Antagonistic potential and biological control mechanisms of Pseudomonas strains against banded leaf and sheath blight disease of maize

doi:10.21203/rs.3.rs-3223916/v1

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Antagonistic potential and biological control mechanisms of Pseudomonas strains against banded leaf and sheath blight disease of maize

https://doi.org/10.21203/rs.3.rs-3223916/v1

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published 11 Jun, 2024

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There is increased interest in developing biological control agents to effectively combat the banded leaf and sheath blight (BL&SB) caused by Rhizoctonia solani, a devastating disease that affects maize and many other crops worldwide. In the current study, we screened 22 rhizobacteria(AS1-AS22) recovered from grassland in the Pithoragarh region of the Central Himalayas for antagonism against Rhizoctonia solanif. sp.sasakii. Two Pseudomonas strains, AS19 and AS21, exhibited the highest in vitro inhibition of fungal mycelium, with cell cultures of AS19 and AS21 showing inhibition was 57.04% and 54.07%, respectively, and with cell-free culture filtrate showing67.41%and 65.74%, respectively. The two bacteria also prevented sclerotium growth. The strains AS19 and AS21were identified as Pseudomonas sp. by 16S rDNA phylogeny and deposited under accession numbers NAIMCC-B-02303 and NAIMCC-B-02304, respectively. The biocontrol mechanisms in the selected strains were investigated in a plate assay and a pot trial. The scanning electron micrograph of the dual culture plate assay showed the intense degradation of fungal mycelium by strains AS19 and AS21. Both strains also produced hydrolytic enzymes. There was a significant reduction in BL&SB disease incidence (DI) with the application of strains AS19 and AS21 in a pot trial using maize crops. The decrease in DI ranged between 25–38.33% in the treatments receiving foliar sprays of two antagonistic isolates 48 h before pathogen inoculation. The results showed that the two Pseudomonas strains act through induced systemic resistance (ISR) and are potent BCAs. The two strains, AS19 and AS21, exhibited broad-spectrum in vitro antifungal activity against Alternaria triticina, Bipolaris sorokiniana, Rhizoctonia maydis, and Fusarium oxysporumf. sp.lentis. Before the commercial-level exploitation of these two BCAs, further studies must be done to validate their biocontrol activity in field trials and to identify a bacterial metabolite responsible for inducing systemic resistance.

Biological sciences/Microbiology

Earth and environmental sciences/Environmental sciences

Pseudomonas strains a promising biocontrol agent for BL& SB disease

Pseudomonas strains induced the resistance in maize against BL& SB disease

Pseudomonas strains may produce a variety of antifungal compounds.

Maize (Zea mays L.), known as the queen of cereals, is one of the most versatile emerging crops, having wider adaptability under varied agro-climatic conditions [1, 2]. The banded leaf and sheath blight (BLSB) disease, caused by Rhizoctonia solanif. sp. sasakii, is a very destructive, challenging-to-manage disease to maize productivity worldwide [3, 4]. In India, the BL&SB disease was first reported by Payak and Renfro in the Terai region of Uttar Pradesh [5], currently in Uttarakhand. Thereafter, the incidences have been documented in Assam, Haryana, Madhya Pradesh, Odisha, Punjab, West Bengal, and the Udaipur district of Rajasthan, and the Mandi district of Himachal Pradesh [6]. Rhizoctonia solanif. sp.sasakii is a necrotrophic fungus that produces phytotoxins during the infection process, forming a necrotic spot on the leaf, sheath, and stem, preferably. In India, BL&SB diminished corn yields by up to 40–70% [7]. The use of biological control agents has recently become more intense due to the hazardous nature of chemical control and the development of fungicide resistance in pathogens [8]. Under favorable environmental conditions, the fungus produces brown sclerotia that survive for long periods, accumulate in the soil, and play an important role in pathogen survival in maize fields. The viability of sclerotia in the soil is affected by soil microorganisms, making biological control an attractive alternative strategy for controlling the BL&SB [9]. The common and effective management option for the BL&SB pathogen is chemical control through foliar application of propiconazole, carbendazim, validamycine, etc. [10]. Considering the damaging environmental impacts of chemical fungicide use, there is a need to look for eco-friendly and sustainable alternatives. In such a case, biological control is the best alternative. Several species of rhizobacteria have been used as biocontrol agents against different soil-borne phytopathogens. These include Bacillus subtilis[11]and Streptomyces spp.[12], fluorescent pseudomonads(FLPs)such as Pseudomonas aeruginosa[13], P. fluorescens[8], and P.putida[14]. These bacteria produce various antibiotics, volatile and non-volatile antifungal metabolites, cell-wall-degrading enzymes, and surfactants, all of which contribute to their antagonistic ability and counteract their negative effect [15]. Several rhizobacteria possess dual actions of biocontrol and plant growth promotion [16]. Among rhizobacteria, FLPs are considered the most promising biocontrol agents (BCAs) because of their higher root colonization ability coupled with antibiotic production in the rhizosphere and persistence throughout the growing season [8, 15].Our study focuses on the potential biocontrol role of fluorescent bacteria from the genus Pseudomonas previously obtained from rhizospheric soils of grassland in the Pithoragarh region of the Central Himalayas.[2] identify and characterize the prominent antagonistic isolates against banded leaf and sheath blight disease. [3] evaluate the efficacy of BCAs on maize BL&SB disease under a greenhouse experiment, and [4] validate the ISR mechanism of biocontrol agents against Rhizoctonia solanif. sp.sasakii. Many strains of root-colonizing microbes are potential elicitors of plant host defenses. Induced systemic resistance (ISR) in host plants against phytopathogens is elicited by various metabolites produced by rhizobacteria [17]. These fluorescent Pseudomonas with biocontrol abilities would be well adapted to agroecosystems when delivered as biofungicides against the BL&SB disease of maize. Pseudomonas species are vigorous niche competitors with a strong capacity for colonization and proliferation, whether in the phylloplane, rhizosphere, or inside plant tissues [18, 19]. Additionally, fungal-pathogen-inhibiting enzymes and a wide range of bioactive metabolites, including antibiotics (DAPG, phenazine, pyrrolnitrin, pyoluteorin, and volatile hydrogen cyanide) and siderophores (pyoverdine, pyochelin, and ferri pyoverdine) which are low molecular weight, high-affinity iron-chelating chemicals, are produced by fluorescent Pseudomonas bacteria with biocontrol properties [20–24]. These rhizospheric bacteria use a variety of strategies, including direct parasitism, antibiosis, and/or resource competition on the infection sites, to eradicate or reduce pathogen growth and colonization. The host’s systemic resistance reactions are elicited by the indirect pathways. There are two mechanisms by which ISR-induced resistance acts systemically not limited to infected parts of the plant, and SAR referred to as systemic acquired resistance [17, 25].Most plants exposed to biocontrol agents induced ISR by altering defense networks and pathways, including phenylpropanoid for resistance against in coming pathogen attacks[17].Systemic-acquired resistance confers perpetuated and broad-spectrum resistance by accumulating the signal molecule salicylic acid(SA) and proteins related to pathogenesis(PRs). Induced systemic resistance is mediated by jasmonic acid and ethylene and is independent of salicyclic acid. Production of JA and ethylene is accomplished by the expression of a set of genes. For example, genes for oxidative enzymes such as peroxidase (PO) and polyphenol oxidase (PPO), oxidative phenols [26], tyrosine ammonia-lyase (TAL), and phenylalanine ammonia-lyase (PAL). Such enzymes have been correlated with defense against pathogens in host plants by a different mechanism [27]. Some biocontrol strains of Pseudomonas sp . induce plant host defenses through a variety of signal translocation pathways [28].P. fluorescens strain WCS417r has been reported to elicit systemic disease resistance in plants through SA-independent, JA-ethylene-dependent signaling, ISR-related gene expression, NPR1-dependent signaling, etc. [29]. The plant’s defense mechanism is induced by PGPR, such as Pseudomonas, Bacillus, and Klebsiella, which resists against the infection. Pseudomonas aeruginosa is the most researched and effective plant symbiont, offering resistance against a broader category of plant pathogens through direct antagonistic interactions or by inducing systemic resistance [30, 31]. The outstanding endo-symbiont P.aeruginosa is widely known for its capacity to promote plant growth and development. It also possesses myco- parasitic abilities, shielding plants against pathogen attack [32]. Enzymes such as peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, chitinase, and β-1,3-glucanase are induced in plants as a defense response [15]. The enzyme peroxidase catalyzes the conversion of phenolic compounds into quinines while releasing hydrogen peroxide. Using an oxidative burst, the antimicrobial molecule H₂O₂ produced causes a huge number of reactive oxygen derivatives, cell death, and inhibition of pathogenic activities. [33]. Rate of polymerization increases phenolic compounds into lignin-like substances which are deposited in cell walls and prevents the further growth and development of the pathogen [19]. Polyphenol oxidases (PPO) catalyze the phenolic compounds to Quinone which have antimicrobial activity [34]. Polyphenol oxidases play defense mechanism roles against plant pathogens [18]. When a pathogen affects the plant, then an immediate rise in PPO indicates quick synthesis of antimicrobials to ward off the pathogens. Phenylalanine ammonia lyase is one of the important enzymes that is responsible for linking the primary metabolism of aromatic amino acids with secondary metabolic products [35, 36]and due to the synthesis of various phenolic compounds as well as anthocyanin, it provides the resistance against the plant pathogens [37].Phenylalanine ammonia lyase (PAL)catalyzes the non oxidative deamination of phenylalanine into trans-cinnamic acid and ammonia which is the initial step in the biosynthesis of phenolic compounds. Changes in PAL activity can take place during pathological events [38]. Phenylalanine ammonia lyase is induced by the plant hormone ethylene and signaling molecules including salicylic acid and jasmonic acid [39.40], and it also can be induced by various biotic and abiotic stresses. There are previous reports of FLPs from the rhizospheric soil of the maize ecosystem possessing antagonistic ability against R. solanif. sp.sasakii, but the effective biocontrol agents have not been commercialized yet [34, 41]. Thus, these indigenous FLP isolates can be effective biocontrol agents against the BL&SB disease.

2.1 Microorganisms

A total of 22 fluorescent pseudomonad isolates (AS1-AS22) recovered from the rhizosphere of various plants growing in different ecosystems of Uttarakhand [42] were retrieved from the culture collection at Rhizosphere biology laboratory, Department of Microbiology, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand Table S1. The pure bacterial isolates were maintained on King’s B Medium containing 20% glycerol at − 80°C.

Rhizoctonia solani f. sp. sasakii causative agent of banded leaf & Sheath blight in maize was obtained from Professor R. P. Singh, Department of Plant Pathology, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand. The culture was maintained on Potato Dextrose agar (PDA).

2.2 In vitro screening of FLPs for antagonism-

2.2.1 Effect of whole cell culture on mycelial growth of R. solani

All 22 FLPs were tested forinvitro antagonistic activity using the dual culture technique on PDA plates [19]. An agar disc (~ 5mm) of actively growing culture of R. solani f. sp. sasakii was placed at the center of the plate. Thereafter, each isolate was streaked equidistantly towards the edge of the plates. In control plate, only fungal culture was inoculated. Plates were incubated at 28 ± 2^OC for 5 days until the mycelium growth reached to the edge of the control plate. Percent mycelial inhibition was calculated according to Jaggi et al.,2020 [43].

2.2.2 Effect of cell free culture filtrates (CFCF) on mycelial growth of R. solani

All 22 FLPs were grown in King’s B broth medium in 250 ml conical flask at 30 ± 2⁰C on rotatory shaker at 120 rpm. After 48 h incubation, culture broths were centrifuged at 10,000 rpm for 10 min to separate the cells. Then the culture filtrates were passed through 0.22µm pore size syringe filters to obtain the cell free culture filtrate. Thereafter, PDA plates were prepared and a disc of actively growing mycelium of R. solani f. sp. sasakii was placed at the center of the plates. Four wells were made with sterile cork borer 3 cm away from center and 80µl of culture supernatant was added to each well. Plate in which wells were filled with King’s B broth served as control. Plates were incubated at 28 ± 2⁰C for 5–7 days and examined for mycelial growth inhibition (%). Growth inhibition was calculated according to Jaggi [43].

2.3 Investigation of potential biological control mechanism-

2.3.1 Detection of extracellular enzymes

Chitinase production was assessed by the formation of a halo zone around the colony following degradation of colloidal chitin [44]. Cellulase production was assayed on carboxymethyl cellulose agar (CMC) plates spot inoculated with the log phase bacterial culture. The formation of the halo zone around the bacterial colony when flooded with 0.1% iodine solution indicated cellulase activity [45]. Protease activity was screened on skim milk agar plates. The formation of a clear zone around the colony based on casein hydrolysis was considered a positive test [46].The assay for amylase activity was done according to Patel [47]. Lipase production was indicated by white precipitation around the bacterial growth on nutrient agar plates supplemented with 1% tween 80 [48].

2.3.2. Production of antifungal metabolite

FLPs were screened for the production of antifungal traits viz., volatile compounds (HCN), and surfactant (rhamnolipid). The production of HCN by bacterial isolates was determined by the method of Bakker and Schipper (1987). Rhamnolipid production was visualized as white halo circle around the well containing bacterial culture on Seigment and Wager agar plates [49].

2.3.3 Test for plant growth promoting traits –

The ammonia-producing ability of selected Pseudomonas strains, AS19 & AS21 was determined by the method of Cappuccino and Sherman[50]. Indole acetic acid (IAA) production was qualitatively detected in nutrient broth (NB) supplemented with 0.1% L-tryptophan. The appearance of a pink color by the addition of Salkowski reagent indicated a positive test [51]. Zinc solubilization was assessed as a halo zone around the colony in the minimal media supplemented with 0.1% zinc carbonate (ZnCO₃). Phosphate-solubilizing activity was qualitatively detected in Pikovaskya agar medium [52]. Siderophore production was determined by the method of Schywn and Neilands[53].

2.4 Morphological and Biochemical characterization of isolates

For morphology identification, isolates were streaked on King’s B agar plates. The plates were incubated at 30 ± 2°C for 12-24h. Thereafter, colony morphology and pigment was observed. The cell shape and arrangement was observed through gram’s staining as described previously by Vincent[54]. Oxidase and catalase tests were performed for bacterial isolates using the method of Nazand Bano [55].The gelatinase test wasper formed according to Smith and Good-ner [56].For H2S production test, the FLP isolates were grown in sulfide indole motility (SIM) broth and incubated at 30 ± 2°C for 2 days. H₂S produced interacts with Ferric ammonium sulphate and forms insoluble black precipitate at subsurface of colony to indicate a positive result. For nitrate reductase test, the isolates were inoculated in nitrate broth and incubated 30⁰C for 24 to 48 h. Thereafter sulfanilic acid and alpha-napthylamine is added. The development of red color indicates positive result.

2.5 Identification of putative antagonistic strains AS19 and AS21

Genomic DNA of AS19 & AS21 was extracted by a modified method of Bazzicalupo and Fani, 1996.(57) The two universal primers GM3f (5’ AGAGTTTGATCMTGGC 3’) and GM4r (5’ TACCTTGTTACGACTT 3’) (58) were used for the amplification of the 1492 bp region of the 16S rDNA gene. The amplification was carried out on a thermal cycler, Gene Amp PCR System 9700 (Applied Biosystems). PCR products were purified using a GeneiPure gel extraction kit (Banglore Genei, India) following the manufacturer’s instruction. The acquired gene sequence was submitted to the NCBI Gene Bank database and accession numbers were obtained. A multiple sequence alignment with the selected reference sequences was performed by the Clustal W algorithm (59), and a phylogenetic tree was constructed using the neighbor joining method (60) with the 1000 bootstrap using MEGA version 7.0 software. Bootstrap replicates used as statistical support for the nodes in the phylogenetic tree (61).

2.6 Scanning microscopy investigation of in vitro co-cultures of pathogenic fungi and potent bacteria

For specimen preparation, small pieces of agar (~ 2 mm²) were taken from the dual cultures and control plates at the interaction zone. Specimens were immersed in 2% glutaraldehyde for 4 h at room temperature. Then, they were washed with 0.1 M phosphate-buffered saline (PBS) (pH- 7.3 to 7.5) four times. The specimens were dehydrated in a graded ethanol series viz., 30%, 50%, 70%, 80%, 90%, and 100% for 15 min each. Then air-dried and sputter-coated with gold-palladium in a Nanotech sputter coating apparatus. Air dried samples were observed in a scanning electron microscope (JEOL, JSM- 6610LV) operated at 15 kV.

2.7 Effect of Pseudomonas spp. AS19 and AS21 on sclerotium germination and development

A log-phase culture of selected bacterial strains was streaked on one edge of a half-strength KB + PDA agar plate. The sclerotium was placed on another edge of the petriplate. The plates were incubated at 28 ± 2°C for 10 days. Thereafter, mycelial growth was recorded. Similarly, to evaluate the effect on sclerotium formation, log-phase bacterial culture was streaked at one edge and a 2–3 day old mycelia disc was placed at another edge of PDA plates. The plates were incubated at 28 ± 2°C for 10 days. Thereafter, the number and weight of sclerotia produced were recorded.

2.8 Efficacy of the strains AS19 and AS21 against R. solani on detached leaf –

A detached leaf assay was a quick method to evaluate biocontrol ability of bacteria [62]. Leaves were collected from the healthy maize plants, then cut transversely into 9 cm-long pieces. The transverse leaf sections were placed on the petri plates containing sterile, moist filter paper. According to the treatments, log-phase bacterial culture (100µl) was spread on the adaxial side of sterile leaf placed on a petri plate Table 1. Subsequently, a 9 mm fungal disc from PDA plates is placed at the center on the adaxial side of the leaf. The plates were incubated at 28⁰C ± 2 for 2 days and observed daily for the appearance of necrotic lesions.

Table 1

Treatments of detached leaf assay
	Treatments
T1	Control(Without FLP culture and pathogenic fungus)
T2	Control ( With FLP strainAS19 alone)
T3	Control (With FLP strain AS21 alone)
T4	Control (Pathogenic fungus alone)
T5	FLP strain AS19 inoculated at 24 hr interval of pathogenic fungus
T6	FLP strain AS21 inoculated at 24 hr interval of pathogenic fungus
T7	Simultaneous inoculation of FLP strain (AS19) and pathogenic fungus
T8	Simultaneous inoculation of FLP strain (AS21) and pathogenic fungus
T9	Pathogenic fungus at 24 hr interval of FLP strain (AS19)
T10	Pathogenic fungus at 24 hr interval of FLP strain (AS21)

2.9 Green house experiment-

A pot trial was carried out in the glass house at the Department of Plant Pathology, G.B.P.U.A. &T., Pantnagar Uttrakhand, India .The pot trial was conducted in three sets (A,B,C) each with different objectives Table S2.

*Collection of all plant material ,or samples comply with relevant institutional ( G.B.P.U.A. &T., Pantnagar Uttrakhand, India) and with all international guidelines and legislation.

2.9.1 Experimental design

A pot study was carried out using the maize variety Pant Sankul Makka (PSM-3) which is susceptible to the banded leaf and sheath blight (BL & SB) pathogen R. solani f. sp. sasakii were collected from G.B. Pant University of Agriculture and Technology, Seed Processing plant district Udham singh Nagar pantnagar,263153 Uttrakhand, India [63]. it has maturity days 80–85.Maize seeds were surface-sterilized with 2% sodium hypochlorite for 2 minutes, followed by 70% alcohol for 60 seconds, and finally washed three times with sterile distilled water to remove traces of chemicals. Surface-sterilized seeds were immersed in bacterial inoculum mixed with 1% carboxymethyl cellulose. Seeds were air-dried for 12 h in an aseptic condition, and sown immediately in sterile plastic pots filled with sterile soil. The pots were prepared in three replicates and arranged in a completely randomized design. Untreated seeds served as the control.

2.9.2 Preparation of bacterial inoculum

Each of the selected antagonistic bacterial cultures (AS19 and AS21) was grown in 150 ml of King’s B broth in a 250 ml Erlenmeyer flask at 30 ± 1°C and 100–120 rpm till a population density of 3x10⁹ CFU ml^− 1 at 36 h. The bacterial cells were centrifuged at 10,000 rpm for 20 min, and the pellets were diluted with sterile distilled water to make OD₆₀₀ of 0.6 which corresponds to a final concentration of 10⁸−10⁹ CFU ml^− 1.

2.9.3 Pot experiment A-

Evaluating biocontrol potential of two antagonistic isolates against R.solani f. sp. sasakii:

About 4.5 kg of sterile soil was filled into a single pot. Thereafter, mass-produced fungal inoculum (10g) was spread on the soil in all pots except absolute control (AB.CON) and mixed with a 10 cm soil layer. Five bacterized maize seeds were sown in pots of A2 and A3 treatments of both isolates. +CONA was sown with carbendazim-treated (2g/kg) seeds. –CONB was sown with untreated seeds. The soil application of 10 ml of bacterial inoculum was given in the A1 and A3 treatments. Randomly Maize leaf samples were collected from glass house in the Department of Plant Pathology, G.B.P.U.A. &T, Pantnagar at 15, 30 and 45 days after infection (DAI) to assess the host-defensive responses like phenylalanine ammonia lyase, polyphenol oxidase and peroxidase, and total phenolic substances [64].Plants were harvested at 85 DAS and observed for disease assessment by disease incidence (DI), lesion length, and plant growth promotion by shoot length (cm), average fresh weight per plant (g) and average biomass per plant (g) were compared with the commercial fungicide (carbendazim) and uninoculated control plants.

2.9.4 Pot experiment B-

Investigation of a biocontrol mechanism

Sterile soil was filled into pots. Five bacterized maize seeds were sown into a pot of B2 and B3 treatments of both the isolates.10 ml of respective bacterial inoculum was applied to the soil of B1 and B3 treatments of both the isolates. Thereafter, a fungal infection was given on 35 DAS with mass produced R. solani f. sp. sasakii on sorghum grains. Infection was produced by inserting the sorghum grains into the leaf sheath. Proper humidity was maintained by spraying water. Leaf samples were collected 15, 30 and 45 days after infection [64] for analysis of host-defensive enzymes like PAL, PPO and PO and total phenolics. After 85 DAS, plants were harvested and observed for shoot length (cm), average fresh weight per plant (g) and average biomass per plant (g).

2.9.5 Pot experiment C

Validating the induced systemic resistance (ISR) mechanism

Five non-bacterized maize seeds were sown per pot in all treatments and + CONC treatments were sown with carbendazim-treated (2g/kg) seeds. A Fungus infection is given at 35 DAS by inserting the sorghum grains into the leaf sheath. Proper humidity was maintained by spraying water. The C1 treatments of AS19 and AS21 were given by foliar spray of the respective bacterial inoculum 24 hours before fungal infection whereas in C2 treatments foliar spray of bacterial inoculum was given 24 hours after fungal infection. Leaf samples were collected 15, 30, and 45 days after infection for analysis of host-defensive enzymes like PAL, PPO, and PO and total phenolic content. After 85 DAS, plants were harvested, and observed for shoot length (cm), average fresh weight per plant (g) and average biomass per plant (g).

2.10 Estimation of Total Phenolic content and Defense related Enzymes

2.10.1 Total phenolic content

The total phenolic content of maize sheath was estimated using the method of Salame and Zieslin [65] Maize sheaths (1g) were homogenized in 10 ml of 80% methanol and agitated for 15 min at 70°C. One ml of the methanolic extract was added to 5ml of distilled water, and 250 µl of 1N folin-ciocalteau reagent was added and maintained at 25°C for 3 min. Then 1 ml of a saturated solution of Na₂SO₃ and 1 ml of distilled water were added, and the reaction mixture was incubated at 25°C for 1 h. The absorbance of the developed blue color was measured using a spectrophotometer at 725 nm. Catechol was used as a standard. The amount of phenolics was expressed as µg catechol per g of fresh tissues.

2.10.2 Defense related enzymes

The sheath samples collected from the different treatments were thoroughly washed with distilled water and dried using blotting paper. They were homogenized with 0.1 N phosphate buffer (PH 6.5) in the ratio of 1:5 (w/v), strained through several layers of cheese cloth, and centrifuged at 6000 rpm, at 4°C for 15 min. The supernatants were used as crude enzymes for the estimation of peroxidase, phenylalanine ammonia lyase, and polyphenol oxidase activities. Polyphenol oxidase activity was assayed using the modified method of Meyer [66]. The standard reaction mixture contained 3mL of 0.1 M phosphate buffer (pH 6.5), 2mL of enzyme preparation, and 1mL of 0.01 M catechol. The reaction mixture was incubated at 28 ± 1°C. The absorbance of reaction mixture was measured at 495 nm at 60 s intervals. The PPO activity was expressed in Ug^− 1 FWmin^− 1. Peroxidase (PO) activity was carried out according to the method described by Srivastava [67]. The reaction mixture contained 1.5 mL of 0.05 N pyrogallol, 0.5 mL of enzyme extract, and 0.5 mL of 1% H₂O₂.The activity was expressed as a change in absorbance at 490 nmat 25°C and was expressed in Ug^− 1 FWmin^− 1 [68]. The assay of PAL activity was conducted according to the method of Zucker [69]. The reaction mixture contained 1mL of 0.1 M borate buffer (pH 8.6), 1mL of phenylalanine solution, and 1mL of enzyme extract. The reaction mixture was kept at 25°C for 30 min. Activity was expressed as a change in absorbance at 290 nm against the blank without adding l-phenylalanine to the reaction mixture. It was determined based on the production end product, i.e., trans-cinnamate.

2.11. Broad-Spectrum Activity of Potent Pseudomonas Strains

AS19 and AS21 were tested for in vitro antagonism against phytopathogenic fungi, viz.,Helminthosporium maydis, Fusarium oxysporumf. sp.lentis, Alternaria alternata, and Bipolaris sorokiniana through a dual culture plate assay. The half-strength KB + PDA plates inoculated with fungal pathogens and bacterial strains were incubated at 30 ± 2°C for 5–7 days. The antagonistic activity was estimated according to the formula given by Hazarika [70]:

% MGI = (A − B)/A × 100

where, A is growth of the test pathogen in the absence of antagonist and B is growth of the test pathogen in the presence of an antagonist.

2.12. Statistical Analysis

The data obtained from different experiments were subjected to statistical analysis using the Statistical Package for Social Studies (SPSS), version 21.0. Data from each experiment were analyzed for variance by a one-way ANOVA test (p < 0.05) and Duncan’s multiple range test (DMRT). All the experiments were conducted in three replications, and the results were expressed as mean ± standard error.

3.1. In Vitro Antagonism Using Whole-Cell Culture(WCC)

Out of 22 isolates, 20 showed different degrees of mycelial growth inhibition of R. solanif.sp.sasakii. Nine isolates (AS3, AS4, AS5, AS7, AS9, AS11, AS12, AS13, and AS20) exhibited 40–50% mycelial inhibition and five isolates (AS1, AS2, AS6, AS8, and AS10) showed 30–40% mycelial inhibition (Table 2). Two isolates, AS19 and AS21, were the most promising antagonists exhibiting 57.4% and 54% mycelial inhibition, respectively (Fig. 1).

3.2 In vitro antagonism using cell-free culture filtrate

The maximum suppression of R. solani f.sp. sasakii mycelial growth was recorded in the CFCF of AS19 (67.41%), followed by AS21 (62.66%) Fig. 1and seven isolates, viz., AS3, AS4, AS7, AS13, AS14, AS16, and AS17, showed 50–60% mycelial inhibition. The remaining eleven isolates showed mycelial inhibition between 40 and 50% Table 2.

Table 2

In vitro antagonistic activity of rhizobacteria (AS1–AS22) against *Rhizoctonia solani* f. sp.sasakii using whole-cell and cell-free filtrates in a dual culture plate assay.
Bacterial isolates	Percent mycelial inhibition (%)
Bacterial isolates	Whole cell culture	Cell free supernatant
AS1	32.78 ± 3.40^b	49.62 ± 2.57^cde
AS2	39.26 ± 3.39^bcd	48.15 ± 2.57^cde
AS3	48.15 ± 3.39^fghi	54.82 ± 3.39^ef
AS4	45.19 ± 3.85^efgh	53.33 ± 3.85^def
AS5	48.89 ± 5.13^ghij	46.67 ± 4.45^bcd
AS6	38.52 ± 5.88^c	49.63 ± 2.57^cde
AS7	44.44 ± 2.22^defg	55.56 ± 2.22^ef
AS8	39.26 ± 3.85^cd	45.93 ± 3.39^bcd
AS9	47.41 ± 5.59^efghi	49.63 ± 2.57^cde
AS10	37.78 ± 3.40^c	42.96 ± 3.39^bc
AS11	48.89 ± 4.63^ghij	48.15 ± 4.63^cde
AS12	42.96 ± 4.45^cdef	43.70 ± 5.59^bc
AS13	49.63 ± 3.40^ghij	57.78 ± 3.85^f
AS14	50.37 ± 2.57^hij	57.78 ± 4.44^f
AS15	00.00 ± 0.00^a	0.00 ± 0.00^a
AS16	52.41 ± 2.57^ijk	59.26 ± 5.59^f
AS17	51.85 ± 4.45^ij	59.26 ± 5.13^f
AS18	00.00 ± 0.00^a	0.000 ± 0.00^a
AS19	57.04 ± 5.59^k	67.41 ± 3.39^g
AS20	42.22 ± 5.59^cde	40.74 ± 5.59^b
AS21	54.07 ± 2.56^jk	65.74 ± 3.39^g
AS22	50.37 ± 2.57^hij	60.00 ± 5.88^f
Standard error mean	0.357	0.480

*Means in each column followed by the same letter were not significantly different (P < 0.05) as determined by the one-way ANOVA and Duncan’s Multiple Range Test (DMRT). Values were the means of three replications ± standard deviation.

3.3 Production of hydrolytic enzymes, PGP traits, and antifungal metabolites

Based on an in vitro antagonism activity against R. solani f. sp. sasakii, AS19 and AS21 showed the maximum percent mycelial inhibition, and these strains were screened for for the production of hydrolytic enzymes, PGP traits, and antifungal metabolites.AS19 and AS21 were positive for several antagonistic attributes, such as antifungal volatile compounds (HCN), cyclic lipopeptides (rhamnolipid), and hydrolytic enzymes (chitinase, gelatinase, and protease). They were positive for PGP traits such as phosphate solubilization, zinc solubilization, siderophore production, and IAA production. Additionally, strain AS21 was positive for reduction of nitrate (Table S3). Therefore, AS19 and AS21 were considered the most promising antagonistic bacterial strains and selected for evaluation of in vivo biocontrol efficacy against R.solani f.sp. sasakii on maize.

3.4. Morphological and Biochemical Characterization

The colonies of isolates AS19 and AS21 were round in shape, creamy in texture, and glistening on King’s B medium (Fig. 2). Under a light microscope, both isolates were Gram-negative, small rods. When biochemically characterized, both isolates were positive for catalase, H₂S, gelatinase, and oxidase, and AS21 was also positive for nitrate reductase.

3.5. Identification of Antagonistic Isolates (AS19 and AS21)

In a phylogenetic tree generated using the 16S rDNA gene sequences, strain AS19 showed 86.62%similarity with the genes of P. aeruginosa and strain AS21 showed 89.27% similarity with P. indoloxidans, respectively (Fig. 3). These strain sequences have been submitted to the Gene Bank database, and accession numbers are MK951710 and MK951711, respectively (Table S4).

3.6. Scanning Electron Microscopy Analysis

Microscopic examination of R. solanif. sp.sasakii mycelium from a dual culture plate assay showed severely distorted hyphae in comparison to the control. The isolates AS19 and AS21 caused the hyphae of R. solanif. sp.sasakii to lyse and deform (Fig. 4). In addition, both were capable of causing a substantial change in the hyphal morphology of the fungus.

3.7. Influence of Pseudomonas Strains on Sclerotial Formation and Germination

The sclerotial germination test showed that Pseudomonas strains AS19 and AS21 were capable of suppressing the sclerotial germination and formation of R. solanif. sp.sasakii(Table S5). Both strains had sclerotia formation rates of 42% and 75%, respectively, and germination rates of 31.9%. On the other hand, in the control plates, germination of the sclerotia was 100% (Fig. 5).

3.8. Detached Leaf Assay for Antagonism

When FLP isolates (AS19andAS21) and pathogenic fungi were simultaneously inoculated on leaves, the% affected area with necrotic lesions was 80.49 ± 0.89% in AS19, and 91.5 ± 2.21% in the AS21 treatment, respectively (Table 3). No disease control was observed in leaves treated with FLPs 24 h post pathogen inoculation. In this case, AS21-treated leaves exhibited 96.40 ± 2.320% necrotic lesions and AS19 exhibited 91.78 ± 0.030% necrotic lesions (Fig. 6). These results demonstrated that leaves pretreated with FLPs before pathogen inoculation exhibited better disease control than R. solanif. sp.sasakii after FLP treatment or simultaneous application of both R. solanif. sp.sasakii and FLPs. This may be due to two possibilities:(1) the antifungal substances produced by FLP isolates inhibit the growth of fungi, and (2) plant-growth-promoting substances produced by bacteria induce systemic resistance against R. solanif.sp.sasakii.

Table 3

Evaluation of antagonistic activity of FLP isolates against *R.solani*f.sp.sasakii through the detached leaf assay.
	Treatments	Total Leaf Area (cm²)	Affected Area (cm²)	% Affected Area
T1	Control (without FLP culture and R. solanif. sp.sasakii)	20.655	0.000	00.00 ± 0.00 ^a
T2	Control (with FLP AS19 alone)	27.280	2.245	08.20 ± 0.10 ^b
T3	Control (with FLP AS21 alone)	28.425	2.425	08.55 ± 0.10 ^c
T4	Control (R. solanif. sp.sasakii alone)	27.750	27.590	99.46 ± 0.10 ^j
T5	FLP AS19 at 24 h interval after fungus	26.825	24.620	91.78 ± 0.10 ^h
T6	FLP AS21 at 24 h interval after fungus	22.365	21.585	96.40 ± 0.10 ⁱ
T7	Simultaneous inoculation of FLP AS19 and R. solanif. sp.sasakii	34.435	27.890	80.49 ± 0.10 ^f
T8	Simultaneous inoculation of FLP AS21 and R. solanif. sp.sasakii	20.500	18.835	91.50 ± 0.10 ^g
T9	R. solanif. sp.sasakii at 24 h after FLP AS19	24.900	23.415	58.39 ± 0.10 ^e
T10	R. solanif. sp.sasakii at 24 h after FLP AS21	23.120	9.135	40.13 ± 0.10 ^d

Means in each column followed by the same letter were not significantly different (p < 0.05) as determined by the one-way ANOVA and Duncan’s multiple range test(DMRT). Values were the means of three replications ± standard deviation.

3.9. Pot Experiment A—Evaluating the Biocontrol Potential of Two Antagonistic Isolates against R. solani f. sp. sasakii

The lowest disease incidence of 40% was observed in combined soil application and seed treatment of AS19 (AS19A3). It was 38.33% less than the negative control (-CON A).

Soil application and seed treatment of AS19 significantly reduced the occurrence of banded leaf and sheath blight compared to seed treatment with the commercial fungicide carbendazim (Table 4). The shoot length of the treatments was in the following descending order: AS19A2, AS21A1, AS21A2, AS19A3, AS21A3, AS19A1, +CONA, -CONA, and AB.CON. There was no correlation between plant growth promotion and% disease incidence. The highest shoot length of 71.60 cm was observed in seed treatment alone with AS19. It was 30.77% higher than the absolute control (AB.CON) and 45.97% higher than the negative control (-CONA). The highest biomass and fresh weight were observed in the soil application plus seed treatment of AS21. It was 9.10 g and 33.20 g, respectively. A comparison of plant growth in -CONA and AB.CON showed that there was a reduction in biomass and shoot length due to BL&SB. Biomass production and fresh weight of the treatments were in the following descending order: AS21A3, AS21A2, AS19A1, AS19A3, AS19A2, AS21A1, +CONA, AB.CON, and -CONA. The fresh weight and biomass were highest in the plants receiving soil application plus seed treatment for AS21. The fresh weight increased by 19.03 g and the biomass by 6.75 g compared to the negative control, whereas they increased by 15.6 g and 5.73 g compared to healthy plants (AB.CON), respectively. From the results, it was confirmed that fungal infection resulted in biomass reduction by 2.404%. This reduction in disease index is positively correlated with the level of antioxidant enzyme activities.

Table 4

Disease assessment and plant-growth-promoting attributes for evaluating the biocontrol potential of two antagonistic isolates against *R.solani*f.sp. *sasakii.*
Treatments	Disease Incidence (%)	Infection Length (cm)	Shoot Length (cm)	Average Fresh Weight/plant (g)	Average Biomass/Plant (g)
AB.CON	0.00 ^a	0.00 ^a	54.75 ± 3.73 ^b	17.60 ± 0.62 ^b	3.37 ± 0.15 ^b
+CONA	43.33 ± 5.77 ^bc	6.35 ± 0.99 ^e	64.03 ± 1.52 ^c	22.45 ± 1.83 ^c	4.79 ± 0.05 ^c
-CONA	78.33 ± 2.89 ^e	6.38 ± 0.46 ^e	49.05 ± 3.03 ^a	14.17 ± 0.76 ^a	2.35 ± 0.07 ^a
AS19A1	53.33 ± 5.77 ^cd	2.82 ± 0.59 ^bc	64.83 ± 2.25 ^c	32.30 ± 2.13 ^f	6.51 ± 0.30 ^e
AS19A2	50.00 ± 5.04 ^bcd	3.68 ± 0.42 ^cd	71.60 ± 2.40 ^d	28.03 ± 1.75 ^de	6.11 ± 0.12 ^de
AS19A3	40.00 ± 0.10 ^b	2.17 ± 0.64 ^b	67.62 ± 2.17 ^cd	30.34 ± 2.99 ^ef	6.14 ± 0.88 ^de
AS21A1	72.22 ± 4.81 ^e	2.74 ± 0.45 ^bc	70.39 ± 3.39 ^d	26.61 ± 1.80 ^d	5.16 ± 0.81 ^cd
AS21A2	56.67 ± 5.77 ^d	4.31 ± 0.68 ^d	69.20 ± 2.77 ^cd	31.56 ± 1.96 ^f	7.87 ± 1.61 ^f
AS21A3	46.67 ± 5.77 ^bcd	2.12 ± 0.24 ^b	67.33 ± 3.69 ^cd	33.20 ± 2.22 ^f	9.10 ± 0.53 ^g
Standard Error Mean	1.044	0.106	0.551	0.367	0.112

Means in each column followed by the same letter were not significantly different (p < 0.05) as determined by the one-way ANOVA and Duncan’s multiple range test (DMRT). Values were the means of three replications ± standard deviation.

Total Phenolic Content and Host Defensive Enzymes

Total phenol content was highest in the plants treated with carbendazim + CONA (i.e., 158.71 mg), followed by plants with soil application of AS19A1 (146.16 mg catechol) at 15 DAI. At 30DAI, phenol content was reduced in the treatments + CONA, AS19A1, AS19A2, AS21A1, AS21A3, B.CON, and increased in treatments -CONA, AS19A3, and AS21A2. At 45 DAI, the phenol content was reduced in treatments + CON A, AS19A1, AS19A2, and AS21A2, and increased in the treatments -CONA, AS19A3, AS21A1, and AS21A3.The total phenol level was lower in healthy plants (AB.CON) (Fig. 7). Similar results, where total phenol was higher in infected plants as compared to healthy ones, have been reported in maize [67]. The amount of total phenol in each treatment was compared with its percent disease incidence. It was observed that the increased phenol content leads to increased plant resistance towards R.solanif.sp.sasakii. The synthesis and accumulation of defense-related enzymes play important roles in plant defense mechanisms. Phenylalanine ammonia lyase plays an important role in the biosynthesis of phenolic phytoalexins [72]. The treatments, in the descending order of PAL activity, wereAS19A3,AS21A3,AS19A2,AS19A1,AS21A2, and AS21A1 at all three sampling schedules of 15, 30,and 45 DAI. It was significantly three times higher (p < 0.05) than diseased plants (-CONA) in the samples of soil application and seed treatment from AS19 and AS21. Peroxidase activity was in the following descending order in treatments:AS19A3, AS21A3, AS21A2, AS19A2,AS21A1, and AS19A1at 15 DAI. Then, at 30 and 45 DAI, no definite trend in the PO activity was observed. The polyphenol oxidase enzyme activity was also higher in the soil and seed treatment of AS19 than in other treatments. Both of the strains also enhanced plant growth promotion. Similar results were reported earlier as well, where Bacillus species induced a multi-fold increase in the level of antioxidant defense enzymes [73]. This shows that plants have evolved reactive-oxygen-species-scavenging antioxidative enzymes to mitigate the harmful effects of oxidative stress. Hence, increased antioxidative enzymes reduced the ROS level and enhanced cell membrane stability [74].

3.10. Pot Experiment B—Evaluating Biocontrol Mechanisms

Percent disease incidence and infection length were observed (Table 5). The soil application plus seed treatment of AS21 (AS21B3) showed the lowest disease incidence (35.56%). Infection length in the same was 1.60 ± 0.09 cm, which was less than infection length in carbendazim-treated plants (+ CONB) (3.80 ± 0.17 cm). The soil plus seed treatment of AS21 exhibited 36.67% disease incidence and 1.47 cm infection length. Soil plus seed treatment of FLPs (AS19 and AS21) gave better disease control than either soil application or seed treatment. The percent disease incidence in soil application plus seed treatment during the pot trial B was less than percent disease incidence in the corresponding treatment during the pot trial A, i.e., the percent disease incidence in AS19B3 was 3.33% lower than AS19A3, whereas in AS21B3, it was 11.11% lower than AS21A3. Hence, the interpretation is that the inoculation of bacterial strains resulted in the induction of systemic resistance. The shoot length values of maize plants in seed treatment with AS21, seed treatment plus soil application of AS21, and AS19 were81.9, 78.10, and 76.33 cm, respectively. These were 40.10, 37.20, and 35.74% higher in the corresponding treatments than the absolute control and 49.63, 47.18, and 45.46% higher than negative control, respectively. The fresh weight values of AS19 and AS21-treated plants were 40.51 g and 37.01 g, respectively. The corresponding biomass values were 8.99 g and 8.83 g, respectively. Fresh weights in AS19 and AS21-treated plants were26.46 g and 22.96 g higher than diseased plants, 22.91 g and19.41 g higher than healthy plants (AB.CON), and 25.73 g and 22.23 g higher than carbendazim-treated plants, respectively. While the biomass values of plants treated with AS19 and AS21 were6.66 g and 6.5 g higher than diseased plants, 5.62 g and5.46 g higher than healthy plants (AB.CON), and 5.83 g and 5.67 g higher than commercial-fungicide-treated plants, respectively. The phenol contents in the soil application plus seed treatment of AS19(173.59) and AS21(153.59) in pot trial B were28.31 and 13.52 mg of catechol/g fresh leaves more than pot trial A, respectively (Fig. 8). Henceforth, it was concluded that the pretreatment of bacterial strains (AS19andAS21) induced a higher phenol content in maize plants. Phenylalanine ammonia lyase activity significantly was fourfold (p ≤ 0.05) higher than that of diseased plants (-CONA) in the soil application and seed treatment samples of AS19 and AS21.Peroxidaseactivity in the treatments was in the following descending order at 15 DAI:AS19B3, AS21B3, AS19B2, AS21B2,AS21B1,and AS19B1.The polyphenol oxidase enzyme activity was significantly higher (p ≤ 0.05)in soil plus seed treatment of AS19 than in other treatments. The levels of PAL, PO, PPO, and total phenol content were high, and disease incidence was lower in bacteria-treated maize plants. The reduction was more in pot trial B. Out of the three methods of application, soil plus seed treatment of strains AS19 and AS21 resulted in the highest reduction of disease incidence. From these results, it was revealed that the treatment of plants with bacterial strains prior to infection increases the total phenol content and activity of host defensive enzymes.CK Chow [75] reported that upregulation of total phenolics and defensive enzymes has been found to be directly or indirectly related to the induction of systemic resistance. Similar results have been reported by Ting [76]. Henceforth, AS19 and AS21 bacterial strains were antagonistic against R. solanif. sp.sasakii through ISR mechanisms. Latha [77] reported that P. fluorescens and B. subtilis increased POD activity in tomato tissues. Hence, BCAs may play a key role in reducing the damage caused by pathogens by increasing the levels of antioxidant enzymes.

Table 5

Disease assessment and plant-growth-promoting attributes for evaluating biocontrol mechanisms.
Treatments	Disease Incidence (%)	Infection Length (cm)	Shoot Length (cm)	Average Fresh Weight/Plant (g)	Average Biomass/Plant (g)
AB.CON	0.00 ^a	0.00 ^a	49.05 ± 3.03 ^a	17.60 ± 0.62 ^b	3.37 ± 0.15 ^b
+CONB	23.33 ± 2.88 ^b	3.80 ± 0.17 ^de	79.85 ± 1.77 ^d	1.78 ± 2.00 ^a	3.16 ± 0.23 ^ab
−CONB	99.50 ± 4.76 ^f	7.20 ± 0.53 ^f	41.25 ± 3.25 ^a	14.05 ± 1.48 ^a	2.33 ± 0.25 ^a
AS19B1	50.00 ± 0.00 ^e	3.98 ± 0.08 ^de	70.47 ± 4.15 ^c	34.81 ± 1.48 ^de	6.46 ± 0.74 ^d
AS19B2	48.89 ± 4.39 ^de	3.33 ± 0.85 ^d	51.20 ± 0.90 ^a	22.48 ± 1.77 ^c	4.46 ± 0.46 ^c
AS19B3	36.67 ± 2.64 ^c	1.47 ± 0.16 ^b	76.33 ± 2.70 ^cd	40.51 ± 1.81 ^f	8.99 ± 0.85 ^e
AS21B1	48.59 ± 4.39 ^de	1.48 ± 0.03 ^b	60.82 ± 1.26 ^b	36.42 ± 1.34 ^e	6.25 ± 0.53 ^d
AS21B2	67.22 ± 3.52 ^e	4.13 ± 0.19 ^e	81.90 ± 1.92 ^e	32.45 ± 1.94 ^d	6.13 ± 0.67 ^d
AS21B3	35.56 ± 2.63 ^c	1.60 ± 0.09 ^b	78.10 ± 1.68 ^d	37.01 ± 0.51 ^e	8.83 ± 0.69 ^e
Standard Error Mean	1.583	0.068	0.258	0.294	0.108

Means in each column followed by the same letter were not significantly different (p < 0.05) as determined by the one-way ANOVA and Duncan’s multiple range test (DMRT). Values were the means of three replications ± standard deviation.

3.11. Pot Experiment C—Validating Induced Systemic Resistance (ISR)-Mediated Control by Foliar Spray of Antagonistic Strains

Percent disease incidence and infection length were observed for validating the induced systemic resistance (ISR) mechanism of foliar spraying of bacterial strains against R.solanif.sp.sasakii.The percent disease incidence by treatment of AS19 and AS21 prior to infection was 38.89% and 41.11%, respectively (Table 6).The results of the trial confirmed that control through foliar spraying of biocontrol agents was ISR-mediated. The pretreatment of both bacterial strains, AS19 and AS21, prior to infection produced the highest shoot lengths of 59.83 cm and 56.40 cm, respectively. They were higher than the absolute control by 10.78 and 7.35 cm and diseased plants by 24.03 and 20.6 cm, respectively. The highest biomass and fresh weight were observed in the treatment of foliar spray of AS19 24 h prior to infection (AS19C1). The treatments had a fresh weight of 25.59 g and a biomass of 5.423g. The phenol content was reduced in all the treatments by 30 and 45 days after induction (Fig. 9). Total phenol content in g fresh leaves was highest in the treatment AS19C1, i.e.,158.03 mg catechol. The phenylalanine ammonia lyase level in 30 DAI samples was significantly decreased from 15 DAI samples in all the treatments except – CON B and absolute control. The PAL activity in C1 treatments of AS19 and AS21 were three fold (p ≤ 0.05) higher than the negative control (− CONB).Then, peroxidase content was significantly decreased from samples of 15 DAI in all treatments at 30 DAI and at 45 DAI. PO activity in the C1 treatments of AS19 and AS21 was significantly seven and six fold (p ≤ 0.05) higher than in the untreated control, respectively. Polyphenol oxidase (PPO)activity of the treatments was in following descending manner, viz., AS19C1, AS21C1, AS19C2, and AS21C2. In conclusion, foliar spraying of AS19 and AS21 bacterial strains 24 h prior to infection of R.solanif.sp.sasakii induced systemic resistance in the plants, viz., in treatments AS19C1 and AS21C1.The percent incidence of BL&SB disease was reduced, whereas the production of host defensive enzymes increased in treatments AS19C1and AS21C1. This result was similar to the earlier study that showed ISR mechanisms can be achieved by the production of host defensive enzymes and increased phenol content [76]. Pot trial C validated that the ISR-mediated control of BL&SB disease through foliar spray of bacterial strains AS19 and AS21. It leads to the added advantage of foliar spraying over soil application plus seed treatment of bacterial strains AS19 and AS21. Foliar spraying can also be given after the establishment of the crop. The ISR-mediated mechanism of control through foliar spraying was one of the best achievements of the study.

Table 6

Disease assessment and plant-growth-promoting attributes for validating ISR through foliar spraying of biocontrol agents.
Treatments	Disease Incidence (%)	Infection Length (cm)	Shoot Length (cm)	Average Fresh Weight/Plant (g)	Average Biomass/Plant (g)
AB.CON	0.00^a	0.00^a	49.05 ± 1.320^c	17.597 ± 0.619^c	3.367 ± 0.153^bc
+CONC	67.22 ± 7.515^d	3.78 ± 0.142^b	36.63 ± 1.320^ab	20.733 ± 0.681^d	4.160 ± 0.246^d
-CONC	82.22 ± 6.777^e	6.70 ± 0.926^c	35.80 ± 3.045^ab	14.233 ± 0.643^b	2.333 ± 0.252^a
AS19C1	43.33 ± 9.547^b	1.37 ± 2.367^a	59.83 ± 4.554^d	25.593 ± 0.665^e	5.423 ± 0.108^e
AS19C2	83.33 ± 8.390^e	5.61 ± 0.317^bc	40.73 ± 1.570^b	11.860 ± 0.740^a	2.087 ± 0.241^s
AS21C1	41.11 ± 4.434^c	1.30 ± 2.252^a	56.40 ± 2.762^d	18.133 ± 0.603^c	4.533 ± 0.153^d
AS21C2	83.33 ± 4.434^e	6.24 ± 0.019^c	35.13 ± 2.839^a	13.017 ± 1.107^ab	3.100 ± 0.265^b
Standard Error Mean	2.553	0.282	0.634	0.162	0.046

Means in each column followed by the same letter were not significantly different (p < 0.05) as determined by the one-way ANOVA and Duncan’s multiple range test (DMRT). Values were the means of three replications ± standard deviation.

3.12. Broad-Spectrum Antagonistic Activity of Pseudomonas spp. AS19 and AS21

AS19 and AS21 inhibited A. triticina mycelium by 49.3% and 53.5%, B. sorokiniana by 50.3% and 51.0%,Helminthosporium maydis by 47.23% and 48.9%, and F. oxysporum f. sp.lentis by 38.9% and 37.9%, respectively (Fig. 10, Table S6).

The banded leaf and sheath blight (BLSB) incited by R. solanif. sp.sasakii is a very destructive disease of maize. Due to the harmful effects of chemicals and widely adapted methods of disease management, researchers are focusing on the application of rhizobacteria as a sustainable approach. The present study was conducted to screen the rhizobacteria recovered from grassland in Uttarakhand against the BL&SB pathogen, to investigate their biocontrol efficacy, and validate the ISR mechanism. Amongst the 22 isolates, AS19, followed by AS21, showed strong antifungal activity against all the tested fungi in the in vitro antagonism tests, using whole-cell culture as well as culture filtrate. The zone of inhibition around tested plant pathogens by AS19 and AS21 may be linked to their ability to produce secondary metabolites such as phenazine, ammonia, and hydrolytic enzymes [43, 79]. Based on 16S rDNA analysis, the results revealed that AS19 belongs to Pseudomonas aeruginosa and AS21 belongs to P. indoloxidans. Pseudomonas spp. have been reported to have great potential for plant growth promotion and plant disease management [80].The pseudomonad group has been extensively studied for their ability to produce hydrolytic enzymes and antimicrobials such as 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, and phenazines [80, 81]. However, these hydrolytic enzymes, such as chitinase, cellulase, protease, and amylase, were evidenced in AS19 and AS21 that inhibit R. solanif.sp.sasakii in vitro. Additionally, Pseudomonas spp. are not typically associated with biocontrol activity but nevertheless contain plant-growth-promoting activity [82]. Therefore, in vitro tests showed the antagonistic as well as plant-growth-promotion activities of the isolates. Microscopic observation of the mycelium showed that the antagonistic strains AS19 and AS21 led to changes in the mycelium of the pathogen. The fungal mycelia in dual culture with the antagonistic bacteria were distorted compared to the control. The Pseudomonas spp. strains AS19 and AS21 suppressed the mycelial growth and germination of sclerotia of R. solanif.sp.sasakii. Management of BL&SB is difficult due to sclerotia formation [83]. Hence, the potential of AS19 and AS21 in inhibiting the germination of sclerotia of R. solanif.sp.sasakii has huge implications for the sustainable management of BL&SB in maize. The effect of plant-growth-promoting bacteria in inhibiting sclerotial germination of R. solani AG-1(IA) has previously been reported [84, 85]. Therefore, bacterial antagonists capable of producing antimicrobial compounds and hydrolytic enzymes with potent inhibitory activity against plant pathogens are more likely to prevent pathogenic fungi and limit the establishment of the disease. Overall, from the above results, we discovered that strains AS19 and AS21 had direct antagonistic effects against the tested pathogen. These two FLP isolates, AS19 and AS21, also showed in vitro antagonism against pathogens of other alternate crops in maize-based cropping systems such as Alternaria triticina, Bipolaris sorokiniana, Rhizoctonia maydis,andFusarium oxysporumf.sp.lentis.After that,the greenhouse experiment was established with three different objectives. The pot trial A revealed that bacterial strains AS19 and AS21 reduced the BL&SB disease incidence in maize in vivo. The soil application plus seed treatment of FLPs (AS19 and AS21) resulted in a higher disease reduction than either soil application alone or seed treatment alone. Seed bio-priming enabled microbial inoculants to establish close contact with the seed and assist in growth promotion and disease reduction [32]. The disease reduction in FLP-treated plants was 38.33 and 31.66%, respectively, compared to the control plants. There was a negative correlation between host defensive enzymes and percent disease incidence. The soil application plus seed treatment of FLPs (AS19 and AS21) also promoted the plant growth. The pot trial B demonstrated that induced systemic resistance was the mechanism of disease control by bacterial strains AS19 and AS21. The pot trial C validated that foliar spraying of bacterial strains AS19 and AS21 was also effective for reducing BL&SB disease. The percent disease reduction in maize plants with foliar sprays of AS19 and AS21 individually given before disease induction was higher than where foliar sprays of BCA were given after disease induction. Hence, biocontrol through foliar spraying of AS19 and AS21 was ISR-mediated. However, bio-priming of the plants with both Pseudomonas strains and subsequent foliar spray of AS19andAS21 significantly activated defense enzymes in the plant and thus restrained the disease progression. The induction and accumulation of antioxidant and defense-related enzymes in pretreated plants enhanced plant health and growth even under stressful circumstances. In the present study, significantly higher accumulation of antioxidant and defense-related enzymes such as activity of PAL, peroxidase, and polyphenol peroxidase were recorded in the plants which were bio-primed with Pseudomonas strains AS19andAS21 and subsequent foliar spraying of these cultures as compared to plants inoculated with the pathogen alone. Substantial increases in phenolics and PAL activities were correlated to enhanced disease resistance in maize via ISR.PAListheessentialenzymethatcausesinducedsystemicresistanceinplantsviathephenylpropanoidpathway.Theperoxidasewastriggeredbytheelevated PALlevel,whichresultedintheproductionoflignin, and the peroxidase and superoxide dismutase assisted in eliminating reactive oxygen species [87].Ligninandotherphenolicsproducedbypolyphenoloxidaseencouragethe cell membranes [64, 86].Increased lignification represents the adaptive mechanism. Though, the results show that disease control in both the Pseudomonas strains is mediated by the induced systemic resistance mechanism in maize against. solani.

This is the first report of the experimental confirmation of Pseudomonas spp. AS19 and AS21 isolated from the grassland rhizosphere located in Uttarakhand, India. They were prominent antagonists against R. solanif.sp. sasakii and were able to produce various antagonistic compounds, including siderophores and hydrolytic enzymes, as well as plant-growth-promotion traits in vitro. The strains of Pseudomonas (AS19 and AS21) also exhibited in vitro broad-spectrum antagonistic activity against phytopathogenic fungi of other crops in the maize-based cropping system. The scanning electron microscopy study revealed that the mycelium of R. solanif.sp. sasakii was distorted in its morphology by these antagonist strains. The bacterial strains AS19 and AS21 also manifested in vivo biocontrol potential against banded leaf and sheath blight in maize. The bio-primed seed with rhizospheric Pseudomonas strains AS19andAS21 and subsequent foliar spray of culture suspension resulted in significant enhancement of antioxidative defense enzymes in the maize plant with a simultaneous decrease in the H₂O₂concentration.Induced systemic resistance was the mechanism for disease control by the activation of the phenylpropanoid pathway, and enhanced accumulation of phenolics significantly decreased the disease severity and infection length with increased accumulation of plant root and shoot biomass in the maize plants pre-inoculated with R. solani. These findings suggest that Pseudomonas strains AS19 and AS21 have the potential to be used as a biocontrol agent in the management of BL&SB in maize. In the future, there will be a need to validate the biocontrol potential of AS19 and AS21 through field studies and identify the metabolite responsible for biocontrol.

Author Contributions: M.S.: conceptualization, methodology, and supervision; N.S.: reviewing and preparation of final draft of manuscript, preparation of graphs, statistical analysis B.NT.: experimentation, recording of results, A.P.: editing and reviewing of manuscript; R.P.S.: inputs for conduct of pot trial and framing of manuscript; V.J., M.K.: conceptualization validation of data;. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.

Funding: The authors gratefully acknowledge the G.B Pant University of Agriculture and Technology, Pantnagar, India, and the Indian Council for Agricultural Research, GOI for providing research support.

Informed Consent Statement: All the co-authors have already given their consent for publication of this manuscript.

Data Availability Statement: The authors declare that all the figures and tables presented in this manuscript are original and the data supporting the findings of this study are available with the article and its supplementary material. Raw data that support the findings are available from the corresponding author upon reasonable request.

Acknowledgements: The authors thank the Department of Plant Pathology, G.B.PUA&T, Pantnagar, Uttarakhand, India for providing polyhouse facilities.

Conflicts of Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Consent to Participate: All the co-authors participated in the preparation of this manuscript as per the requirement.

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Antagonistic potential and biological control mechanisms of Pseudomonas strains against banded leaf and sheath blight disease of maize

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Abstract

Figures

Highlights

1. Introduction

2. Material and Methods

2.1 Microorganisms

2.2 In vitro screening of FLPs for antagonism-

2.2.1 Effect of whole cell culture on mycelial growth of R. solani

2.2.2 Effect of cell free culture filtrates (CFCF) on mycelial growth of R. solani

2.3 Investigation of potential biological control mechanism-

2.3.1 Detection of extracellular enzymes

2.3.2. Production of antifungal metabolite

2.3.3 Test for plant growth promoting traits –

2.4 Morphological and Biochemical characterization of isolates

2.5 Identification of putative antagonistic strains AS19 and AS21

2.6 Scanning microscopy investigation of in vitro co-cultures of pathogenic fungi and potent bacteria

2.7 Effect of Pseudomonas spp. AS19 and AS21 on sclerotium germination and development

2.8 Efficacy of the strains AS19 and AS21 against R. solani on detached leaf –

2.9 Green house experiment-

2.9.1 Experimental design

2.9.2 Preparation of bacterial inoculum

2.9.3 Pot experiment A-

2.9.4 Pot experiment B-

2.9.5 Pot experiment C

2.10 Estimation of Total Phenolic content and Defense related Enzymes

2.10.1 Total phenolic content

2.10.2 Defense related enzymes

2.11. Broad-Spectrum Activity of Potent Pseudomonas Strains

2.12. Statistical Analysis

3. Results

3.1. In Vitro Antagonism Using Whole-Cell Culture(WCC)

3.2 In vitro antagonism using cell-free culture filtrate

3.3 Production of hydrolytic enzymes, PGP traits, and antifungal metabolites

3.4. Morphological and Biochemical Characterization

3.5. Identification of Antagonistic Isolates (AS19 and AS21)

3.6. Scanning Electron Microscopy Analysis

3.7. Influence of Pseudomonas Strains on Sclerotial Formation and Germination

3.8. Detached Leaf Assay for Antagonism

3.10. Pot Experiment B—Evaluating Biocontrol Mechanisms

3.11. Pot Experiment C—Validating Induced Systemic Resistance (ISR)-Mediated Control by Foliar Spray of Antagonistic Strains

3.12. Broad-Spectrum Antagonistic Activity of Pseudomonas spp. AS19 and AS21

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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