Traceability Assay and Bioinoculant Effects of Two Plant Growth-Promoting Bacillus altitudinis Strains Isolated from Ilex paraguariensis St. Hil

Abstract

Ilex paraguariensis St. Hil. (yerba mate) is a native tree to the Atlantic forest of South America, where Argentina is the world’s largest producer. In a previous study, two strains of Bacillus spp. were isolated from I. paraguariensis St. Hil. seedlings and selected for their ability to promote plant growth. To gain a better understand this, a traceability assay was performed to verify the bioinoculant effects of Bacillus spp. on I. paraguariensis St. Hil. seedlings. A manual search for unique genome sequences of B. altitudinis T5S-T4 and 19RS3 strains was performed to develop strain-specific primers. The traceability assay was performed in a nursery with I. paraguariensis St. Hil. seedlings that were inoculated with B. altitudinis T5S-T4, 19RS3, or a combination of both. Growth parameters, including height, number of leaves, neck diameter, and chlorophyll index, were measured every 15 days in the nursery. Fresh and dry weights of the aerial parts, leaves, roots, and leaf area were measured in the laboratory. The substrate DNA was extracted using a commercial kit. B. altitudinis T5S-T4 and 19RS3 were identified in the substrate 14 and 28 days after the first inoculation, respectively, to day 112 of the assay. The bioinoculation effect of the two-strain combination was confirmed by significant increases in growth parameters, including height, neck diameter, chlorophyll index, and wet weight of aerial parts. The designed and synthesized strain-specific primers allowed the monitoring of the colonization and persistence of B. altitudinis T5S-T4 and B. altitudinis 19RS3 on I. paraguariensis St. Hil seedling substrate inoculated under nursery conditions. The properties of B. altitudinis T5S-T4 and 19RS3 in consortium make them excellent candidates for the formulation of bioinoculants for I. paraguariensis St. Hil.