Comparative Study of Bacillus-Based Plant Biofertilizers: A Proposed Index

Abstract

The market for bacteria as agricultural biofertilizers is growing rapidly, offering plant-growth stimulants; biofungicides; and, more recently, protectors against extreme environmental factors, such as drought. This abundance makes it challenging for the end user to decide on the product to use. In this work, we describe the isolation of a strain of Bacillus velezensis (belonging to the operational group Bacillus amyloliquefaciens) for use as a plant-growth-promoting rhizobacterium, a biofungicide, and a protector against drought. To compare its effectiveness with other commercial strains of the same operational group, Bacillus amyloliquefaciens, we analyzed its ability to promote the growth of pepper plants and protect them against drought, as well as its fungicidal activity through antibiosis and antagonism tests, its ability to solubilize potassium and phosphates, and its ability to produce siderophores. Finally, we used a probit function, a type of regression analysis used to model the outcomes of analyses, to quantify the biostimulatory effectiveness of the different plant-growth-promoting rhizobacteria, developing what we have called the Agricultural Protection Against Stress Index, which allowed us to numerically compare the four commercial strains of the operational group Bacillus amyloliquefaciens, based on a Delphi method-a type of regression analysis that can be used to model a cumulative normal distribution-and integrate the results from our panel of tests into a single value.

Keywords: agricultural protection against stress index; biofertilizer efficiency; biofungicides; microbial biodiversity.

Figure 1

Relative water content of pepper plants subjected to drought and inoculated with different bacterial isolates. Relative water content of plants subjected to drought and inoculated with different isolates. The name indicated on the axis refers to the strain used. Only plants presenting values greater than 0.8 are displayed. Controls of plants inoculated with the drought-protectant Microbacterium koreensis 3J1 and the non-protective control, Pseudomonas putida KT2440, are also shown.

Figure 2

Optical, transmission, and scanning microscopy of the A6 isolate. Spore, endospore, and vegetative cell forms of isolate A6 cultured on TSB for 24 h. (A) Optical microscopy image, ×1000 magnification, where spores and endospores are stained with malachite green stain and appear light blue/green, while vegetative cells are stained with safranin and appear pink/red. (B) Scanning electron microscopy of vegetative cells of strain A6. (C) Transmission electron microscopy of spores of the A6 strain, showing the ultrastructure’s coat, corte, inner membrane, and core. Magnification is shown at the bottom of the electronic microscopy image.

Figure 2

Optical, transmission, and scanning microscopy of the A6 isolate. Spore, endospore, and vegetative cell forms of isolate A6 cultured on TSB for 24 h. (A) Optical microscopy image, ×1000 magnification, where spores and endospores are stained with malachite green stain and appear light blue/green, while vegetative cells are stained with safranin and appear pink/red. (B) Scanning electron microscopy of vegetative cells of strain A6. (C) Transmission electron microscopy of spores of the A6 strain, showing the ultrastructure’s coat, corte, inner membrane, and core. Magnification is shown at the bottom of the electronic microscopy image.

Figure 3

Taxonomic comparison of the A6 isolate with closely related Bacillus strains. Neighbour-joining phylogenetic tree based on 16S rRNA sequence (GenBank acc. no. SAMN40153660) comparisons of isolate A6 and its 35 closest relatives. Strains DSM7, D747, and FZB42 were used as the outgroups. The numbers at bifurcations indicate how many times each species coincided in this position as a percentage, and only values > 50% are shown. Bar, 0.01 changes per nucleotide position.

Figure 4

Tomato plants treated with different Bacillus strains under biostimulation conditions. Appearance of the plants inoculated with the different Bacillus strains at 31 days after inoculation. (A) Stem length (B), root length (C), and dry weight of the plants (D) are represented for each group of plants at 0 days (represented in light gray) and at 31 days (represented in dark gray) after inoculation. Significant differences are indicated by the presence of letters, with one letter corresponding to a period of 0 days and two letters corresponding to a period of 31 days. aa indicates that not there are differences with non-inoculated plants. bb and cc indicate higher dry weight values with respect to non-inoculated plants. dd indicates lower dry weight values than non-inoculated plants.

Figure 4

Tomato plants treated with different Bacillus strains under biostimulation conditions. Appearance of the plants inoculated with the different Bacillus strains at 31 days after inoculation. (A) Stem length (B), root length (C), and dry weight of the plants (D) are represented for each group of plants at 0 days (represented in light gray) and at 31 days (represented in dark gray) after inoculation. Significant differences are indicated by the presence of letters, with one letter corresponding to a period of 0 days and two letters corresponding to a period of 31 days. aa indicates that not there are differences with non-inoculated plants. bb and cc indicate higher dry weight values with respect to non-inoculated plants. dd indicates lower dry weight values than non-inoculated plants.

Figure 5

Pepper plants treated with different Bacillus strains under xeroprotection conditions. Appearance of the plants inoculated with the different Bacillus 32 days after sowing (A). Root lengths (B), stem lengths (C), fresh weights (D) and total dry weights (E) are represented for each group of plants at 0 days (represented in light gray) and at 31 days (represented in dark gray). Significant differences are indicated by the presence of letters, with one letter corresponding to a period of 0 days and two letters corresponding to a period of 32 days. aa indicates that not there are differences with non-inoculated plants. bb and cc indicate higher parameter values with respect to non-inoculated plants.

Figure 5

Pepper plants treated with different Bacillus strains under xeroprotection conditions. Appearance of the plants inoculated with the different Bacillus 32 days after sowing (A). Root lengths (B), stem lengths (C), fresh weights (D) and total dry weights (E) are represented for each group of plants at 0 days (represented in light gray) and at 31 days (represented in dark gray). Significant differences are indicated by the presence of letters, with one letter corresponding to a period of 0 days and two letters corresponding to a period of 32 days. aa indicates that not there are differences with non-inoculated plants. bb and cc indicate higher parameter values with respect to non-inoculated plants.

Figure 6

(A) Photograph of the antagonism test of Bacillus genus strains on F. oxysporum. The growth of the fungus F. oxysporum in the presence of the different strains after 15 days is shown. (B) Antagonism of Bacillus genus strains on F. oxysporum. The diameter growth of the F. oxisporum fungus is shown in the presence of the different Bacillus strains at day 5 (dark gray), day 10 (gray), and day 15 (light gray). The statistical tests that are represented with one letter correspond to the time of 5 days, those with two letters to the time of 10 days, and those with three letters to the time of 15 days. a, b and c indicate that the diameter of F. oxysporum is significantly smaller than that of the control.

Figure 6

(A) Photograph of the antagonism test of Bacillus genus strains on F. oxysporum. The growth of the fungus F. oxysporum in the presence of the different strains after 15 days is shown. (B) Antagonism of Bacillus genus strains on F. oxysporum. The diameter growth of the F. oxisporum fungus is shown in the presence of the different Bacillus strains at day 5 (dark gray), day 10 (gray), and day 15 (light gray). The statistical tests that are represented with one letter correspond to the time of 5 days, those with two letters to the time of 10 days, and those with three letters to the time of 15 days. a, b and c indicate that the diameter of F. oxysporum is significantly smaller than that of the control.

Figure 7

(A) Photograph of the antagonism test of Bacillus genus strains on B. cinerea. The growth of the fungus B. cinerea in the presence of the different strains after 15 days is shown. (B) Antagonism of Bacillus genus strains on B. cinerea. The diameter growth of the B. cinerea fungus is shown in the presence of the different Bacillus strains at day 5 (dark gray), day 10 (gray), and day 15 (light gray). The statistical tests that are represented with one letter correspond to the time of 5 days, those with two letters to the time of 10 days, and those with three letters to the time of 15 days. b and c indicate size significantly smaller than the control.

Figure 7

(A) Photograph of the antagonism test of Bacillus genus strains on B. cinerea. The growth of the fungus B. cinerea in the presence of the different strains after 15 days is shown. (B) Antagonism of Bacillus genus strains on B. cinerea. The diameter growth of the B. cinerea fungus is shown in the presence of the different Bacillus strains at day 5 (dark gray), day 10 (gray), and day 15 (light gray). The statistical tests that are represented with one letter correspond to the time of 5 days, those with two letters to the time of 10 days, and those with three letters to the time of 15 days. b and c indicate size significantly smaller than the control.

Figure 8

Diameters of halos produced by the solubilization of phosphates. Halo diameter values (cm) produced by the solubilization of phosphates of each strain. The Arthrobacter koreensis 5J12A strain was used as a negative control. a indicates that there are no differences in the halo diameter in the presence of KT2440. b and c indicate diameters significantly smaller than the KT2440 control.

Figure 9

Production of 3-indoleacetic acid, indole butyric acid, and giberellic acid A3. Indole acetic acid production values for each strain are shown at 24 (light gray bars) and 72 h (dark gray bars) of incubation (A). Indole butyric acid production values for each strain at 72 h of incubation are depicted (B). Giberellic acid A3 production values for each strain at 24 h of incubation are also shown (C). a indicates no significant differences with the KT2440 control, while b, c, d, and e indicate significant differences with the KT2440 control.

Figure 9

Production of 3-indoleacetic acid, indole butyric acid, and giberellic acid A3. Indole acetic acid production values for each strain are shown at 24 (light gray bars) and 72 h (dark gray bars) of incubation (A). Indole butyric acid production values for each strain at 72 h of incubation are depicted (B). Giberellic acid A3 production values for each strain at 24 h of incubation are also shown (C). a indicates no significant differences with the KT2440 control, while b, c, d, and e indicate significant differences with the KT2440 control.