Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

Abstract

bAzospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N 2 fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (ϳ10 7 CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available.A zospirillum is one of the most important genera of plant growth-promoting rhizobacteria found worldwide under a variety of environmental and soil conditions (1). The diazotroph Azospirillum brasilense is the best-studied species of the genus, is found in close association with many agriculturally important crops, and exerts beneficial effects on plant growth and productivity (2-4). Nitrogen fixation (5, 6) and the production of the auxin 3-indoleacetic acid (IAA) by many representatives of the genus Azospirillum are related to the growth promotion effects observed in inoculated plants, such as increases in root length and the numbers of root hairs and lateral roots (3).The biotechnological use of A. brasilense inoculants in Latin American and in Brazil, in particular, has increased in recent years (7). Strain FP2 is a spontaneous mutant of A. brasilense Sp7 (8). Strain Sp7 has been shown to be capable of stimulating the growth of several members of the family Poaceae and increasing the productivities of wheat and maize crops (2). Strain FP2 can also promote the growth of wheat (9) and enhance maize and wheat productivity under field conditions (unpublished data). Most of the A. brasilense inoculants in B...

Application of Bacillus velezensis NJAU-Z9 Enhanced Plant Growth Associated with Efficient Rhizospheric Colonization Monitored by qPCR with Primers Designed from the Whole Genome Sequence

“…Results from natural soil using qPCR based on the strain primers showed that P3 and P4 primers were identified as NJAU-Z9-specific quantitative PCR primers in combination with the plate counts. Similar to our results, Stets et al [ 41 ] also used the plate count data as a reference for determining primer pairs when monitoring Azospirillum brasilense FP2 on wheat roots in sterile and non-sterile conditions. For bacteria, there is a similar relationship between the number of single-copy genes and the number of colonies (CFU), and in the short-term inoculation experiment, the use of plate count data is a powerful reference for the effectiveness of specific primers [ 42 ].…”

“…Primer verification assays have been performed using genomic DNA sequences from six other Bacillus species and our target strain as templates, and except primers P5, the others are strain specific which cannot amply any gene segment. In accordance with our results, four primer pairs were designed from Azospirillum brasilense FP2 whole genome, and after verification assays, three were left as strain primer pairs [ 41 ]. Thus, WGS offers genetic information with the highest resolution to explore novel strategies for monitoring the target microbes.…”

Response of Soil Bacterial Community and Pepper Plant Growth to Application of Bacillus thuringiensis KNU-07

“…On the other hand, culture-independent methods, such as reporter nucleic acid-based, gene-based, and immunological methods, are capable of detecting less abundant, slow-growing, and unculturable bacteria [8][9][10]. However, most culture-independent methods are incapable of monitoring population dynamics at a species level, making it difficult to determine the fate of some strains [23,24].…”

Effectiveness of Plant Beneficial Microbes: Overview of the Methodological Approaches for the Assessment of Root Colonization and Persistence

“…In fact, strain-specific primers recovered from draft genome sequence analysis were employed for qPCR to quantify Az. brasilense FP2 in wheat roots as well as to assess its competitiveness following coinoculation with other PGPR (Stets et al, 2015; Table 3). All of these works demonstrate the high effectiveness and specificity of this culture-independent approach based on the use of strain-specific primers, allowing rapid and inexpensive detection of bioinoculants in the plant rhizosphere for monitoring and quantification purposes, which is also useful in nonsterile and uncontrolled conditions.…”