Bioactive specialized metabolites produced by the emerging pathogen Diplodia olivarum

A new cleistanthane nor-diterpenoid, named olicleistanone (1), was isolated as a racemate from the culture filtrates of Diplodia olivarum an emerging pathogen involved in the aetiology of branch canker and dieback of in several plant species typical of the Mediterranean maquis in Sardinia, Italy. When the fungus was grown in vitro on Czapek medium olicleistanone was isolated together with some already known phytotoxic diterpenoids identified as sphaeropsidins A, C, and G and diplopimarane (2-5). Olicleistanone was characterized by spectroscopic methods (essentially 1D and 2D NMR and HR ESIMS) as 4-ethoxy-6a-methoxy-3,8,8-trimethyl-4,5,8,9,10,11-hexahydrodibenzo[de,g]chromen-7(6aH)-one. When the fungus was grown on mineral salt medium showed to produce (-)-mellein (6), sphaeropsidin A and a very low amount of sphaeropsidin G and diplopimarane. Olicleistanone (1) exhibited remarkable activity against Artemia salina L. (100% larval mortality) at 100 µg/mL. In addition, it did not exhibit phytotoxic, antifungal and antioomycetes activity. Among the metabolites isolated (1-6), the sphaeropsindin A (2) proved to be active in all bioassay performed exhibiting remarkable phytotoxicity on Phaseolus vulgaris L., Juglans regia L. and Quercus suber L. leaves at 1 mg/mL. Moreover, it completely inhibited the mycelial growth of Athelia rolfsii, Diplodia corticola, Phytophthora cambivora and P. lacustris at 200 µg/plug. It was also active in the Artemia salina assay. In this latter assay, diplopimarane (4) and sphaeropsidin G (4) were active (100% larval mortality). Diplopimarane also showed antifungal and antioomycetes activities. Athelia rolfsii was the most sensitive species to diplopimarane. Sphaeropsidin C (3) and (-)-mellein (6) were found to be inactive in all bioassays. Results obtained in this study have allowed us to expand the knowledge on the metabolic profile of Botryosphaeriaceae members and characterize the main secondary metabolites secreted in vitro by D. olivarum for the first time.

Succinic Acid Production as Secondary Metabolite from Bacillus megaterium ELI24

Background: Succinic acid can be used as a precursor of many industrially important chemicals and has an extensive application range to apply in different industrial areas, such as pharmaceutical and food industry. Objective: In this work, we isolated a strain of B. megaterium which produces succinic acid in optimized czapek medium for its production. Methods: A study of the production of this organic acid was realized in different media adding insoluble phosphate sources to czapek medium. Results: Two Bacillus strains ELI 24 and ELI30 that were isolated from soil in Mexico were fully characterized as Bacillus megaterium and B. thuringiensis. B. megaterium ELI 24 was proved to secrete succinic acid which was clearly identified by spectroscopic methods, such as 1H and 13C NMR, and X-ray analysis. Conclusion: This organic acid could improve the solubility of insoluble phosphate, which could help in the growth of plants.

Acţiunea erbicidului Trifluralin asupra micromicetelor

50 micromycete strains from NCNM were tested and cultivated on Czapek medium with 20% glucose in the presence of trifluralin in concentrations of: 50; 100 and 200 mg / l. It was found that all strains growth on all tested media regardless of the concentration of trifluralin supplemented in the agar medium. In order to better highlight the action of trifluralin on micromycetes in subsequent research, glucose was excluded from the Czapek medium and the dose of trifluralin was increased from 200 mg / l to 300 and 500 mg / l, so it was shown that out of 12 strains studied 6 no growth on glucose-free Czapek medium, and supplementation of this medium with trifluralin helps to stimulate their growth.

The effect of fungicides on growth of Fusarium fungi in vitro

Four different fungicides that recommended to control seed-borne pathogens, were tested in this study. Three fungicides contain singly pyraclostrobin (200 g/L), thiram (400 g/L), fludioxonil (25 g/L), and one fungicide contains together imazalil, metalaxyl, and tebuconazole (50, 40, and 30 g/L, respectively). Comparative assessment of the fungicides at four concentrations of active substance (10, 100, 1 000, and 10 000 ppb) on the growth of ten strains of Fusarium spp. was studied in vitro tests. To visualize the effect of fungicides on fungal growth the strains were cultivated in 50 mL liquid Czapek medium containing fungicides in the range of concentrations. The fungicide based on pyraclostrobin was the most effective in growth inhibition of Fusarium fungi. Broadly, the using of this fungicide is confident way to control Fusarium species, which are serious threat to crop production. The sensitivity of F. acuminatum, F. graminearum, F. semitectum, F. culmorum, F. sporotrichioides, F. equiseti strains to fungicides was higher in compare with the strains belonging to F. oxysporum, F. solani, F. verticillioides, and F. proliferatum.

Expanding the chemical diversity of an endophytic fungus Bulgaria inquinans, an ascomycete associated with mistletoe, through an OSMAC approach

OSMAC approach on endophytic Bulgaria inquinans by addition of a mixture of salts (MgSO4, NaNO3 and NaCl) to solid Czapek medium induced the accumulation of new secondary metabolites.

Pantoea ananatis Converts MBOA to 6-Methoxy-4-nitro-benzoxazolin-2(3H)-one (NMBOA) for Cooperative Degradation with its Native Root Colonizing Microbial Consortium

6-Methoxy-benzoxazolin-2(3 H)-one (MBOA) is a degradation product derived from 2,4-Dihydroxy-7-methoxy-2 H-1,4-benzoxazin-3(4 H)-one (DIMBOA), one of the bioactive compounds found e.g., in maize. Here we present hitherto unknown 6-methoxy-4-nitro-benzoxazolin-2(3 H)-one (NMBOA) produced in Czapek medium by Pantoea ananatis (Enterobacteriaceae). P. ananatis is a member of a microbial consortium dominated by the zygomycete Actinomucor elegans, which was isolated from roots of Abutilon theophrasti. NMBOA was identified by NMR spectra and HR-ESI-MS analyses, revealing an unusual position of the nitro group at C-4. Nitration of MBOA initiates the degradation of the compound that is almost completed within three days by the entire consortium and isolated P. ananatis. The yeast Papiliotrema baii, another member of the consortium, is unable to degrade NMBOA but stored it at the surface of its polysacchararide capsule. NMBOA has negative effects on microbial growth in liquid medium whereas seedlings of Brassica oleracea var. gongylodes L. (kohlrabi) or Lepidium sativum (cress) are not impaired up to 500 μM. Degradation via nitration may be important to understand the behavior of microbial species and effects of microbiomes when exposed to MBOA.

PULSED ELECTRIC FIELD FOR PASTEURIZATION OF FRESH SUGARCANE JUICE

Pasteurization methods using pulsed electric fields (PEFs) have proved efficient at non-thermal inactivation of microorganisms and pathogens. The methods usually use a series of high intensity, narrow band electric fields that pass through target mediums where they induce irreversible damage to cellular membranes leading to the death of microorganisms by internal content loss breakage of cell homeostasis and other effects. This research proposed a simple, lab-scale PEF pasteurization equipment that uses common output electric voltages (220V and 110V, which provide electric power of 5.5 and 2.75 kV/m, respectively) with a simple designed Human-Machine Interface control. At a voltage of 110V, the equipment was tested with non-sterilized Czapek medium which showed more than 1.42-log reduction in the microbial density at 30s of the treatment and temperature below 60 o C. A test on suspended E. coli in modified Czapek medium also indicated an inactivation of most of bacterial cells after 40s of the treatment. We then applied the equipment to pasteurize fresh sugarcane juice and, as a result, the content of native microorganisms in sugarcane juice was reduced 1.2-log cycle, compared to that of original juice before the treatment.

Lack of correlation between micro fungi species and chemical control method of Atta treated with toxic baits

ABSTRACT: Atta sexdens rubropilosa (leaf-cutter ants) has a symbiotic association with a fungus and has a negative interaction with other fungi due to parasitism of the fungus cultivated by ants; also, there are several other fungi with no exact known role occurring in their cultivated fungus garden. In the present study, we use the ITS region (internal transcribed spacer) to identify fungi in colonies treated with toxic baits. Experiments using two toxic baits were carried out: 0.75g of sulfluramid [0.3%] and 0.75g fipronil [0.003%]. Samples of fungi were collected and cultured in Czapek medium for seven days to allow fungal growth and subsequent identification. Total DNA was isolated from 100-150 mg of mycelium using the CTAB method and using PCR, with the universal primers (ITS4 and ITS5), to amplify the ITS region. Sequencing was performed using the Sanger method. Sequences were subjected to BLAST, allowing the identification of nine different species of the orders Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales and Tremellales showing a variation in identity of 96-100%. Using “The Automatic Barcode Gap Discovery” analysis, nine groups were identified, corresponding to species described in NCBI. The K2P distances were used to generate a tree using Neighbour-joining, demonstrating that the species were grouped according to phylogenetic groups. We concluded that leaf-cutter ant colonies exhibited a wide variety of fungi and this study suggested that there is no correlation between the species of fungi isolated with the control method used on the ant nest.

Testing the efficacy of phytohormones of microbial origin in promoting plant growth

Mustard Is an Important Oilseed Which Is Used For many Purposes Like Culinary, Religious And Cultural Purposes By Humanity Since Time Immemorial. To Increase The Production of Mustard, Phytohormones Can Be Used Which Are Pro1duced With The Help Of Microorganisms. Phytohormones are the signaling or organic compound which helps in the development and growth of plant. Rhizobium and Bacillus sp were used to produce phytohormones which are isolated from different sources and investigated for their ability to produced auxin and cytokinin in PM2 and Czapek medium respectively. Thin layer chromatography and High performance liquid chromatography were performed to detect and quantify the amount of hormones in the production medium. Rhizobium produced the highest auxin (0.087mg/l) and cytokinin (0.047mg/ml) in the production medium. Then these microbiologically synthesized auxin and cytokinin were treated with mustard seeds to check their ability in promoting growth development. Auxin produced from microorganisms comparatively showed the fastest shoot development (7.9cm length) and leaf (length-1.8cm and width-1.9cm) than cytokinin and control.

Production of Ligninolytic Enzymes by Cultures of White Rot Fungi

Some Basidiomycota were chosen for studies of key ligninases synthesis (25°C, 30 days) in modified medium (shaken or not cultures) with added wheat straw. Liquid Czapek medium with straw yielded a higher amount of laccase than peroxidase, ground straw induced enzyme worse than chopped straw. With peroxidase the reverse dependencies were observed. Laccase of Lentinus edodes synthesized two enzyme isoforms (ca 30 and 16 kDa). In T. versicolor culture active laccase protein with highest molecular mass ca 65 kDa was found. P. sajor-caju yielded three different peroxidase isoforms. Ligninase biosynthesis depended on strain, straw fragmentation extent, culture method and growth medium.