R)-7-hydroxy-10-methyl-3,4,7,8,9,10-hexahydro-2H-oxecin-2-one and (Z)-6-hydroxy-3,5-dimethyloct-2-en-4-one, respectively. The geometry of the double bond of truncatenolide was assigned by the value of olefinic proton coupling constant and that of truncatenone by the correlation observed in the corresponding NOESY spectrum. The relative configuration of each stereogenic center was assigned with the help of 13C chemical shift and 1H-1H scalar coupling DFT calculations, while the absolute configuration assignment of truncatenolide was performed by electronic circular dichroism (ECD). When tested on soybean seeds, truncatenolide showed the strongest phytotoxic activity. Tyrosol and N-acetyltyramine also showed phytotoxicity to a lesser extent, while truncatenone weakly stimulated the growth of the seed root in comparison to the control. When assayed against Macrophomina phaseolina and Cercospora nicotianae, other severe pathogens of soybean, truncatenolide showed significant activity against M. phaseolina and total inhibition of C. nicotianae. Thus, some other fungal nonenolides and their derivatives were assayed for their antifungal activity against both fungi in comparison with truncatenolide. Pinolidoxin showed to a less extent antifungal activity against both fungi, while modiolide A selectively and totally inhibited only the growth of C. nicotianae. The SAR results and the potential of truncatenolide, modiolide A, and pinolidoxin as biofungicides were also discussed.

Bacterial Lipodepsipeptides and Some of Their Derivatives and Cyclic Dipeptides as Potential Agents for Biocontrol of Pathogenic Bacteria and Fungi of Agrarian Plants.

Biotic stresses (fungi, bacteria, insects, weeds, etc.) are some of the most important causes of the decrease in the quality and quantity of crops that could become an emergency due to a noteworthy increase in the world population. Thus, to overcome these problems, massive use of chemical pesticides has been carried out with heavy consequences for environmental pollution and food safety. An eco-friendly alternative can be using natural compound-based biopesticides with high efficacy and selectivity. Some bacterial lipodepsipeptides (tolaasins I, II, A, D, and E and WLIP together with hexacetyl- and tetrahydro-tolaasin I and WLIP methyl ester) and cyclic dipeptides (cyclo(l-Pro-l-Tyr), cyclo(d-Pro-l-Tyr), cyclo(l-Pro-l-Val), and cyclo(l-Pro-l-Leu)) were assayed against several pathogenic bacteria and fungi of important agrarian plants. Lipodepsipeptides showed strong growth inhibition of all microorganisms tested in the range of 0.1-0.8 µg/mL, while cyclodipeptides, despite preserving this ability, showed a noteworthily reduced antimicrobial activity being active only in the range of 15-900 µg/mL. Among the lipodepsipeptides and cyclic dipeptides assayed, tolaasin d and cyclo(l-Pro-l-Tyr) (also named maculosin-1) appeared to be the most toxic compounds. Some structure-activity relationships of lipodepsipeptides were also discussed along with their practical application as biopesticides in agriculture.

Pseudomonas fluorescens Showing Antifungal Activity against Macrophomina phaseolina, a Severe Pathogenic Fungus of Soybean, Produces Phenazine as the Main Active Metabolite.

Pseudomonas fluorescens 9 and Bacillus subtilis 54, proposed as biofungicides to control Macrophomina phaseolina, a dangerous pathogen of soybean and other crops, were grown in vitro to evaluate their ability to produce metabolites with antifungal activity. The aim of the manuscript was to identify the natural compounds responsible for their antifungal activity. Only the culture filtrates of P. fluorescens 9 showed strong antifungal activity against M. phaseolina. Its organic extract contained phenazine and mesaconic acid (1 and 2), whose antifungal activity was tested against M. phaseolina, as well as Cercospora nicotianae and Colletotrichum truncatum, other pathogens of soybean; however, only compound 1 exhibited activity. The antifungal activity of compound 1 was compared to phenazine-1-carboxylic acid (PCA, 3), 2-hydroxyphenazine (2-OH P, 4), and various semisynthetic phenazine nitro derivatives in order to perform a structure-activity relationship (SAR) study. PCA and phenazine exhibited the same percentage of growth inhibition in M. phaseolina and C. truncatum, whereas PCA (3) showed lower activity against C. nicotianae than phenazine. 2-Hydroxyphenazine (4) showed no antifungal activity against M. phaseolina. The results of the SAR study showed that electron attractor (COOH and NO2) or repulsor (OH) groups significantly affect the antifungal growth, as well as their α- or ß-location on the phenazine ring. Both PCA and phenazine could be proposed as biopesticides to control the soybean pathogens M. phaseolina, C. nicotianae, and C. truncatum, and these results should prompt an investigation of their large-scale production and their suitable formulation for greenhouse and field applications.

Phenazine-1-Carboxylic Acid (PCA), Produced for the First Time as an Antifungal Metabolite by Truncatella angustata, a Causal Agent of Grapevine Trunk Diseases (GTDs) in Iran.

The phytopathogenic fungus Truncatella angustata, associated with grapevine trunk diseases (GTDs) in Iran, produces the well-known secondary metabolite isocoumumarin (+)-6-hyroxyramulosin and surprisingly also phenazine-1-carboxylic acid (PCA). PCA, identified by spectroscopic (essentially 1H NMR and ESI MS) spectra, is a bacterial metabolite well known for its antifungal activity and was found for the first time in T. angustata culture filtrates. The antifungal activity of PCA was assayed against four different fungi responsible for GTDs, Phaeoacremonium minimum, Phaeoacremonium italicum, Fomitiporia mediterranea, involved in grapevine esca disease, and Neofusicoccum parvum, responsible for Botryosphaeria dieback. The activity was compared with that of the known commercial fungicide, pentachloronitrobenzene, and the close phenazine. PCA and phenazine exhibited strong antifungal activity against all phytopathogenic fungi, inhibiting the fungal growth by about 90-100% and 80-100%, respectively. These results suggested that T. angustata could use PCA to compete with other phytopathogenic fungi that attack grapevine and thus PCA could be proposed as a biofungicide against the fungi responsible for grapevine esca and Botryosphaeria dieback diseases.

Genomic and Physiological Characterization of Bacilli Isolated From Salt-Pans With Plant Growth Promoting Features.

Massive application of chemical fertilizers and pesticides has been the main strategy used to cope with the rising crop demands in the last decades. The indiscriminate use of chemicals while providing a temporary solution to food demand has led to a decrease in crop productivity and an increase in the environmental impact of modern agriculture. A sustainable alternative to the use of agrochemicals is the use of microorganisms naturally capable of enhancing plant growth and protecting crops from pests known as Plant-Growth-Promoting Bacteria (PGPB). Aim of the present study was to isolate and characterize PGPB from salt-pans sand samples with activities associated to plant fitness increase. To survive high salinity, salt-tolerant microbes produce a broad range of compounds with heterogeneous biological activities that are potentially beneficial for plant growth. A total of 20 halophilic spore-forming bacteria have been screened in vitro for phyto-beneficial traits and compared with other two members of Bacillus genus recently isolated from the rhizosphere of the same collection site and characterized as potential biocontrol agents. Whole-genome analysis on seven selected strains confirmed the presence of numerous gene clusters with PGP and biocontrol functions and of novel secondary-metabolite biosynthetic genes, which could exert beneficial impacts on plant growth and protection. The predicted biocontrol potential was confirmed in dual culture assays against several phytopathogenic fungi and bacteria. Interestingly, the presence of predicted gene clusters with known biocontrol functions in some of the isolates was not predictive of the in vitro results, supporting the need of combining laboratory assays and genome mining in PGPB identification for future applications.

Structural studies on the O-specific polysaccharide of the lipopolysaccharide from Pseudomonas donghuensis strain SVBP6, with antifungal activity against the phytopathogenic fungus Macrophomina phaseolina.

An O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide (LPS) of Pseudomonas donghuensis SVBP6, a bacterium with a broad-spectrum antifungal activity in vitro, particularly that against Macrophomina phaseolina. This latter is one of the most virulent and dangerous pathogens of plants, including soybean which is an economically important crop in Argentina today. The OPS was studied by sugar analysis and spectroscopy (1D and 2D 1H and 13C NMR) showing the following trisaccharide repeating unit: →6)-ɑ-D-ManpNAc-(1 â†’ 3)-ß-l-Rhap-(1 â†’ 4)-ß-D-Glcp-(1→. The crude LPS, the purified LPS and the O-chain were assayed for their antifungal activity against M. phaseolina at 25, 50, 100, and 200 µg plug-1. The results showed that the crude LPS best inhibition was at 200 µg plug-1, able to inhibit the fungus growth by about 45%, while purified LPS and the corresponding OPS, in the same condition, reduced fungus growth by 65%, and 75%, respectively. Furthermore, the purified LPS and OPS significantly reduced the growth of M. phaseolina already at 100 µg plug-1 compared to the crude LPS. The structure of the O-chain is unique among the bacterial LPS and this is the first time that both the antifungal activity of a bacterial LPS and its corresponding O-chain were described.

Plant Growth Promotion Function of Bacillus sp. Strains Isolated from Salt-Pan Rhizosphere and Their Biocontrol Potential against Macrophomina phaseolina.

In recent decades, intensive crop management has involved excessive use of pesticides or fertilizers, compromising environmental integrity and public health. Accordingly, there has been worldwide pressure to find an eco-friendly and safe strategy to ensure agricultural productivity. Among alternative approaches, Plant Growth-Promoting (PGP) rhizobacteria are receiving increasing attention as suitable biocontrol agents against agricultural pests. In the present study, 22 spore-forming bacteria were selected among a salt-pan rhizobacteria collection for their PGP traits and their antagonistic activity against the plant pathogen fungus Macrophomina phaseolina. Based on the higher antifungal activity, strain RHFS10, identified as Bacillus vallismortis, was further examined and cell-free supernatant assays, column purification, and tandem mass spectrometry were employed to purify and preliminarily identify the antifungal metabolites. Interestingly, the minimum inhibitory concentration assessed for the fractions active against M. phaseolina was 10 times lower and more stable than the one estimated for the commercial fungicide pentachloronitrobenzene. These results suggest the use of B. vallismortis strain RHFS10 as a potential plant growth-promoting rhizobacteria as an alternative to chemical pesticides to efficiently control the phytopathogenic fungus M. phaseolina.

Phaseocyclopentenones A and B, Phytotoxic Penta- and Tetrasubstituted Cyclopentenones Produced by Macrophomina phaseolina, the Causal Agent of Charcoal Rot of Soybean in Argentina.

Two new penta- and tetrasubstituted cyclopentenones, named phaseocyclopentenones A and B (1 and 2), together with guignardone A (3), were isolated from Macrophomina phaseolina cultures. The phytopathogenic fungus was isolated from infected soybean tissues showing charcoal rot symptoms in Argentina. Charcoal rot is a devastating disease considering that soybean is one of the main legumes cultivated in the world. Phaseocyclopentenones A and B were characterized by 1D and 2D 1H and 13C NMR spectroscopic and HRESIMS spectrometric data and chemical methods as 4-benzoyl-3,4,5-trihydroxy-2-phenylcyclopent-2-enone and 3,5-dihydroxy-2,4-diphenylcyclopent-2-enone, respectively. The relative configuration of phaseocyclopentenones A and B was assigned by 1H and NOESY NMR methods, while their absolute configurations were assigned by electronic circular dichroism methods. When assayed on a nonhost plant (Solanum lycopersicum L.) by the leaf puncture assay, phaseocyclopentenones A and B and guignardone A showed phytotoxic activity, while only 1 and 2 were toxic when tested on cuttings of the same plant. No phytotoxicity or antifungal activity was detected for the three compounds on the host plant soybean (Glycine max L.) and against some of its fungal pathogens, namely, Cercospora nicotianae and Colletotrichum truncatum, also isolated from infected soybean plants in Argentina.

The temperature of growth and sporulation modulates the efficiency of spore-display in Bacillus subtilis.

BACKGROUND: Bacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: (i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and (ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. It has been recently shown that B. subtilis builds structurally and functionally different spores when grown at different temperatures; based on this finding B. subtilis spores prepared at 25, 37 or 42 °C were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach. RESULTS: Immune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42 °C. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42 °C slightly more efficient than 25 °C spores in adsorbing at least some of the model proteins tested. CONCLUSION: Our results indicate that the temperature of spore production allows control of the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.