A Drought Resistance-Promoting Microbiome Is Selected by Root System under Desert Farming

<div><h3>Background</h3><p>Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of “reverse desertification”. Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress.</p> <h3>Methodology/Principal Findings</h3><p>We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (<em>Capsicum annuum</em> L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. <em>Bacillus</em> spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by <em>Klebsiella</em> spp. (61% and 44% respectively). Most of the isolates (95%) presented <em>in vitro</em> multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for <em>Bacillus</em> and the rhizobacteria strains. We show that the <em>C. annuum</em> rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress.</p> <h3>Conclusions/Significance</h3><p>Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a “resource island” able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.</p> </div

Diversity indexes of the microbial collection.

<p>The indexes were calculated for the sequences of bacterial strains isolated from the different fractions of the pepper root system and the non-cultivated arid soil. Sequences have been grouped in OTUs based on nucleotide similarity at 99%.</p

PGP potential of the microbial collection.

<p>Percentage distribution of plant growth promoting activities and tolerance to abiotic stress among the isolates of the bacterial collection obtained from the different fractions of the pepper root system and the non-cultivated arid soil.</p><p>Auxin  =  auxin production; P Sol.  =  inorganic phosphate solubilization; EPS  =  exopolysaccharide release; Sid. = siderophores production; NH₃  =  ammonia production; Prot.  =  protease activity; PEG = poly-ethylen-glycol.</p

Rhizobacteria increased plant resistance to drought stress.

<p>Abbreviations for the figure: CP, (positive) abiotic control, irrigated at the water holding capacity of the soil along all the experiment; NC, (negative) abiotic control, subjected to drought by interrupting water supply for 12 days. (A) Representative images of plants exposed to rhizobacteria compared to untreated plants eight days after the induction of drought. (B) Leaf physiological parameters in treated and untreated plants eight days after the induction of drought.Abbreviations: Pn, net photosynthesis; E, evapo-transpiration; Gs, stomatal conductance; Ci, internal carbon dioxide (CO₂). Student t-test was adopted to statistically analyse the data. *:p≤0,05; **:p≤0,01; ***:p≤0,001. The data reported in the graphs are representative of one replicate experiment. (C) Percentage increase in root fresh weight (FW) and root length (L) of water stressed plants, compared to the abiotic stressed control, set as 0%.</p

Cluster analysis of total microbial communities according to 16S rRNA DGGE profiles.

<p>The cluster analysis of the plot line was obtained from 16S rRNA PCR-DGGE bacterial community profiles, according to Pearson correlation. The analyzed fractions were root tissues (E), rhizosphere (R), root-surrounding soil (S) and bulk soil (B) of three replicate plants of pepper.</p

Abundance of culturable bacteria associated to the different fractions of the pepper root system.

<p>The isolation was performed on different cultivation media. In the table it is reported the amount of bacterial isolates composing the strain collection associated to pepper endosphere and root-associated soil fractions. E, Endosphere; R, rhizosphere; S, root-surrounding soil; B, non-cultivated arid soil.</p

Rhizocompetence of <i>gfp</i>-labelled bacteria on different plant models.

<p>Plant root colonization experiments performed with a <i>Klebsiella pneumoniae</i> strain isolated from the pepper rhizosphere genetically labeled with a <i>gfp</i>. (A) and (B) colonization of <i>Arabidospis thaliana</i> rhizoplane; (C) and (D) colonization of the pepper rhizoplane. Red spots represent root autofluorescence as acquired through the TRICT filter. The scale bars of the different images in the figure correspond to 100 µm.</p

Bacterial genera distribution of the PGP potential.

<p>Percentage distribution of plant growth promoting activities and tolerance to abiotic stress according to the genera of the bacterial collection obtained from the pepper root system and the non-cultivated arid soil.</p><p>Auxin  =  auxin production; P Sol.  =  inorganic phosphate solubilization; EPS  =  exopolysaccharide release; Sid. = siderophores production; NH₃  =  ammonia production; Prot.  =  protease activity; PEG = poly-ethylen-glycol.</p

Cluster analysis of the cultivable bacteria associated to pepper fractions.

<p>The cultivable fraction of pepper-associated bacteria was compared to uncultivated soil, by performing a cluster analysis according to Pearson correlation.</p

Distribution of microbial taxa in the collection of culturable bacterial isolates associated to pepper plants.

<p>Numbers indicate the number of strains assigned to each genus or species, respectively.</p><p>The numbers in parentheses are the total number of isolates for each fraction.</p