|Plant growth-promoting rhizobacteria (PGPR) and other beneficial bacteria are increasingly used as biofertilizers for enhancing crop production. After PGPR-treated seeds are planted, root colonization is considered to be an essential first step in plant growth-promotion. Plant roots exude diverse organic compounds, including sugars, and successful bacterial colonization hinges on nutrient uptake from the host plants through extracellular enzymatic activity. Strains of Bacillus amyloliquefaciens subsp. plantarum (Bap) colonize plant roots, and many have been used as PGPR during the past decades.
Pectin is a complex structural polysaccharide found in the middle lamella and primary cell wall of plant cells. Little is known about the possible role of pectin in root colonization and plant growth-promotion. It is possible that Bap strains obtain some carbon via production of extracellular pectate lyase enzymes that degrade pectin in plant root cells. Bacterial genes are involved in degradation and transport of the pectin-associated compounds D-glucuronate and D-galacturonate which can be used as energy and carbon sources. Pectin is broken down by a series of pectinolytic enzymes of soil and rhizosphere bacteria. Some pectinolytic bacteria are plant pathogens which over-produce pectinolytic enzymes, thereby causing plant cell death. Other nonpathogenic soil and rhizosphere bacteria can also degrade pectin in plant rhizospheres without causing damage to plants. Hence, understanding the expression of pectin-associated genes and their involvement in pectin degradation, transport, and utilization by PGPR is critical.
The overall objective of this study was to determine the potential role of pectin degradation and utilization in root colonization and plant growth-promotion of soybean by specific PGPR strains. Experiments were designed to evaluate the potential effects of pectin degradation and utilization by Bap strains on root colonization and plant growth-promotion using pectin amendment to soil. Objective 1 consisted of two parts: a) to identify Bacillus amyloliquefaciens subsp. plantarum strains with gyrB primers (UP-1 & UP-2r), and b) to identify exuT and uxuB genes with exuT and uxuB primers in the Bap strains. The hypotheses for objective 1 were i) gyrB primers (UP-1 & UP-2r) will identify Bap strains, and ii) exuT and uxuB primers will identify exuT and uxuB genes in Bap strains for utilization of sugars derived from pectin. Of 79 Bacillus species strains screened via gyrB universal primers, 59 were confirmed as Bacillus amyloliquefaciens subsp. Plantarum, and the remaining 20 were identified as B. mojavensis, B. sonorensis, B. tequilensis, Bacillus subtilis, and B. pumilis based on the gyrB phylogenetic tree. None of the strains was found to cluster together with Bacillus amyloliquefaciens subsp. amyloliquefaciens. The Bap strains were then screened for the presence of the exuT and uxuB genes. Out of 59 Bap strains, 57 were found to contain exuT and 52 contained uxuB.
Objective 2 had two parts: a) to screen a large collection of Bap strains for pectin degradation and utilization as a sole carbon source, b) to conduct a comparative genomic analysis that includes Bap strains that lack the capacity for pectin utilization. The hypotheses for objective 2 were a) pectin degradation capacity of Bap strains is linked to utilization of D-Glucuronate of D-galacturonate as an energy source, and b) whole genome sequence analysis will reveal all the pectin-associated and defective genes by RAST and CLC genomics software. Pectin degradation and utilization activity of 59 Bap strains were tested in vitro on Pectate Agar (PA) and Tris-Spizizen Salts (TSS) medium. The highest pectate lyase activities were observed in Bap strains AP193, AP203, AP299, AP80, AP102, and AP52, while the lowest activities were with Bap strains AP 194, AP214, AP215, and AP305. Twelve Bap strains (AP67, AP71, AP77, AP78, AP85, AP102, AP108, AP135, AP143, AP189, AP192, and AP193) grew vigorously on TSS medium. Six Bap strains (AP194, AP204, AP214, AP216, AP219, and HD73) had lower growth compared to other Bap strains on TSS medium. The whole genome sequence was determined for Bap strains AP194 and AP214 using an Illumina MiSeq sequencer based on in vitro test results.
Objective 3 was to evaluate the effect of pectin amendments to soil, together with selected pectinolytic Bap strains, for potential enhancement in root colonization, and the magnitude of Bap-induced plant growth of soybean plants. The hypothesis was that bacterial utilization of D-Glucuronate as a carbon source will improve root colonization and increase root and shoot biomass. Rifampicin-resistant mutants of AP143, AP193, and Btk strain HD73 were selected and used to evaluated effects of pectin supplement on root colonization and plant growth-promotion of soybean in greenhouse tests. The combination of Bap rifR strains with pectin amendment resulted in enhanced nodule formation and weights of soybean shoots and roots weights, compared to the same Bap rifR strains without pectin amendment. In contrast, Btk rifR strain did not exhibit plant growth-promotion activity when used with or without pectin amendment. In summary, inoculation of selected pectinolytic Bap rifR strains together with pectin amendments to soil enhanced root colonization and plant-growth promotion of soybean plant. In the greenhouse tests using nonsterilized field soil, pectin amendment with Bap rifR strains also enhanced formation of nodules of indigenous Bradyrhizobium japonicum on soybean roots. In conclusion, the results demonstrate that the combination of selected pectinolytic Bap strains and soil amendment with pectin enhanced plant growth and nodulation by indigenous symbiotic N-fixing bacteria.