Untargeted and Targeted LC-MS/MS Based Metabolomics Study on In Vitro Culture of Phaeoacremonium Species

Grapevine (Vitis vinifera L.) can be affected by many different biotic agents, including tracheomycotic fungi such as Phaeomoniella chlamydospora and Phaeoacremonium minimum, which are the main causal agent of Esca and Petri diseases. Both fungi produce phytotoxic naphthalenone polyketides, namely scytalone and isosclerone, that are related to symptom development. The main objective of this study was to investigate the secondary metabolites produced by three Phaeoacremonium species and to assess their phytotoxicity by in vitro bioassay. To this aim, untargeted and targeted LC-MS/MS-based metabolomics were performed. High resolution mass spectrometer UHPLC-Orbitrap was used for the untargeted profiling and dereplication of secondary metabolites. A sensitive multi reaction monitoring (MRM) method for the absolute quantification of scytalone and isosclerone was developed on a UPLC-QTrap. Different isolates of P. italicum, P. alvesii and P. rubrigenum were grown in vitro and the culture filtrates and organic extracts were assayed for phytotoxicity. The toxic effects varied within and among fungal isolates. Isosclerone and scytalone were dereplicated by matching retention times and HRMS and MS/MS data with pure standards. The amount of scytalone and isosclerone differed within and among fungal species. To our best knowledge, this is the first study that applies an approach of LC-MS/MS-based metabolomics to investigate differences in the metabolic composition of organic extracts of Phaeoacremonium species culture filtrates.

Isolation and Identification of Lipopeptide-Producing Bacillus velezensis Strains from Wheat Phyllosphere with Antifungal Activity against the Wheat Pathogen Zymoseptoria tritici

Septoria tritici blotch, caused by the fungal pathogen Zymoseptoria tritici, is a highly significant disease on wheat crops worldwide. The objective of the present study was to find out new bacterial strains with bio-antimicrobial activity against Z. tritici. Two phyllospheric bacteria (S1 and S6) were isolated from wheat ears and identified as Bacillus velezensis strains according to 16S rRNA Sanger sequencing. Antagonistic assays performed with either living strains or cell-free culture filtrates showed significant in vitro antifungal activities against Z. tritici. For the culture filtrates, the half-maximal inhibitory dilution and the minimal inhibitory dilution were 1.4% and 3.7% for the strain S1, and 7.4% and 15% for the strain S6, respectively. MALDI—ToF analysis revealed that both strains synthesize cyclic lipopeptides but from different families. Interestingly, only strain S1 produces putative bacillomycin D. Such differential lipopeptide production patterns might explain the difference observed between the antifungal activity of the culture filtrates of the two strains. This study allows the identification of new lipopeptide-producing strains of B. velezensis with a high potential of application for the biocontrol of Z. tritici.

Development of next-generation formulation against Fusarium oxysporum and unraveling bioactive antifungal metabolites of biocontrol agents

Biocontrol agents serve as a sustainable means of controlling wilt caused by the widespread plant pathogen, Fusarium oxysporum f. sp. lycopersici. The present study aimed to develop water dispersible granules (WDG) using response surface methodology (RSM) for Bacillus subtilis MTCC 2274 and Trichoderma harzianum MTCC 3928, and to compare their antifungal efficacy with other formulations. Further, characterization of the bioactive metabolites responsible for biocontrol was performed. A new microbial formulation, WDG, was developed in the present study with talcum powder (substrate), alginic acid (dispersing agent) and acacia gum (wetting agent) (suspensibility 82.23%; wetting time 2.5 min; dispersion time 10.08 min) that fulfilled the guidelines of Collaborative International Pesticides Analytical Council (CIPAC). In planta study demonstrated that WDG of B. subtilis showed maximum reduction in disease incidence (48%) followed by talc formulation of B. subtilis (44%) and WDG of T. harzianum (42%) with profound effect on plant growth promotion. B. subtilis and T. harzianum demonstrated protease (929 and 846 U ml−1 min−1), chitinase (33.69 and 154 U ml−1 min−1), and β-1,3-glucanase (12.69 and 21.47 U ml−1 min−1) activities. Culture filtrates of B. subtilis and T. harzianum exhibited significant inhibition against mycelial growth of pathogen. The compounds present in the culture filtrates were identified with GC–MS as fatty acids, alkanes, phenols, benzene, pyran derivatives etc. The major non-volatile compounds in bioactive antifungal fraction were identified as derivatives of morpholine and piperdine for T. harzianum and B. subtilis, respectively. The findings propose a multivariate biocontrol mechanism against phytopathogen by production of hydrolytic enzymes, volatile and non-volatile compounds, together with development of an efficient next-generation formulation.

Enhanced Yield of Pepper Plants Promoted by Soil Application of Volatiles From Cell-Free Fungal Culture Filtrates Is Associated With Activation of the Beneficial Soil Microbiota

Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants’ responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota.

Endophytic Fungi as Potential Bio-Control Agents against Root Knot Nematode, Meloidogyne incognita in Banana

A Survey was conducted in 12 districts of Assam to collect 92 healthy banana root samples. A total of 55 fungal isolates were successfully isolated from commercial banana cultivars. The culture filtrates were extracted from 55 endophytic fungal isolates and screened against root knot nematode, Meloidogyne incognita in vitro and pot culture studies. Among them, five fungal isolates viz., EF4, BF7, BF27, BF28 and BF35 showed 100% inhibition of egg hatching and 96.33 to 81.33% juvenile mortality of M. incognita with an exposure period of 72h when compared to other isolates and control. On paddy seed treatment with endophytic fungi of five promising isolates, two isolates viz., BF7 and BF28 significantly enhanced germination percentage (82.67%, 73.33%) and vigour index (62.91, 47.24%), respectively. The selected five endophytic fungal isolates were evaluated for their efficacy against M. incognita in banana under pot culture conditions. The study revealed that culture filtrates of BF7 and BF28 significantly reduced the soil and root nematode population, number of adult females, egg masses and root gall index of M.incognita compared to untreated control. The isolates BF7 and BF28 also significantly increased the growth parameters viz., pseudostem height, root length and pseudostem girth. These promising endophytic fungal isolates viz., BF7 and BF28 were identified as non-pathogenic Fusarium oxysporum strains (Accession no. MN567668) and (Accession no. MN567710), respectively by PCR -18S rRNA of ITS region of gene sequence and phylogenetic tree construction.

Reacția unor linii de tomate la izolatele fungului Alternaria alternata

The paper presents the results of the appreciation of some tomato lines based on the resistance to the fungal pathogen Alternaria alternata. Culture filtrates (CF) of pathogen in the most cases did not significantly influence for the seed germination. A more obvious impact was manifested in the case of the root and the stem, the deviations from the control constituting -38.2…. -69.6% for the root and -40.7… -72.6% - for the stem. It was found that the greatest importance in the reaction to A. alternata isolates for seed germination had the genotypic factor, and for root and stem growth - the isolation factor - 95.7-91.0%.

Isolation, characterization, and molecular identification of soil bacteria showing antibacterial activity against human pathogenic bacteria

Background The present study dealt with the screening of soil bacteria with antibacterial activity from different locations in Bangalore, India. Antibiotics play the role of self-defense mechanism for the bacteria and are produced as secondary metabolites to protect themselves from other competitive microorganisms. The need for new antibiotics arose as the pathogenic bacteria acquire resistance to various antibiotics meant for treating human diseases. Given the importance of antibiotics of bacterial origin, standard techniques have been used to isolate and characterize the soil bacteria which showed antibacterial activity. Results The isolated bacteria were tested against human pathogenic bacteria like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae by primary and secondary screening methods. The isolates PR1, PR2, and PR3 were confirmed to have antibacterial activity against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae by both methods. Studies on the effect of filter sterilization, autoclaving, and proteinase K treatment on culture filtrates showed filter sterilization as the best method. The effect of different carbon and nitrogen sources on the antibacterial activity showed that preference by each isolate differed for carbon and nitrogen requirements. The isolates PR1, PR2, and PR3 were identified as Bacillus aryabhattai strain PR-D07, Arthrobacter humicola strain PR-F07, and Neomicrococcus lactis strain PR-F11 through 16S rRNA sequencing. Conclusion Findings from this research work are encouraging and could proceed further to applied aspects. Only 3 bacterial isolates out of 263 isolates from soil samples displayed antibacterial activity against human pathogens S. aureus, E. coli, P. aeruginosa, and K. pneumoniae. They were identified as B. aryabhattai, A. humicola, and N. lactis by 16S rRNA studies and all of them are Gram-positive. Each isolate preferred different carbon and nitrogen sources for their enhanced antibacterial activity. Efficacy of the culture filtrates of these isolates was tested by filter sterilization, autoclaving, and proteinase K treatment. Filter-sterilized culture filtrates showed higher antibacterial activity than other treatments. A comparison of the antibacterial activity of culture filtrates and antibiotic streptomycin produced an inhibition zone of 18.5 mm and 15.5 mm respectively. This is the first report on the antibacterial activity of all the 3 bacterial strains (B. aryabhattai strain PR-D07, A. humicola strain PR-F07, and N. lactis strain PR-F11), against all the human pathogens, mentioned earlier. It is also found that the antibiotic factor is proteinaceous as proteinase K considerably reduced the antibacterial activity of the culture filtrates. With the above significant results, these 3 bacteria are considered to be promising candidates for the isolation of new antibacterial agents.

Bacterial Culture Filtrates Antinematode Activity of Abomasum Bacterial Culture Filtrates against Haemonchus contortus in Small Ruminants

Haemonchosis is a parasitic disease of small ruminants that adversely affects livestock production. Haemonchus contortus is one of the most prevalent nematode parasites that infect the abomasum of small ruminants. This parasite reduces milk production, overall growth and sometimes causes the death of the infected animals. The evaluation of the biocontrol potential of some abomasum bacterial isolates against H. contortus is investigated in this study. Out of which, three isolates—Comamonas testosteroni, Comamonas jiangduensis, Pseudomonas weihenstephanesis—show significant effect against the nematode L3, adult, and egg hatch inhibition assays. Various concentrations of metabolites from these bacteria are prepared and applied in different treatments compared with control. In the case of adult mortality assay, 50% metabolites of C. testosteroni and P. weihenstephanesis show 46% adult mortality, whereas C. jiangduensis shows 40% mortality. It is observed that decreasing the concentration of bacterial metabolite, lowers nematode mortality. The minimum nematode mortality rate is recorded at the lowest filtrates concentration of all the bacterial isolates. The same trend is observed in egg hatch inhibition assay, where the higher concentration of bacterial culture filtrates shows 100% inhibition of H. contortus egg. It is concluded that the effect of bacterial culture filtrates against H. contortus is dose-dependent for their activity against nematode L3, adult, and inhibition of egg hatchment.