Bio efficacy of indigenous biological agents and selected fungicides against branch canker disease of (Macrophoma theicola) tea under field level.

BACKGROUND: Branch canker caused by Macrophoma theicola is a major stem disease of tea plants (Camellia spp.). In tea plantations, this disease causes crop loss and it is one of the major limiting factor for yield stagnation. In very few instances it causes considerable damage in new clearings (about 3 or 4 years old) and large number of bushes have been killed. As there is no control measures for branch canker disease in south Indian tea plantation, this field study was conducted in naturally infected pruned tea field at UPASI Tea Research Institute (Good Agricultural Practice), Valparai, Tamil Nadu, India. METHODS: The chemical fungicides, biological agents and bio products were evaluated under naturally infected field of seedling plants for two consecutive disease seasons (2014-2015) and there was 11 treatments with three applications. All the treatments were carried out in the time of February-March and October-November (2014-2015). The two set of application was conducted per year. Each set contains eight rounds during the month of February-March as well as October-November (2014-2015). The chemical fungicides, biological agents and commercial bio products were measured as per UPASI- TRF, recommendation viz., COC (50 g/ha and 0.2 g/plot), Companion (20 g/ha and 0.08 g/plot), biological agent of Bacillus amyloliquefaciens, Tichoderma harzianum, Gliocladium virens and Beauveria bassiana (5 kg/ha and 20.8 g/plot) and bio product of Tari (1 L/ha and 4.2 ml/plot) and Tricure (1 L/ha and 4.2 ml/plot). RESULTS: The present investigation revealed the integrated application of Companion/Bacillus amyloliquefaciens showed superior control of branch canker disease followed by the treatment with Companion alone under field condition. Copper oxychloride/Bacillus amyloliquefaciens was moderately effective followed by Copper oxychloride. The significantly reduced canker size was recorded with treatment of Bacillus amyloliquefaciens followed by commercial organic fungicides of Tari (Organic Tea Special) and Tricure (0.03% Azadirachtin). The least canker size was observed with Gliocladium virens followed by Beauveria bassiana. Branch canker disease incidence was increased in untreated control plants when compared to treated plants. CONCLUSION: Among these 11 treatments, the integrated treatment of companion at rate of 0.08 g and Bacillus amyloliquefaciens (20.8 g) showed the most significantly decreased canker size (DPL, 5.76) followed by another treatment with companion (0.08 g) (DPL, 4.11). The moderate reduction of canker size was observed by the treatment with Copper oxychloride (0.2 g)/Bacillus amyloliquefaciens (20.8 g) (DPL, 3.05) followed by the treatment of copper oxychloride alone (DPL, 1.74). Therefore, the integrated application of Companion/Bacillus amyloliquefaciens proved significantly effective in the management of branch canker disease under the field conditions.

Biotransformation of quercetin by Gliocladium deliquescens NRRL 1086.

With an attempt to synthesize high-value isoquercitrin (quercetin-3-O-ß-D-glucopyranoside), we carried out the biotransformation of quercetin (1) by Gliocladium deliquescens NRRL 1086. Along with the aimed product quercetin 3-O-ß-D-glycoside (2), three additional metabolites, 2-protocatechuoyl-phlorogucinol carboxylic acid (3), 2,4,6-trihydroxybenzoic acid (4), and protocatechuic acid (5), were also isolated. The time-course experiments revealed that there were two metabolic routes, regio-selectivity glycosylation and quercetin 2,3-dioxygenation, co-existing in the culture. Both glycosylation and oxidative cleavage rapidly took place after quercetin feeding; about 98% quercetin were consumed within the initial 8 h and the oxdized product (2-protocatechuoyl-phlorogucinol carboxylic acid) was hydrolyzed into two phenolic compounds (2,4,6-trihydroxybenzoic acid and protocatechuic acid). We also investigated the impact of glucose content and metal ions on the two reactions and found that high concentrations of glucose significantly inhibited the oxidative cleavage and improved the yield of isoquercitrin and that Ca2+, Fe2+, Mn2+, Mg2+, and Zn2+ inhibited glycosylation. To test the promiscuity of this culture, we selected other four flavonols as substrates; the results demonstrated its high regio-selectivity glycosylation ability towards flavonols at C-3 hydroxyl. In conclusion, our findings indicated that the versatile microbe of G. deliquescens NRRL 1086 maitained abundant enzymes, deserving further research.

Diversity synthesis of tetrahydroprotoberberines glycosides by combined chemical and microbial catalysis.

The present study was designed to construct the structurally diverse library of tetrahydroprotoberberines (THPBs) by combining the methods of chemical nonselective demethylation and microbial glycosylation. HPLC-MS/MS analyses tentatively identified 12 de-methylated and 9 glycosylated derivates of THPBs and 5 rarely oxidized glycosides of THPBs in the library. Through this effort, we achieved not only a variety of the THPBs and their glycosides but also tested the catalytic characteristics and capabilities of G. deliquescens NRRL 1086.

Glycosylation and sulfation of emodin by Gliocladium deliquescens NRRL 1086.

The present study was designed to explore the substrate scope and biocatalytic capability of Gliocladium deliquescens NRRL 1086 on phenolic natural products. Emodin was subjected to the fermentation culture of Gliocladium deliquescens NRRL 1086 according to the standard two-stage protocol. The biotransformation process was monitored by HPLC-DAD-MS, the main product was isolated by column chromatography, and the structure was elucidated on the basis of NMR spectroscopy. Emodin could be fully metabolized by Gliocladium deliquescens NRRL 1086, resulting in high yield of emodin 6-O-ß-D-glucopyranoside and small amount of sulfated product. In conclusion, our results may provide a convenient method to prepare emodin 6-O-ß-D-glucopyranoside and the microbe catalyzed glucosylation/sulfation will give an inspiration to pharmacokinetic model studies in vitro.

Glycosylation and sulfation of emodin by Gliocladium deliquescens NRRL 1086 / 中国天然药物

The present study was designed to explore the substrate scope and biocatalytic capability of Gliocladium deliquescens NRRL 1086 on phenolic natural products. Emodin was subjected to the fermentation culture of Gliocladium deliquescens NRRL 1086 according to the standard two-stage protocol. The biotransformation process was monitored by HPLC-DAD-MS, the main product was isolated by column chromatography, and the structure was elucidated on the basis of NMR spectroscopy. Emodin could be fully metabolized by Gliocladium deliquescens NRRL 1086, resulting in high yield of emodin 6-O-β-D-glucopyranoside and small amount of sulfated product. In conclusion, our results may provide a convenient method to prepare emodin 6-O-β-D-glucopyranoside and the microbe catalyzed glucosylation/sulfation will give an inspiration to pharmacokinetic model studies in vitro.

Identification and Characterization of Gliocladium viride Isolated from Mushroom Fly Infested Oak Log Beds Used for Shiitake Cultivation

A green mold species that has not previously been reported in Korea was isolated from oak log beds used for shiitake (Lentinula edodes) cultivation that were infested by mushroom flies. In this study, we identify the mold species as Gliocladium viride (an anamorph of Hypocrea lutea) and describe its mycological properties. The fungus was cottony on both potato dextrose agar (PDA) and Czapek yeast extract agar (CYA), but was colored white on PDA and became yellowish green and brown on CYA. Mycelial growth on PDA attained a diameter of 73 mm at 30degrees C after 5 days. The fungus grew faster on malt extract agar (> 80 mm, 5 days at 25degrees C) compared to CYA and PDA (< 68 mm, 5 days at 25degrees C). Penicillate conidiophores of the fungus are hyaline, smooth walled, branching above typically in four stages, and 120~240 microm in length. Club-shaped or slender phialides are formed on the metulae. Conidia of the fungus were ovate and elliptic, yellowish brown and green, and 2.5~3.0 microm x 1.8~2.3 microm in size. Typically, slimy conidia are formed in a mass and colored brown to dark green to almost black. The internal transcribed spacer rDNA and translation elongation factor 1 alpha gene sequences of the fungus isolated here show 99% identity with previously identified G. viride strains.

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists.

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.

[Secondary metabolites of Gliocladium sp., a growth accelerating fungus for Anoectochilus roxburghii (Wall.) Lindl].

OBJECTIVE: To study the secondary metabolites of fungus Gliocladium sp. that helps accelerate the growth of A. roxburghii. METHOD: Compoud isolation by chromatography and structure elucidation by chemical and spectral analyses. RESULTS: Five compounds were obtained and elucidated as: 8(E)-N-(2'-hydroxypalmityl)-1-O-beta-gly-copyranosyl-3-hydroxyl-9-methyl-2- octodecanine-4, 8-diene (I), N-(2'-hydroxytetracosanoyl)-1,3,4-trihydroxy-2-octodecanine(II), 7, 22-diene-3-hydroxy-6,9-epidioxyergosta(III), ergostol(IV) and alpha-palmitin(V). CONCLUSION: I, II, III were obtained from Gliocladium sp. for the first time.