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Postdoc, Nanjing Agricultural University

PINBOARD SUMMARY

Biocontrol of phytopathogens through rhizobacterial volatile organic compounds have gained much attention recently. We have been working on finding out potential volatiles against multiple phytopathogens for many years. In this regard, we have evaluated recently a Bacillus atrophaeus strain FA12, originated from the rhizospheric soil of Pakistan, for its in vitro antifungal volatile activity against a soil-borne devastating phytopathogen, Rhizoctonia solani. The results have showed that FA12 has proposed a significant role of its producing 7 out of 15 VOCs against R. solani, identified through GC-MS analysis. Moreover, the antifungal activity of these compounds suggested a potential biocontrol mechanism against R. solani over short and long distances. Our research would reveal new insights among the on-going interactions of microorganisms in the soil.

7 ITEMS PINNED

Isolation, identification and characterization of Pediococcus pentosaceus LB44 and Weissella confusa LM85 for the presence of bacteriocin-like inhibitory substances (BLIS)

Abstract: Lactic acid bacteria (LAB) were isolated from different soil samples such as dairy, garden, rhizospheric soil and sediments of lake. Thirty nine strains of bacteria were isolated using direct spreading method but only one showed the characteristics of LAB. Whereas, using accumulation with incubation method, fifty seven strains were found to be LAB out of sixty eight strains of bacteria isolated. Therefore, accumulation with incubation method was found to be more effective and faster. On the basis of higher growth and antimicrobial activity, two potential isolates LB44 and LM85 were selected for the further characterization. Strain LB44 and LM85 were identified using 16S rDNA amplification and sequencing as Pediococcus pentosaceus LB44 and Weissella confusa LM85, respectively. Cell free supernatant (CFS) of these strains showed antimicrobial activity, stability at higher temperatures, acidic pH and in the presence of organic solvents, detergents and surfactants. Antimicrobial activity was not affected by treatment with catalase and lipase but reduced after treatment with proteinase K reveals the presence of bacteriocin-like inhibitory substance in CFS. CFS inhibited the growth of other Gram-positive and Gram-negative bacteria such as Micrococcus luteus, Lactobacillus delbrueckii, L. curvatus, L. plantarum, L. acidophilus, Enterobacter cloaceae, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Bacillus subtilis, B. licheniformis, Salmonella typhi and Escherichia coli. Lactic acid bacteria (LAB) were isolated from different soil samples such as dairy, garden, rhizospheric soil and sediments of lake. Thirty nine strains of bacteria were isolated using direct spreading method but only one showed the characteristics of LAB. Whereas, using accumulation with incubation method, fifty seven strains were found to be LAB out of sixty eight strains of bacteria isolated. Therefore, accumulation with incubation method was found to be more effective and faster. On the basis of higher growth and antimicrobial activity, two potential isolates LB44 and LM85 were selected for the further characterization. Strain LB44 and LM85 were identified using 16S rDNA amplification and sequencing as Pediococcus pentosaceus LB44 and Weissella confusa LM85, respectively. Cell free supernatant (CFS) of these strains showed antimicrobial activity, stability at higher temperatures, acidic pH and in the presence of organic solvents, detergents and surfactants. Antimicrobial activity was not affected by treatment with catalase and lipase but reduced after treatment with proteinase K reveals the presence of bacteriocin-like inhibitory substance in CFS. CFS inhibited the growth of other Gram-positive and Gram-negative bacteria such as Micrococcus luteus, Lactobacillus delbrueckii, L. curvatus, L. plantarum, L. acidophilus, Enterobacter cloaceae, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Bacillus subtilis, B. licheniformis, Salmonella typhi and Escherichia coli.Pediococcus pentosaceusWeissella confusaMicrococcus luteus, Lactobacillus delbrueckii, L. curvatus, L. plantarum, L. acidophilus, Enterobacter cloaceae, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Bacillus subtilis, B. licheniformis, Salmonella typhiEscherichia coli

Pub.: 01 Sep '16, Pinned: 09 Oct '17

Biofilm of Aureobasidium pullulans var. pullulans on winter wheat kernels and its effect on other microorganisms

Abstract: Winter wheat, grown under greenhouse conditions, was protected four times with a cell suspension of Aureobasidium pullulans var. pullulans during the growing season. After harvest, the distribution and survival rates of the studied biocontrol agent were analyzed under a scanning electron microscope. The abundance of filamentous fungi, yeasts, pseudomonads and Azotobacter bacteria was determined by inoculation onto selective agar media. A. pullulans produced mostly unicellular chlamydospores on the surface and in the brush of kernels. Multicellular blastospore conglomerates secreted extracellular polymeric substances (EPS), and their biofilms were found in the brush and crease of kernels. The application of a cell suspension of A. pullulans with the density of 104 CFU to winter wheat spikes, repeated four times, inhibited the growth of pseudomonads, Azotobacter bacteria and filamentous fungi. Winter wheat, grown under greenhouse conditions, was protected four times with a cell suspension of Aureobasidium pullulans var. pullulans during the growing season. After harvest, the distribution and survival rates of the studied biocontrol agent were analyzed under a scanning electron microscope. The abundance of filamentous fungi, yeasts, pseudomonads and Azotobacter bacteria was determined by inoculation onto selective agar media. A. pullulans produced mostly unicellular chlamydospores on the surface and in the brush of kernels. Multicellular blastospore conglomerates secreted extracellular polymeric substances (EPS), and their biofilms were found in the brush and crease of kernels. The application of a cell suspension of A. pullulans with the density of 104 CFU to winter wheat spikes, repeated four times, inhibited the growth of pseudomonads, Azotobacter bacteria and filamentous fungi.Aureobasidium pullulansAzotobacterA. pullulansA. pullulans4Azotobacter

Pub.: 01 Sep '16, Pinned: 09 Oct '17

Antibiotic activity of bacterial endobionts of basidiomycete fruit bodies

Abstract: Abstract Bacterial strains (93 isolates) capable of growth on full-strength nutrient media were isolated from 86 fungal fruit bodies collected in the Moscow region. Antimicrobial activity of the endobiont isolates against 12 bacterial and fungal test strains (including drug-resistant ones) was studied in submerged cultures. Most of the strains (84.9%) were found to produce antibiotic compounds with different antimicrobial properties, including antifungal activity in 18.3% of the strains. Morphological characteristics and analysis of the 16S rRNA gene sequences were used to determine the taxonomic position of 16 bacterial strains of the following 10 species: Bacillus subtilis, Ewingella americana, Pseudomonas sp., Stenotrophomonas maltophilia, as well as Achromobacter spanius, B. licheniformis, Hafnia paralvei, Micrococcus terreus, Nocardia coeliaca, and St. rhizophila, which have not been previously known to be endobionts of basidiomycete fruit bodies. Antimicrobial activity of A. spanius, E. americana, H. paralvei, M. terreus, N. coeliaca, and St. rhizophila has not been reported previously. Complex mechanisms of symbiotic relations between fungi and bacteria, including those associated with antibiotic formation, probably developed in the course of co-evolution.AbstractBacterial strains (93 isolates) capable of growth on full-strength nutrient media were isolated from 86 fungal fruit bodies collected in the Moscow region. Antimicrobial activity of the endobiont isolates against 12 bacterial and fungal test strains (including drug-resistant ones) was studied in submerged cultures. Most of the strains (84.9%) were found to produce antibiotic compounds with different antimicrobial properties, including antifungal activity in 18.3% of the strains. Morphological characteristics and analysis of the 16S rRNA gene sequences were used to determine the taxonomic position of 16 bacterial strains of the following 10 species: Bacillus subtilis, Ewingella americana, Pseudomonas sp., Stenotrophomonas maltophilia, as well as Achromobacter spanius, B. licheniformis, Hafnia paralvei, Micrococcus terreus, Nocardia coeliaca, and St. rhizophila, which have not been previously known to be endobionts of basidiomycete fruit bodies. Antimicrobial activity of A. spanius, E. americana, H. paralvei, M. terreus, N. coeliaca, and St. rhizophila has not been reported previously. Complex mechanisms of symbiotic relations between fungi and bacteria, including those associated with antibiotic formation, probably developed in the course of co-evolution.Bacillus subtilisEwingella americanaPseudomonasStenotrophomonas maltophiliaAchromobacter spaniusB. licheniformisHafnia paralveiMicrococcus terreusNocardia coeliacaSt. rhizophilaA. spaniusE. americanaH. paralveiM. terreusN. coeliacaSt. rhizophila

Pub.: 01 Nov '16, Pinned: 09 Oct '17

Native isolate of Trichoderma: a biocontrol agent with unique stress tolerance properties.

Abstract: Species of Trichoderma are widely recognized for their biocontrol abilities, but seldom studied collectively, for their plant growth promotion, abiotic stress tolerance and bioremediation properties. Our study is a concentrated effort to establish the potential of native isolate Trichoderma harzianum KSNM (T103) to tolerate biotic (root pathogens) and abiotic stresses [high salt (100-1000 mM); heavy metal (chromium, nickel and zinc: 1-10 mM); pesticides: malathion (100-600 ppm), carbofuran (100-600 ppb)], along with its ability to support plant growth. In vitro growth promotion assays with T103 treated Vigna radiata, Vigna mungo and Hordeum vulgare confirmed 'non-species specific' growth promotion effects of T103. At lower metal concentration, T103 treatment was found to completely negate the impact of metal stress [60 % increase in radicle length (RL) with no significant decrease in %germination (%G)]. Even at 10 mM metal, T103 inoculation gave 80 % increase in %G and >50 % increase in RL. In vitro experiments confirmed high metal reduction capacity (47 %-Cr, 35 %-Ni and 42 %-Zn) of T103 at concentrations as high as 4 mM. At maximum residual concentrations of malathion (440 ppm) and carbofuran (100 ppb) reported in agricultural soils, T103 maintained 80 and 100 % survivability, respectively. T103 treatment has improved %G and RL in all three hosts challenged with pesticide. Isolate T103 was found to effectively suppress growth of three major root pathogens: Macrophomina phaseolina (65.83 %) followed by Sclerotium rolfsii (19.33 %) and Fusarium oxysporum (19.18 %). In the light of these observations, native T. harzianum (T103) seems to be a competent biocontrol agent for tropical agricultural soils contaminated with residual pesticides and heavy metals.

Pub.: 25 Jun '16, Pinned: 09 Oct '17

Understanding Colonization and Proliferation Potential of Endophytes and Pathogen in planta via Plating, Polymerase Chain Reaction and Ergosterol Assay

Abstract: This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb) introduced into oil palm ramets (host model). The endophytes selected were Diaporthe phaseolorum (WAA02), Trichoderma asperellum (T2), and Penicillium citrinum (BTF08). Ramets were first inoculated with 100 mL of fungal cells (106 cfu mL-1) via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR) detected and identified the isolates from the plant tissues. The ergosterol assay (via High Performance Liquid Chromatography, HPLC) confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49 days were however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

Pub.: 03 Nov '16, Pinned: 09 Oct '17

Characterizing antagonistic activities and host compatibility (via simple endophyte-calli test) of endophytes as biocontrol agents of Ganoderma boninense

Abstract: This study characterized the antagonistic activities of five fungal endophytes (Aspergillus calidoustous BTF07, Penicillium citrinum BTF08, Trichoderma asperellum T2, Diaporthe phaseolorum WAA02, Diaporthe phaseolorum MIF01) and evaluated their endophyte-host compatibility with the host plant (oil palm). The antifungal activities of the endophytes towards Ganoderma boninense (GB) were first established using the dual culture test, revealing antagonistic nature of endophytes via production of non-volatiles, volatiles and competitive exclusion. Endophyte-host compatibility was then assessed using a simple but rapid endophyte-calli dual-culture assay, and results validated using endophyte-ramet test. Results revealed that endophytes elicited different responses in oil palm calli. BTF08 had growth promoting effects towards the host tissues with the highest calli weight (1013 mg) obtained, while BTF07 appeared to inhibit calli (1006 mg) leading to browning and necrosis. This endophyte-calli test also revealed the influence of calli on endophyte growth. Isolates BTF08 and GB benefited from host association, with increased radial growth (2.36 cm and 2.31 cm, respectively) compared to growth in the absence of calli (2.10cm and 2.15 cm, respectively). Endophytes and GB were also isolated from host tissues, suggesting compatibility and ability to colonize host tissues (root, stem, leaf). This suggested the reliability of the endophyte-calli test as a rapid assay to provide an insight on the endophyte-host compatibility.

Pub.: 03 Dec '16, Pinned: 09 Oct '17