Isolation and characterization of rhizobacteria and their effects on root extracts of Valeriana officinalis. Australian journal of crop science,

Abstract Plant growth promoting rhizobacteria (PGPR) are a group of microorganisms which can enhance growth parameters of host plants and can be used as biofertilizers. Valerian (Valeriana officinalis) is a perennial herb which is used as a medicinal plant. In the current study 40 colonies of bacteria were isolated from the rhizosphere of valerian by growing in various enriched and selective media including S1 and King B, crystal violet agar and methyl red agar. Furthermore, the ability of bacteria to produce siderophore, indoleacetic acid (IAA), hydrogen cyanide (HCN), lipase and protease were tested in vitro. Identification of isolates was performed by using Bergey's manual of systematic bacteriology. Additionally, the effects of seven isolated bacteria (belong to Pseudomonas genus) that showed a high potential of siderophore, IAA, HCN, lipase and protease production on the quantity of root extracts were investigated under greenhouse condition. Results showed that the population of Pseudomonas was the highest in comparison to other genera in the rhizosphere of plant. Isolated bacteria could mostly produce siderophore, lipase, HCN and protease. Two isolates (belong to Pseudomonas genus) significantly increased the amount of valerenic acid in the root extract. The results revealed that PGPR increased shoot length and could also enhance quantity of root extract

A novel baiting microcosm approach used to identify the bacterial community associated with <i>Penicillium bilaii</i> hyphae in soil

<div><p>It is important to identify and recover bacteria associating with fungi under natural soil conditions to enable eco-physiological studies, and to facilitate the use of bacterial-fungal consortia in environmental biotechnology. We have developed a novel type of baiting microcosm, where fungal hyphae interact with bacteria under close-to-natural soil conditions; an advantage compared to model systems that determine fungal influences on bacterial communities in laboratory media. In the current approach, the hyphae are placed on a solid support, which enables the recovery of hyphae with associated bacteria in contrast to model systems that compare bulk soil and mycosphere soil. We used the baiting microcosm approach to determine, for the first time, the composition of the bacterial community associating in the soil with hyphae of the phosphate-solubilizer, <i>Penicillium bilaii</i>. By applying a cultivation-independent 16S rRNA gene-targeted amplicon sequencing approach, we found a hypha-associated bacterial community with low diversity compared to the bulk soil community and exhibiting massive dominance of <i>Burkholderia</i> OTUs. <i>Burkholderia</i> is known be abundant in soil environments affected by fungi, but the discovery of this massive dominance among bacteria firmly associating with hyphae in soil is novel and made possible by the current bait approach.</p></div

Colonization of <i>Penicillium bilaii</i> hyphae by bacteria after incubation for A: 6 days and B: 8 days in soil.

<p>Hyphae and bacteria were stained by SYBR Green and the microscopy image was obtained at 400 times magnification.</p

Visualization of <i>P</i>. <i>bilaii</i> hyphal growth and hyphal viability.

<p>Glass cover slips were recovered from soil microcosms at day 0, 4, 6 and 8. Column A: Calcofluor White M2R was used to stain the cell walls of <i>P</i>. <i>bilaii</i>. Columns B, C and D: FUN 1 stain was used to determine metabolically active (B) or inactive (D) hyphae, as well as active and inactive spores (C). Killed hyphae were set as a negative control (Column D, Day 0). All images were obtained at 630 times magnification.</p

Schematic diagram showing the microcosm setup for isolation of hypha-associated bacteria from soil.

<p>Schematic diagram showing the microcosm setup for isolation of hypha-associated bacteria from soil.</p

Neighbor-joining tree of bacteria associated with <i>P</i>. <i>bilaii</i> hyphae belonging to the genus <i>Burkholderia</i>.

<p>The tree was constructed from 375 unambiguously aligned nucleotides from the V3-V4 regions of the 16S rRNA gene. Sequences derived from hyphae-associated bacteria from the microcosms are designated OTU HB. Bootstrap values are based on 1000 pseudo-replications, and only values > 50% are shown.</p

Composition of microbial communities in A: Bulk soil or B and C: Attached to the hyphae of <i>P</i>. <i>bilaii</i>.

<p>Relative abundances of taxa are shown on phylum level in panel A and B (<i>Proteobacteria</i> are displayed at the class level), while panel C depicts the distribution of proteobacterial genera from the hyphae-associated community. “Others” represent taxa with < 1% relative abundance. Data for either microbial community is obtained from two replicates.</p