Induced Systemic Resistance (ISR) in Arabidopsis thaliana by Bacillus amyloliquefaciens and Trichoderma harzianum Used as Seed Treatments

The Trichoderma fungal species and the bacteria Bacillus species were described as inducers of plant systemic resistance in relation to their antagonistic activity. The objective of this study was to evaluate the effect of selected strains of Bacillus amyloliquefaciens (I3) and Trichoderma harzianum (A) on inducing systemic resistance in Arabidopsis thaliana as a model for plant molecular genetics. The microorganisms were identified and were confirmed for their antagonistic potential in vitro and in vivo in previous studies. In order to explore this mechanism, two mutants of A. thaliana carrying a PR1 promoter (a conventional marker of salicylic acid (SA) pathway) and LOX2 promoter (a marker triggering jasmonic acid (JA) pathway activation) were analyzed after inoculating antagonists. Transgenic reporter line analysis demonstrated that B. amyloliquefaciens I3 and T. harzianum A induce A. thaliana defense pathways by activating SA and JA at a high level compared to lines treated with chemical elicitors of references (acibenzolar-S-methyl (Bion 50 WG (water-dispersible granule)), SA, and methyl jasmonate). The efficacy of B. amyloliquefaciens I3 and T. harzianum A in inducing the defense mechanism in A. thaliana was demonstrated in this study.


Introduction
Inducing the plant defense mechanism is considered one of the most effective sustainable biocontrol solutions that can be implemented as a promising approach in plant protection. As a matter of fact, the plant defense mechanism can be stimulated by beneficial microorganisms that have the potential to induce a distant induced systemic resistance (ISR) in the plant. The potential to activate ISR is assigned to antagonistic microorganisms [1,2] or to their secondary metabolites [3,4]. The plant defense mechanism is typically related to ISR activation [5][6][7][8], the signaling pathways of which depend on cultivars, pathogen, and microorganism inducer [9,10]. Bacillus amyloliquefaciens I3 isolated from wheat leaves and Trichoderma harzianum A isolated from Argan tree rhizosphere in Morocco demonstrated high antagonistic potential against Zymoseptoria tritici in vitro and in greenhouse conditions [11]. Buensanteai et al. [12] reported that B. amyloliquefaciens strain KPS46 induces ISR in soybean against Xanthomonas axonopodis pv. glycines. Also, it was demonstrated that surfactine extracted from B. amyloliquefaciens strain S499 triggers ISR in wheat against Z. tritici [3]. Furthermore, Harman et al. [13] proved that T. harzianum strain T39 triggers ISR in many plant species against a broad range of diseases. Likewise, T. harzianum Tr6 was able to induce resistance in cucumber and A. thaliana against Pseudomonas sp. Ps14 [14].
In this study, the objective was to evaluate the potential of B. amyloliquefaciens I3 and T. harzianum A to induce and activate defense pathways in Arabidopsis (A. thaliana) by seed treatment. This property was described in vitro and in vivo using mutant lines of A. thaliana which report the activity of gene promoters PR1 and LOX2, markers of salicylic acid (SA) and jasmonic acid (JA) defense pathways, respectively. Qualitative characterization by spatiotemporal monitoring (histochemical tests) of this type of induction in planta and quantitative analysis from protein extract assays at different stages of the culture show a role of these antagonists in the induction of defense mechanisms.

Biological Material
Mutant lines pPR1::GUS and pLOX2::GUS (N6357 and N57953, respectively) were used as reporter lines for SA and JA activity, respectively. These lines were provided by Nottingham Arabidopsis Stock Center.
In-plant analysis of β-glucuronidase (GUS) activity in Arabidopsis lines, genetically transformed by transcriptional fusions of the UIDA gene with promoters of the PR1 (PR1 line) and LOX2 (LOX2 line) genes allows the activation of these promoters already described as markers of induction of the SA and JA pathways, respectively [15,16].

Preparation of Antagonists Inocula
Two strains with antagonistic potential were used in this study: strain I3 of B. amyloliquefaciens and strain A of T. harzianum [11]. T. harzianum inocula were prepared using cultures growing in PDA media (Potato Dextrose Agar) incubated seven days at 25 • C in the dark. B. amyloliquefaciens I3 inocula were plated in Luria Bertani (LB) Agar [17] and incubated 72 h at 27 • C in the dark.

Arabidopsis Seedling Preparation
Ten milligrams of seeds (6000 Arabidopsis seeds) were stirred in 1 mL of sterilizing solution (ethanol 70%, sodium hypochlorite 10%) for 10 min, subsequently rinsed three times with ethanol 70%, and dried up using Whatman papers disinfected with ethanol.
Arabidopsis was cultivated in vitro and in vivo for studying its different early growth stages (germination, cotyledons, leaf production, and rosette production); the study focuses also on late growth stages (flowering and silique ripening). In vitro seedlings were grown in Murashige and Skoog basal salt mixture agar media in petri plates of 90 mm in diameter. In vivo seedlings were sown in pots of 7 × 7 × 8 cm, with three seeds in each pot containing 2/3 peat and 1/3 perlite.
Seeds were stratified at 4 • C in the dark before adding the antagonist suspensions. Afterward, 10 µL of the antagonist suspensions (10 9 colony-forming units (cfu)/mL) were added on the top of each seed in in vitro conditions. In the in vivo conditions, the substrate was inoculated with 8 mL of the antagonist suspensions (10 9 cfu/mL). Both plates and pots were transferred to a growth room kept at long-day conditions (19 • C/21 • C, 16/8-h photoperiod, 60% relative humidity, and 150 µE (microEinstein) of luminance).
In this study, four controls were made: a negative control in which seeds were treated with sterile milliQ water, and positive controls where seeds were treated with acibenzolar-S-methyl at 925 µM (Bion 50 WG, Syngenta company), SA at 140 µg/mL which induced PR1 activity, and methyl jasmonate (MeJa) at 60 µM which induced LOX2 activity in reporter cell lines. All plants in controls were treated using foliar spray 24 h before being stained with GUS solution.
Ten plants were used to study every growth stage modality cultivated in vitro, and three plants were used to study in vivo growth modality in pots. Plants were dipped in GUS mixture for 12 h and incubated in the dark at 37 • C while stirring for 150 rotations per minute. Tissues were then washed with ethanol 70%. Stained tissues were studied with a binocular loupe, and the ratio of surface area stained over total surface area of each plant was estimated using ImageJ software. This assay was repeated three times. Protein extraction from roots, leaves, and floral stems was performed at the flowering stage. The amount of proteins in floral stems was also determined at the silique ripening stage. The test was repeated three times. Samples for each extraction were snap-frozen in liquid nitrogen and conserved at −80 • C. Floral stems and leaves were ground in the mortar using liquid nitrogen, while roots were lyophilized before grinding with small glass beads. In total, 300 mg of floral stem and leaf powder was weighed in 15-mL laboratory tubes and, subsequently, 3 mL of extraction buffer was added. All tubes were incubated in ice for 30 min, mixed every 10 min using a vortex, and then placed on a cold centrifuge (4 • C) for 10 min at 3000× g. The supernatant was re-centrifuged for 20 min and other 10 min at 20,000× g. The final volume of supernatant was conserved at 4 • C until chip analysis. For roots, 500 µL of extraction buffer was added to 100 mg of plant powder. The suspension was mixed in a vortex and placed in a cold centrifuge (4 • C) for 30 min at 14,000× g.

Preparing Protein Micro-Plate
On a 96-well micro-plate, a range of standards from 0 µg/µL to 5 µg/µL of BSA (bovine serum albumin) at 0.1 mg/mL was deposited. In each well of the micro-plate, 1 µL of plant extract, 49 µL of sterile milliQ water, and 200 µL of reactive dye Bio-Rad Protein Assay diluted at 1/4 were added. Samples were established in triplicate in the same micro-plate. The micro-plate was analyzed using a spectrophotometer at 595 nm as described in the Bradford method [18].

Statistical Analysis
SPSS 21 statistic software was applied for histochemical data analysis. Statistical analyses were established at two-factor level; the first corresponded to treatments (B. amyloliquefaciens I3, T. harzianum A, Bion 50 WG, SA, MeJa, and negative control) and the second factor indicated the growth stages (germination, cotyledons, leaf production, rosette, and flowering). ANOVA analysis was applied for analysis of mean data variation, while the Duncan test was used for the comparison of means at p = 0.05.
The value of β-glucuronidase activity was transformed to square roots for data analysis. The applied factors were treatments (B. amyloliquefaciens I3, T. harzianum A, Bion 50 WG, SA, MeJa, and negative control), growth stage (flowering, silique ripening), and vegetative organs (roots, leaves, and floral stems). Variance analysis was utilized to study the variation of averages in the amount of β-glucuronidase activity using SPSS 21. Means were compared with the Duncan test at p = 0.05.

β-Glucuronidase Expression by GUS Staining
The antagonist inoculation in transgenic Arabidopsis (PR1 and LOX2 reporter lines) demonstrated the induction of SA (PR1 line) and JA (LOX2 line) pathways. Results of β-glucuronidase (GUS) staining in both reporter lines (Figures 1 and 2) whose seeds were treated by B. amyloliquefaciens I3 and T. harzianum A revealed the effective activation of both LOX2 and PR1 promoters and, thus, the induction of SA and JA pathways. Similar GUS staining results were shown in lines treated with chemical elicitors (Bion 50 WG, MeJa, and SA) at different growth stages (germination, cotyledons, leaf production, rosette, and flowering). The value of β-glucuronidase activity was transformed to square roots for data analysis. The applied factors were treatments (B. amyloliquefaciens I3, T. harzianum A, Bion 50 WG, SA, MeJa, and negative control), growth stage (flowering, silique ripening), and vegetative organs (roots, leaves, and floral stems). Variance analysis was utilized to study the variation of averages in the amount of β-glucuronidase activity using SPSS 21. Means were compared with the Duncan test at p = 0.05.

β-Glucuronidase Expression by GUS Staining
The antagonist inoculation in transgenic Arabidopsis (PR1 and LOX2 reporter lines) demonstrated the induction of SA (PR1 line) and JA (LOX2 line) pathways. Results of βglucuronidase (GUS) staining in both reporter lines (Figures 1 and 2) whose seeds were treated by B. amyloliquefaciens I3 and T. harzianum A revealed the effective activation of both LOX2 and PR1 promoters and, thus, the induction of SA and JA pathways. Similar GUS staining results were shown in lines treated with chemical elicitors (Bion 50 WG, MeJa, and SA) at different growth stages (germination, cotyledons, leaf production, rosette, and flowering).     The transgenic Arabidopsis line carrying pLOX2::GUS, planted in vitro, did not show any GUS stain pattern when treated with both antagonists during all the studied developmental stages (germination, cotyledon, leaf production, rosette). On the other hand, when this line was treated with methyl-jasmonate, the GUS stain patterns ranged from 79% in germination to 81% in the rosette stage ( Figure 4). Plants presented highly significant GUS stain differences in all the treatments in the flowering stage. The GUS stain patterns covered 84% of the leaf surfaces when plants were treated with B. amyloliquefaciens I3. The GUS stain patterns covered 82% and 78% of the leaf surfaces when treated with MeJa and T. harzianum A, respectively. On the other hand, the negative controls were stained on less than 2% of the total surface ( Figure 4).  The transgenic Arabidopsis line carrying pLOX2::GUS, planted in vitro, did not show any GUS stain pattern when treated with both antagonists during all the studied developmental stages (germination, cotyledon, leaf production, rosette). On the other hand, when this line was treated with methyl-jasmonate, the GUS stain patterns ranged from 79% in germination to 81% in the rosette stage ( Figure 4). Plants presented highly significant GUS stain differences in all the treatments in the flowering stage. The GUS stain patterns covered 84% of the leaf surfaces when plants were treated with B. amyloliquefaciens I3. The GUS stain patterns covered 82% and 78% of the leaf surfaces when treated with MeJa and T. harzianum A, respectively. On the other hand, the negative controls were stained on less than 2% of the total surface ( Figure 4). The transgenic Arabidopsis line carrying pLOX2::GUS, planted in vitro, did not show any GUS stain pattern when treated with both antagonists during all the studied developmental stages (germination, cotyledon, leaf production, rosette). On the other hand, when this line was treated with methyl-jasmonate, the GUS stain patterns ranged from 79% in germination to 81% in the rosette stage ( Figure 4). Plants presented highly significant GUS stain differences in all the treatments in the flowering stage. The GUS stain patterns covered 84% of the leaf surfaces when plants were treated with B. amyloliquefaciens I3. The GUS stain patterns covered 82% and 78% of the leaf surfaces when treated with MeJa and T. harzianum A, respectively. On the other hand, the negative controls were stained on less than 2% of the total surface ( Figure 4).

Quantitative Analysis of Promoter Activation by Enzymatic Dosage of GUS in Protein Extracts
The dosage of GUS activity in the transgenic Arabidopsis line, carrying pPR1::GUS treated with both antagonists, demonstrated a significantly higher activity in roots and floral stems compared to that observed in leaves ( Figure 5). β-Glucuronidase activity measured in the root extract was 0.78 µg/µL, 0.82 µg/µL, and 0.89 µg/µL when plants were treated with Bion 50 WG, SA, and milliQ water, respectively. However, the measured activity was 2.03 µg/µL and 1.97 µg/µL in the root extract of plants treated with B. amyloliquefaciens I3 and T. harzianum A, respectively. β-Glucuronidase activity in the root extract of plants treated with B. amyloliquefaciens I3 and T. harzianum A was comparable to that measured in floral stems. The measured activity was 2.54 µg/µL when plants were treated with B. amyloliquefaciens I3 and 2.07 µg/µL when treated with T. harzianum A. The β-glucuronidase activity in the root extract was higher than that measured in leaves.

Quantitative Analysis of Promoter Activation by Enzymatic Dosage of GUS in Protein Extracts
The dosage of GUS activity in the transgenic Arabidopsis line, carrying pPR1::GUS treated with both antagonists, demonstrated a significantly higher activity in roots and floral stems compared to that observed in leaves ( Figure 5). β-Glucuronidase activity measured in the root extract was 0.78 µg/µL, 0.82 µg/µL, and 0.89 µg/µL when plants were treated with Bion 50 WG, SA, and milliQ water, respectively. However, the measured activity was 2.03 µg/µL and 1.97 µg/µL in the root extract of plants treated with B. amyloliquefaciens I3 and T. harzianum A, respectively. β-Glucuronidase activity in the root extract of plants treated with B. amyloliquefaciens I3 and T. harzianum A was comparable to that measured in floral stems. The measured activity was 2.54 µg/µL when plants were treated with B. amyloliquefaciens I3 and 2.07 µg/µL when treated with T. harzianum A. The β-glucuronidase activity in the root extract was higher than that measured in leaves. The dosage of GUS activity in Arabidopsis lines carrying pLOX2::GUS showed significantly different results in all the treatments and in different tissues during plant development. The measured β-glucuronidase activity in the root extract was 1.68 µg/µL in plants treated with T. harzianum A and 1.55 µg/µL in plants treated with B. amyloliquefaciens I3. Additionally, the GUS activities were more significant in the antagonist treatments compared to samples treated with MeJa and water. The measured GUS activity in floral stems treated with T. harzianum A, B. amyloliquefaciens I3, and MeJa were 1.88 µg/µL, 2.24 µg/µL, and 2.20 µg/µL, respectively, whereas it did not exceed 1.13 µg/µL in samples treated with water. The activity in the leaf extract treated with different treatments was low compared to that of floral stems ( Figure 6).  The dosage of GUS activity in Arabidopsis lines carrying pLOX2::GUS showed significantly different results in all the treatments and in different tissues during plant development. The measured β-glucuronidase activity in the root extract was 1.68 µg/µL in plants treated with T. harzianum A and 1.55 µg/µL in plants treated with B. amyloliquefaciens I3. Additionally, the GUS activities were more significant in the antagonist treatments compared to samples treated with MeJa and water. The measured GUS activity in floral stems treated with T. harzianum A, B. amyloliquefaciens I3, and MeJa were 1.88 µg/µL, 2.24 µg/µL, and 2.20 µg/µL, respectively, whereas it did not exceed 1.13 µg/µL in samples treated with water. The activity in the leaf extract treated with different treatments was low compared to that of floral stems ( Figure 6).

Dosage of β-Glucuronidase Proteins in Floral Stems at Silique Ripening Stage of pPR1::GUS and pLOX2::GUS Lines
The β-glucuronidase activity in total extracts of plants treated with T. harzianum A and B. amyloliquefaciens I3 at the silique ripening stage was much higher in floral stems of transgenic lines carrying pLOX2::GUS than in plants treated with water ( Figure 7). The activity was 1.43 µg/µL, 1.92 µg/µL, and 1.64 µg/µL when the pLOX2::GUS line was treated with MeJa, B. amyloliquefaciens I3, and T. harzianum A, respectively. When pPR1::GUS plants were treated with Bion 50 WG or SA, the activity of β-glucuronidase was 1.90 µg/µL compared to those treated with B. amyloliquefaciens I3 and T. harzianum A with an activity of 1.82 µg/µL and 1.45 µg/µL respectively.   The β-glucuronidase activity in total extracts of plants treated with T. harzianum A and B. amyloliquefaciens I3 at the silique ripening stage was much higher in floral stems of transgenic lines carrying pLOX2::GUS than in plants treated with water ( Figure 7). The activity was 1.43 µg/µL, 1.92 µg/µL, and 1.64 µg/µL when the pLOX2::GUS line was treated with MeJa, B. amyloliquefaciens I3, and T. harzianum A, respectively. When pPR1::GUS plants were treated with Bion 50 WG or SA, the activity of β-glucuronidase was 1.90 µg/µL compared to those treated with B. amyloliquefaciens I3 and T. harzianum A with an activity of 1.82 µg/µL and 1.45 µg/µL respectively.

Dosage of β-Glucuronidase Proteins in Floral Stems at Silique Ripening Stage of pPR1::GUS and pLOX2::GUS Lines
The β-glucuronidase activity in total extracts of plants treated with T. harzianum A and B. amyloliquefaciens I3 at the silique ripening stage was much higher in floral stems of transgenic lines carrying pLOX2::GUS than in plants treated with water ( Figure 7). The activity was 1.43 µg/µL, 1.92 µg/µL, and 1.64 µg/µL when the pLOX2::GUS line was treated with MeJa, B. amyloliquefaciens I3, and T. harzianum A, respectively. When pPR1::GUS plants were treated with Bion 50 WG or SA, the activity of β-glucuronidase was 1.90 µg/µL compared to those treated with B. amyloliquefaciens I3 and T. harzianum A with an activity of 1.82 µg/µL and 1.45 µg/µL respectively.

Discussion
Various studies were conducted on the induction of systemic resistance by beneficial microbes leading to a decrease in incidence or severity of diseases in different plant pathosystems [19,20]. It was reported that different species of Trichoderma spp. and Bacillus spp. colonize the rhizosphere of different plants, protecting them against a wide range of aerial and soilborne pathogens [21][22][23]. In recent years, increasing attention was accorded to the study of direct interactions between Trichoderma spp./Bacillus spp. and plants, including molecular studies on plant defense mechanisms that allowed a better understanding of how antagonists interact with plants [12,[24][25][26]. In the present study, the induction of SA and JA pathways by B. amyloliquefaciens I3 and T. harzianum A from the cotyledons to the flowering stage was demonstrated using GUS staining patterns (Figures 1 and 2). The GUS staining pattern in the plant surface was significantly higher (p = 0.05) when plants were treated with both antagonists at the flowering stage compared to the percentage of GUS dyes in plants treated with the commonly tested chemical references, Bion 50 WG and SA (Figure 3). There was no visible GUS staining in pLOX2::GUS lines observed in in vitro cultivated plants; however, in the flowering stage, the GUS staining was observed. On the other hand, the GUS staining pattern was very high in plant leaves treated with B. amyloliquefaciens I3 and T. harzianum A, which demonstrates the potential of strains A and I3 in inducing the JA pathway ( Figure 4). It is important to note that both transgenic plants exhibited a positive GUS staining and were also induced in the flowering and the silique ripening stages, while the antagonist inoculation was established in the substrate during the early developmental stages (Figures 2 and 7). In addition to GUS staining, the activity of β-glucuronidase (GUS) in different plant tissues in the flowering stage revealed SA and JA pathway induction in roots, leaves, and floral stems in both pPR1::GUS and pLOX2::GUS lines, with a higher activity in floral stems ( Figures 5  and 6). Furthermore, the β-glucuronidase activity was significantly higher at p = 0.05 in floral stems of pLOX2::GUS than that of the pPR1::GUS line in the silique ripening stage.
Our results are in agreement with the study conducted by Niu et al. [27], who demonstrated that B. cereus AR156 induces ISR in A. thaliana, by simultaneously activating SA and JA pathways. Similarly, van Wees et al. [10] proved that strain WCS417r of Pseudomonas fluorescens induces ISR in tomato and A. thaliana against Pseudomonas syringae pv. tomato and Fusarium oxysporum f. sp. raphanin, respectively. Ryu et al. [28] also demonstrated that different plant growth-promoting rhizobacteria (PGPR) (Serratia marcescens 90-166, Bacillus pumilus SE34, P. fluorescens 89B61, P. fluorescens 89B27, B. amyloliquefaciens IN937, and B. subtilis IN937) induce SA and JA pathways in cucumber. However, it is important to note that surfactines and fengycines, which are lipo-peptides produced by the genus of Bacillus, may interact with plant membrane cells and disturb them (provoking stress), leading to a similar defense reaction in the plant that may be confused with ISR induction [4].
In a previous study, Yoshioka et al. [29] revealed that Trichoderma asperellum colonizing A. thaliana roots induced ISR through the SA signaling cascade. Additionally, plants treated with Trichoderma filtrates induced SA and jasmonate/ethylene (JA/ET) pathways. Alizadeh et al. [14] confirmed that T. harzianum Tr6 activated SA and JA pathways in transgenic A. thaliana myb72 and sid2 against Botrytis cinerea. A similar result was obtained by Elsharkawy et al. [6], who demonstrated that T. asperellum SKT-1 strain induced ISR (SA and JA/ET) in A. thaliana against the Y strain of the cucumber mosaic virus (CMV-Y).
Our results demonstrated the potential of the two antagonists to systemically induce SA and JA pathways in A. thaliana. According to the biocide effect, the antagonists may offer more sustainable plant protection in field conditions using the treated seeds with B. amyloliquefaciens I3 and T. harzianum A. This was confirmed by Barakat et al. [30], who showed the antagonistic potential of I3 and A strains against Z. tritici by seed coating in greenhouse and in field conditions (unpublished data).
Author Contributions: B.I. conceived, designed, and performed experiments and wrote the paper; C.N. supervised, contributed in data analysis and translation, and reviewed the paper; G.P. was responsible experiment validation, supervision, and review; E.B. and E.G.M. were responsible for supervision and review; A.S. contributed in protein extraction; N.M. was responsible for protein extraction and dosage; M.M. supervised the study.