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Highlights:
The Bacillus genus was the most representative isolated in the rhizosphere of Jatropha curcas.
Eight rhizobacteria showed antagonistic activity against C. lunata and Fusarium equiseti.
The antifungal effect (30 to 79 % inhibition) was due to volatile and diffusible compounds.
First report of bacteria isolated from J. curcas to improve the germination and growth in tomato plants.
Introduction
Jatropha curcas L. is a perennial tropical species native to Mexico that has recently received much attention because of its role in the production of bio-diesel, an eco-friendly, biodegradable, and non-toxic fuel (Mazumdar, Singh, Babu, Siva, & Harikrishna, 2018). The oil extracted from J. curcas seeds is an important source of the fatty acids that are used to produce biofuel, and successful biofuel production depends heavily on the physicochemical properties and moisture content of J. curcas seeds (Keneni, Hvoslef-Eide, & Marchetti, 2019).
Previous studies have demonstrated that the quality of J. curcas seeds is affected by water activity, storage time and phytopathogenic fungi (Dharmaputra, Worang, Syarief, & Miftahudin, 2009). It has been shown that phytopathogenic fungi caused changes in the fatty acid content, which could affect the production of biofuels (Oluwatoyin & Anthony, 2019). In addition, it has been demonstrated that diverse fungal species, including Aspergillus flavus, Rhizopus nigricans, Fusarium equiseti, and Curvularia lunata, affect the germination of J. curcas seeds (Anjorin, Omolewa, & Salako, 2011; Pabón-Baquero, Velázquez-del Valle, Evangelista-Lozano, León-Rodriguez, & Hernández-Lauzardo, 2015). Traditionally, phytopathogenic fungi have been controlled with chemical fungicides. Nonetheless, it has been suggested that chemical fungicides represent a potential risk to the environment and human health, with the additional problem of generating fungal resistance (Yang et al., 2019). An alternative strategy is the use of antagonistic rhizobacteria as biological control agents for phytopathogenic fungi.
There are few studies of the microbial diversity of the rhizosphere of J. curcas. However, the isolation of diverse plant growth-promoting rhizobacteria (PGPR) capable of solubilizing phosphates and producing hydrogen cyanide and siderophores has been reported previously (Jha & Saraf, 2012). In recent studies, these metabolites have been associated with protection against fungal diseases in many crops (Abo-Elyousr, Khalil Bagy, Hashem, Alamri, & Mostafa, 2019). Currently, to our knowledge, there is only one report on the use of antagonistic rhizobacteria isolated from J. curcas to control phytopathogenic fungi of this crop in India (Latha et al., 2011).
In addition to potential antifungal effects, a study has shown that bacteria isolated from the rhizosphere of J. curcas promote plant growth (Jha, Patel, & Saraf, 2012). This is an important possible application, since improper or excessive use of chemical fertilizers to promote growth causes environmental and health damage, so a development formulated based on bacteria is needed to offer better alternatives for reducing the consumption of chemical products. This effect could occur in plants other than J. curcas itself, such as the tomato, of which Mexico is the largest exporter worldwide (Saavedra, Figueroa, & Cauih, 2017). Thus, the aim of this study was to isolate and identify bacteria from rhizospheric soil of J. curcas with antagonistic ability against two phytopathogenic fungi species (C. lunata and F. equiseti) of this crop and to evaluate its capacity to promote the growth of tomato plants. Bacterial strains with potential use as fertilizers may be offered as an alternative more environmentally friendly and useful to farmers.
Materials and methods
Isolation of rhizobacteria from Jatropha curcas and source of phytopathogenic fungi
Samples were collected in Yautepec, Morelos, Mexico (18° 49’ N, 99° 05’ W) from rhizospheric soil of J. curcas from five sampling sites (one point in the centre and four on the edges of one field of 4 000 m2). Using sterile equipment, 100 g of rhizosphere soil from up to 15 cm depth at each point were collected. From homogenized samples, 10 g of rhizospheric soil was taken, deposited in 90 mL of sterile distilled water and stirred for one minute. Serial dilutions were carried out and 0.1 mL was spread on nutrient agar (NA). After 24 h of incubation at 28 ± 2 °C, single bacterial colonies with different morphological appearances were counted, purified and stored at 4 °C in NA. The phytopathogenic fungi C. lunata and F. equiseti were isolated from J. curcas seeds in previous work by Pabón-Baquero et al. (2015) and belong to the fungal collection of the Laboratory of Phytopathology, CEPROBI-IPN. The fungal strains were maintained on potato dextrose agar (PDA) plates at 28 ± 2 °C.
Evaluation of antagonistic activity of bacterial isolates
The antagonistic activity of 70 bacterial isolates was assessed against C. lunata or F. equiseti by the dual culture technique on PDA. A 5-mm mycelial disc of fungal was placed at the centre of the Petri dishes. Five microliters (3 x 108 CFU∙mL-1) of four bacterial isolates were placed equidistant from the centre at four points of the Petri dishes. PDA plates inoculated with the pathogen alone were used as a control. The plates were incubated at 28 ± 2 °C until the mycelium reached the edges of the control plates. Bacterial isolates that showed inhibition of mycelial growth in the area of the agar were selected as potential antagonists (Karimi, Amini, Harighi, & Bahramnejad, 2012).
In vitro antagonistic activity by volatile compounds
The methodology described by Karimi et al. (2012) was carried out with some modifications. The bacterial isolated were streaked on NA, and at the same time, a 5 mm disc of a 7-d-old culture) of each fungal species was placed at the centre of another Petri dishes with PDA medium. Then both half-plates were placed face to face preventing physical contact between the fungi and the bacterium and were sealed with Parafilm to prevent loss of volatiles compounds. The plates were incubated at 28 ± 2 °C and the inhibition zone was measured with a digital calliper. The mean radius (mm) of fungal growth in the presence of each strain was compared to that of the control cultures (containing only a disc of each fungal species) to determine the percent inhibition of the mycelial growth using the formula (C‒T)/C * 100 %, where C = control and T = treatment.
In vitro antagonistic activity by diffusible compounds
The antagonistic activity of the bacterial isolates was assessed against C. lunata and F. equiseti by the dual culture technique on PDA. A 5-mm mycelial disc of each fungus was placed at the centre of the Petri dishes. Five microliters (3 x108 CFU∙mL-1) of each bacterial isolate was placed at four points equidistant from the centre of the Petri dishes. PDA plates inoculated with the pathogen alone were used as controls. The plates were incubated at 28 ± 2 °C until the control plates showed full growth. The percent inhibition of the mycelial growth was determined as described previously (Karimi et al., 2012).
Morphological and molecular identification of antagonistic bacteria
Bacterial isolates that showed antagonistic activity towards the phytopathogenic fungi tested were selected for identification by morphological and molecular procedures. Genomic DNA was extracted and purified using the ZR Fungal/Bacterial DNA MiniPrepTM kit. Amplification of the 16S rRNA gene sequences of antagonistic isolates was carried out as described by Weisburg, Barns, Pelletier, and Lane (1991). Additionally, the PCR amplification of the internal portion of the sodA gene sequences in some isolates was achieved according to Gatson et al. (2006). To identify the Bacillus thuringiensis isolate, which harbours any of the cry1, cry2 and/or cry7 genes, PCR analysis was carried out with specific primers (Ben-Dov et al., 1997; Ceron, Ortíz, Quintero, Güereca, & Bravo, 1995; Sauka, Cozzi, & Benintende, 2005). The nucleotide sequences of the 16S rRNA and sodA were compared with sequences from the NCBI database (National Center for Biotechnology Information, 2020) using the BLAST tool. The alignment of multiple sequences and maximum likelihood phylogenetic analysis were performed with the program MEGA 5 (Tamura et al., 2011). The reliability of the phylogenetic tree was evaluated using a bootstrap test with 1 000 replicates.
Production of antifungal compounds by antagonistic bacteria
Each antagonistic bacterial isolate was tested directly to produce hydrogen cyanide (HCN), siderophores, proteases, and chitinases as follows. Production of HCN was assessed on nutrient agar medium and bacterial culture were streaked on the surface, then sterilized filter papers were soaked in 2.0 % Na2CO3 in 5.0 % (w/v) picric acid and placed in the lid of the Petri dish. The Petri dishes were sealed with parafilm and incubated at 30 °C for 4 days. A change in the color of the filter paper from yellow to reddish-brown was positive for cyanogenic activity (Karimi et al., 2012). Siderophore production was determined with a chrome azurol sulfonate (CAS) agar plate assay using Grimm Allen medium as a base (Baakza, Vala, Dave, & Dube, 2004; Schwyn & Neilands, 1987). Protease activity was determined from clearing zones in skimmed milk agar (SMA), according to Chantawannakul, Oncharoen, Klanbut, Chukeatirote, and Lumyong (2002). Chitinase activity was determined by streaking each strain on a culture medium containing 6.7 g∙L-1 yeast nitrogen base, 5 g∙L-1 colloidal chitin, and 15 g∙L-1 agar. Chitinase production was assessed by visual examination of cleared zones developed around colonies incubated at 28 ± 2 °C for 72 h.
Screening for plant growth-promoting activities by rhizobacteria
Phosphate solubilization and quantification of indole-3-acetic acid (IAA)
Phosphate solubilization was determined from clearing zones in the culture medium. Bacterial culture was streaked onto the surface of inorganic phosphate agar (10 g∙L-1 glucose, 0.5 g∙L-1 (NH4)2SO4, 0.3 g∙L-1 NaCl, 0.3 g∙L-1 KCl, 0.03 g∙L-1 FeSO4, 0.03 g∙L-1 MnSO4, 0.3 g∙L-1 MgSO4, and pH 7.2) with calcium phosphate (1 g∙L-1 Ca3(PO4)2) as the sole P source (Qian et al., 2010). The Petri dishes were incubated for 3-7 days (28 ± 2 °C).
The production of IAA was determined as described by Karimi et al. (2012). Briefly, bacterial strains were inoculated into nutrient broth (5 g∙L-1 casein peptone, 1.5 g∙L-1 yeast extract, 1.5 g∙L-1 beef extract, and 5 g∙L-1 NaCl) with or without tryptophan (500 µg∙mL-1) and incubated at 30 °C for 3 days. A 5-mL culture was removed from each tube and centrifuged at 12 000 x g for 15 min. A 2 mL aliquot of supernatant was transferred to a fresh tube to which 100 µL of 10 mM orthophosphoric acid and 4 mL of reagent (1 mL of 0.5 M FeCl3 in 50 mL of 35 % HClO4) were added. The mixture was incubated at room temperature for 30 min, and the absorbance was read at 530 nm.
Germination and growth promotion
Tomato seeds (Solanum lycopersicum L. “Bola”) were disinfected with ethanol and sodium hypochlorite. Briefly, seeds were selected by the buoyancy method and then disinfected for 1 min in 5 % commercial sodium hypochlorite, then immersed in 70 % ethanol for 1 min, and finally, washed three times with sterile distilled water.
The bacterial strains A1, A2, A3, A4, A5, A6 and A8 were inoculated on Luria-Bertani agar (LB agar: 10 g∙L-1 casein peptone, 5 g∙L-1 yeast extract, 10 g∙L-1 NaCl, 15 g∙L-1 agar and 7.2 pH adjusted and sterilized) and incubated for 24 h at 30 °C. According to the 0.5 Mcfarland scale (1.5 x 108 UFC∙mL-1), bacterial suspension was prepared in 0.9 % saline solution as inoculum for the germination and growth promotion tests. The bacterium P. aeruginosa was not tested in in vivo bioassays to avoid contaminating the environment with this opportunist pathogen to humans.
Twenty seeds per strain were used for the germination bioassay. Seeds were immersed in the bacterial suspension with agitation for 2 h at 180 rpm. The negative control seeds were immersed in 0.9 % saline solution with the same conditions described above. Seeds were sown in a seedbed with sterilized soil substrate, and germination percentage was recorded for the next 15 days.
To evaluate growth promotion, the germinated seeds were placed in a cup with sterilized soil substrate in a greenhouse at 30 ± 2 °C and 70 ± 5 % relative humidity with a 10 h light/14 h dark photoperiod. Fertilization and bacterial suspension inoculation were performed as follows: after seedlings were transplanted, the positive controls were fertilized with 5 mL of Nitrofoska Blaukorn® classic (3 g∙L-1; 18 % N, 8 % P, 16 % K, and other nutrients); the treatments consisted of 5 mL of 0.5 Mcfarland scale of bacterial suspension of each strain, and the negative control was performed with sterilized distilled water. After six weeks of the bioassay, the stem diameter, plant length, number of leaves, plant fresh weight, and root fresh weight were recorded.
Experiments were conducted in a completely randomized design; three replicates with four repetitions each were performed. The data were analysed with ANOVA and means were compared with the Tukey test (P < 0.05) using SigmaPlot version 10.0 (Systat Software, Inc., 2007).
Results
Isolation and antagonistic activity of rhizobacteria from Jatropha curcas
A total of 124 isolates were obtained in nutrient agar medium from the rhizospheric soil of J. curcas. Only 70 isolates grew in the PDA medium. Colony appearance varied, coinciding with common bacterial morphotypes. In the antagonism tests, of the 70 bacteria isolates tested, eight isolates inhibited mycelial growth of C. lunata and F. equiseti, which were selected for their antagonistic activity (Table 1) and were designated as A1 to A8.
Effect of volatile and diffusible bacterial compounds on fungal mycelial growth
The results of the antagonistic effect of volatile or diffusible compounds on the mycelial growth of F. equiseti and C. lunata are shown in Table 1. In general, all isolates significantly inhibited the growth of fungal mycelia. However, the inhibition percentage varied with the fungal species and type of compounds evaluated. The A7 isolate showed the strongest inhibitory effect (48.7 to 79.8 %) on both fungal species and by both types of compounds. It is followed by the strains A5, A4, and A3 which showed inhibition of mycelial growth between 30 % and 57 % (Table 1).
Table 1 Inhibition of mycelial growth of Curvularia lunata and Fusarium equiseti by volatile and diffusible compounds (experiments were incubated at 28 °C) of antagonistic bacterial isolates obtained from rhizospheric soil of Jatropha curcas.
| Bacterial isolates | Inhibition of mycelial growth (%) | |||
|---|---|---|---|---|
| Volatile compounds | Diffusible compounds | |||
| C. lunata | F. equiseti | C. lunata | F. equiseti | |
| Control | 0 a | 0 a | 0 a | 0 a |
| A1 | 32.8 b | 31.3 b | 51.9 d | 40.9 c |
| A2 | 29.1 b | 32.1 b | 51.6 d | 41.4 c |
| A3 | 27.0 b | 21.5 b | 57.0 e | 48.4 c |
| A4 | 43.0 bc | 25.6 b | 54.7 e | 49.5 c |
| A5 | 35.0 b | 31.2 b | 55.3 e | 45.8 c |
| A6 | 31.3 b | 23.1 b | 33.7 c | 22.8 b |
| A7 | 66.6 c | 48.7 c | 76.3 f | 79.8 d |
| A8 | 31.0 b | 24.8 b | 20.9 b | 27.2 b |
Different letters within columns indicate significant differences at P < 0.05, according to the Tukey test.
Morphological characteristics and molecular identification of antagonistic rhizobacteria
Morphological characteristics and molecular identification of antagonistic rhizobacteria are shown in Table 2. The eight antagonistic bacteria isolated consisted of seven Gram-positive rods (A1-A6 and A8) and one Gram-negative rod (A7). Molecular identification was consistent with morphological characteristics for all isolates. The 16S rRNA gene sequences were deposited in GenBank and compared with those already found in the database. The results showed four different antagonistic rhizobacteria species (Bacillus subtilis, Bacillus mojavensis, Bacillus thuringiensis, and Pseudomonas aeruginosa).
The BLAST search revealed that the 16S rRNA gene sequence of A1 and A2 isolates were 100 % identical to B. subtilis JX123316. The 16S rRNA sequences of A3, A4, and A5 isolates showed 100 % matches with B. mojavensis KC519442 (A3 and A4) and JX126863 (A5). The A6 and A8 isolates were identified as two different strains of B. thuringiensis (100 % matching with B. thuringiensis KC683724 and KC692184, respectively). In addition, PCR analysis demonstrated that both isolates of B. thuringiensis contained cry genes. The taxonomic identification of the A7 isolate was confirmed with a 100 % identity with the 16S rRNA sequence of P. aeruginosa KC787580. Moreover, the sodA nucleotide sequences of A1 and A2 were found to be 99 % identical to sodA of B. subtilis CP002468 and CP003329, respectively. A3, A4, and A5 isolates were 99 % similar to sodA of B. mojavensis AY197618. The accession numbers obtained in this study from 16S rRNA and sodA nucleotide sequences are shown in Table 2.
Table 2 Morphological and molecular identification of antagonistic bacterial isolates obtained from rhizospheric soil of Jatropha curcas.
| Isolates | Morphological characteristics | Molecular identification | ||||||
|---|---|---|---|---|---|---|---|---|
| 16S rRNA | sodA | |||||||
| Gram reaction | Cell shape | GenBank (accession number) | Identity (%) | Identification (accession number to GenBank) | GenBank (accession number) | Identity (%) | Identification (accession number to GenBank) | |
| A1 | + | rod | JX123316 | 100 | Bacillus subtilis | CP002468 | 99 | Bacillus subtilis |
| KF255983 | KF313554 | |||||||
| A2 | + | rod | JX123316 | 100 | Bacillus subtilis | CP003329 | 99 | Bacillus subtilis |
| KF255984 | KF313555 | |||||||
| A3 | + | rod | KC519442 | 100 | Bacillus mojavensis | AY197618 | 99 | Bacillus mojavensis |
| KF255985 | KF313556 | |||||||
| A4 | + | rod | KC519442 | 100 | Bacillus mojavensis | AY197618 | 99 | Bacillus mojavensis |
| KF255986 | KF313557 | |||||||
| A5 | + | rod | JX126863 | 100 | Bacillus mojavensis | AY197618 | 99 | Bacillus mojavensis |
| KF255987 | KF313558 | |||||||
| A6 | + | rod | KC683724 | 100 | Bacillus thuringiensis | - - | - - | - - |
| KF255988 | ||||||||
| A7 | - | rod | KC787580 | 100 | Pseudomonas aeruginosa | - - | - - | - - |
| KF255989 | ||||||||
| A8 | + | rod | KC692184 | 100 | Bacillus thuringiensis | - - | - - | - - |
| KF255990 | ||||||||
Positive reaction (+) and negative reaction (-). Samples not analyzed (- -)
Phylogenetic analysis
The phylogenetic trees constructed from the 16S rRNA and sodA gene sequences are shown in Figures 1 and 2, respectively. The 16S rRNA tree showed three distinct clusters: cluster 1 contained B. mojavensis and B. subtilis strains, cluster 2 contained B. thuringiensis, and cluster 3 contained P. aeruginosa (Figure 1). To separate and corroborate the identification of the two Bacillus species in cluster 1, sodA sequence analyses were carried out on the A1-A5 isolates. The sodA tree clearly defined two different clusters: cluster 1 containing A1 and A2 (B. subtilis) and cluster 2 containing A3, A4 and A5 (B. mojavensis) (Figure 2).
Figure 1 Phylogenetic tree using the 16S rRNA gene nucleotide sequences of eight bacterial isolates (letters bold: Bacillus mojavensis, B. subtilis, B. thuringiensis, and Pseudomonas aeruginosa) from rhizospheric soil of Jatropha curcas. *Borrelia burgdorferi (L40596) was included as an outgroup. The tree was constructed using the Neighbor-Joining method with 1 000 bootstrap replications. The scale bar indicates the number of substitutions per nucleotide position.
Figure 2 Phylogenetic tree based on sodA gene sequences of five bacterial isolates (bold type: Bacillus mojavensis and B. subtilis) from rhizospheric soil of Jatropha curcas. *Lactobacillus casei (HE970764) was included as an outgroup. The tree was constructed using the neighbor-joining method with 1 000 bootstrap replications. The scale bar indicates the number of substitutions per nucleotide position.
Antifungal compounds produced by bacterial strains
The eight strains were tested to determine their biocontrol activity (Table 3). The results demonstrated different behaviour of each bacterial species, with all strains having some degree of antagonistic effect in the mycelial growth of C. lunata and F. equiseti. All Bacillus spp. produced hydrolytic enzymes. Protease activity was detected in B. subtilis, B. mojavensis and B. thuringiensis, while chitinase activity was only observed in B. thuringensis strains (A6 and A8). All strains except B. thuringiensis produced siderophores. Only P. aeruginosa produced HCN (Table 3).
Table 3 Bacterial isolates from rhizospheric soil of Jatropha curcas: characterization of compounds with biological control and plant growth promotion potential.
| Strains | Biological control | Plant growth promotion | ||||
|---|---|---|---|---|---|---|
| Proteases | Chitinases | Siderophores | HCN | Phosphate solubilization | IAA (µg∙mL-1) | |
| Bacillus subtilis (A1) | + | - | + | - | - | 4.85 ± 0.004 c |
| Bacillus subtilis (A2) | + | - | + | - | - | 5.06 ± 0.006 c |
| Bacillus mojavensis (A3) | + | - | + | - | - | 4.30 ± 0.003 c |
| Bacillus mojavensis (A4) | + | - | + | - | - | 4.35 ± 0.010 c |
| Bacillus mojavensis (A5) | + | - | + | - | - | 4.55 ± 0.006 c |
| Bacillus thuringiensis (A6) | + | + | - | - | - | 15.31 ± 0.054 b |
| Pseudomonas aeruginosa (A7) | - | - | + | + | + | 18.73 ± 0.001 a |
| Bacillus thuringiensis (A8) | + | + | - | - | - | 10.56 ± 0.068 c |
Presence (+) or absence (-) of the compound is indicated for all substances except indole-3-acetic acid (IAA), where mean ± standard deviation of concentration is shown. HCN: hydrogen cyanide. Different letters within columns indicate significant differences at P < 0.05, according to the Tukey test.
Phosphate solubilization and quantification of indole-3-acetic acid in bacterial strains
The phosphate solubilization and IAA production abilities of different bacterial strains are shown in Table 3. Among the eight strains evaluated, only P. aeruginosa showed phosphate solubilization activity, but all strains tested produced IAA. While most of the isolates produced similar levels of IAA, P. aeruginosa (A8, 18.73 µg∙mL-1) and B. thuringiensis (A6, 15.31 µg∙mL-1) had significantly higher IAA production than the remaining isolates.
Germination evaluation and plant growth-promoting activity
Germination percentage was recorded 15 days after planting. The negative control showed the lowest percentage germination, followed by the strains of B. mojavensis (A5, A3) and B. subtilis (A2) with around 50 to 60 percent germination. The strains B. subtilis (A1), B. mojavensis (A4) and B. thuringiensis (A6) showed the highest germination with between 80 and 88 % (Table 4).
Growth promotion by the isolated strains was evaluated using size variables, which differed slightly in their results. In the plant length variable, the strains B. mojavensis (A4), B. subtilis (A1), and B. thuringiensis (A6) showed statistically significant differences (P < 0.05) with between 15.4 and 19.8 cm, followed by B. mojavensis (A3, A5), B. subtilis (A2) and B. thuringiensis (A8) with 14.0 to 15.3 cm. These latter strains did not differ significantly (P > 0.05) from the positive control according to the Tukey test (Table 4). In the stem diameter, the strains B. subtilis (A1) and B. thuringiensis (A6) had the largest stems, with 5 and 4.98 mm, respectively. Although the other strains also had larger stem diameters than the positive control, the Tukey test did not show significant differences (P > 0.05) (Table 4). For the number of leaves, treatments with the bacteria B. subtilis (A1), B. mojavensis (A3, A4, A5) and B. thuringiensis (A8) had up to two leaves more than the positive control (Table 4). In terms of the plant’s fresh weight, B. mojavensis (A4), B. thuringiensis (A6) and B. subtilis (A1) had the best results. Finally, according to the Tukey test fresh root weight did not differ significantly (P > 0.05) between treatments and positive control. For all tests, the smallest sizes and weights occurred in plants from the negative control group (without treatment) (Table 4).
Table 4 Bacillus strains treatments inoculated on tomato seeds to evaluated germination and plant growth promotion.
| Strain | Germination (%) | Plant length (cm) | Stem diameter (mm) | Leaf number | Plant fresh weight (g) | Root fresh weight (g) |
|---|---|---|---|---|---|---|
| A1 | 86.67 ± 2.89 a | 16.94 ± 0.71 ab | 5.00 ± 0.51 a | 7.00 ± 0.57 a | 6.29 ± 1.41 ab | 1.05 ± 0.46 a |
| A2 | 61.67 ± 2.89 dc | 15.34 ± 0.69 bc | 4.15 ± 0.33 ab | 6.42 ± 0.97 ab | 4.27 ± 1.50 bc | 0.77 ± 0.47 a |
| A3 | 58.33 ± 7.64 dc | 15.18 ± 1.55 bc | 4.52 ± 0.48 ab | 6.85 ± 0.90 a | 4.95 ± 1.43 ac | 0.76 ± 0.23 a |
| A4 | 81.67 ± 2.89 ab | 19.81 ± 2.10 a | 4.77 ± 0.61 ab | 7.42 ± 0.78 a | 7.93 ± 1.21 a | 1.21 ± 0.21 a |
| A5 | 53.33 ± 5.77 d | 15.35 ± 2.86 bc | 4.58 ± 0.50 ab | 6.85 ± 0.69 a | 5.77 ± 2.08 ac | 0.87 ± 0.40 a |
| A6 | 88.33 ± 2.89 a | 15.44 ± 1.41 b | 4.98 ± 0.59 a | 6.42 ± 0.53 ab | 6.55 ± 2.41 ab | 1.02 ± 0.61 a |
| A8 | 70.00 ± 5.00 bc | 14.07 ± 1.73 bc | 4.34 ± 0.68 ab | 6.85 ± 0.37 a | 5.20 ± 1.53 ac | 0.86 ± 0.27 a |
| NTF | NA | 12.14 ± 2.73 c | 3.76 ± 1.08 b | 5.57 ± 0.78 bc | 3.51 ± 1.90 c | 0.61 ± 0.34 ab |
| NC | 50 ± 5.00 d | 8.37 ± 2.07 d | 2.58 ± 0.43 c | 5.00 ± 0.57 c | 1.39 ± 0.74 d | 0.24 ± 0.13 b |
*NTF = nitrofoska fertilizer; CN = negative control; NA = not apply. Different letters within columns indicate significant differences at P < 0.05, according to the Tukey test.
Discussion
There are few scientific reports on the isolation, identification, and characterization of microbial communities of J. curcas. A previous report revealed that the rhizosphere of this perennial crop contains rich microbial biodiversity and demonstrated the importance of obtaining PGPR isolates to understand their ecological and social impact (Jha, Patel, Rajendran, & Saraf, 2010). It is therefore essential to evaluate the functional potential of the isolates obtained from J. curcas crops. In this study, eight rhizobacteria isolates showed antagonistic activity against C. lunata and F. equiseti, phytopathogenic fungi of J. curcas seeds. This antifungal effect on the mycelial growth of the two fungal species was demonstrated by the presence of the volatile and diffusible compounds produced by the bacterial isolates.
Morphological and molecular identification showed that these antagonistic bacteria belong to the Bacillus and Pseudomonas genera. The phylogenetic trees showed distinct clusters that contain different Bacillus species (B. mojavensis, B. subtilis and B. thuringiensis) and one strain of P. aeruginosa. The work of Wong-Villarreal et al. (2019) reported various strains of P. aeruginosa, bacteria with biotechnological potential isolated from the J. curcas rhizosphere. In that study, different strains of P. aeruginosa were able to degrade aromatic hydrocarbons and promoted growth in Zea mays L. Although the centre of origin and domestication of J. curcas is Mexico (Mazumdar et al., 2018), the potential of its rhizospheric bacteria is little known. Considering the incidence of pests and diseases reported in J. curcas (Góngora-Canul et al., 2018; Saragih, Fajri, Mahmudy, Abadi, & Anggodo, 2018), it is important to evaluate the potential for biological control and plant growth promotion by rhizobacteria strains.
In this study, the antagonistic rhizobacteria demonstrated their ability to produce antifungal metabolites. B. subtilis strains produced proteases and siderophores, and they had substantial antagonistic activity against the mycelial growth of C. lunata and F. equiseti. The results are consistent with those reported in studies carried out with B. subtilis isolated from J. curcas in India and Mexico (Hernández-Guerra et al., 2016; Latha et al., 2011), which produced siderophores and inhibited mycelial growth of the phytopathogenic fungi Lasiodiplodia theobromae and Fusarium verticillioides. The B. mojavensis strains isolated here also produced proteases and siderophores and inhibited the mycelial growth of the two fungal species tested. In previous studies, B. mojavensis inhibited the mycelial growth of three important phytopathogenic fungi: Ceratocystis fimbriata, Pestalotiopsis microspora and F. verticilloides (Hernández-Guerra et al., 2016; Mohamad et al., 2018). Another significant bacterial species isolated in this study was B. thuringiensis. This strain produced hydrolytic enzymes such as proteases and chitinases and had cry genes, essential for the biocontrol of insect pests. The chitinase activity of B. thuringiensis and its antifungal effect against phytopathogenic fungi have been previously demonstrated (Martínez-Zavala, Barboza-Pérez, Hernández-Guzmán, Bideshi, & Barboza-Corona, 2020). Finally, we isolated a strain of P. aeruginosa, which was the isolate with the strongest antagonistic effect on the mycelial growth of two fungal species tested. It (along with other isolates from this study) produced siderophores and was the only rhizobacterium that produced HCN. HCN is a secondary metabolite that acts as an inhibitor of cytochrome c oxidase, and P. aeruginosa contributes to the suppression of plant diseases by HCN production (Abo-Elyousr et al., 2019).
Currently, rhizobacteria are known to be efficient biofertilizers in different crops, providing phytohormones like auxin, cytokinin, gibberellin, and ACC deaminase, in addition to providing nutrients by nitrogen fixation, phosphorus solubilization, and sequestration of iron by the production of siderophores. On the one other hand, the plants’ roots provide essential molecules for bacteria metabolism such as sugars, organic acids, and amino acids (Olanrewaju, Glick, & Babalola, 2017). All of the strains analysed in this study can produce IAA. Numerous reports have indicated that IAA production is related to plant growth stimulation by Bacillus sp. and Pseudomonas sp., among other microorganisms (Jha & Saraf, 2012; Uzair et al., 2018). Previous studies have indicated that Bacillus spp. and Enterobacter cancerogenus can enhance the growth of J. curcas (Desai et al., 2007; Jha et al., 2012). Here, only P. aeruginosa showed phosphate solubilization activity and this species exhibited the highest beneficial potential. It has been reported that P. aeruginosa solubilizes phosphates significantly and produces IAA, siderophores, and HCN and that it can be used as a bio-inoculant to increase productivity of legumes (Ahemad & Khan, 2012). Currently, there are few works about the bacteria B. subtilis, B. thuringiensis and B. mojavensis as promoters of the growth on tomato plants and germination inductors. The study by Cabra-Cendales, Rodríguez-González, Villota-Cuasquer, Tapasco-Alzate, and Hernández-Rodríguez (2017) demonstrated that one strain of B. subtilis induced the germination of around 86.7 % of tomato seeds, however, it did not include statistical comparisons with uninoculated seeds. In this study the germination percentage of all strains showed statistically significant differences compared to the negative control (uninoculated seeds had 50 % germination). The study of Cabra-Cendales et al. (2017) coincides with our results; in both studies the bacteria increased the stem measurements as well as fresh mass. Qi, Aiuchi, Tani, Asano, and Koike (2016) tested the promotion growth activity of B. thuringiensis on tomato plants and seed germination and found larger stem length and plant fresh weight compared to an untreated control, and the strains promoted tomato seed germination. On the other hand, B. mojavensis has no previous reports of growth promoting effects in tomato plants, though the study by Pyo, Shrestha, Park, and Kang (2014) demonstrated the potential promotion growth activity of one strain of B. mojavensis by spraying bacterial suspension on the leaves of altari radish and lettuce. The foliar treatments increased the quantity, length, and weight of leaves and roots in both altari radish and lettuce crops.
Nowadays, it is known the pollution caused by the excessive usage of synthetic products in the agricultural activity, which decrease the quality of water and soil. Furthermore, these products affect the human health, which is why the application of pesticides in relation to their harmful effects is a matter of concern worldwide (Leong et al., 2020). In this regard, Mexico is the largest producer of tomatoes in the world and the possible use of PGPB may be an option to reduce the consumption of chemicals. Studies using PGPB have shown them to be an alternative as biofertilizers and disease suppressants, which can be more environmentally friendly than chemicals. Overall, in this study it was demonstrated that several of the rhizobacteria of J. curcas produce compounds of potential importance for biological control of pests and diseases and improvement of plant growth.
Conclusions
Eight strains of rhizobacteria isolated from rhizospheric soil of Jatropha curcas showed antagonistic activity against Curvularia lunata and Fusarium equiseti by the effects of the volatile and diffusible compounds. The rhizobacteria Bacillus mojavensis, B. thuringiensis, and Pseudomonas aeruginosa can suppress mycelial growth by production of antifungal compounds, indicating their potential in biocontrol against diseases of J. curcas. In addition, to our knowledge, this is the first report of rhizobacteria isolated of J. curcas that demonstrated their capacity to improve the germination seeds and promote growth of tomato plants.
