Introduction

Soybean (Glycine max L.) is an important legume crop and an excellent source of oil and protein (Singh et al. 2007). Macrophomina phaseolina causes charcoal rot root and seedling blight, affecting more than 500 crop and non-crop species, especially prevalent in the arid regions of the world (Kunwar et al. 1986). Annual losses of 30–50% have been reported in soybean due to charcoal rot (Yang and Navi 2005). Khan (2007) demonstrated decrease in stem diameter and root weight due to lesions caused by charcoal rot on taproot and basal portion of the stem of soybean plants measured during reproductive stage. Fungus overwinters in dry soil on residue of host plants and contaminate the seed. The sclerotia germinate on the surface of roots and form germ tubes that cause infection. In the early stages of pathogenesis, the mycelium penetrates the root epidermis of 7–42 days old soybean plants and is particularly confined to the inter-cellular spaces of the cortex of the primary roots (Gupta et al. 2012). Microsclerotia of M. phaseolina have been usually observed in the epidermis, inside taproots and lower stems of wilted plants. The earliest symptoms of charcoal rot include leaf rolling, reduction in leaf size and wilting of leaves (Hershman 2011).

The fungicides used to kill the M. phaseolina cause adverse effects on both aquatic and terrestrial ecosystems (Wightwick et al. 2010). Biological control offers ecofriendly approach to combat diseases. Plant growth promoting rhizobacteria act as biocontrol agent through production of siderophores, hydrogen cyanide, antibiotics, inhibits fungal growth through production of fungal cell wall lysing enzymes and also decreases shelf life of the soil borne pathogens (Beneduzi et al. 2012). PGPR help to elicit induced systemic resistance (ISR) with enhanced defensive response exhibited by host plant (Sangeetha et al. 2010). Enzymes responsible for inducing resistance comprise of chitinase, lipoxygenase, peroxidase, superoxide dismutase and catalase among others. These defense-related enzymes represent an important protective system for plants against pathogenesis (Singh et al. 2013). Various bacterial strains e.g. Pseudomonas putida, Enterobacter cloacae, Acinetobacter calcoaceticus, Ochrobactrum haematophilum, Bacillus amyloliquefaciens and Bacillus cereus have been reported to possess antagonistic activity against pathogenic fungi in soybean plants (Zhao et al. 2018).

Soil microbial communities maintain the fertility of soil by mediating both synthesis and decomposition of soil organic matter and therefore, influence cation exchange capacity, soil N, P and S reserve, soil acidity and soil water holding capacity. Plant growth promoting rhizobacteria participate in decomposition, mineralization, phosphate solubilization and nutrient availability and thus influence the efficiency of nutrient cycles (Singh et al. 2011).

The present study was aimed to evaluate physiological mechanism mediated by P. putida and P. stutzeri inoculations on the control of charcoal rot disease caused by Macrophomina phaseolina and further to demonstrate their effects on soil physicochemical properties.

Materials and methods

Isolation of antagonistic bacteria

Post harvested rhizospheric soil (1 g) was collected from the rhizosphere of soybean plants grown in the fields of National Agriculture Research Centre (NARC), Islamabad and suspended in 10 ml autoclaved water. The suspension was stirred for 1 h with magnetic stirrer and centrifuged for 15 min at 3000 rpm. The supernatant was diluted using decimal dilution technique. An aliquot (20 μl) of 107 dilution was spread on the Petriplate containing Luria-Bertani (LB) medium. The bacterial colonies appearing in the plates were further streaked to get the pure colony (Vidhyasekaran and Muthamilan 1995).

Fungal culture

The M. phaseolina isolate used in this study was provided by Dr. Shahzad Asad (Crop Disease Research Institute (CDRI), NARC, Islamabad). The pathogen was isolated from infected roots of sunflower. Macrophomina phaseolina (isolate MP5) was cultured on potato dextrose agar (PDA) plates and incubated at 30 °C for 4–5 days to obtain the mycelium.

Quantification of metabolites produced by PGPR

PGPR metabolites viz. protease, catalase and hydrogen cyanide (HCN) produced by P. stutzeri (KX574858) and P. putida (KX574857) were quantified by growing them in respective medium as described below:

Protease activity

The bacteria were allowed to grow in LB broth and cell free supernatant was obtained. The supernatant was precipitated by adding 50–55% ammonium sulphate and precipitate was used as crude enzyme extract. The crude enzyme extract and substrate (1.0% casein) solution were mixed in 1:1 (v/v) ratio and incubated at 40 °C for 20 min. The reaction was stopped by adding 3 ml of 10% tri-chloroacetic acid. Absorbance was measured at 280 nm and quantified by comparing the standard curve drawn between the absorbance and concentration of tyrosine. The activity was expressed as amount of the enzyme that released 1μg of tyrosine ml−1 min−1 under the assay conditions (Yang and Huang 1994).

Catalase activity

The catalase activity was evaluated by adding 0.2 ml of cell extracts to 1.4 ml of freshly prepared 13.2 mM H2O2 in 0.05 M K2HPO4 (pH 7.0). Absorbance was observed at 240 nm by recording optical density (OD) for 1 to 3 min (Pine et al. 1984).

HCN production

Bacteria were grown in Kings B broth amended with glycine (4.4 gl−1). Uniform strips of filter paper were soaked in alkaline picrate solution and kept hanging inside the conical flask. After incubation at 28 ± 2 °C for 48 h, the sodium picrate in the filter paper was reduced to a reddish compound in proportion to the amount of HCN evolved. The colour was eluted by placing the filter paper in a test tube containing 10 ml of distilled water and its absorbance was read at 625 nm (Sadasivam and Manickam 1992).

Antifungal activity of rhizobacteria

Antifungal activities of P. stutzeri and P. putida were evaluated by using the agar tube dilution method (Sutter et al. 1979). Screw capped test tubes containing 10 ml solution of potato dextrose agar (PDA) were autoclaved for 21 min at 121 °C. Test tubes were then allowed to cool at 50 °C and PDA was loaded with 67 μl of bacterial broth. The test tube contents were solidified at room temperature in slanting position. Test tubes having solidified media containing bacterial broth of different dilutions were inoculated with 4 mm piece of fungal inoculum. One plugged test tube having DMSO was used as positive control. Test tube without bacterial broth was taken as negative control. The incubation was done for 7 days at 28 °C. Reading was noted after 7 days by measuring linear length (mm) of the fungus in slant and inhibition of fungal growth was calculated with reference to negative control.

$$ \mathrm{Percent}\ \mathrm{inhibition}\ \mathrm{of}\ \mathrm{mycelial}\ \mathrm{growth}=100-\frac{\mathrm{Linear}\ \mathrm{growth}\mathrm{in}\ \mathrm{test}\ \left(\mathrm{mm}\right)}{\mathrm{Linear}\ \mathrm{growth}\mathrm{in}\ \mathrm{control}\ \left(\mathrm{mm}\right)}\times 100 $$

Plant materials

Seeds of two soybean varieties; cvv. Ajmeri and NARC were obtained from Crop Disease Research Institute (CDRI), National Agricultural Research Centre, Islamabad.

Preparation of bacterial inocula and seed treatment

The antagonistic bacteria P. stutzeri and P. putida were cultured in nutrient broth on a shaker incubator (ECELLA E23, USA) at 120 rpm for 24 h at 28 °C. The bacterial cultures were centrifuged at 6000 rpm for 15 min and re suspended in phosphate buffer (pH 7.4). The supernatant was discarded and the bacterial pellet was diluted with distilled water and its optical density (OD) was adjusted to 1 at 600 nm (Thompson et al. 1996). Surface sterilization of seeds were performed with 0.1% HgCl2 and successively washed with double distilled water. Soybean seeds were soaked in bacterial inoculum for 3–4 h. The seeds soaked in autoclaved distilled water served as control.

Disease induction

Inocula of M. phaseolina was prepared following the method of Doubledee et al. (2018). Sorghum seeds (140 g) were soaked in distilled water overnight and autoclaved for 20 min at 121 °C and 15 psi pressure. Fresh culture of M. phaseolina (100 microsclerotia per gram dry weight of soil) was mixed with autoclaved sorghum seeds and kept in incubator for 14 days at 30 °C. These inoculated sorghum seeds were then mixed in the soil, 4 h before sowing soybean seeds. The population of M. phaseolina was recorded as 1.2 × 107 colony forming units per gram (CFU g−1) of sorghum using dilution plate technique (Mihail and Taylor 1995).

Experiment design

The experiment was conducted in greenhouse under control conditions. Soybean seeds planted in plastic pots (25 × 40 cm2), containing sterilized soil and sand (3:1) were allowed to grow in greenhouse at 20/25 °C (night/day) during March at 50%–60% relative humidity for 14 h. Treatments were made as; untreated un-inoculated control plants (T1), seeds inoculated with P. stutzeri (T2), seeds inoculated with P. putida (T3), seeds inoculated with P. stutzeri and soil infested with M. phaseolina (T4), seeds inoculated with P. putida and soil infested with M. phaseolina (T5) and soil infested with M. phaseolina (T6). The experiment was repeated thrice. Three replications were used per treatment and each replicate consisted of four plants per pot.

Physicochemical characteristics of soil

Electrical conductivity (EC) and total dissolved solids (TDS)

The EC and TDS were analyzed following the methods of Corwin and Yemoto (2017).

Soil organic matter and field capacity

Soil organic matter provides nutrients and maintains the soil structure and field capacity that changes under biotic stresses and PGPR treatments (Singh 2011). Soil organic matter content and field capacity of rhizospheric soil were calculated following the method of Walkley and Black (1934) and Staple and Lehane (1962) respectively.

Nutrient contents of soil sample

The nutrients balance was disturbed due to PGPR and pathogen attack (Jamal et al. 2018). Hence the nutrient contents were determined following the atomic absorption spectrometer using microwave-assisted digestion method developed by Kingston et al. (1998).

Plant growth parameters

Germination index (GI)

The number of germinating seeds were counted for seven days at a regular interval of 24 h. The germination index was evaluated by using the following formula (Khan and Ungar 1996).

$$ \mathrm{Germination}\ \mathrm{index}=\frac{\mathrm{No}.\mathrm{of}\ \mathrm{seeds}\ \mathrm{germinated}\ }{\mathrm{Days}\ \mathrm{of}\ \mathrm{first}\ \mathrm{count}}+\frac{\mathrm{No}.\mathrm{of}\ \mathrm{seeds}\kern0.5em \mathrm{germinated}\ }{\mathrm{Days}\ \mathrm{of}\ \mathrm{second}\ \mathrm{count}}+\dots +\frac{\mathrm{No}.\mathrm{of}\ \mathrm{seeds}\ \mathrm{germinated}\ }{\mathrm{Days}\ \mathrm{of}\ \mathrm{final}\ \mathrm{count}} $$

Fresh weight and dry weight estimation

Fresh weight was obtained by weighing the fresh harvested plant sample. Plant samples were heated in an oven for 72 h at 80 °C for dry weight estimation (Godbold and Huttermann 1988).

Estimation of chlorophyll content

Chlorophyll content was determined by chlorophyll meter (SPAD 502 Plus Konica, Minolta SENSING, INC, JAPAN) from fully expanded mature leaf (Markwell et al. 1995).

Assessment of disease intensity

The disease incidence and disease severity index were recorded on 90th day of sowing.

Disease incidence (DI)

The disease incidence was recorded following the formula given by Groth et al. (1999).

$$ \mathrm{Disease}\ \mathrm{Incidence}=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{infected}\ \mathrm{plants}\ }{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{plants}\ \mathrm{assessed}} \times 100 $$

Disease severity index (DSI)

The disease severity index was calculated using the formula of Chiang et al. (2017). The DSI ranges from 0 to 11 point scale (Where 0 = no symptom; 1 = 0 to 3%, 2 = 3 to 6%, 3 = 6 to 12%, 4 = 12 to 25%, 5 = 25 to 50%, 6 = 50 to 75%, 7 = 75 to 87%, 8 = 87 to 94%, 9 = 94 to 97%, 10 = 97 to 100%, and 11 = 100%) (Mengistu et al. 2007).

$$ \mathrm{Disease}\ \mathrm{Severity}\ \mathrm{Index}=\frac{\mathrm{Sum}\ \mathrm{of}\ \mathrm{in}\mathrm{dividual}\ \mathrm{numerical}\ \mathrm{rating}}{\mathrm{Total}\ \mathrm{Number}\ \mathrm{of}\ \mathrm{Plants}\ \mathrm{assessed}\times \mathrm{Maximum}\ \mathrm{score}\ \mathrm{in}\ \mathrm{scale}}\times 100 $$

Assay of defense-related enzymes

The changes in defense-related enzymes such as polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were determined by measuring their activities (Ganeshamoorthi et al. 2008).

Polyphenol oxidase activity (PPO)

Fresh leaves (0.5 g) were homogenized in 4 ml chilled phosphate buffer (pH 6.8). Homogenate was centrifuged at 10,000 rpm for 20 min at 4 °C. The PPO activity was determined by incubating a reaction mixture containing 500 μl of enzyme, 1500 μl of assay buffer and 50 mM pyrogallol at 30 °C for 5 min. The reaction was stopped by adding 500 μl of 5% sulphuric acid. The absorbance of purpurogallin formed was recorded at 420 nm using an ultraviolet spectrophotometer (U-1500 Spectrophotometer HITACHI Germany). The PPO activity was expressed as change in absorbance of 0.1 min−1 mg−1 of protein (Meyer et al. 1974).

Phenylalanine ammonia-lyase activity (PAL)

Fresh leaves (1 g) were ground at 4 °C in 5 ml of 0.1 M sodium borate buffer (pH 8.8). Homogenate was centrifuged at 4 °C and the resultant supernatant was used as enzyme extract. Enzyme extract (200 μl) was incubated with 0.5 ml of 0.02 M phenylalanine and 2 ml of boric acid buffer at 30 °C for 30 min. The reaction was stopped by addition of 200 μl hydrochloric acid (HCl) and the absorbance of reaction mixture was measured at 290 nm. The PAL activity was expressed as nmol of cinnamic acid produced min−1 mg−1 of protein (Whetten and Sederoff 1992).

Assay of antioxidant enzymes

Antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) that change significantly during biotic stresses and PGPR treatments were estimated by measuring their activities (Rais et al. 2017).

Superoxide dismutase activity (SOD)

The plant tissue (0.2 g) was pulverized at 4 °C in 4 ml of phosphate buffer (pH 7.8) solution containing 1 g polyvinylpyrrolidone (PVP) and 0.0278 g Na2EDTA in 100 ml phosphate buffer (pH 7.8). Homogenate was centrifuged at 4 °C for 10 min and the volume of the supernatant was adjusted to 8 ml. The SOD was calculated by modifying the protocol of Beauchamp and Fridovich (1971). The absorbance was recorded at 560 nm against the blank (phosphate buffer pH 7). One unit enzyme activity (U) was defined as the amount of SOD, which reduced the absorbance by 50% as compared to the control (lacking enzyme). The SOD activity was expressed as U mg−1 of protein.

Peroxidase activity activity (POD)

Fresh leaves (1 g) were macerated using 5 ml of ice cold calcium chloride (0.5 M) solution. The mixture was centrifuged at 1000 x gravity for 8 min. Reaction mixture comprised of 0.1 ml enzyme extract, 1.4 ml MES, 0.5 ml p-Phenylenediamine and 0.45 ml of H2O2. The POD activity was calculated according to the protocol described by Van Assche et al. (1988). The difference in initial and final absorbance readings was observed at 510 nm in spectrophotometer for 2 min.

Catalase activity (CAT)

Fresh leaves (0.5 g) were homogenized in 2 ml of 50 mM potassium phosphate buffer (pH 7.0) and 2% polyvinylpolypyrrolidone. The resulting lysate was centrifuged at 3000 rpm for 10 min. Catalase activity was estimated by modifying the protocol of Aebi (1984). The reaction mixture (0.2 ml enzyme extract +2.7 ml reaction water +0.3 ml of 0.1 M H2O2) was incubated in a water bath for 30 min. The enzyme activity was determined by monitoring the decrease in absorbance at 240 nm due to the consumption of H2O2.

Determination of proline content

Proline is an osmoprotectant in plants and has significant roles in cell signaling and energy production (Liang et al. 2013). Proline was increased in response to infection and was analyzed following the procedure of Bates et al. (1973). Reaction mixture comprised of 2 ml of enzyme extract, 2 ml of glacial acetic acid and 1 ml of ninhydrin. Reaction mixture was heated in a water bath at 100 °C for 1 h until brick red color developed. 4 ml of toluene was added after cooling the reaction mixture and then transferred to separating funnel. The absorbance was read at 520 nm in spectrophotometer against toluene blank.

Estimation of total soluble proteins

Proteins are directly or indirectly involved in plant defense (Souza et al. 2017). The total soluble proteins in leaves of plants were estimated according to Lowry et al. (1951). Plant leaves (0.1 g) were macerated in 1 ml of phosphate buffer (pH 7.5) and centrifuged at 3000 rpm for 10 min. The volume of supernatant (0.1 ml) was raised upto 1 ml by adding distilled water in a separate test tube. After 30 min incubation, the absorbance of each sample was determined at 650 nm. The bovine serum albumen of different concentrations viz. 20, 40, 60, 80 and 320 mg were prepared and used as standards.

Nutrient content of plant leaves

The nutrient contents (Mg, Ca, Na and K) of soybean plants were determined by the wet acid digestion method of Jones (2001). Dry plant leaves (0.5 g) were allowed to digest overnight with 10 ml of nitric perchloric acid, the temperature of fume hood was gradually raised to 235 °C till all the traces of nitric acid disappeared. The digested material was allowed to cool for few minutes and few drops of distilled water were added. The extract was filtered through Whatmann (No. 42) filter paper. The volume of filtrate was raised to 50 ml with distilled water. The reagent blank was prepared.

$$ \mathrm{Nutrient}\ \mathrm{Contents}\ \mathrm{in}\ \mathrm{Plant}=\frac{\left(\mathrm{ppm}\ \mathrm{in}\ \mathrm{extract}-\mathrm{Blank}\right)\times \mathrm{A}\times \mathrm{dilution}\ \mathrm{factor}}{\mathrm{W}} $$
A:

Total volume of extract (ml)

W:

weight of dry plant part

Statistical analysis

The results of experiments were subjected to analysis of variance (ANOVA) using Statistix 8.1 version. Treatment of means were compared using least significance test (LSD) at P < 0·05. Principal component analysis correlation (PCA) was applied on the mean values of all variables using XL-STAT 2010. The experiment was repeated 3 times using three replicates.

Results

Biochemical characterization of P. stutzeri and P. putida

P. stutzeri and P. putida produced HCN, exhibited protease and catalase activities (Table 1). P. stutzeri exhibited maximum protease (25.3 U ml−1) and catalase (2 U mg−1 of Protein) activities. Highest HCN production (1.6 mg l−1) and antifungal activity (55%) was also showed by P. stutzeri.

Table 1 Biochemical characteristics of P. stutzeri and P. putida
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Physicochemical parameters of soil

There was 33% decrease in electrical conductivity in response to infestation over control (Table 2). Rhizospheric soil of plants previously inoculated with P. stutzeri and P. putida, receiving infestation of Macrophomina, showed 133% and 100% increase, respectively, in EC as compared to control and 250% and 200% increase, respectively, as compared to infested soil.

Table 2 Physicochemical characteristics of rhizospheric soil of soybean plants
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Rhizospheric soil of plants treated with P. stutzeri showed 10% increase in total dissolved solids as compared to control and infested soil (Table 2). Macrophomina infection in PGPR inoculated plants significantly (P < 0.05) enhanced the TDS of rhizospheric soil. P. stutzeri being more effective than P. putida resulted in 102% increase in TDS over control and infested soil.

The organic matter was 14% and 11% higher in the rhizospheric soil of plants inoculated with P. stutzeri and P. putida, respectively, over control (Table 2). Both PGPRs significantly (P < 0.05) enhanced the OM of the rhizospheric soil after infestation with Macrophomina. P. stutzeri, being more stimulatory, showed 32% increase in OM over control and infested soil.

Infestation reduced the field capacity by 32% as compared to control (Table 2). Inoculation with P. putida following infestation showed 37% increase in field capacity over control and 103% increase over infested soil whereas, there was 62% and 138% increase in field capacity in control and infested soil, respectively, due to P. stutzeri inoculation.

Soil infestation resulted in 40%, 25%, 20% and 9% decrease in Ca, Na, K and Mg contents, respectively, in rhizosphere soil over control (Fig. 1). Both PGPRs ameliorated the inhibitory effects of infection on nutrient contents of rhizospheric soil. Inoculation with P. stutzeri resulted in 71%, 100%, 59% and 55% increase in Mg, K, Na and Ca contents, respectively, as compared to control.

Fig. 1
figure 1

T1 (Uninfected uninoculated control), T2 (Rhizospheric soil of plants previously inoculated with P. stutzeri), T3 (Rhizospheric soil of plants previously inoculated with P. putida), T4 (Rhizospheric soil of plants previously inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Rhizospheric soil of plants previously inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina.). Post harvested soil collected after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Effect of Macrophomina phaseolina on growth parameters

Infection resulted in 24% and 39% decrease in germination index in cv. Ajmeri and cv. NARC, respectively, over control (Table 3). Maximum increase of 147% and 115% in GI were recorded in plants inoculated with P. stutzeri following infection with Macrophomina in cv. Ajmeri and cv. NARC, respectively, over control.

Table 3 Effect of Macrophomina phaseolina on growth parameters of soybean plants
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The root area was reduced by 34% and 50% while root length was reduced by 18% and 45% in cv. Ajmeri and cv. NARC, respectively, following infection (Table 3). Plants treated with P. stutzeri following infection showed a significant increase of 227% and 188% in root area and 131% and 86% increase in root length in cv. Ajmeri and cv. NARC, respectively, as compared to control.

Infection resulted in 34% and 36% decrease in shoot height in cv. Ajmeri and cv. NARC, respectively, over control (Table 3). Plants inoculated with P. stutzeri exposed to infection exhibited significant increase of 117% and 103% in shoot height in cv. Ajmeri and cv. NARC, respectively, over control.

Infection with Macrophomina reduced chlorophyll content in cv. Ajmeri and cv. NARC by 49% and 31%, respectively (Table 3). Inoculation with P. putida following infection resulted in 62% increase in chlorophyll content in cv. NARC over control whereas, P. stutzeri inoculated plants on exposure to infection showed 89% increase in chlorophyll content in cv. Ajmeri over control.

Infection decreased the shoot fresh weight by 34% and 49% and shoot dry weight by 30% and 48% in cv. Ajmeri and cv. NARC, respectively, over control (Fig. 2). Plants inoculated with P. stutzeri following infection showed maximum increase in shoot fresh weight which was 120% and 100% in cv. Ajmeri and cv. NARC, respectively, as compared to control whereas, shoot dry weight was increased by 160% and 122% in cv. Ajmeri and cv. NARC, respectively, over control.

Fig. 2
figure 2

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Root fresh weight was reduced by 20% and 30% and root dry weight was reduced by 50% and 77% in cv. Ajmeri and cv. NARC, respectively, in response to infection as compared to control (Fig. 2). Inoculation with P. stutzeri being more effective resulted in 80% and 60% increase in root fresh weight and 150% and 100% increase in root dry weight in cv. Ajmeri and cv. NARC, respectively, over control.

Disease incidence and disease severity index

As compared to control, infection resulted in 46% and 55% increase in disease incidence in cv. Ajmeri and cv. NARC, respectively (Table 4). Plants inoculated with P. stutzeri, experiencing Macrophomina infection, in cv. Ajmeri showed 95% reduction in disease incidence over infected plants.

Table 4 Effect of Macrophomina phaseolina on disease severity index and disease incidence of soybean plants
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Soybean plants infected with Macrophomina presented 60% and 70% increase in disease severity index in cv. Ajmeri and cv. NARC, respectively, over control (Table 4). Plants inoculated with P. stutzeri on exposure to infection in cv. Ajmeri and cv. NARC resulted in 76% and 60% decrease, respectively, in disease severity index over infected plants. Plants inoculated with P. putida in cv. Ajmeri and cv. NARC on infection with M. phaseolina showed 23% and 22% reduction, respectively, in disease severity index over infected plants.

Defense-related enzymes and antioxidant enzymes

There was linear increase with time in PPO and PAL activity from 30th - 90th day after infection (Fig. 3 and Fig. 4). Measurements made 30 days after infection revealed that cv. Ajmeri had lower PPO and PAL activities than that of cv. NARC in control. On 60th day, the PPO and PAL activities of cv. Ajmeri were increased to the level of cv. NARC which did not show any significant increase. On 90th day after infection, both varieties showed significant increase in PPO (203% and 179% in cv. Ajmeri and cv. NARC respectively) and PAL (114% and 80% in cv. Ajmeri and cv. NARC respectively) activities as compared to control. Both PGPRs, under uninfected conditions, had lesser effect on PPO and PAL activities. Under infection, both PGPRs significantly (P < 0.0.5) increased PPO and PAL activities in both varieties by 2–3 folds. Plants inoculated with P. stutzeri showed maximum increase of 435% and 386% in PPO activity and 257% and 180% in PAL activity in cv. Ajmeri and cv. NARC, respectively, as compared to control. The pattern of response was similar for CAT, SOD and POD in infection and other treatments.

Fig. 3
figure 3

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Fig. 4
figure 4

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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There was 637% and 391% increase in CAT activity in cv. Ajmeri and cv. NARC, respectively, in response to infection as compared control (Fig. 5). Plants inoculated with P. stutzeri on infection induced higher accumulation of catalase enzyme (1867% and 1424% in cv. Ajmeri and cv. NARC, respectively) as compared to control.

Fig. 5
figure 5

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Infection resulted in 140% and 100% increase in SOD activity and 533% and 433% increase in POD activity in cv. Ajmeri and cv. NARC, respectively, as compared to control (Fig. 6 and Fig. 7). Plants inoculated with P. stutzeri, infected with Macrophomina resulted in 290% and 240% increase in SOD activity and 733% and 666% increase in POD activity in cv. Ajmeri and cv. NARC, respectively, after 90 days of infection as compared to control.

Fig. 6
figure 6

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Fig. 7
figure 7

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Total protein and proline content of leaves

Plants exposed to infection resulted in 170% and 140% increase in protein content and 250% and 200% increase in proline content in cv. Ajmeri and cv. NARC, respectively, as compared to control (Fig. 8). No significant effect of P. stutzeri inoculation was observed under uninfected condition. Inoculation with P. stutzeri following infection showed 400% and 300% increase in total protein content in cv. Ajmeri and cv. NARC, respectively, as compared to control. Proline content was also increased by 530% and 400% in P. stutzeri treated plants following infection in cv. Ajmeri and cv. NARC, respectively, over control.

Fig. 8
figure 8

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Plant nutrient contents

Soybean plants infected with Macrophomina resulted in 35%, 29%, 23% and 20% reduction in Na, K, Mg and Ca contents, respectively, in cv. Ajmeri while cv. NARC showed 46%, 42%, 31% and 30% decrease in Mg, Ca, Na and K contents, respectively, over control (Fig. 9). Plants inoculated with P. stutzeri subsequently infected with Macrophomina resulted in 431%, 153%, 125% and 78% increase in Ca, K, Mg and Na contents, respectively, in cv. Ajmeri while cv. NARC showed 308%, 137%, 122% and 72% increase in Ca, K, Mg and Na contents, respectively, over control. Plants inoculated with P. putida, infected with Macrophomina, also showed similar pattern of increase but the percentage increase was relatively lower in both varieties with greater percentage decrease in cv. NARC over control.

Fig. 9
figure 9

T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina). Plants were harvested after 90th day of sowing, infestation with Macrophomina and inoculation with PGPR. Means followed by the different letters are significantly different as determined by the LSD test (p < 0.05). Standard error is also recorded. The data is expressed as the average of three replications and three repetitions

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Correlation between disease severity, plant growth parameters and defense enzymes

There was a significant positive correlation (alpha = 0.05) between CAT, SOD, POD, PPO and PAL activities, proline and protein content, shoot fresh and dry weight and root fresh and dry weight as indicated by principal component analysis (Fig. 10). Disease severity was positively correlated with CAT, SOD, POD, PPO and PAL activities and proline and protein content but was negatively correlated with shoot fresh and dry weight and root fresh and dry weight.

Fig. 10
figure 10

The biplot between axes, F1 and F2, shows 97.74% variation in which contribution of F1 is 77.03% and F2 is 20.71%. Red dots indicate correlation between plant growth parameters, antioxidant and defense enzymes and disease severity. Blue dots indicate the effect of treatments on plant parameters. The variables positively correlated are present in the same quadrant. S.F.W (Shoot fresh weight), S.D.W (Shoot dry weight), R.F.W (Root fresh weight), R.D.W (Root dry weight), POD (Peroxidase), SOD (Superoxide dismutase), PAL (Phenylalanine ammonia-lyase), PPO (Polyphenol oxidase), CAT (Catalase), DSI (Disease severity index), T1 (Uninfected uninoculated control), T2 (Plants inoculated with P. stutzeri), T3 (Plants inoculated with P. putida), T4 (Plants inoculated with P. stutzeri and soil infested with M. phaseolina), T5 (Plants inoculated with P. putida and soil infested with M. phaseolina), T6 (Soil infested with M. phaseolina)

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Correlation between PGPR metabolites and defense enzymes

Inoculation with P. stutzeri showed significant effects on antioxidant and defense enzyme activities in infected plants as indicated by principal component analysis (Fig. 11). The SOD, POD, catalase, PPO and PAL activities of soybean leaves were positively correlated (alpha = 0.05) with protease, catalase, HCN and antifungal activity of P. stutzeri.

Fig. 11
figure 11

The biplot between axes (F1 and F2) shows 99.09% variation in which contribution of F1 is 94.27% and F2 is 4.82%. The variables positively correlated are present in the same quadrant. Red dots represent correlation between plant defense and antioxidant enzymes and bacterial metabolites (HCN, protease, catalase and antifungal activity). Blue dots represent the effect of bacterial treatments on antioxidant and defense enzymes. POD (Peroxidase), SOD (Superoxide dismutase), PAL (Phenylalanine ammonia-lyase), PPO (Polyphenol oxidase), CAT (catalase), AFA (Antifungal activity). T4 (P. stutzeri), T5 (P. putida)

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Discussion

Both PGPRs produced HCN, had protease, catalase as well as antifungal activities against Macrophomina but the magnitude of these activities was higher in P. stutzeri than P. putida. Moreover, positive correlation existed between the activities of protease and catalase, HCN production and antifungal activity of P. stutzeri and activities of antioxidant and defense enzymes in soybean plants. This indicated the role of these metabolites in activation of antioxidant enzyme system under Macrophomina infection. Rais et al. (2017) demonstrated that metabolites produced by Bacillus sp. were significantly positively correlated with enhanced antioxidant enzymes in rice against P. oryzae indicating the role of the metabolites in eliciting systemic resistance against fungal pathogens.

Significant increase in the field capacity, electrical conductivity, total dissolved solids and organic matter content recorded by the PGPRs helped to combat disease. Organic matter helps in maintaining soil structure because of its higher EC, water holding capacities and chelation ability (Orhan et al. 2006). The use of organic amendments is one of the most successful control methods of soil-borne diseases. Karthikeyan et al. (2006) reported that organic amendments along with biocontrol agents minimized the incidence of root rot instigated by M. phaseolina in groundnut. Jamal et al. (2018) reported that B. amyloliquefaciens, when used as an inoculant, increased soil EC, moisture content, organic matter, cation exchange capacity and nutrient contents of soil which improved the growth of pepper plants. Optimum EC is required for the availability of nutrients in soil. Cotxarrera et al. (2002) used sewage sludge compost for the repression of Fusarium wilt of tomato, as higher EC reduces pathogens in soil. Soybean plants at field capacity had lower risk of charcoal rot as high moisture content suppresses the effects of disease. Arias et al. (2013) stated that Pinus radiata maintained at 100% field capacity had no seed mortality by M. phaseolina and remained asymptomatic until the end of the experiment. Furthermore, other bioactive compounds such as antibiotics and certain phenolic acids produced by these bacteria have positive effect on soil fertility, decrease soil pathogens and therefore improve crop growth (Gouda et al. 2018). Macrophomina is a nutrient scavenger, especially in soil with nutrient deficiencies and at high soil temperatures (Mengistu et al. 2016). The observations indicated that P. stutzeri and P. putida significantly enhanced the uptake of Mg, Ca, Na and K contents in plants and their availability in rhizospheric soil which subsequently reduced the damaging effects of infection. Nutrient uptake was 2–3 times higher in plants inoculated with PGPRs under infected conditions. Hashem et al. (2017) demonstrated that B. subtilis enhanced the uptake of N, Mg, K, Ca, Zn, Cu, Mn and Fe in Macrophomina effected mung bean plants.

Both PGPRs significantly overcame the infection induced decrease in the germination index (GI) of both varieties. Also, both varieties (cv. Ajmeri and cv. NARC) responded differently to the combined treatment of infection and PGPR (P. stutzeri and P. putida). P. stutzeri was more effective and the response of cv. Ajmeri was greater than cv. NARC. Similar trend was followed for root length, root area, shoot height and chlorophyll content. The results were parallel with Khare and Arora (2010) where Pseudomonas aeuroginosa significantly enhanced the shoot and root length in Macrophomina infected chickpea plants. P. stutzeri was also reported to possess antifungal activity against Botrytis cinerea and promoted shoot height, root length, chlorophyll content, and total fresh weight in tomato plants (Rojas-Solís et al. 2018). Hernandez-Montiel et al. (2017) observed increase in plant height, stem diameter and root biomass of tomato plants due to production of indoleacetic acid by P. putida

Both disease incidence and severity were significantly decreased in PGPR treated plants which possibly attributed to HCN production, possession of antifungal activity and presence of catalase and protease enzymes. Pastor et al. (2016) reported that P. putida PCI2 not only significantly increased the shoot and root biomass of tomato plants but also suppressed the disease by 30% elicited by F. oxysporum. Noteworthy, cv. NARC, the sensitive variety, had higher incidence of disease with greater severity as compared to cv. Ajmeri. The lower disease severity index in cv. Ajmeri, the tolerant variety, may be attributed to the accumulation of significantly higher phenolics that played a vital role in plant disease control as compared to cv. NARC (Siranidou et al. 2002). Therefore, cv. NARC exposed to infection had much greater reduction in shoot and root biomass. Scandiani et al. (2014) found that roots and foliar symptoms in soybean were higher with Fusarium tucumaniae in susceptible cultivar and there were significant differences between disease-associated variables (root biomass, shoot height and shoot biomass) in inoculated and uninoculated genotypes. The principal component analysis revealed that disease incidence and disease severity index were negatively correlated with shoot and root biomass. In addition to this, there existed a positive correlation between disease severity and activities of antioxidant and defense enzymes. Akladious and Abbas (2014) reported that increase in disease severity was positively correlated with antioxidant enzymes in chickpea plants against Fusarium wilt suggesting that induction of defense response suppresses the disease incidence as well as severity.

Proline is an important osmolyte that protects subcellular structures under osmotic stress (Kishor et al. 2005). P. stutzeri significantly reduced the deleterious effects of infection by augmenting the production of proline and protein contents in the leaves. Altinok et al. (2013) resorted that Pseudomonas and Bacillus isolates accumulated higher quantity of proline in eggplant against Fusarium oxysporum. Romeiro et al. (2010) suggested that Bacillus cereus synthesized protein that can elicit increased resistance in tomato leaves against Corynespora cassiicola, which may give protection against multiple pathogens in tomato.

Both P. stutzeri and P. putida, may be implicated as bio fungicides (Pham et al. 2017). The PGPR mediated control of disease is ecofriendly and sustainable, therefore it can be used as a supplement with fungicides to minimize their harmful effects.