Phosphate solubilizing Pseudomonas and Bacillus combined with rock phosphates promoting tomato growth and reducing bacterial canker disease

Abstract

Nowadays, sustainable agriculture approaches are based on the use of biofertilizers and biopesticides. Tomato (Solanum lycopersicum L.) rhizosphere could provide rhizobacteria with biofertilizing and biopesticide properties. In this study, bacteria from the rhizosphere of tomato were evaluated in vitro for plant growth promotion (PGP) properties. Five Pseudomonas isolates (PsT-04c, PsT-94s, PsT-116, PsT-124, and PsT-130) and one Bacillus isolate (BaT-68s), with the highest ability to solubilize tricalcium phosphate (TCP) were selected for further molecular identification and characterization. Isolates showed phosphate solubilization up to 195.42 μg mL^-1. All isolates showed phosphate solubilization by organic acid production. The six isolates improved seed germination and showed effective root colonization when tomato seeds were coated with isolates at 10⁶ cfu g^-1 in axenic soil conditions. Furthermore, the selected isolates were tested for beneficial effects on tomato growth and nutrient status in greenhouse experiments with natural rock phosphate (RP). The results showed that inoculated tomato plants in the presence of RP have a higher shoot and root lengths and weights compared with the control. After 60 days, significant increases in plant Ca, Na, P, protein, and sugar contents were also observed in inoculated seedlings. In addition, inoculated tomato seedlings showed an increase in foliar chlorophyll a and b and total chlorophyll, while no significant changes were observed in chlorophyll fluorescence. In greenhouse, two Pseudomonas isolates, PsT-04c and PsT-130, showed ability to trigger induced systemic resistance in inoculated tomato seedlings when subsequently challenged by Clavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker. High protection rate (75%) was concomitant to an increase in the resistance indicators: total soluble phenolic compounds, phenylalanine-ammonia lyase, and H₂O₂. The results strongly demonstrated the effectiveness of phosphate-solubilizing bacteria adapted to rhizosphere as biofertilizers for tomato crops and biopesticides by inducing systemic resistance to the causal agent of tomato bacterial canker disease.

Keywords: Bacillus; Pseudomonas; biofertilization; induced systemic resistance; plant growth promotion; rock phosphate; solubilization.

Figure 1

Solubilization of phosphorus, potassium, and zinc insoluble forms on NBRIP (purple medium), Alexandrov (green medium), and Bunt and Rovira media (translucid medium) respectively by three solubilizing strains PsT-04c (A), PsT-116 (B), and PsT-130 (C) compared with a non- solubilizing strain PsT-e75 (D).

Figure 2

C18-HPLC profiles of the organic acids secreted by the six selected bacterial isolates PsT-04c, BaT-68s, PsT-94s, PsT-116, PsT-124, and PsT-130. GA, gluconic acid; CA, citric acid; MA, maleic acid; ND, not determined.

Figure 3

Effect of treatments by selected bacterial isolates on protein and sugar contents of tomato seedlings after 60 days. Different letters above the bars indicate the differences are significant at P < 0.05.

Figure 4

Principal component analysis (PCA) of tomato plants subjected to phosphate solubilizing bacteria. The variables (agro-morphological, physio-biochemical, and minerals including phosphorus) are presented in blue while bacterial treatments are presented in red. Control: absence of the phosphate solubilizing bacteria, PsT-04c, PsT-94s, PsT-116, PsT-124, and PsT-130: treatments with phosphate solubilizing Pseudomonas sp. isolates. BaT-68s: treatment with phosphate solubilizing Bacillus isolate. SL, shoot Lenght; RL, root length; SFW, shoot fresh weight; RFW, root fresh weight; S-Ca, shoot calcium content; S-Na, shoot sodium content; S-K, shoot potassium content; R-Ca, root calcium content; R-Na, root sodium content; R-K, root potassium content; Phos, plant leaf phosphorus; Sug, total soluble sugar content; Prot, protein content; Chl a, chlorophyll a; Chl b, chlorophyll b; Total Chl, total chlorophyll.

Figure 5

Representative pictures showing biocontrol of tomato bacterial canker after 60 days. (A) Control tomato seedling, (B) Tomato seedlings inoculated with the Pseudomonas isolate PsT-04c and susbesquently infected with Clavibacter michiganensis, (C,D) Decaying tomato seedlings infected with Cmm showing bacterial canker symptoms.

Figure 6

The mortality rate recorded after 120 days in tomato seedling treated with water (negative control), in tomato seedlings artificially infected with Cmm (positive control), and in tomato seedlings inoculated with isolates PsT-04c and PsT-130 before infection by Cmm. Different letters above the bars indicate that the differences are significant at p < 0.05.

Figure 7

Mechanisms of defense induced in bacterized tomato seedlings by the two Pseudomonas isolates PsT-04c and PsT-130 compared with negative control and Cmm-infected seedlings. (A) Phenolic compounds contents, (B) Phenylalanine ammonia-lyase (PAL) activity, and (C) H₂O₂ content. Cmm: Clavibacter michiganensis positive control, Cmm + PsT-04c, and Cmm + PsT-130: seedlings treated, respectively, with PsT-04c and PsT-130 and post challenged with Cmm. Different letters above the bars indicate that the differences are significant at p < 0.05.

Figure 8

Unrooted consensus phylogenetic trees reconstructed by the neighbor-joining method (Saitou and Nei, 1987) based on almost-complete 16S rRNA gene sequences (1,500 bp). (A) phylogenetic relationships between five fluorescent Pseudomonad isolates (PsT-04c, PsT-94s, PsT-116, PsT-124, and PsT-130) and 25 closely related Pseudomonas type species, (B) phylogenetic relationships between BaT-68s and 13 closely related Bacillus species. Closest species were derived using NCBI BLAST search tool (Altschul et al., 1990). The GenBank accession numbers of species are placed in parenthesis. Sequences were aligned using Muscle, and Evolutionary analyses were conducted in MEGA 11 software (Stecher et al., 2020; Tamura et al., 2021). Bootstrap support values: percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown above the branches (Felsenstein, 1985). The evolutionary distances are computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The scale bar represents nucleotide substitutions per site. There were 1,558 positions for Pseudomonas and 1,580 for Bacillus in the final dataset. Isolates PsT-04c, PsT-94s, and PsT-116 shared >99% sequence identity with the type strains of Pseudomonas aeruginosa. Isolate PsT-124 shared >99% sequence identity with P. qingdaonensis. Isolate PsT-130 showed high similarity level to P. azotoformans, isolate BaT-68s shared >99% sequence identity with Bacillus paramycoides.