Carbendazim-resistance associated β2-tubulin substitutions increase deoxynivalenol biosynthesis by reducing the interaction between β2-tubulin and IDH3 in Fusarium graminearum

Microtubule is a well-known structural protein participating in cell division, motility and vesicle traffic. In this study, we found that β₂-tubulin, one of the microtubule components, plays an important role in regulating secondary metabolite deoxynivalenol (DON) biosynthesis in Fusarium graminearum by interacting with isocitrate dehydrogenase subunit 3 (IDH3). We found IDH3 negatively regulate DON biosynthesis by reducing acetyl-CoA accumulation in F. graminearum and DON biosynthesis was stimulated by exogenous acetyl-CoA. In addition, the expression of IDH3 significantly decreased in the carbendazim-resistant mutant nt167 (Fgβ₂^F167Y). Furthermore, we found that carbendazim-resistance associated β₂-tubulin substitutions reducing the interaction intensity between β₂-tubulin and IDH3. Interestingly, we demonstrated that β₂-tubulin inhibitor carbendazim can disrupt the interaction between β₂-tubulin and IDH3. The decreased interaction intensity between β₂-tubulin and IDH3 resulted in the decreased expression of IDH3, which can cause the accumulation of acetyl-CoA, precursor of DON biosynthesis in F. graminearum. Thus, we revealed that carbendazim-resistance associated β₂-tubulin substitutions or carbendazim treatment increases DON biosynthesis by reducing the interaction between β₂-tubulin and IDH3 in F. graminearum. Taken together, the novel findings give the new perspectives of β₂-tubulin in regulating secondary metabolism in phytopathogenic fungi.     

Biological properties of the wild rhizosphere strain Pseudomonas fluorescens 2137 and its derivatives marked with the gusA gene

he natural wild rhizosphere strain P. fluorescens 2137 was marked with the β-glucuronidase gene gusA. The introduction of this gene influenced the viability of the wild strain, as well as its certain physiological parameters, such as cultural characteristics, biochemical properties, and antagonistic activity against the phytopathogenic fungi Fusarium culmorum, F. oxysporum, F. graminearum, and Verticillum nigrescens. The gusA-marked derivative strains that deviate the least from the wild strain in biological properties can be used to monitor populations of P. fluorescens 2137 cells in the plant rhizosphere.     

Development of a powder formulation based on <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">cereus</Emphasis> sensu lato strain B25 spores for biological control of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> in maize plants

Maize is an economically important crop in northern Mexico. Different fungi cause ear and root rot in maize, including Fusarium verticillioides (Sacc.) Nirenberg. Crop management of this pathogen with chemical fungicides has been difficult. By contrast, the recent use of novel biocontrol strategies, such as seed bacterization with Bacillus cereus sensu lato strain B25, has been effective in field trials. These approaches are not without their problems, since insufficient formulation technology, between other factors, can limit success of biocontrol agents. In response to these drawbacks, we have developed a powder formulation based on Bacillus B25 spores and evaluated some of its characteristics, including shelf life and efficacy against F. verticillioides, in vitro and in maize plants. A talc-based powder formulation containing 1 × 10⁹ c.f.u. g^?1 was obtained and evaluated for seed adherence ability, seed germination effect, shelf life and antagonism against F. verticillioides in in vitro and in planta assays. Seed adherence of viable bacterial spores ranged from 1.0 to 1.41 × 10⁷ c.f.u. g^?1. Bacteria did not display negative effects on seed germination. Spore viability for the powder formulation slowly decreased over time, and was 53 % after 360 days of storage at room temperature. This formulation was capable of controlling F. verticillioides in greenhouse assays, as well as eight other maize phytopathogenic fungi in vitro. The results suggest that a talc-based powder formulation of Bacillus B25 spores may be sufficient to produce inoculum for biocontrol of maize ear and root rots caused by F. verticillioides.     

The effect of <Emphasis Type="Italic">Baccharis glutinosa</Emphasis> extract on the growth of mycotoxigenic fungi and fumonisin B<Subscript>1</Subscript> and aflatoxin B<Subscript>1</Subscript> production

The aim of this study was to evaluate the effect of Baccharis glutinosa isolated extract on the growth of Aspergillus flavus and Aspergillus parasiticus, and their aflatoxin B₁ production; and growth of Fusarium verticillioides, and their fumonisin B₁ production. The three fungi were exposed to an antifungal fraction, designated as fraction F6-1, isolated from B. glutinosa by methanolic extraction followed by silica gel chromatography. The growth of the fungi was evaluated in kinetics of radial extension growth, kinetics of spores germination, length and diameter of hyphae, spores diameter, as well as in aflatoxin B₁ and fumonisin B₁ production. Fraction F6-1 caused radial growth inhibition of the three fungi mainly F. verticillioides. Spores germination of A. flavus and A. parasiticus was delayed in the early stage of the incubation time, although they completely germinated at 27 h. In contrast, spore germination of F. verticillioides was inhibited 87.7% up to 96 h. The lengths and diameters of hyphae, and spore diameters of the three fungi, were significantly smaller in comparison with those of the controls, and several morphological alterations were observed. Concerning aflatoxin B₁ and fumonisin B₁, fraction F6-1 did not show any inhibition effect at the concentration used. Fraction F6-1 was able to significantly inhibit the development of the three fungi, mainly F. verticillioides. The strong inhibitory effect of F6-1 on hyphae and spores suggests that it interacted with the fungi cell walls, which caused severe deformities. Nevertheless, this fraction was unable in inhibiting mycotoxin production from the three fungi at the concentration tested.     

Laccase activity and putative laccase genes in marine-derived basidiomycetes

Studies of laccases from marine-derived fungi are limited. In the present work, putative laccase genes from three marine-derived basidiomycetes and their laccase activities were evaluated. High amounts of laccase were produced by the fungal strains Marasmiellus sp. CBMAI 1062 (971.2 U L⁻¹) and Peniophora sp. CBMAI 1063 (709.03 U L⁻¹) when grown for 21 d at 28 °C in MA2ASW medium prepared with artificial seawater. Marine-derived basidiomycetes produced multiple distinct laccase sequences of about 200 bp with 73–90 % similarity to terrestrial basidiomycete laccases. Marasmiellus sp. CBMAI 1062 and Tinctoporellus sp. CBMAI 1061 showed the greatest laccase gene diversity with three and four distinct putative laccase sequences, respectively. This is the first report of laccase genes from marine-derived fungi, and our results revealed new putative laccases produced by three basidiomycetes.     

Molecular characterization of a novel β-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum

Trichoderma harzianum, a soil-borne filamentous fungus, is capable of parasitizing several plant pathogenic fungi. Secretion of lytic enzymes, mainly glucanases and chitinases, is considered the most crucial step of the mycoparasitic process. The lytic enzymes degrade the cell walls of the pathogenic fungi, enabling Trichoderma to utilize both their cell walls and cellular contents for nutrition. We have purified a 110 kDa novel extracellular β-1,3-exoglucanase from T. harzianum, grown with laminarin or in dual cultures with host fungi. The corresponding gene, lam1.3, and its cDNA were isolated and their nucleotide sequences determined. The deduced amino-acid sequence predicted a molecular mass of 110.7 kDa of a mature protein excluding a signal peptide. LAM1.3 showed high homology to EXG1, a β-1,3-exoglucanase of the phytopathogenic fungus Cochliobolus carbonum, and a lower homology to BGN13.1, a β-1,3-endoglucanase isolated from T. harzianum. However, it contains a unique C-terminal embodying cysteine motifs. The expression of lam1.3 in growth with laminarin, but not with glucose, was found to be a result of differential accumulation of the corresponding mRNA.     

Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment

An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.     

Extracellular hydrolases of strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the β-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and β-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only β-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

The new bacterial strain <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. IB-1: A producer of exopolysaccharide and biologically active substances with phytohormonal and antifungal activities

The bacterial strain IB-1, which exhibits antagonism towards phytopathogens and stimulates the growth of agricultural plants, was isolated from the soil. Analysis of the cultural, morphological, physiological, and biochemical features and nucleotide sequences of the genes 16S rRNA and gyrB, as well as the fatty acid composition, made it possible to attribute the strain IB-1 to the genus Paenibacillus; however, the results did not provide an unambiguous conclusion on its species. The strain Paenibacillus sp. IB-1 possesses nitrogenase activity, the ability to synthesize indoleacetic acid and cytokinin-like compounds, and antagonistic activity towards phytopathogenic fungi, which indicates prospects for its use as a biological product for agricultural purposes. A high-viscous exopolysaccharide was isolated from the cultural fluid of Paenibacillus sp. IB-1. Based on the data from IR and NMR spectroscopy, it was shown to be a heteropolymer comprised of one to four linked α-L-guluronic acid and β-D-mannuronic acid residues. The exopolysaccharide was successfully tested as an adhesive for presowing treatment of barley and wheat seeds with biofungicides.     

Synthesis of novel fenfuram-diarylether hybrids as potent succinate dehydrogenase inhibitors

Twelve novel fenfuram-diarylether hybrids were designed, synthesized and characterized by ¹H NMR and MS. Their in vitro antifungal activities were evaluated against five phytopathogenic fungi by mycelial growth inhibition method. Most compounds showed significant antifungal effect on Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 1c exhibited the most potent antifungal effect on R. solani with an EC₅₀ value of 0.242 mg/L, superior to the commercial fungicide boscalid (EC₅₀ = 1.758 mg/L) and the lead fungicide fenfuram (EC₅₀ = 7.691 mg/L). Molecular docking revealed that compound 1c featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 2-chlorophenyl group of compound 1c formed a π-π stacking with D/Tyr-128 and a Cl-π interaction with B/His-249, which made compound 1c more active than fenfuram against SDH.     

Analysis of β-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7?μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1?μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin.     

Mesoionic compounds with antifungal activity against Fusarium verticillioides

Background

Fungi contaminate the food of humans and animals, are a risk to health, and can cause financial losses. In this work, the antifungal activities of 16 mesoionic compounds (MI 1–16) were evaluated against mycotoxigenic fungi, including Aspergillus spp., Fusarium verticillioides and Penicillium citrinum. Furthermore, the decreased ergosterol in the total lipid content of Fusarium verticillioides was investigated.

Results

F. verticillioides was the most sensitive fungus to the mesoionic compounds. Among the evaluated compounds, MI-11 and MI-16 presented higher antifungal effects against F. verticillioides, with MIC values of 7.8 μg/ml, and MI-2 and MI-3 followed, with MICs of 15.6 μg/ml. The most active compounds were those with heterocyclic ring phenyl groups substituted by electron donor moieties (MI-11 and MI-16). Among some compounds with higher activity (MI-2, MI-11 and MI-16), decreased ergosterol content in the total lipid fraction of F. verticillioides was demonstrated. MI-2 reduced the ergosterol content approximately 40% and 80% at concentrations of 7.8 μg/ml and 15.6 μg/ml, respectively, and MI-11 and MI-16 decreased the content by 30% and 50%, respectively, when at a concentration of 7.8 μg/ml.

Conclusion

These findings indicate that mesoionic compounds have significant antifungal activity against F. verticillioides.     

Dual biocontrol potential of the entomopathogenic fungus Akanthomyces muscarius against Thaumetopoea pityocampa and plant pathogenic fungi

Akanthomyces spp. species are known for their capacity to biocontrol of certain insects and plant pathogens; however, their ability to biocontrol the pine processionary (Thaumetopoea pityocampa) and certain phytopathogenic fungi belonging to the genera Fusarium and Curvularia have not been studied before. In this study, a strain from Akanthomyces muscarius was isolated from wheat grains and then identified by morphological and molecular tests. The strain was further studied for its capacity to control Thaumetopoea pityocampa larvae through dose-mortality tests, and its ability to control some phytopathogenic fungi strains of the genera Fusarium and Curvularia was studied through direct confrontation tests. Dose-mortality tests at three concentrations of Akanthomyces muscarius against the first instar larvae revealed a mortality of 92.15% after 11 days for the concentration of 2.3 × 10⁶ conidia.ml⁻¹, with a median lethal concentration of 7.6 x10³ conidia.ml^1. Our isolate also showed antifungal activity against these phytopathogenic fungi with inhibition rates ranging from 39.61% to 52.94%. Akanthomyces muscarius proved to be a promising biocontrol agent for plant pests and diseases.     

Characterization of Fusarium spp. associated with pineapple fruit rot and leaf spot in Peninsular Malaysia

Pineapple (Ananas comosus) is one the important fruit crops planted in Malaysia, and this study was conducted to determine Fusarium spp. associated with diseases of the fruit crop as Fusarium is prevalent in tropical countries. Our objective was to identify and characterize Fusarium spp. associated with pineapple fruit rot and leaf spot mainly found on the fruits and leaves in Peninsular Malaysia. Fusarium isolates (n = 108) associated with pineapple fruit rot and leaf spot were characterized by morphological, molecular and phylogenetic analyses, a mating study and pathogenicity testing. TEF‐1α sequence analysis identified Fusarium proliferatum, Fusarium verticillioides, Fusarium sacchari and Fusarium sp. Mating was successful only between tester strains of F. proliferatum and F. verticillioides. Sexual crosses with standard tester strains showed that 82 isolates of F. proliferatum produced fertile crosses with mating population D (Gibberella intermedia) and three isolates of F. verticillioides were fertile with the tester strain of mating population A (Gibberella moniliformis). All isolates were pathogenic, causing pineapple fruit rot and leaf spot, thus fulfilling Koch's postulates.     

Efficacy assessment of antifungal metabolites from Chaetomium globosum No.05, a new biocontrol agent,against Setosphaeria turcica

Northern corn leaf blight (NCLB), an important and potentially destructive corn foliar disease, is caused by Setosphaeria turcica. The intent of this study was to evaluate antifungal metabolites from Chaetomium globosum (Cg) strain No.05 to suppress NCLB in maize. This strain significantly suppressed mycelial growth of numerous phytopathogenic fungi especially S. turcica on potato dextrose agar medium. The secondary metabolites of the strain inhibited mycelial growth and conidial germination of S. turcica. When co-inoculated at three droplets (5 μL/droplet) of conidial suspension (5 × 10⁴ conidia/mL) on each 8-cm-long detached leaf, 20% culture filtrates completely suppressed disease incidence of northern corn leaf blight. The application of the culture filtrates at 2 h post-inoculation (hpi) of S. turcica in greenhouse studies showed a 81.9% inhibition of NCLB on the seedlings, while culture filtrates applied before pathogen inoculation showed even higher rates of disease reduction. The application of the culture filtrates had no observed effects on the treated maize leaves or seedlings. Two active compounds, isolated from the extracts, were identified as chaetoglobosin A and chaetoglobosin C based on the spectroscopic analysis. Both in vitro and in planta bioassay experiments showed that chaetoglobosin A displayed potent biocontrol efficiency against S. turcica. To the best of our knowledge, this is the first report of the evaluation of the inhibitory effects of C. globosum and chaetoglobosin A against S. turcica both in vitro and on detached maize leaves.     

Molecular cloning and biochemical characterization of α- and β-tubulin from potato plants (Solanum tuberosum L.)

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) α-tubulin and β-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of α- and β-tubulin were 2.0–3.5 mg l^?1 and 1.3–3.0 mg l^?1 of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant α- and β-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.     

The synthetic strigolactone GR24 influences the growth pattern of phytopathogenic fungi

Strigolactones that are released by plant roots to the rhizosphere are involved in both plant symbiosis with arbuscular mycorrhizal fungi and in plant infection by root parasitic plants. In this paper, we describe the response of various phytopathogenic fungi to the synthetic strigolactone GR24. When GR24 was embedded in the growth medium, it inhibited the growth of the root pathogens Fusarium oxysporum f. sp. melonis, Fusarium solani f. sp. mango, Sclerotinia sclerotiorum and Macrophomina phaseolina, and of the foliar pathogens Alternaria alternata, Colletotrichum acutatum and Botrytis cinerea. In the presence of this synthetic strigolactone, intense branching activity was exhibited by S. sclerotiorum, C. acutatum and F. oxysporum f. sp. melonis. Slightly increased hyphal branching was observed for A. alternata, F. solani f. sp. mango and B. cinerea, whereas suppression of hyphal branching by GR24 was observed in M. phaseolina. These results suggest that strigolactones not only affect mycorrhizal fungi and parasitic plants, but they also have a more general effect on phytopathogenic fungi.     

Diesel degradation potential of endophytic bacteria isolated from Scirpus triqueter

Scirpus triqueter, a dominant species in wetland of Huangpu-Yangtze estuary can be used for phytoremediation. Endophytic bacteria from S. triqueter were investigated to evaluate the ability to degrade diesel. Bacterial incubation experiments were established to screen diesel degradation of endophytic microorganisms from S. triqueter. The oil-degrading bacterial strain J4AJ was discovered in vivo of S. triqueter and identified as Pseudomonas sp. U-3 bacterial strain also isolated from the root and stem of S. triqueter was identified to be the potent producer of biosurfactant as Bacillus subtilis. The minimum surface tension of U-3 was 30.9 mN/m. The bacterium manifested outstanding adaptation ability at extremes of temperature, pH and salinity. The removal ratio of J4AJ could be greatly improved by adding U-3 strain. However, the degradation ratio of J4AJ was obviously decreased by the addition of Alkyl Polysaccharide Glycoside (APG). The results suggested that J4AJ and U-3 inoculated were conducive to the degradation of diesel, which can be used for the remediation of oil-contaminated ecological environment.