Introduction

Saffron is the most expensive spice of the world with extensive application in food, medicine and beverages (Menia et al. 2018; Jafari et al. 2020; Bhagat et al. 2021). Though the demand for saffron is increasing steadily, the traditional area under cultivation and production is declining concomitantly, leading to a lot of adulteration (Qadri 2017; Cardone et al. 2020). Saffron production is on a decline in Jammu and Kashmir, the only saffron cultivating union territory in India, due to various biotic and abiotic factors (Menia et al. 2018; Gupta et al. 2021). The biotic factors include various corm rot causing fungal pathogens; however rot caused by Fusarium oxysporum and Fusarium solani is the most destructive (Gupta and Vakhlu 2015; Bhagat et al. 2022). Chemical fungicides used to manage corm rot have a deleterious effects on the environment and also leads to the development of resistance phytopathogens (Burketova et al. 2015; Pandin et al. 2017); hence, there is a need to replace them with eco-friendly technology, such as the use of plant growth promoting bacteria i.e., PGPB (Mishra et al. 2018; Ramakrishna et al. 2019; Ahirwar et al. 2020). PGPB influence plant growth and development by various direct mechanisms such as phosphate solubilization, siderophore production, nitrogen fixation, phytohormone production (Batista et al. 2021; Ajijah et al. 2023). Indirectly PGPB protects the plant from deleterious organisms, as they have inherent antagonistic properties which include competing for food, niche or production of chemical compounds lethal for the growth of pathogens or by eliciting a defense response in plants called induced systemic resistance i.e., ISR (Shafi et al. 2017; Kour and Sodhi 2022; Selari et al. 2023).

Among all known plant growth promoting bacteria (PGPB), Bacillus and Pseudomonas spp. are most preferred (Bhagat et al. 2021). However, members of Bacillus are more preferred as they have the capacity to form heat resistance spores, secrete secondary metabolites (antibiotics, siderophores and volatile compounds) and cell wall degrading enzymes, such as β-1,3-glucanase and chitinase (Radhakrishna et al. 2017). Bacillus species also induces defense related plant genes by induction of systemic resistance (ISR), along with genetic and structural modifications of the host plant (Sharaf-Eldin et al. 2008; Shafi et al. 2017; Mahapatra et al. 2022). ISR is a phenomenon that enhances the host plant’s defense by strengthening the plant cell wall, activating various signal transduction pathways and by enhancing the synthesis of plant defense enzymes when challenged by the pathogen (Sansinenea 2019; Yu et al. 2022). In plants, ISR is reported to be involved in disease suppression caused by various fungal pathogens such as Colletotrichum truncatum, Fusarium oxysporum f.sp. lycopersici (Fol), Fusarium oxysporum f.sp. niveum (Fon), Fusarium solani, Xanthomonas campestris pv. Vesicatoria, etc. (Elanchezhiyan et al. 2018; Gowtham et al. 2018; Akram et al. 2013; Jiang et al. 2019; Chandrasekaran et al. 2017). ISR is associated with increased activity of defense related enzymes such as phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), lipoxygenase (LOX), chitinase (Chi) and β,1-3 glucanase (GLU) (Palani et al. 2016; Elanchezhiyan et al. 2018; Jiang et al. 2019; Can et al. 2022).

All these enzymes have been reported to increase the defense of the host plant against various pathogens such as in tomato against Fusarium oxysporum and Fusarium solani (Rashid et al. 2021a, b), in bean against Fusarium oxysporum (Garces-Fiallos et al. 2022), in melon against Fusarium oxysporum f.sp. melonis race 1.2, (Sadeghpour et al. 2022), in pigeon pea against Fusarium udum (Hussain et al. 2023). PAL enzyme catalyses the committed step of the phenylpropanoid pathway resulting in the production of various secondary metabolites (Abdollahi et al. 2022). The enzyme PPO and PO provide defense barrier and resistance to host plant against pathogens by producing oxidative phenols and by detoxifying the reactive oxygen species produced during the reactive burst in plants, respectively (Garces-Fiallos et al. 2022). GLU enzyme hydrolyses the glucan which is an important structural component of fungal cell wall and LOX enzyme provides resistance to host plant by producing antifungal compounds via polyunsaturated fatty acids (PUFA) (Singh et al. 2022; Ye et al. 2023).

In the present study, a comparative evaluation of two native Bacillus species Bar D5 and Bam W2 has been done to select the better biostimulant of the two for field trials and subsequent commercialization.

Material and Methods

Corm Samples, Fungal and Bacterial Strains, and Culture Conditions

Saffron corms were collected in sterile polythene bags from Pampore town of Pulwama district (34.02° N longitude, 74.93° E latitude and 1574 m altitude from sea level), Jammu and Kashmir, India in July 2019. The pathogenic Fusarium oxysporum R1 (Fox R1) (GenBank Accession number: KF663598) used in the present study, has been reported earlier by our group (Gupta and Vakhlu 2015). The Fox R1 culture was cultivated on potato dextrose agar (PDB Himedia, India) at 25 °C for 7 days. Further, Fox R1 agar plug was inoculated in potato dextrose broth (PDB, Himedia) and incubated at 28 °C for 7 days at 180 rpm in incubator shaker (Scigenics, India). After 7 days the culture was filtered through five layers of muslin cloth for harvesting the spores. The filtrate was centrifuged at 10,000 g for 10 min at 4 °C and a concentration of 1012 spores/ml was maintained with sterile distilled.

Bacterial strains used in present study have also been characterized earlier by our group; Bacillus sp. strain D5 (GenBank Accession number: KT228251) (Magotra et al. 2021), Bacillus amyloliquefaciens W2 (GenBank Accession number KF663600) (Gupta and Vakhlu 2015). Both the cultures were maintained on nutrient agar (Himedia, India) at 37 °C for 24 h. The loop full of active culture were inoculated in nutrient broth and incubated at 37 °C for 48 h with shaking at 180 rpm till stationery phase. After 48 h, the cell count was maintained at 108 cells/ml (Magotra et al. 2021).

In-Vitro Plant Growth Promotion (PGP) Activities

Phosphate solubilization

The phosphate released by the isolates was quantified by chlorostannous reduced molybdophosphoric method (Thakur et al. 2019). 0.5 ml of Bacillus culture with 1 × 108 cells/ml spore concentration was inoculated in National Botanical Research Institute's phosphate growth medium (NBRIP) broth and incubated at 37 °C for 48 h in shaking incubator at 180 rpm. The cultures were centrifuged at 10,000 rpm at 25 °C for 15 min and supernatant was collected. 100 µl of the supernatant was added to 10 ml of chloromolybdic reagent (15 g (NH4)2MoO4 in 400 ml distilled water and 342 ml of concentrated HCl, diluted up to 1 L) and then diluted up to 40 ml by the addition of distilled water. 5–6 drops of chlorostannous acid reagent (10 g SnCl2.2H2O in 25 ml concentrated HCl) was added and mixed properly. Bacillus converts phosphate in the complex form as ortho-phosphate which reacts with chloromolybedic reagent and forms phosphomolybedic acid complex during the reaction which gives blue colour as a result of formation of KH2PO4. The blue colour formed was measured at 660 nm against blank in UV/visible spectrophotometer (Thermo Scientific™ GENESYS™ 10S UV–Vis Spectrophotometer) using KH2PO4 standard curve (Taktek et al. 2017).

Siderophore Production

The quantification of siderophore produced was measured by chrome azurol S (CAS) shuttle assay by inoculating 0.5 ml (1 × 108 cells/ml) of Bacillus culture into Luria–Bertani (LB) broth medium (Arora and Verma 2017) and inoculated for 24 h. Cultures were centrifuged at 10,000 rpm at 25 °C for 15 min. 1 ml of the supernatant was added into 1 ml CAS solution and incubated at 25 °C for 5 min. 10 µl of shuttle solution (0.2 M 5-sulfosalicylic acid) was added to it. Siderophore produced by the Bacillus scavenge iron from Fe-CAS complex and subsequently CAS dye is released into the medium (Pahari et al. 2017).

Siderophore content as % was calculated as:

$${\text{Siderophore }}\% = \frac{{\left( {{\text{Ar}} - {\text{As}}} \right)}}{{{\text{Ar}}}} \times 100$$

Ar = Absorbance of reference at 630 nm (Uninoculated culture medium with CAS solution and shuttle solution) As = Absorbance of test sample at 630 nm.

Indole-3-Acetic Acid Quantification

The amount of IAA produced by bacteria was quantified using Salkowski’s method (Glickmann and Dessaux 1995). 0.5 ml (1 × 108 cells/ml) of bacterial culture was inoculated into Luria–Bertani (LB) broth containing tryptophan (100 µg/ml) and incubated for 48 h at 37 °C. After incubation cultures were centrifuged at 12,000 rpm for 5 min at 25 °C. 2 ml of Salkowski’s reagent was added to the 1 ml supernatant, ferric chloride ions in the salkowski’s reagent reacts with indole moiety present in the supernatant and pink colored was produced that was measured at 535 nm. The concentration of Indole-3-acetic acid (IAA) was obtained from IAA standard curve (Walitang et al. 2017).

Ammonia Production

Bacillus isolates were tested for the production of ammonia using peptone water medium. 0.5 ml (1 × 108 cells/ml) of bacterial cultures was inoculated into peptone water (10 ml) and incubated at 37 °C for 36 h. After incubation, cultures were centrifuged for 20 min at 1800 rpm at 4 °C. 1 ml of Nessler’s reagent was added to 9 ml of supernatant, iodide ions present in Nessler’s reagent react with ammonia under alkaline conditions and colored complex is formed that was measured at 530 nm. The concentration of ammonia produced was determined by (NH4)2SO4 standard curve by varying the known concentration of substrate (Sahoo and Chaudhuri 2019).

Amylase, Cellulase and Protease Activity

1% skim milk powder (Asha and Palaniswamy 2018), 1% soluble starch (Luang-In et al. 2019) and 1% Carboxy Methyl Cellulose (CMC) (Croos et al. 2019), was added to LB media for the production of protease, amylase, and cellulase, respectively. Bacillus cultures were inoculated in protease, amylase, and cellulase production media and incubated at 37 °C for 24 h at 180 rpm. Supernatant from all the cultures were collected by centrifugation at 5000 rpm for 15 min at 4 °C.

Amylase assay was performed by using 1% starch solution in 0.1 M Citrate buffer (pH 5) and 100 μl supernatant at 45 °C for 10 min. The produced reducing sugars were quantified by adding 3 ml of 3,5-dinitrosalicyclic acid (DNS) and heated for 15 min in boiling water bath. 1 ml of 40% potassium sodium tartarate (Rochelle salt) was added and final volume was raised up to 10 ml by adding distilled water. Reducing sugars produced were measured at 575 nm. The concentration was measured in reference to standard curve made by varying the concentrations of glucose. One unit of amylase was defined as the amount of enzyme required to release 1 μmol of sugar in 1 min (Kalyani and Rajesh 2018).

Endo-β-1,4-glucanase activity of cellulase enzyme was determined by DNS (3,5-dinitrosalicylic acid) method. 1% CMC substrate solution was prepared in citrate buffer 1 N (pH 5.0). 100 μl of supernatant, 1 ml citrate buffer and 1 ml CMC solution were mixed and incubated for 30 min at 45 °C. DNS was added to stop the reaction; solution was placed in boiling water for 10 min, and then cooled for color stabilization. Glucose released during reaction was measured at 540 nm. One unit of cellulase activity was the amount of enzyme required to produce 1 μmol of glucose per minute (Viswanath et al. 2018).

Protease assay was performed using casein as a substrate prepared in carbonate-bicarbonate buffer 20 mM (pH 10). 0.25 ml of supernatant (cell free extract) was added to 0.5 ml of buffer containing 0.5% substrate and incubated for 30 min at 37 °C. After incubation 0.75 ml of 10% trichloroacetic acid was added and incubated at 25 °C for 30 min to terminate the reaction. Supernatant was collected by centrifugation for 10 min at 12,000 rpm 0.6 ml of 1 N NaOH was added to 0.6 ml of the supernatant and incubated at 25 °C for 15 min. Absorbance was measured at 450 nm. One unit of protease activity was defined as the amount of enzyme which produces 0.001 changes in absorbance by hydrolyzing casein per mg of substrate per minute under assay conditions (Lim et al. 2019).

In-Planta Pot Assays for Growth Parameters

Bioformulation preparation was done by the method described by Magotra et al. (2021). In brief, nutrient broth of Bam W2 and Bar D5 containing (108 cells ml−1) individually, were mixed with autoclaved calcium carbonate in the ratio of 1:2 (v/w) and dried for 4 days. Finally 1% sterile carboxymethyl cellulose (CMC) was added to it. Slurry for priming of the corms was prepared by mixing 100 g dried bioformulation in 100 ml sterile distilled water. Priming of the corms was done by dipping corms in the slurry for 5 min, and then air drying them for 5 h before sowing in open in the laboratory. Pot trials were laid in complete randomized block design (CRBD) method. Following treatments were given to 6 corm sets with 10 corms in each set:

  • C-Mock primed corms (primed with slurry of calcium carbonate and CMC in distilled water) planted in sterile soil

  • B1-Bam W2 primed corms planted in sterile soil

  • B2-Bar D5 primed corms planted in sterile soil

  • CF-Mock primed corms planted in Fox R1 inoculated soil

  • T1-Bam W2 primed corms planted in Fox R1 inoculated soil

  • T2-Bar D5 primed corms planted in Fox R1 inoculated soil

Inoculation with Fox R1 was done by mixing 1 ml of 1012 ml−1 Fox R1 spores to 200 g of autoclaved sand: soil mixture (1:1 w/w) soil that makes final spore count in soil to 5 × 1010 spores g−1 of soil. 10 pots were laid for each experiment with 1 corms/pot and in total, 60 pots were laid for the 6 treatments. After planting the corms in pots, pots were incubated in plant growth chamber, under 16 h light/8 h dark cycle at 26 ± 2 °C with 80% relative humidity for 30 days.

After 30 days, all the corms along with the shoot and roots developed in this period were taken out and evaluated for various growth parameters and disease severity index (DSI). DSI was calculated in each treatment by the method (Gupta and Vakhlu 2015) and disease reduction percentage (DRP) by the formula:

$${\text{DRP}} = \frac{{{\text{DSI of CF}} - {\text{DSI of T}}}}{{\text{DSI of CF}}} \times 100$$

CF-corms planted in Fox R1 (1012 spores ml−1) inoculated soil and T (Corms primed with respective bacterial bioformulation and planted in Fox R1 inoculated soil).

In-Planta Estimation of Induction of Defense Related Enzymes

A separate experiment was conducted for evaluation of induction of defense related enzymes in corms. In saffron, Fusarium oxysporum penetrates the roots and corms only through wounds or injury (Bhagat et al. 2022; Mansotra et al. 2023). So, for the induction of defense mechanism of saffron plant, injury was given using sterile tip in order to mimic natural wounds. A total of 150 corms were taken and divided in to three sets (S1, S2, and S3; 50 corms/set). S1-Mock primed corms, S2-Bam W2 primed corms and S3-Bar D5 primed corms (Fig. 1). A total of 150 pots were laid maintaining 1 corm/pot. All the 150 corms were grown for a period of 30 days so that corms get primed in the presence of Bar D5 and Bam W2 before inoculating them with pathogen Fox R1. Corms were sown in autoclaved sand: soil mixture (1:1 w/w) in pots and were incubated in plant growth chamber, under 16 h light/8 h dark cycle at 26 ± 2 °C with 80% relative humidity for 30 days. After 30 days of incubation, all the corms were taken out and 50 corms of each set was further divided into two sets (25 corms in each set) so in total six sub sets (S1a, S1b, S2a, S2b, S3a, S3b) were made. All the corms were injured with sterilized needle (3 × 1 cm) and placed in their respective pots. The pots of sub sets (S1b, S2d and S3b) were inoculated with Fox R1 and inoculation was done by mixing 1 ml of 1012 ml−1 Fox R1 spores to 200 g of soil that makes final spore count in soil to 5 × 1010 spores g−1 of soil as mentioned earlier. The remaining subsets (S1a, S2a, and S3a) were inoculated with 1 ml of sterile distilled water that served as control for each treatment, respectively. The details of the different subsets have been shown in Fig. 1. Plantlets at 1, 3, 5, 8, and 11 days post inoculation from all the treatments were taken out and washed under running tape water for 15 min to remove soil particles adhered to the corm surface, leaves and roots followed by tunic removal. The clean corms were then used for the following tests:

Fig. 1
figure 1

Experiment layout for the in-planta interaction of Bacillus species (Bar D5 and Bam W2) and pathogen (Fox R1) in saffron corms. Bar D5-Bacillus sp. strain D5, Bam W2-Bacillus amyloliquefaciens W2 and Fox R1-Fusarium oxysporum R1

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In-Planta Inhibition of Fox R1

Fox R1 load was determined in different treatments; CF, T1, and T2 (Fig. 1) and inhibition % was calculated in presence of both Bam W2 and Bar D5. Two different methods were used for determining the antifungal potential (inhibition %) of Bar D5 and Bam W2 against Fox R1.

  1. 1.

    Visual symptoms: In this method, a lesion diameter was measured (mm) at 1, 3, 5, 8, 11 dpi and inhibition percentage was calculated as:

    $$\frac{{{\text{Diameter of CF lesion}} - {\text{Diameter of T lesion}}}}{{\text{Diameter of CF lesion}}} \times 100 = {\text{Inhibition percentage}}$$

    CF-Fox R1 inoculated corms, T (Bam W2 and Bar D5 primed and Fox R1 inoculated corms)

  2. 2.

    Fox R1 load: Fox R1 load was determined in CF, T1, and T2 (Fig. 1) at day 1, 3, 5, 8, 11 dpi by calculating the colony forming units (CFU). Fusarium specific Komada medium was used for the culturing of Fox R1 from infected part of corm (Komada 1975). 100 mg of the tissue was taken and washed with sterile distilled water under sterile conditions. Then it was crushed using sterile pestle mortar and 1 ml of distilled water was added to the crushed tissue. Serial dilution of the suspension was prepared and 10–2 dilution was spread on the media plates and incubated at 28 °C for 5 days. After 5 days, colonies were counted and the load was determined. Inhibition percentage was calculated as

    $$\frac{{{\text{Load in CF}} - {\text{Load in T}}}}{{\text{Load in CF}}} \times 100 = {\text{Inhibition percentage}}$$

    All the experiments were replicated independently three times at different time intervals.

Dry Matter Accumulation

Washed corms from different treatments were taken and air dried for 1 h at 25 °C. Fresh weight (FW) of each corm was determined. Corms were dried in the hot air oven at 60 °C for 48 h (Gaspar et al. 2017). Dry weight (DW) was determined. The dry matter percentage (DM %) was calculated using formula:

$${\text{DM}}\% = \frac{{{\text{DW}}}}{{{\text{FW}}}} \times 100$$

Defense Enzyme Activity

Crude protein was extracted by the method developed by Abdel-Monaim et al. (2012) with some modifications. 1 g of fresh corm tissue at 1, 3, 5, 8, 11 dpi was ground with liquid nitrogen in a mortar and pestle at 25 °C. The resulting powder was added to 50 mM ice-cold potassium phosphate buffer (pH 6.8) (4 ml) containing 1 mM ethylene diamine tetra-acetate (EDTA), 1% polyvinylpyrrolidone (PVP) and 1 M NaCl. The solution was centrifuged at 8000 rpm for 25 min at 4 °C. The supernatant was used for activity of defense enzymes.

Phenylalanine-Ammonia Lyase (PAL)

PAL activity was measured according to the method of Astaneh et al. (2018). The reaction mixture consists of 4 ml of 100 mM Tris–HCL buffer (pH 8.8), 0.2 ml of 40 mM L-phenylalanine and 0.2 ml crude enzyme extract. PAL activity leads to release of trans-cinnamic acid by its activity on phenylalanine and was measured at 290 nm after 5 min of start of reaction. 10,900 M−1 cm−1 was employed as molar extinction coefficient for enzyme activity.

Peroxidase (PO)

PO activity was determined according to Uarrota et al. (2016) with some modifications. The reaction mixture was prepared by adding 25 μl of crude enzyme extract, 2 ml of solution containing 1% guaiacol, 1% hydrogen peroxide, and 50 mM potassium phosphate buffer (pH 6.8). PO activity leads to release of oxidized guaiacol from guaiacol and oxygen, which was measured after 15 min of start of reaction at 436 nm and 26.6 mM−1 cm−1 employing molar extinction coefficients (e) for enzyme activity calculation.

Polyphenol Oxidase (PPO)

PPO activity was determined by method given by Nguyen et al. (2018). The reaction mixture was prepared by adding 50 μl of crude protein extract, 3 ml of solution containing 100 mM potassium phosphate buffer (pH 6.8) and catechol (25 mM). PPO activity leads to release of ortho-quinones from catechol that was measured at 410 nm after 10 min of start of reaction at 30 °C and 1300 M−1 cm−1 was employed as molar extinction coefficient (e) for enzyme quantification.

Lipoxygenase (LOX)

LOX activity was determined according to Garcia et al. (2017). The reaction mixture was prepared by adding 3 ml of 50 mM sodium phosphate buffer pH 6.0, 60 μl of 10 mM linoleic acid, and 30 μl of crude protein extract. LOX activity leads to release of hydroxyperoxides from linoleic acid was measured at 234 nm after 10 min of start of reaction at min at 25 °C and 25,000 M−1 cm−1 was employed as molar extinction coefficient (e) for quantification of enzyme activity.

β,1-3 Glucanase (GLU)

GLU activity was determined by Laminarin-DNS method (Gowtham et al. 2018) using laminarin (Sigma-Aldrich) as a substrate. The reaction mixture was prepared by adding 62.5 μl of crude protein extract and 62.5 μl of 4% laminarin prepared in 50 mM sodium acetate buffer (pH 5.0) and incubated at 37 °C for 1 h. Reaction was stopped by the addition of 375 μl of DNS (dinitrosalicyclic acid) reagent and the mixture was incubated at 40 °C for 10 min then 4.5 ml of distilled water was added. The glucose released from the laminarin was measured at 540 nm. Enzyme activity was quantified as mg équivalent of glucose per min per gram of fresh weight and expresed as IU/g of fresh tissue.

RNA Isolation and cDNA Preparation

Total RNA was isolated from 100 mg of corm tissue at 5 dpi using TRIZOL reagent (Invitrogen; Cat No. 15596026) according to the manufacturer’s protocol with some modifications. RNA samples were treated with DNase (Promega, USA Cat No. M6101) to remove DNA contamination. The purity and concentration of the total RNA was determined using spectrophometer (Thermo Scientific™ GENESYS™ 10S UV–Vis Spectrophotometer). RNA sample with ratio 1.8–2.0 was used for further study. cDNA was synthesized from 10 μg of the total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied biosystems Cat No. 4368814) following the manufacturer’s protocol and stored at − 20 °C for further use. Gene specific primers for RT-PCR (Table 1) were designed by using Primer Express (v3.0) software (Applied Biosystems®, USA) based on the sequences retrieved from National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov).

Table 1 Gene specific primers used in present study for quantitative RT-PCR expression
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Quantitative Real Time PCR (qPCR)

The RT-PCR was performed in 8-well strips using SYBR Green-based assay (Thermo-Scientific, Cat No. 4309155) on 7500 Real Time PCR System (Applied Biosystems® model). The 10 μl reaction mixture consisted of SYBR Green Master Mix (5 μl), cDNA template (1 μl) and gene specific primers (0.5 μM each; 0.5 μl forward primer and 0.5 μl reverse primer). The PCR program followed was; for holding stage 95 °C for 10 min, followed by 40 cycles of amplification with following thermal cycle: denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, and extension at 72 °C for 30 s. The relative quantification was done by 2−ΔΔCT method (Pfaffl 2006) and saffron tubulin gene was taken as an internal control for normalization.

Statistical Analysis

The experiment for plant growth promotion and disease severity was conducted using 10 biological replicates. All other experiments were conducted using five biological replicates and each biological replicate was evaluated based on three technical replicates. Results were expressed as mean ± standard deviation. Data were statistically analyzed by ANOVA using IBM SPSS statistics version 26. The Multiple Duncan range test was performed for analyzing differences between mean values at significant level (p < 0.05). All the experiments were replicated independently three times at three different time intervals.

Results

Comparison of In-Vitro Plant Growth Promotion Activities of Two Bacillus Species

Bar D5 and Bam W2 were evaluated for PGP activities under same conditions. Both produced phosphatase, siderophores, indole acetic acid, ammonia, protease, amylase and cellulase in-vitro; but production was higher in Bar D5, except for siderophores that was higher in Bam W2 (Table 2). Phosphate solubilization by Bar D5 was 482.68 µg ml−1, IAA production 213.15 µg ml−1, ammonia production 3.9 mg ml−1, amylase activity 30.8 IU ml−1 and cellulase activity was 42.3 IU ml−1. Whereas in Bam W2, phosphate solubilization was 22.31 µg ml−1, IAA production 139.84 µg ml−1, ammonia production 2.8 mg ml−1, amylase activity 11.2 IU ml−1 and cellulase activity was 30.0 IU ml−1. Siderophore production by Bam W2 was 30.1 μg ml−1 that is 1.5 times (56%) more than Bar D5. Protease enzyme activity was almost equal i.e., 5.6 IU ml−1 in Bar D5 and 5.7 IU ml−1 in Bam W2 (Table 2).

Table 2 Quantitative evaluation of in-vitro plant growth promoting activities of Bacillus sp. strain D5 and B. amyloliquefaciens W2 and in other reported Bacillus species
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Growth Parameters

The effects of Bam W2 and Bar D5 on the growth parameters of saffron plant in different treatments were monitored after 30 days of incubation. The results have been tabulated in Table 3. Both Bam W2 and Bar D5 increased the shoot number (1.3 ± 0.56, 1.6 ± 0.75 folds), shoot length (1.1 ± 0.03, 1.2 ± 0.07 folds), root number (1.0 ± 0.03, 1.2 ± 0.05 folds), root length (1.0 ± 0.02, 1.1 ± 0.05 folds), number of leaves (1 ± 0.08, 1.2 ± 0.03 folds), shoot dry matter percentage (1.0 ± 0.01, 1.2 ± 0.04 folds), and root dry matter percentage (1.0 ± 0.05, 1.3 ± 0.07 folds) as compared to untreated control (C). In pathogen treated corms (CF), the shoot number decreased by 0.7 ± 0.01 folds, shoot length by 0.9 ± 0.02 folds, root number by 1.0 ± 0.06 folds, root length by 1.0 ± 0.03 folds, number of leaves by 0.9 ± 0.08 folds, shoot dry matter percentage by 0.9 ± 0.09 folds, and root dry matter percentage by 0.8 ± 0.02 folds compared to untreated corms (C) (Table 3). The results indicated that both the strains promoted the growth of the saffron plant though Bar D5 showed better results as compared to Bam W2. As expected, in the presence of pathogen plant growth was reduced (Table 3).

Table 3 Effect of Bacillus species (Bar D5 and Bam W2) on the growth of saffron in presence and absence of pathogen (Fusraium oxysporum R1) in pot assays
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Disease Severity Index and Disease Reduction Percentage

Disease severity index and disease reduction percentage was calculated in all the treatments after 30 days post inoculation and has been tabulated in Table 4. Bar D5 and Bam W2 significantly reduced the disease severity in plants (T2 by 72.4% and T1 by 55.1%) as compared to pathogen control CF (Table 4). These results indicated that Bar D5 was more efficient as compared to Bam W2, in reducing the corm rot symptoms.

Table 4 Disease severity index and Disease reduction percentage of corm rot symptoms caused by Fusarium oxysporum R1 in saffron plant with different treatments in pot assays
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In-Planta Inhibition of Fox R1

Severe corm rot symptoms were observed in corms inoculated with Fox R1 (CF). Lesion diameter kept on increasing from 1 to 11 dpi i.e., maximum symptoms was observed at 11 dpi. In T1 (corms primed with Bam W2 and inoculated with Fox R1) diameter of lesion was less compared to CF. However, in T2 (corms primed with Bar D5 and inoculated with Fox R1) less symptoms of corm rot (less lesion diameter) were observed compared to CF and T1. In control, corms inoculated with sterile distilled water, no lesions were observed. The inhibition % has been calculated and tabulated in Table 5. The maximum lesion diameter in all the treatments were at 11 dpi but maximum inhibition was at 5 dpi in both T1 (40.8%) and T2 (60.9%) compared to CF (Table 5). Similar pattern of results were obtained in case of Fox R1 load determined by CFU method. The maximum load was at 11 dpi but maximum inhibition was at 5 dpi in T1 (Bam W2 treated), it was 48.7% and T2 (Bar D5 treated) it was 70.5% compared to CF, and then decrease in inhibition rate was observed. Although, the pattern of inhibition was same in both the assays, the actual value of inhibition % varied in both (Table 5).

Table 5 In-planta inhibition of Fox R1 by Bam W2 and Bar D5
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Dry Matter Accumulation in Corms

Corms were evaluated for the accumulation of dry matter in the presence and absence of Bacillus species and pathogen up to 11 dpi. At 0 dpi, Bacillus primed corms (S2 and S3) accumulated 1.17 ± 0.05 and 1.23 ± 0.02 fold more dry matter, respectively, compared to mock primed corms (S1). At 1, 3, 5, 8, and 11 dpi, maximum biomass accumulation were in B2 (Bar D5) and B1 (Bam W2) primed corms followed by T2 and T1 (Bar D5 and Bam W2 primed and Fox R1 inoculated corms) compared to untreated corms (Fig. 2), whereas in CF there was significant folds decrease in biomass i.e., 1.07 ± 0.08 (1 dpi), 1.09 ± 0.03 (3 dpi), 1.12 ± 0.06 (5 dpi), 1.13 ± 0.05 (8 dpi), 1.15 ± 0.07 (11 dpi) compared to C (Fig. 2).

Fig. 2
figure 2

The dry matter percentage accumulation in saffron corms with different treatments at different days post inoculations (1, 3, 5, 8 and 11 days post inoculation). C-Mock primed corms, CF-Fox R1 inoculated corms, T1-Bam W2 primed corms and Fox R1 inoculated corms, T2-Bar D5 primed and Fox R1 inoculated corms, B1-Bam W2 primed corms only and B2-Bar D5 primed corms only. Bar D5-Bacillus sp. strain D5, Bam W2-Bacillus amyloliquefaciens W2, and Fox R1-Fusarium oxysporum R1. Error bars represent the standard deviation (SD). ANOVA was performed at significant level (p < 0.05). Means with same superscript letters (within each day) indicate no significant difference between according to the multiple Duncan test (p < 0.05)

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Plant Defense Related Enzymes Activity

The activity of plant defense related enzymes PAL, PO, PPO, LOX, and GLU was quantified in corm tissues of saffron plant, as per experimental design shown in Fig. 1 at 1, 3, 5, 8, and 11 dpi. The activities of all defense enzymes increased rapidly after inoculation of Fox R1 and maximum activities were observed at 5 dpi in all the treatments (Fig. 3a–e, Table 6) then it was found to be decreased at 8 and 11 dpi. Among different treatments, T2 showed the maximum activities of all the tested enzymes at 5 dpi i.e., PAL expression (2.8 ± 0.66 folds), PO (6 ± 0.47 folds), PPO (5 ± 1.1 folds), LOX (3.4 ± 0.71 folds), and GLU (3.4 ± 0.31 folds) increase as compared to C; whereas in T1, PAL (2.2 ± 0.37 folds), PO (2.58 ± 0.20 folds), PPO (3.5 ± 0.57 folds), LOX (2.2 ± 0.41 folds), and GLU (2.1 ± 0.58 folds), increase compared to C. To summarize, induction of all defense enzymes estimated that the activity was highest in T2 (Bar D5 + Fox R1), followed by T1 (Bam W2 + Fox R1) and last in CF (corms challenged with Fox R1 alone) and C (basal activity in corms without infection) (Fig. 3a–e).

Fig. 3
figure 3

The activities of different defense related enzymes. a Phenylalanine ammonia lyase (PAL), b peroxidase (PO), c β-1,3 glucanase (GLU), d Lipoxygenase (LOX), e polyphenol oxidase (PPO) in saffron corm with different treatments at different days post inoculations (1, 3, 5, 8, and 11 days post inoculation). C-Mock primed corms, CF-Fox R1 inoculated corms, T1-Bam W2 primed and Fox R1 inoculated corms, T2-Bar D5 primed and Fox R1 inoculated corms, B1-Bam W2 primed corms only and B2-Bar D5 primed corms only. Bar D5-Bacillus sp. strain D5, Bam W2-Bacillus amyloliquefaciens W2 and Fox R1-Fusarium oxysporum R1. Error bars represent the standard deviation (SD). ANOVA was performed at significant level (p < 0.05). Means with same superscript letters (within each day) indicate no significant difference between according to the multiple Duncan test (p < 0.05)

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Table 6 In-planta induction of defense enzymes [fold increase compared to untreated corms (control)] and maximum activity (dpi) of Bar D5 and Bam W2 and other reported Bacillus species
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Plant Defense Enzyme Related Genes Expression

Differential expression of genes, encoding above defense related enzymes were studied by qPCR at 5 days post inoculation (5 dpi) as the maximum enzyme activity was observed at 5 dpi. The pattern of expression of these genes was in tune with the enzyme production. Among all the treatments the maximum expression was observed in T2 (Bar D5 + Fox R1) i.e., priming with Bar D5 significantly increased the transcription of defense related enzymes when challenged with Fox R1. Peroxidase gene was highest expressed gene with 10.1 ± 0.95 folds increase, followed by PPO (8.1 ± 0.62 folds), GLU (4.9 ± 0.30 folds), PAL (4.5 ± 0.11 folds) and LOX (3.5 ± 0.32 folds) as compared to C (mock primed corms). In T1 (Bam W2 + Fox R1) as well, peroxidase was highest expressed gene with 5.6 ± 0.32 fold increase followed by PPO with 5 ± 0.43 folds, GLU with 3.4 ± 0.35 folds, PAL with 3.6 ± 0.2 folds, and LOX with 3 ± 0.62 folds increase as compared to C (mock primed corms). In CF (corm challenged with pathogen) the activity fold increase was 4.1 ± 0.3 folds in PO, 3.4 ± 0.35 folds in PPO, 2.3 ± 0.26 folds GLU in 3.1 ± 0.41 folds in PAL and 2.6 ± 0.87 folds in LOX as compared to C (Fig. 4). A comparison of fold change of activity and expression of all the 5 enzymes at 5 dpi has been tabulated in Table 7.

Fig. 4
figure 4

Quantitative reverse transcriptase PCR expression of defense related genes, Phenylalanine ammonia lyase (PAL), Peroxidase (PO), Polyphenol oxidase (PPO), Lipoxygenase (LOX), β-1,3 glucanase (GLU) in saffron corm at day 5 post inoculation in different treatments. C-Mock primed corms, CF-Fox R1 inoculated corms, T1-Bam W2 primed and Fox R1 inoculated corms, T2-Bar D5 primed and Fox R1 inoculated corms, B1-Bam W2 primed corms only and B2-Bar D5 primed corms only. Bar D5-Bacillus sp. strain D5, Bam W2-Bacillus amyloliquefaciens W2 and Fox R1-Fusarium oxysporum R1. Error bars represent the standard deviation (SD). ANOVA was performed at significant level (p < 0.05). Means with same superscript letters (within each enzyme) indicate no significant difference between according to the multiple Duncan test (P < 0.05)

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Table 7 Comparative fold of defense related activities and gene expression of different enzymes in saffron corms treated with bacilli challenged with pathogen Fusarium oxysporum R1
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Discussion

Plant growth promoting bacteria (PGPB) can promote plant growth directly and/or indirectly (Batista et al. 2021; Danesh et al. 2021). The direct promotion is by phosphate solubilization, nitrogen fixation, siderophore production, enhancement of root proliferation for mineral uptake from soil and by the production of various phytohormones (Enebe and Babalola 2019; Bhattacharyya et al. 2020). Among rhizospheric bacteria, Bacillus species are the most common and commercialized PGPB. They are also most commercialized biological control agents, against various phytopathogens, due to their direct and indirect role in plant growth promotion and defense (Shafi et al. 2017; Timmusk et al. 2017). In addition, Bacillus species also possesses faster replication rate, root colonization ability and longer field life, on account of the spore formation (Aloo et al. 2019). Direct biological control ability of Bacillus species is a result of production of various antibiotics and extracellular enzymes that directly inhibit the growth of pathogens and indirect biocontrol activity is by induction of host plant defense system (Sahu et al. 2019; Bhusal and Mmbaga 2020; Vishwanathan et al. 2020). Bacillus species has been previously reported as PGPB in saffron (Gupta and Vakhlu 2015; Kour et al. 2018; Magotra et al. 2021), tomato (Abdallah et al. 2018; Masmoudi et al. 2021), sugarcane (Xia et al. 2020), rice (Kumar et al. 2021), wheat (Rashid et al. 2021a, b), maize (De Sousa et al. 2021) etc.

Native Bacillus sp. strain D5 isolated from cormosphere (corm sheath) of saffron corm (Magotra et al. 2021) and B. amyloliquefaciens W2 from bulk soil of saffron, have been isolated and characterized as PGPB earlier by our group (Gupta and Vakhlu 2015). These Bacillus species solubilize phosphate, produce siderophores, ammonia and various other enzymes in-vitro (Table 2). Amongst the two Bacillus strains, Bar D5 exhibited higher in-vitro PGP activities, except for siderophore production, that was 1.9-folds higher in Bam W2. Various Bacillus species are reported to possess these activities that have been tabulated in Table 2. The Phosphate solubilization activity of bacteria (PGPB) results in the uptake of phosphorous from the soil that is an important element for the growth of plants (Asril et al. 2021). Siderophores are the low molecular weight iron chelating compounds that help in the uptake of iron from the soil that is vital for plant growth and renders them less available to the pathogenic organism present in the soil (Sultana et al. 2021). Plant growth promoting bacteria are known to produce many plant hormones that play major role in the many developmental stages, of the plant’s life cycle. In the present study, both the Bacillus species produced IAA that is important for plant growth and development and modifies root structure for better absorption of nutrients (Ismail et al. 2021). Ammonia production is another strategy used by PGPB that helps in increasing the fresh weight of the plant as well as the shoots and roots (Ismail et al. 2021).

In pot assays, as well, both the Bacillus species significantly increased the growth of the saffron corms, as compared to untreated corms. In addition, both reduce the disease severity, as compared to pathogen control (Tables 3, 4). Both the Bacillus inoculated plants had growth due to the PGP properties possessed by Bar D5 and Bam W2, as they make the unavailable nutrients present in the soil available to plants. As reported earlier, Bam W2 significantly inhibits increased the growth of Fox R1 by 40% in in-vitro plate assays and disease by 57.1% in pot assays (Gupta and Vakhlu 2015). Bar D5 significantly increased the growth of the saffron plant in pot trials compared to untreated control and also reduced the disease incidence by 71.4% compared to pathogen control (Magotra et al. 2021). Sharaf-Eldin et al. (2008) have reported that the commercially available Bacillus subtilis (FZB24) significantly increased the leaf length, stigma weight, crocin, picrocrocin, and safranal content in saffron and reduced the sprouting time in saffron corms, as compared to untreated control (Sharaf-Eldin 2008). B. aryabhattai SRB02 from the soyabean rhizosphere, is reported to enhance root and shoot length of the plant, in pots by the production of various phytohormones (Park et al. 2017). Abdallah et al. (2018) reported Bacillus amyloliquefaciens subsp. Plantarum 32A significantly increased the germination level and growth of the tomato plant and reduced the symptoms of crown gall disease in the root of the tomato plant, in pot assays. Bacillus xiamenensis PM14 from sugarcane rhizosphere increased the fresh weight (30%), plant length (5%), cane length (27.5%), root length (37%), and reduced red rot disease incidence, in greenhouse experiments (Xia et al. 2020). In the present study, Bam W2 and Bar D5 significantly reduced the disease severity in the saffron plant (T1 by 55.1% and T2 by 72.4%) as compared to pathogen control (CF). Similar to the present study, Gupta et al. (2021) have reported that different biocontrol agents such as Tricoderma asperellium (68%), Bacillus subtilis (42.9%), Bacillus pumilus (20.6%), and Bacillus straptosphericus (14.2%) inhibits the growth of Fusarium oxysporum and reduce the disease incidence of corm rot in saffron.

To evaluate the potential of Bar D5 over Bam W2 as a biostimulant, the experiments were conducted in-planta in pot assays. One of the direct ways to monitor plant growth is to measure the effect of the biostimulant on biomass accumulation. Various species of Bacillus are known to increase the dry matter in crop plants (Kang et al. 2014). Though the experiments were initiated with corms of similar weight (~ 10 g) after 30 days of incubation with both the biostimulants, there was a clear indication of an increase in biomass in corms primed with Bar D5 and Bam W2 as compared to mock primed corms. Bam W2 (B1) and Bar D5 (B2) primed corms had maximum biomass accumulation in comparison to mock primed corms (C) (Fig. 2). However, Bam W2 and Bar D5 primed corms after challenged with Fox R1 (T1 and T2) accumulated less biomass than B1 and B2 but more than mock primed corms (C) and pathogen inoculated corms (CF) up to 11 dpi (Fig. 2). The order of biomass accumulation among different treatments was B2 > B1 > T2 > T1 > C > CF. This result indicates that even after infection both the biostimulant help plant to accumulate biomass. It is known fact that as pathogen invades the plant, it channels its energy/resources towards defense, similar results were observed in present case as well. Before infection with the pathogenic Fox R1, the primed corms with Bar D5 and Bam W2 had similar biomass on day zero but at 11 dpi, the accumulation was less in pathogen infected corms as compared to the corms that were not infected (Fig. 2). The decrease in biomass accumulation in bacilli primed, pathogen inoculated corms could be a result of stress caused by the pathogen. It was concluded that both Bar D5 and Bam W2 stimulate biomass accumulation, however upon infection, the accumulation is decreased relatively. Further, pathogen severely affects the biomass accumulation as uninfected corm accumulates more biomass in comparison to infected corm (Fig. 2). Similarly, Bacillus subtilis FZB24® has been reported to significantly increase the biomass and yield in the saffron plant (Sharaf-Eldin et al. 2008), though in this study no disease or pathogen was studied. B. simplex has been reported to increased the yield of kiwifruit (Erturk et al. 2010), B. subtilis ALB629 enhances the dry matter in cacao (Falcäo et al. 2014) and B. megaterium mj1212 increased the fresh weight in mustard plants (Kang et al. 2014).

In the present study, both Bar D5 and Bam W2 induced production of defense related enzymes by increased expression of the genes encoding these enzymes in the presence of Fox R1 (Figs. 3a–e, 4). The maximum activity of defense related enzymes was in T2 (Bar D5 primed and Fox R1 inoculated corms) followed by T1 (Bam W2 primed and Fox R1 inoculated corms) as compared to pathogen inoculated corm (CF) and mock primed corm (C) at 5 dpi. Biocontrol agents inhibit the pathogen attack either by direct inhibition, resource scavenging or by induced systemic resistance (Charpe et al. 2019; Palani et al. 2016). ISR is the response of a plant to pathogen invasion, by enhanced production of defense related enzymes, that is augmented many fold by PGPB (Borris et al. 2019; Chandrashekaran et al. 2017). These PGPB makes host plant more tolerant to pathogens attack. Except for B. amyloliquefaciens (FZB24) against Fusarium wilt in tomato plant (Elanchezhiyan et al. 2018) and B. velezensis F21 in watermelon against Fusarium oxysporum f. sp. niveum (Fon) (Jiang et al. 2019), most of the studies have reported maximum activity of defense related enzymes at 5 dpi (Table 6). The production of defense enzymes by Bar D5 was more than Bam W2 (Fig. 3a–e). These defense enzymes are effective in controlling the disease cause by various pathogens. These enzymes are induced by Bacillus species in various plants but their production increases after challenge with pathogens (Table 6). These enzymes are present in plants at the basal level, at a given point of time but get enhanced on interaction with both pathogen and/or biocontrol (Chandrashekaran and Chun 2016). However, different kind of responses has been reported in different plant. The comprehensive data has been tabulated in Table 6.

PAL is an enzyme of the phenylpropanoid pathway that converts L-phenylalanine to trans-cinnamic acid contributing to the production of secondary metabolites, including lignins, flavonoids, coumarins and jasmonate resulting in plant cell wall lignifications (Astaneh et al. 2018; Garces-Fiallos et al. 2022). Similarly, PPOs produce quinones from ortho-diphenolic compounds that are highly reactive and by using molecular oxygen they cross link the proteins in the cell, creating a protein deficient environment for the pathogens (Taranto et al. 2017). Likewise, reactive oxygen species produced by plants as first line of defense against pathogens and are detoxified by enzyme PO, which is reported to be involved in cell wall lignifications and defense against oxidative stress (Hanaka et al. 2018; Thiebaut et al. 2022). Furthermore, β-1,3-glucanases are the lytic enzymes that hydrolyze the fungal cell wall components, β-1,3-glucan (Xu et al. 2016; Ye et al. 2023). Moreover, LOXs are non-heme dioxygenases that produce unsaturated fatty acid hydroperoxides, such as oxylipins, (from polyunsaturated fatty acids by adding molecular oxygen) and oxylipins have antimicrobial activity hence are involved in plant defense (Babenko et al. 2017; Gonzalez-Gordo et al. 2022).

In the present study, control corms have some basal level of these enzymes production, but once challenged with the pathogen, the production of enzyme increased. There is a clear indication of some cross talk between all the three participants that is plant-Bacillus-pathogen. Similar results have also been reported by (Elanchezhiyan et al. 2018 and Jiang et al. 2019; Sadeghpour et al. 2022; Hussain et al. 2023), but the underlying molecular mechanism and signaling pathway in saffron is a matter of further investigation. One underlying response in all the cases was that biocontrol agents and pathogens induce systemic response, but the magnitude of defense response varies in different plants. In certain reports, the induction of defense genes was highest in biocontrol treated plants alone (Chandrashekaran et al. 2017; Jiang et al. 2019) and in other plants the highest activity was in combination of biocontrol and pathogen (Elanchezhiyan et al. 2018 and Gowtham et al. 2018; Naz et al. 2021). This is the first report of pathogen-biocontrol interaction and induction of defense response in saffron.

The defense enzyme production profile was complemented with analysis of real time expression of the genes encoding them, by qPCR at 5 dpi only because the defense enzyme production was observed maximum at 5 dpi. Similar to results of defense enzyme production, maximum expression was found in corms primed with Bar D5 and inoculated with Fox R1 (T2) followed by Bam W2 primed and Fox R1 inoculated corms (T1) compare to CF, C, B1 and B2. The gene expression profiles for these defense enzymes were in sync with the enzyme production profiles, though the fold increase in expression of transcripts was more than that of enzymes (Table 7). Chandrashekaran et al. (2017) have reported maximum expression at 4 dpi of β,1-3 glucanase and PAL gene, in the tomato plant treated with B. subtilis CBR05 and challenged with Erwinia carotovora sub sp. carotovora (Chandrashekaran et al. 2017). In another study, B. subtilis CBR05 significantly induced the activity and expression of GLU and PAL enzymes, in tomato plant against Xanthomonas campestrispv versicatoria (XCV) and maximum activity and expression were at 72 h (Chandrashekaran et al. 2017). Interestingly, among all the defense enzymes studied in the present study, peroxidase (PO) was expressed maximum both at translational and transcriptional levels. Peroxidase is reported to detoxify reactive oxygen species generated during stress, therefore reactive burst is the major defense during stress conditions in many plants (Bhattacharjee 2019), which could be the probable reason of defense against Fox R1 in Crocus sativus as well.

In the present study, there was significant inhibition of Fox R1 in-planta by Bam W2 and Bar D5 (Table 5). However, Bar D5 substantially reduced the load of Fox R1 as compared to Bam W2. The maximum inhibition was observed at 5 dpi and then the inhibition rate decreased at 8 and 11 dpi. The maximum inhibition at 5 dpi can be correlated with the maximum activity of defense related enzymes at 5 dpi. In lesion diameter method the inhibition % was (40.8 ± 2.79 in T1 and 60.9 ± 2.04 in T2) and in CFU method the inhibition % was (48.7 ± 2.03 in T1 and 70.5 ± 1.37 in T2) (Table 5). As the activity of enzymes decreased, there was a sudden rise in Fox R1 load. Similarly, bacterium Rhodopseudomonas palustris strain GJ-22 in-planta inhibited the growth of pathogen Phytophthora Infestans causing late blight in potato by 75% at 5 dpi (Zhang et al. 2020). Murolo et al. (2019) have checked the effect of three bioformulation in-planta against Cryphonectria parasitica, causing chestnut blight disease in sweet chestnut. In the co-inoculation method, B. subtilis reduced the necrotic areas by > 70% and in dual inoculations, B. subtilis reduced the size of cankers on chestnut stems (29–67%), Trichoderma spp. (36–65%) and Glomus spp. (31%–63%) at 15 dpi (Murolo et al. 2019). In the present study, it can be concluded that based on in-vitro PGP activities, pot assays for plant growth promotion, induction of defense related enzyme activity and expression and inhibition of Fox R1, Bar D5 has the potential to be used as a commercial PGP and biocontrol agent against Fusarium oxysporum R1 pathogen causing corm rot of saffron. However, commercialization of this Bacillus strain as a biostimulant or biocontrol will need standardization of formation methods for its large scale production and toxicological studies to certify it as safe to use.

Conclusion

Since replacement and/or complementation of chemical fertilizers by biological agents is the need of the hour, and in the case of saffron chemical augmentation is not effective anymore, as the yield is on decline year after year. Despite the availability of chemical fungicides, corm rot caused by Fusarium oxysporum is a major threat to saffron production world over. Based on comparison of in-vitro activity, in-planta pot assay for growth parameters, dry matter accumulation, defense enzyme production and expression of the corresponding genes, Bar D5 comes out to be the most suited biostimulant, in comparison to Bam W2. It has a clear advantage for its use as a bioinoculant for Crocus sativus due to its nativeness and its biocontrol activity through ISR. However, the actual molecular cross talk between the Bacillus-corm-pathogen and its signaling needs further investigation. Consequently, it may be evaluated for plant growth promotion and induction of systemic resistance against fungal pathogens, in other plants as well.