Plant growth-promoting rhizobacteria associated with avocado display antagonistic activity against <i>Phytophthora cinnamomi</i> through volatile emissions

<div><p>Rhizobacteria associated with crops constitute an important source of potentially beneficial microorganisms with plant growth promoting activity or antagonistic effects against phytopathogens. In this study, we evaluated the plant growth promoting activity of 11 bacterial isolates that were obtained from the rhizosphere of healthy avocado trees and from that of avocado trees having survived root rot infestations. Seven bacterial isolates, belonging to the genera <i>Bacillus</i>, <i>Pseudomonas</i> and <i>Arthrobacter</i>, promoted <i>in vitro</i> growth of <i>Arabidopsis thaliana</i>. These isolates were then tested for antagonistic activity against <i>Phytophthora cinnamomi</i>, in direct dual culture assays. Two of those rhizobacterial isolates, obtained from symptomatic-declining trees, displayed antagonistic activity. Isolate A8a, which is closely related to <i>Bacillus acidiceler</i>, was also able to inhibit <i>P</i>. <i>cinnamomi</i> growth <i>in vitro</i> by 76% through the production of volatile compounds. Solid phase microextraction (SPME) and analysis by gas chromatography coupled with mass spectrometry (GC-MS) allowed to tentatively identify the main volatiles emitted by isolate A8a as 2,3,5-trimethylpyrazine, 6,10-dimethyl-5,9-undecadien-2-one and 3-amino-1,3-oxazolidin-2-one. These volatile compounds have been reported to show antifungal activity when produced by other bacterial isolates. These results confirm the significance of rhizobacteria and suggest that these bacteria could be used for biocontrol of soil borne oomycetes through their volatiles emissions.</p></div

Molecular identification of avocado rhizobacteria with plant growth promoting activity by 16S rRNA sequencing.

<p>Taxonomic assignment was determined with the rdp classifier tool. Sequence closest matches were based on the NCBI database “16S ribosomal RNA sequences (Bacteria and Archaea)”.</p

Maximum-Likelihood tree of partially sequenced 16S rRNA genes.

<p>Bold letters indicate rhizobacterial isolates that were obtained in this study. Values above nodes are bootstrap values obtained from 1000 replicates.</p

Direct antagonism assays of avocado rhizobacteria against <i>Phytophthora cinnamomi</i>.

<p>Mycelial growth was measured at fifth day after dual culture.</p

Localization and characteristics of sampling site.

<p>Geographic projection of Veracruz state and the Municipality of Huatusco (a); view of sampling zones (b), the symptomatic zone A is surrounded by yellow ovals; a representative healthy or symptomatic-declining tree is shown (c, d); map (a) was created using ArcMap 10.2.2. The authors generated digital information such as polygons and contours (public domain).</p

Co-cultivation of <i>Arabidopsis thaliana</i> seedlings with rhizobacterial isolates from symptomatic-declining avocado trees.

<p>Representative photographs of Arabidopsis Col-0 seedlings transferred to non-inoculated (Control) fresh media, or inoculated with rhizobacterial isolates at 2.5 cm (long distance) and 1 cm (close distance) from root tip. Primary root length (b), lateral root number (c) and fresh weight accumulation (d) were the developmental parameters analyzed. Data values represent the mean of 30 seedlings ± SE per treatment; different letters in graphs indicate significant differences (<i>P</i> < 0.05).</p

Antagonical activity of isolate A8a, phylogenetically close to <i>Bacillus acidiceler</i>, against <i>Phytophthora cinnamomi</i>.

<p>Disks of <i>P</i>. <i>cinnamomi</i> mycelium were grown on agar PDA-containing plates. Radial growth was monitored for 11 days in non-inoculated conditions (a). Confrontation with <i>B</i>. <i>acidiceler</i> was performed by directly co-cultivating mycelial disks with bacterial inoculum, at a distance of 2 cm (b), or indirectly, growing them on the opposite halves of the same Petri dish (c). Assays were carried out by triplicate. Representative plates were photographed at day 5 and 11 after inoculation (dai). At 7 dai, the inhibition percentage of mycelial radial growth by direct (light gray bars) or indirect (dark gray bars) antagonism was analyzed (d); hyphal deformations induced by indirect inoculation were visually analyzed by stereoscopic observations at 7 dai (e). Values shown in (d) represent the mean of three replicates ± SD; asterisks indicate significant inhibition (<i>P</i> ≤ 0.05).</p

Tentative identification of volatiles emitted by bacterial isolate A8a through SPME and GC-MS^†.

<p>Tentative identification of volatiles emitted by bacterial isolate A8a through SPME and GC-MS<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194665#t003fn001" target="_blank">^†</a>.</p

Scanning electron microscopy of <i>P</i>. <i>cinnamomi</i> mycelium antagonized by volatiles emitted by isolate A8a, phylogenetically close to <i>Bacillus acidiceler</i>.

<p>After 7 d, control mycelium (a) was compared with mycelium indirectly exposed to bacteria-inoculated plates (b).</p