Beneficial effect of Bacillus sp. P12 on soil biological activities and pathogen control in common bean

Beneficial effect of Bacillus sp. P12 on soil biological activities and pathogen control in common bean

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Journal Pre-proofs Beneficial effect of Bacillus sp. P12 on soil biological activities and pathogen control in common bean Daniela C. Sabaté, Gabriela Petroselli, Rosa Erra-Balsells, M. Carina Audisio, Carolina Pérez Brandan PII: DOI: Reference:

S1049-9644(19)30631-0 YBCON 104131

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Biological Control

Received Date: Revised Date: Accepted Date:

16 August 2019 9 October 2019 22 October 2019

Please cite this article as: Sabaté, D.C., Petroselli, G., Erra-Balsells, R., Carina Audisio, M., Brandan, C.P., Beneficial effect of Bacillus sp. P12 on soil biological activities and pathogen control in common bean, Biological Control (2019), doi:

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Beneficial effect of Bacillus sp. P12 on soil biological activities and pathogen control in common bean Daniela C. Sabatéa,b, Gabriela Petrosellic,d, Rosa Erra-Balsellsc,d, M. Carina Audisiob,e Carolina Pérez Brandana


Instituto Nacional de Tecnología Agropecuaria (INTA)-Estación Experimental Salta. Ruta

Nacional 68 Km 172. 4403. Cerrillos. Salta, Argentina. b

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad

Nacional de Salta. Av. Bolivia 5150. 4400 Salta, Argentina. c

Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de

Química Orgánica. Pabellón II, 3er P., Ciudad Universitaria, 1428 Buenos Aires, Argentina. d

CONICET, Universidad de Buenos Aires. Centro de Investigación en Hidratos de

Carbono (CIHIDECAR). Facultad de Ciencias Exactas y Naturales Pabellón II, 3er P. Ciudad Universitaria, 1428 Buenos Aires, Argentina. e Instituto

de Investigaciones para la Industria Química (INIQUI),

Corresponding authors: [email protected] (D.C. Sabaté), [email protected] (C. Pérez Brandán).

Abstract Soil is the foundation for agriculture and the medium where almost all food-producing plants are grown. Extensive use of fertilizers and pesticides has affected soil quality, with dangerous ecological effects; therefore, environmentally friendly biological alternatives have been widely recommended. This study explored the potential of Bacillus sp. P12 strain for improving soil quality and consequently favoring growth of common bean (Phaseolus vulgaris L.) and, on the other hand, for controlling infection with the plant pathogen Macrophomina phaseolina, which induces charcoal rot. P12 inhibited in vitro the development of six native M. phaseolina strains isolated from Salta region, with fungal inhibition ranging between 55 and 70 %. This bacilli synthesized different isoforms of the lipopeptides kurstakin, surfactin, iturin, polimyxin and fengycin in the presence of the plant pathogen, as determined by MALDI-TOF. Under greenhouse conditions, incidence of M. phaseolina was 100% in white common bean cv. Alubia in the control treatment, whereas seeds inoculated with P12 showed a 40 % reduction of the pathogen effect at the end of the trial. Activities of the soil enzymes FDA, DHA and AP were increased (22, 31 and 6.2 %, respectively) in the rhizosphere where P12-treated seeds were grown. P12 treatment decreased metabolic quotient, suggesting greater metabolic efficiency. P12 also enhanced the glomalin-related soil protein in the rhizosphere soil. Moreover, populations of the potential native biocontrol agents Trichoderma spp. and Gliocladium spp. increased in the rhizosphere where P12 treatment was applied. Here, Bacillus sp. P12 was found to not only is a potential biocontrol agent, but also improves soil quality, which would enhance properties required for maintaining a healthy soil, rich in nutrients and beneficial microorganisms, thereby improving agricultural production. Keywords: Bacillus, Phaseolus vulgaris L., biological control, lipopeptide

1. Introduction Agricultural production is one of the most important issues of Argentina’s economy. The vast territory and diverse climates present in Argentina offer suitable conditions for a wide array of agricultural production activities in the different regions. Soils are the basis of agriculture and the medium where almost all food-producing plants are grown. However, the increasing development of agricultural production has involved undesirable soil management practices that have had negative consequences on soil properties; indeed, those practices usually degrade soil fertility, structure and health, reducing yields of production systems. One of the main crops grown in Argentina is common bean (Phaseolus vulgaris L.), a food legume widely appreciated for its nutritional value in developed and developing countries. It is mainly cultivated in north-western Argentina (NWA), with Salta province being the most important producer, with an estimated cultivated area of about 350,000 ha in the last years 2017-2018 (Vale et al., 2018), whereby, the crop is very important for the regional economy. Common bean is scarcely consumed in our country and 95% of the total production is exported; thus, this crop is very important in the international trade, generating high export revenues for Argentina (Casalderrey, 2018). Besides soil properties, other factors may negatively affect bean production and reduce crop yield and quality, such as pathogenic microorganisms. The fungus Macrophomina phaseolina (Tassi) Goid is a soilborne necrotrophic pathogen widely distributed worldwide, which causes charcoal rot disease in different plant species, including various important crops, such as common bean (Kaur et al., 2012; Perez-Brandán et al., 2012). The fungus forms microsclerotia, which can persist within the plant residue and soil; both microsclerotia and infected seeds serve as the primary source of inoculum. This mode of reproduction makes it difficult to reduce pathogen incidence or damage (Meyer et al., 1973; Papavizas, 1977; Baird et al., 2003). In our region, root rot of M. phaseolina has been controlled effectively by fumigating the soil or seeds (Casalderrey, 2018); however, the ever increasing cost of the application, restricted use of fungicides and concerns about environmental impact clearly indicate the need to search for effective control management strategies. Research during the last decades has explored biological control as a feasible option for the management of plant

pathogens. The use of microorganisms for biological control provides an environmentally safe and potentially stable alternative to chemical control. Bacillus sp. P12, a potential biocontrol agent and bean crop growth promoter, was isolated from soil samples taken from NWA (Sabaté et al., 2018). This bacilli was found to promote the growth of this crop, synthesize metabolites, such as auxins, siderophores and lipopeptides, improve seed phytosanitary state, and reduce the incidence of Slerotinia slerotiorum, a plant pathogen of regional concern. The current study was conducted to explore the potential of using Bacillus sp. P12 strain for improving soil quality and, consequently, promoting crop growth, and on the other hand, for controlling the infection of common bean plants with M.phaseolina. 2. Materials and Methods 2.1. Effect of Bacillus P12 as a biocontrol agent 2.1.1. Fungal growth inhibition assays A dual culture technique was used for these trials (Landa et al., 1997). The inhibitory activity of the P12 strain was evaluated against the phytopathogenic native fungal strains Macrophomina phaseolina Tassi (Goid) 02, 03, 06, 27, 32 and Campichuelo, isolated from NWA, belonging to the culture collection of Agricultural Microbiology Laboratory of the Estación Experimental Agropecuaria EEA-INTA-Salta, Argentina. The different strains were grown on Potato Dextrose Agar (PDA, Britania), at an incubation temperature of 28 °C for 7 days. Five-day-old fungal discs (4 mm diameter) of each tested fungus were placed in the center of 9-cm-diameter Petri dishes containing PDA medium. Then, 10 µl obtained from 24-hold cultures of the isolated bacteria were placed equidistant from one another. After incubation at 28 ºC for 7 days, the mycelial growth diameter of each phytopathogen was measured and the percentage of fungal inhibition (FI) was calculated according to Royse and Ries (1978): FI (%) = RGI x 100 RGI= (C-T) / C,

where T is the average diameter of mycelial growth in presence of the Bacillus sp. strain, C is the average diameter of mycelial growth without bacterial samples, RGI: radial growth inhibition. The assays were performed in triplicate. 2.1.2. MALDI-MS analysis of lipopeptides involved in the antifungal activity Lipopeptide synthesis by P12 strain was analyzed on PDA medium with the six previously mentioned strains of M. phaseolina. The interaction of bacilli with each fungus produced an inhibition zone of a large diameter. A portion of 4 mm (sample) was removed from the inhibition zone and kept at -20 ºC. Finally, each sample was resuspended in 0.5 ml of water at pH 8 and vigorously shaken for 30 s. The samples were analyzed by MALDI-MS, as described by Torres et al. (2016). Spectra were recorded on a Bruker Ultraflex II TOF/TOF (Bruker Daltonics, Bremen, Germany). As MALDI matrix, 9H-pyrido [3,4b] indole (norharmane, nHo) was used (Sigma-Aldrich, USA). For MALDI-MS experiments dry droplet sample preparation or sandwich method was used according to Nonami et al. (1997), loading successively 0.5 μl of matrix solution, analyte solution and matrix solution after drying each layer at normal atmosphere and room temperature. Mass spectra were acquired in linear positive ion modes. External mass calibration was made using aqueous solution (1 mg/ml) β-cyclodextrin (MW 1134; [M+Na]=1157.35730 [M+K]= 1173.33010. Spectra were obtained and analyzed with the programs FlexControl and FlexAnalysis, respectively. 2.1.3. Effect of Bacillus sp. P12 cell culture on common bean seeds grown in soils contaminated with M. phaseolina Seeds from the white common bean cv. Alubia were used. They were initially sterilized in 70 % alcohol for 30 seconds and then in 1% sodium hypochlorite solution for 1 min. After this treatment, the seeds were inoculated by submerging them for 30 minutes in the 48-hold cell culture of P12 at a concentration of 1 x 108 cells/ml. In addition, the effect of the chemical fungicide frequently used in the region (Maxim®Evolution), applied according to the manufacturer’s instruction, was evaluated. Non-inoculated seeds were used as control.

In addition, an inoculum of M. phaseolina was prepared on rice, according to the method proposed by Castellanos et al. (2011). Trays of sterilized soil (loam soil with 2.91% organic matter, 0.17% total nitrogen, pH 6.9, 32% sand, 44% silt and 24% clay, typic ustorthents according to USDA Soil Taxonomy) were prepared as substrate, which were then artificially infected with the M. phaseolina culture obtained from the rice (0.5 g of inoculum per Kg of soil). 70 white bean cv. Alubia seeds were sown per tray using the following treatments: P12, the chemical fungicide, and control (non-inoculated seed). The trays were incubated at 23-26 ºC for 21 days. In these assays, germination energy (GE) at five days from seeding, and pathogen incidence were determined. Disease incidence was determined visually as the percentage of plants showing characteristic symptoms. 2.1.4. Area under the disease progress curve Visual assessments of charcoal rot incidence were registered three times between 5 and 21 days after seeding (same as the previous experiment). A modified version of area under the disease progress curve (AUDPC) (equation 1) (Wilcoxson et al., 1975), was used to evaluate disease incidence over time, as follows:


where R1 to R3 are incidence ratings corresponding to times t1 to t3. 2.2. Effect of Bacillus sp. P12 on soil microbiological properties 2.2.1. Experimental design The experiments were conducted in the greenhouse at EEA-INTA-Salta; temperature was about 24 ºC and relative humidity ranged between 60 and 75%. Seeds were sown in pots (three seeds per pot) in a completely randomized design. Two treatments were established: seeds inoculated with P12 (as mentioned in section 2.2.3) and non-inoculated control seeds. Five replications were performed per treatment. The soil employed for the experiment was taken from a long-term trial where cover crops were evaluated on a degraded area of Lerma Valley, established in 2010 (Pérez Brandán et al., 2017). Soil texture is loamy (32% sand, 44% lime, 24% clay), with 2.91% organic

matter, Udic Ustocrept (USDA Soil Taxonomy). The experiments were maintained under greenhouse up to R6 crop stage. 2.2.2. Soil sampling Soil sampling was conducted before flowering of common bean. Rhizosphere soil samples were taken from the roots of one plant per pot, which was considered a single sample (either P12 treatment or control). A total of five single samples of rhizosphere soil were collected per treatment (i.e. five replications per treatment). Roots were gently shaken to remove loosely adhering soil and placed in plastic bags. Soil samples were sieved at field moisture (2 mm), homogenized, air-dried and stored at 4 °C for further analysis. 2.2.3. Soil microbial activities Microbial activity was estimated by hydrolysis of fluorescein diacetate activity (FDA), according to Adam and Duncan (Adam and Duncan, 2001). Briefly, 2 g of soil and 15 ml of 60 mM potassium phosphate buffer pH 7.6 were placed in a 50 ml conical flask. Substrate (FDA, 1000 mg ml-1) was added to start the reaction. The flasks were placed in an orbital incubator at 30 °C and 100 rpm for 20 min. Once removed from the incubator, 15 ml of chloroform/methanol (2:1 v/v) was immediately added to stop the reaction. The contents of the conical flasks were then centrifuged at 447 × g for 5 min. Finally, the supernatant was filtered and measured at 490 nm on a spectrophotometer. Acid phosphatase (AP) was assayed using 1 g soil, 4 ml 0.1 M universal buffer (pH 6.5), and 1 ml 25 mM p-nitrophenyl phosphate (Tabatabai and Bremner , 1969). After incubation at 37±1 °C for 1 h, the enzyme reaction was stopped by adding 4 ml 0.5 M NaOH and 1 ml 0.5 M CaCl2 to prevent the dispersion of humic substances. Absorbance was measured in the supernatant at 400 nm. Dehydrogenase activity (DHA) was determined according to García et al. (1997). Briefly, 1 g of soil at 60% field capacity was exposed to 0.2 ml of 0.4% INT (2-p-iodophenyl-3-pnitrophenyl-5-phenyltetrazolium chloride) in distilled water at 22 °C for 20 h in the dark. The INTF (iodonitrotetrazolium formazan) formed was extracted with 10 ml of methanol by shaking vigorously for 1 min and filtering through a Whatman No. 5 filter paper. INTF was measured spectrophotometrically at 490 nm.

2.2.4. Microbial biomass and respiration Microbial biomass C (MBC) was determined using the chloroform fumigation-incubation technique (Jenkinson and Powlson, 1976). Soil microbial respiration was determined as potentially mineralizable C (CO2-C respiration) (Alef, 1995). The amount of CO2 released was measured from chloroform-treated and untreated soil samples (ca. 20 g). Treated samples were previously fumigated with chloroform, inoculated with fresh soil, and incubated with NaOH 0.2 M at room temperature in the dark for no longer than two weeks. Released CO2 was estimated using HCl 0.2 N. For the quantification of microbial respiration, flasks without soil served as the control treatment. The metabolic quotient (qCO2) was then determined by calculating the relationship between respiration and MBC values. 2.2.5. Glomalin-related soil protein (GRSP) GRSP was determined in the easily extractable glomalin form, according to Wright and Upadhyaya (1996). Soil samples were sieved (0.2 mm mesh size), and GRSP was extracted with 20 mmol·l−1 sodium citrate (pH 7.0) at a rate of 250 mg of aggregates to 2 ml of buffer, which was autoclaved at 121 °C for 30 min. All supernatants from a sample were combined, the volume was measured, an aliquot was centrifuged at 10000g for 15 min to remove soil particles, and Bradford reactive easily extractable protein was measured. Fresh soil was employed to take these measurements. Soil aliquots were oven-dried (104 °C) for 3 days, with the purpose of referring the values of GRSP to dry soil. 2.2.6. Determination of native beneficial microorganisms Microbial populations were determined by soil dilution plating on various agar media. Fungal groups Trichoderma spp. and Gliocladium spp. populations were quantified by mixing a soil sample (10 g) in flasks containing 90 ml of sterile distilled water. To make the corresponding dilutions, 1 ml was taken from this suspension and serially diluted in sterile distilled water. An aliquot of 400 µl was taken from the 10-3 dilution tube and spread on potato dextrose agar supplemented with rose bengal (20 mg·l-1), streptomycin (100 mg·l-1), and chloramphenicol (300 mg·l-1). Plates were incubated at 25 °C, with 8 h of light. After

incubation, colony forming units (CFU) of Trichoderma spp. and Gliocladium spp. were quantified (Vargas Gil et al., 2009). Trichoderma spp. and Gliocladium spp. were identified based on colony characteristics, and morphological traits were determined by microscopic observations. Bacterial groups Actinomycetes and fluorescent pseudomonads were quantified by weighing 1 g of soil per sample, which was placed in flasks containing 100 ml of sterile distilled water. The solution was vigorously shaken for 30 min and then serially diluted in sterile distilled water. An aliquot of 100 µl was taken from the last dilution (10-2) and spread onto agar. Actinomycetes were determined using modified Küster medium (Vargas-Gil et al., 2009) and fluorescent pseudomonads were quantified using selective King’s medium B (King et al., 1954). Both groups of microorganisms were quantified because they contain species that can act as potential biocontrol agents. Plates were incubated at 25 °C in the dark. Data were expressed as the number of colony forming units (CFU·(g dry soil)-1). Fresh soil was employed to take these measurements. Soil aliquots were oven-dried (104 °C) for 3 days, with the purpose of referring the values of CFU to dry soil. 2.3. Statistical analysis Data were calculated and statistically analyzed using Microsoft Office Excel and INFOSTAT software (Di Rienzo et al., 2012) for Windows. Analyses of variance (ANOVA) with LSD (least significant difference) were used to test differences in incidence M. phaseolina, enzymatic activity and other biological attributes. In all cases, residuals were tested for normality via the Shapiro-Wilks’ test. To test for differences between means, an LSD test at a significance level of P≤ 0.05 was used. 3. Results 3.1. Fungal growth inhibition assays The analysis of the effect of P12 strain on the different M. phaseolina strains showed a similar effect on the six plant pathogens (Fig. 1). The plant pathogen strain 32 was the most sensitive one, since it was inhibited with approximately 70% of FI. The most resistant

pathogen strain was Campichuelo, with a FI close to 55%, showing significant differences respect strain 32. Strains 02, 03, 06 and 27 were similarly inhibited by the bacilli, with no significant differences among them. 3.2. MALDI-MS analysis of lipopeptides involved in antifungal activity MALDI-TOF mass spectra analysis revealed signals compatible with surfactin, iturin, kurstakin, polymyxin and fengycin homologues in samples of Bacillus sp. P12 strain incubated in PDA medium in presence of six strains of M. phaseolina (Table 1). As shown in Figures S1- S6 (see Supplementary material) signals observed are clearly located in two different m/z regions: first group were observed in m/z 980-1200 and the second group located in the region m/z 1400-1600. Accordingly to data in the literature (Vater et al., 2002; Yang et al. 2006; Price et al., 2007; Pathak et al., 2012; Torres et al., 2015) in the first region signals can be assigned mainly to kurstakin, surfactins and iturins, and in the second m/z region to fengycins.

Lipopeptide isoforms were detected as protonated

[M+H]+ and/or sodiated [M +Na]+ and/or potassiated [M+K]+ adduct (Table 1). P12








C50H86N7O13, C51H88N7O13, C52H90N7O13 and C53H92N7O13) in the presence of all strains of M. phaseolina and another one (C54H94N7O13) in presence of strains 03, 27 and







C48H74N11O15, C49H76N11O15 and C52H82N11O15) were detected in presence of strains 02, 03, 06 and Campichuelo. One isoform of kurstakin (C44H74N7O11) was detected in presence of 02,03,27, 32 and Campichuelo. Polymyxin D1 was detected in all samples as very low intensity signals (m/z 1166 and m/z 1182; Table 1). The synthesis of four isoforms of fengycin (C72H110N12O20, C73H112N12O20, C74H114N12O20 and C75H116N12O20) was also determined in the presence of all phytopathogenic strains, with the exception of strain 27, where the isoforms C75H116N12O20 was not detected. No significant differences were observed in the production of lipopeptides (number of homologues) by P12 against the 6 phytopathogenic strains used. In all cases signals relative intensity was higher for surfactins homologues than for fengycins. Moreover, signal assigned to the surfactin isoform C52H90N7O13 was the highest in all samples, sometimes

detected as [M+K]+ aspecies at t m/z=1059 (in presence of strains 02, 03, 06 and Campichuelo), or as the [M+Na]+ species at m/z=1043 ( experiments with strains 27 and 32). 3.3. Effect of Bacillus cell culture on common bean seeds grown in soils contaminated with M. phaseolina The M. phaseolina strain Campichuelo was selected for these assays because it was one of the most resistant strains against the bacilli, as indicated by the %FI. Untreated common bean seeds (control) sown in soil infested with M. phaseolina Campichuelo exhibited a GE of 8%. After 15 days of seeding, disease incidence was 100% in the control (Fig. 2), with infected seedlings showing the typical disease signs. Seeds treated with the chemical fungicide and sown in M. phaseolina-infested soil exhibited a GE of 30% and disease incidence was the 50%, 15 days after seeding. Seeds treated with P12 showed a GE of 28%, and lower disease incidence which was 25%, 15 days after seeding, statistically significant decrease of disease incidence respect to the control (Fig. 2). At the end of the trials, the incidence was of 60 and 65%, in seeds inoculated with P12 and chemical fungicide, respectively, which was statistically significant decrease of disease respect to the control. Throughout the assay it was detected the initial symptoms of charcoal root rot on emerged seedlings such as dark and irregular lesions of different sizes on the cotyledons and on the stem tissues of the bean. Infected cotyledons always remained attached to the stems, and bright and systemic chlorosis was also observed on young leaves above the site of infection. Disease progress in P12 treatments was significantly lower than in the treatments with chemical fungicide and the control, with an AUDPC of 406, 665 and 1120, respectively. 3.4. Soil microbial activities Inoculation of seeds with P12 increased soil enzyme activities (Fig. 3). FDA activity was 22% higher in P12 treatment soil than in control treatment (Fig. 3A). The treatment with the bacilli also increased DHA and AP activities by 31 and 6.2%, respectively, compared with the control (Fig. 3B and C).

3.5. Microbial biomass and respiration Microbial respiration increased significantly (65%) in P12 treatment respect to the control (Table 2). Mean MBC value was also higher in P12 treatment (0.15 mg g−1) than in the control (0.11 mg g-1). Metabolic quotient value was lower in P12 treatment (1.61) than in the control (2.1). 3.6. Glomalin-related soil protein (GRSP) Inoculation of common bean seeds with P12 increased GRSP in rhizosphere soil by 27% with respect to values detected in untreated soils (Fig. 4). 3.7. Determination of native beneficial microorganisms Inoculation of seeds with Bacillus sp. P12 induced a significant increase of beneficial microorganisms present in the soil. An increase of 13% was recorded for Trichoderma spp. populations and of 44% for actinomycetes colony count in treatments with P12 respect to control (Fig. 5). However, the presence of Gliocladium spp. or Pseudomonas spp. strains was not detected in the treatment with the bacilli or in the control. 4. Discussion In this study, we determined that Bacillus sp. P12 native strain, isolated from soils of Salta province, reduced charcoal rot in common bean (Phaseolus vulgaris L.) caused by Macrophomina phaseolina under greenhouse conditions. The use of beneficial microorganisms is considered one of the most promising methods for safe crop management practices, since it maintains soil health and quality. However, few attempts have been made to find bacterial biocontrol agents for controlling diseases that affect the common bean crop in the Salta province, the main producer in north-western Argentina (NWA) region (Torres et al., 2016; Torres et al., 2017; Sabaté et al., 2017; Sabaté et al., 2018). The potential effect of B. amyloliquefaciens B14 on charcoal rot was previously determined in black bean cv. Nag. 12 (Sabaté et al., 2017). In the present study, P12 strain inoculated in white common bean cv. Alubia seeds was found to significantly (approximately 40%) reduce disease incidence under greenhouse conditions, also showing a greater effect than that of the commercial fungicide usually used in the region. The

detected effect of P12 on charcoal rot is very important and should be explored more deeply, since bean is one of the most widely cultivated crops in the region and the effects of charcoal rot reduces bean yield and quality, causing significant economic losses (Perez Brandán et al., 2012). In this study, P12 strain significantly inhibited pathogen growth in vitro. Numerous studies have demonstrated that the antifungal effect of Bacillus strains against phytopathogenic fungi is due to lipopeptide synthesis (Kumar et al., 2012; Cawoy et al., 2014; Li et al., 2014; Liu et al., 2014; El Arbi et al., 2016; Torres et al., 2016; Kumar et al., 2016; Torres et al., 2017; Sabaté et al., 2017; Sabaté 2018). Previous studies demonstrated the synthesis of surfactin, iturin and fengycin by B. amyloliquefaciens PGPBacCA1 when tested against M. phaseolina (Torres et al., 2016), as well as against F. solani and S. sclerotiorum (Torres et al., 2017). Previous works also reported the capacity of B. amyloliquefaciens B14 to synthesize surfactins, iturins, and fengycins, and to co-produce kurstakins and polymyxins grown in the presence of M. phaseolina (Sabaté et al., 2017) and of P12 grown in the presence of S. sclerotiorum (Sabaté et al., 2018). In this study, the capacity of P12 to synthesize kurstakins, surfactins, iturins, polimyxin and fengycins against M. phaseolina when the strains are in contact with the fungus was also demonstrated. The lipopeptide surfactin has not only a primary antibacterial activity but also a synergistic effect on iturin (Thimon et al., 1992) and fengycin (Koumoutsi et al., 2004). Iturins, fengycins and kurstakins are known for their antifungal activity (Hathout et al., 2000; Ongena and Jacques, 2008; Béchet et al., 2012; Falardeau et al., 2013); therefore, these metabolites might be responsible for the antagonistic activity of P12 against the plant pathogens used in this study. Soil microorganisms and enzymes are the primary mediators of soil biological processes, including organic matter degradation, mineralization, nutrient recycling and transformation, all of which play an important role in maintaining soil quality and ecosystem functionality. Soil enzyme activities and microbial communities are considered to be reliable biological indicators of soil function and health. FDA hydrolysis has been widely used as an indicator of overall microbial activity, since the ubiquitous lipase, protease, and esterase are involved in this reaction (Achuba and Peretiemo-Clarke, 2008), and all these enzymes play a role in soil organic matter decomposition (Nannipieri et al., 2003). Our results demonstrate that

P12 enhanced FDA activity; thus, the consequent greater availability of nutrients would increase soil nutrient capacity and would therefore favor crop growth. Phosphorus (P) plays a key role in crop productivity, and its availability depends, among other factors, on P mineralization from soil organic matter. This enzymatic process is performed by a group of phosphatases, such as AP, which provide inorganic P to the soil solution (Nannipieri et al., 1982; Tabatabai, 1994; Richardson, 2001). In this work, P12 was found to increase AP activity; this increase would allow a greater availability of soluble P, favouring crop growth. On the other hand, seed inoculation with P12 was found to increase DHA activity in the rhizosphere. DHA activity is also considered an indicator of oxidative metabolism in soils; since this enzyme is exclusively intracellular, it is linked to viable cells and, therefore, to microbiological activity (Skujins 1973). In a previous work, P12 was found to increase crop germination potential of common bean by 14.5 % compared to control seeds, as well as root length (15%) and stem length (30%) (Sabaté et al., 2018). The recorded beneficial effect of P12 on soil enzyme activity, which would in turn improve soil fertility, might be related to crop growth promotion induced by this bacilli. Glomalin-related soil proteins (GRSP) are glycoproteins abundantly produced by arbuscular mycorrhizal fungi in roots and soil. In this work, inoculation of common bean seeds with P12 was found to increase GRSP content by 27% in rhizosphere soil. This result is very important, since GRSP efficiently bind mineral particles together, thereby improving soil structure and related properties, such as porosity, hydraulic conductivity, and resistance to capping, crusting, compaction, and erosion (Wright et al., 1996; Singh, 2012). Therefore, GRSP preservation should be a goal in every soil management program. Another parameter indicating soil quality is metabolic quotient (qCO2). A low qCO2 reflects improved soil biophysical conditions, whereas a high qCO2 indicates that microorganisms require more energy to maintain biomass (Anderson, 1994; Insam and Domsch, 1988). The decrease in qCO2 observed in P12 treatments suggests greater metabolic efficiency in the rhizosphere. Rhizosphere microbial communities have strong effects on plant growth and health, including nutrition, disease suppression, and resistance to both biotic and abiotic stresses (Berendsen et al., 2012; Hacquard et al., 2015; Andreote and Silva, 2017; Jacoby et al.,

2017). In this work, inoculation with P12 was found to increase populations of beneficial soil rhizosphere microorganisms, such as Trichoderma spp. and actinomycetes, which are frequently found in the rhizosphere of agricultural crops. Endophytic Trichoderma spp. are used as an attractive option for management of some plant diseases and other beneficial effects on plants, including growth promotion and tolerance to abiotic stresses (LópezBucio et al., 2015; Mendoza-Mendoza et al., 2018; De Silva et al., 2019; Adnan et al., 2019) and has been found to stimulate common bean growth (Hoyos-Carvajal et al. 2009). In addition, it has been reported that actinobacterias strains present in soils can support plants not only by increasing soil nutrient content but also by controlling plant pathogens such as M. phaseolina (Gopalakrishnan et al., 2011; Vurukonda et al., 2018; Chaurasia et al., 2018). The present results suggest that Trichoderma spp. and actinomycetes populations, along with P12 strain inoculated in seeds, might act synergistically, increasing their individual beneficial effect. This is the first work demonstrating the beneficial effects of a Bacillus strain used as potential PGPR in common bean crop from NWA on the soil biological activities. This result highlights the importance of these types of studies as a tool for the development of sustainable agricultural practices that can be easily applied in our region. Bacillus sp. P12 not only is a potential biocontrol agent, but also contributes to improve soil quality, which would enhance properties required for maintaining a healthy soil, rich in nutrients and beneficial microorganisms, thereby improving agricultural production. Acknowledgements The authors would like to thank CIUNSa: Consejo de Investigación de la Universidad Nacional de Salta (C-2453; A-2311). University of Buenos Aires (UBACyT 20020170100110BA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET PIP 0072CO) and to the Agencia Nacional de Promoción Científica y Tecnológica (PICT 2016-0130) for financila support. We also thank the members of the Recursos Naturales Department (EEA INTA Salta) for their help in soil sampling and processing, especially to Marcos David Alvarez. Audisio MC, Sabaté DC, ErraBasells R and Petroselli G, are Research Members of CONICET. The Ultraflex II (Bruker) TOF/TOF mass spectrometer was supported by a grant from ANPCYT, PME2003 No.125, CEQUIBIEM, DQB, FCEN, UBA.

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Figure 1.






a c


% FI

50 40 30 20 10 0 2




M. phaseolina



Figure 2. a 100

Incidence (%)



b b



40 20 0

a a


b 7 days

14 days

21 days

Figure 3.

FDA (µg fluorescein/g soil/h)

50 40


a b

30 20 10 0 Control


AP (µmol p-nitrophenol/g soil/h)





b 8


6 Control




DHA (mg INTF/g soil/h)

a 14




8 Control




Figure 4.

GRSP (mg/g soil)


a b


0 Control



Figure 5.

Log CFU/g soil






Trichoderma spp. spp. Trichoderma


Figure captions Figure 1. Percentage of fungal inhibition (% FI) of Bacillus sp. P12 against different strains of M. phaseolina Tassi (Goid) 02, 03, 06, 27, 32 and Campichuelo. Figure 2. Incidence of M. phaseolina on common bean under different treatments: seeds inoculated with P12


with chemical

fungicide --■--, and control -♦-. Figure 3. Mean values of fluorescein diacetate (FDA) (A), acid phosphatase (AP) (B) and dehydrogenase (DHA) (C) activities in P12 and control treatments. Figure 4. Values obtained from Glomalin-related soil protein (GRSP) in P12 and control treatments. Figure 5. Soil populations of native beneficial microorganisms (log CFU/g soil) in the rhizosphere with P12 (...) and control (-) treatments.


Chemical formula


[C44H74N7O11 [C48H82N7O13Na]+ [C49H84N7O13Na]+ [C50H86N7O13Na]+ [C51H88N7O13Na]+ [C52H90N7O13Na]+ [C47H72N11O15Na]+ [C52H90N7O13K]+ [C48H74N11O15Na]+ [C53H92N7O13K]+ [C49H76N11O15Na]+ [C54H94N7O13K]+ [C49H76N11O15K]+ [C52H82N11O15H]+

971.54 987.59 1001.60 1015.62 1029.63 1043.65 1053.50 1059.62 1067.52 1073.64 1081.54 1087.65 1097.51 1101.61

Polimyxin D1

[C50H93N15O15Na]+ [C50H93N15O15K]+

1166.69 1182.66

Fengycin Fengycin Fengycin Fengycin Fengycin Fengycin Fengycin Fengycin Fengycin

[C73H112N12O20H]+ [C72H110N12O20Na]+ [C74H114N12O20H]+ [C73H112N12O20Na]+ [C75H116N12O20H]+ [C74H114N12O20Na]+ [C73H112N12O20K]+ [C74H114N12O20K]+ [C75H116N12O20K]+

Kurstakin Surfactin Surfactin Surfactin Surfactin Surfactin Iturin Surfactin Iturin Surfactin Iturin Surfactin Iturin Iturin Not assigned Not assigned Not assigned Polimyxin D1


1477.82 1485.78 1491.83 1499.80 1505.85 1513.82 1515.77 1529.79 1543.81

02 972.87 986.89 1000.83 1016.79 1030.80 1044.58 1052.65 1059.21 1066.81 1074.87 1082.74 n.d. 1097.87 1102.74 n.d. 1120.76 n.d. 1166.22 1182.02 n.d. 1486.11 1492.29 1500.45 n.d. 1513.79 1515.94 1529.88 1544.02

03 972.59 986.74 1000.73 1016.73 1030.76 1044.67 1052.66 1058.83 1066.72 1074.75 1082.76 1088.78 1097.78 1102.81 n.d. 1120.64 n.d. 1166.88 1182.65 n.d. 1486.83 n.d. 1500.02 n.d. 1513.87 1515.69 1530.29 1544.40

Macrophomina phaseolinaa 06 27 32 b n.d. 972.81 972.60 986.57 986.75 986.67 1000.68 1000.69 1000.69 1016.69 1016.76 1016.60 1030.73 1030.83 1030.80 1044.47 1043.81 1044.0.2 1052.65 1052.55 n.d. 1058.85 1058.92 1058.60 1066.83 1066.73 1066.90 1074.66 1074.82 1074.44 1082.69 1082.73 1082.80 n.d. 1088.85 n.d. 1097.70 n.d. 1097.05 1102.69 n.d. 1102.83 n.d. 1112.79 1112.76 n.d. 1120.61 1120.68 n.d. n.d. 1134.84 1166.74 1165.64 1166.62 1182.79 1182.78 1181.96 n.d. n.d. 1479.11 1485.88 1486.92 1486.06 n.d. n.d. 1492.02 1499.91 1499.99 1500.86 1507.17 n.d. 1507.87 1513.75 1513.96 1513.74 1515.87 1515.91 1515.09 1529.22 1530.06 1529.77 n.d. n.d. 1543.66

Table 1. Lipopeptides synthesized by Bacillus sp. P12 against M. phaseolina characterized by MALDI-MS a

m/z values obtained for each experiment are listed. For details see Materials and Methods

Campichuelo 972.67 986.32 1000.74 1016.70 1030.74 1044.73 1052.75 1058.76 1066.75 1074.78 1082.76 1088.93 1097.74 1102.74 1112.82 1120.74 1134.73 n.d. 1182.82 n.d. 1485.85 n.d. 1500.03 1507.76 1514.02 1515.87 1529.98 1544.92


n.d. no detected


Table 2. Correlation analysis between microbial functionality parameters Microbial

MBC (mg g-1)


Respiration (mg g-1)

2 3 4





Bacillus sp. P12




Different letters indicate values that are significantly different (p ≤ 0.05).

6 7

Research Highlights


1. P12 reduced the incidence of M. phaseolina in seedlings of common bean.


2. P12 synthesizes surfactins, iturins, fengycins, kurstatin and polymyxin.

10 11 12 13 14 15

3. P12 treatment decreased metabolic quotient and increase activities of the soil enzymes FDA, DHA and AP.