Date Published: February 15, 2018
Publisher: Public Library of Science
Author(s): Hajeewaka C. Mendis, Varghese P. Thomas, Patrick Schwientek, Rauf Salamzade, Jung-Ting Chien, Pramuditha Waidyarathne, Joseph Kloepper, Leonardo De La Fuente, Sudisha Jogaiah.
Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan® probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104–105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.
Plant growth-promoting rhizobacteria (PGPR) are a group of bacteria that colonize plant roots and provide beneficial effects on plant growth and development . Many diverse bacterial genera such as Alcaligenes, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, Bacillus, Burkholderia, Clostridium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, and Serratia include specific strains which are reported as PGPR [2–5]. PGPR have direct or indirect effects on plant growth promotion and improved crop yield. Direct effects of PGPR include providing plants with fixed nitrogen and phytohormones, increasing the availability of nitrogen, soluble phosphate and minerals in the soil and control or inhibition of the activity of plant pathogens [6–9]. Some PGPR are also responsible for promoting plant growth indirectly by eliciting Induced Systemic Resistance (ISR) [10–13].
The aim of this study was to develop a qPCR-based protocol to quantitate total root colonization by two PGPR strains, B. firmus I-1582 and B. amyloliquefaciens QST713 in corn seedlings grown in natural soil. Quantification of root colonization by PGPR is an essential part of tracking inoculants in the field, allowing studies of persistence and understanding of environmental fate of introduced microbes applied in the soil, as well as discovering and evaluating potential PGPR strains. With more interest in preserving soil health and exploiting soil microbiome, there are more microbe-based seed and soil products being made available to the growers, therefore differentiating strains and products is fundamental for the evaluation of the environmental impact of these inoculants. Despite several microscopy and PCR based quantification methods described recently and in the past, dilution-plate counting is still the most common method used in quantification of root colonization by PGPR bacteria in greenhouse and in field under non-sterile soil. One of the common drawbacks of PCR based methods is that these methods are not applicable to non-sterile conditions in greenhouse or in field . However, dilution-plate counting is laborious, and sample processing and plating has to be carried out immediately after sampling. Therefore, a faster and more convenient quantification method to quantitate root and rhizosphere colonization of PGPR is required. The method we describe here to quantitate root colonization of PGPR can successfully replace dilution-plate counting method and has the advantage of being strain-specific.