Introduction

Powdery mildew of grapevine (Vitis vinifera L.) is caused by Erysiphe necator (earlier Uncinula necator (Schw.) Burn). This pathogen is biotropic in nature and can infect all green tissues of the grapevines under warm and humid weather. India is largely a table grape producer and most of the commercial varieties are highly susceptible to powdery mildew. The disease can be present almost through the year, except for a brief period during summer when the hot and dry weather becomes unfavourable for powdery mildew infections (Sawant et al. 2015). Infection reduces vine productivity, yield and quality of fruits; hence a number of chemical and biological interventions are needed to protect the crop and avoid significant yield losses. Grapevines are trained on extended ‘Y’ trellises and complete coverage of the foliage during spray applications is not achieved. The pathogen continues to grow and sporulate on the unprotected foliage and there is thus a continuous availability of inoculum to start fresh infections as soon as the protective effect of the applied control measure has diminished. Increasing use of chemical fungicides causes several negative effects including development of fungicide resistance in the pathogen and presence of objectionable levels of residues at harvest (Sawant et al. 2016b). There was thus a need for more effective, alternative methods of plant protection.

Most of the plants possess inducible defense mechanisms against a number of pathogens, which can be activated upon treatment with specific microorganisms and this state of enhanced defensive capacity can be seen in tissues spatially distant from the site of application of the microorganism, hence it is called as induced systemic resistance (Walters 2009; van Loon 2000). The induction of systemic resistance in plants against soil borne and foliar diseases by microorganisms applied to the soil has been reviewed with respect to its potential use in crop protection (Kloepper et al. 1999). Induced systemic resistance (ISR) was described earlier in carnation plants that were systemically protected by Pseudomonas fluorescens strain WCS417r against Fusarium oxysporum f. sp. dianthi (Van Peer et al. 1991) and in cucumber plants against Colletotrichum orbiculare by select strains of plant growth-promoting rhizobacteria (Wei et al. 1991). In studies under greenhouse and field conditions, specific strains of plant-growth–promoting rhizobacteria (PGPR), especially Bacillus species elicited systemic protection of bell pepper, tomato, sugar beet, water melon, musk melon, cucumber against a range of pathogens (Kloepper et al. 2004; Wei et al. 1996; Yan et al. 2002).

Plant growth promoting rhizobacteria (PGPR), too, provide protection to plants from diseases by employing different mechanisms which include increases in defense related enzyme activities such as phenylalanine ammonia-lyase, peroxidase, polyphenoloxidase, ß-1,3-glucanase and chitinase; as well as induction of specific pathogen related proteins (PRs) in leaves of plants; production of siderophore; indole acetic acid, hydrogen cyanide; nutrient solobilization etc. which has been aptly reviewed by Vejan et al. (2016) and Choudhary et al. (2007). While peroxidase and polyphenoloxidase are catalysts in the formation of lignin, PAL and other enzymes are involved in the formation of phytoalexins. The microbial metabolite, hydrogen cyanide (HCN), promotes plant nitrogen accumulation and root elongation (Marques et al. 2010) and may play a role in biological control of pathogens (Defago and Haas 1990).

Chitin and glucan are cell wall components of many pathogenic fungi and de-polymerization of cell wall by the combined activity of chitinases and glucanases could kill fungi. Several specific strains of Bacillus species, viz. B. amyloliquifaciens, B. subtilis, B. mycoides, B. pasteurii, B. pumilus, and B. sphaericus were shown to elicit significant reduction in the incidence or severity of various diseases on a diversity of crop plants (Choudhary and Johri 2009). The efficacy of PGPR-ISR isolates against a disease can be enhanced slightly when they are used in mixtures; furthermore their efficacy in controlling multiple diseases on same host species can also be improved (Raupach and Kloepper 1998). However, registration of formulations containing two or more organisms may not always be feasible.

The objective of this study was to identify bacterial isolates which can be gainfully used in vineyards for induction of systemic resistance against powdery mildew of grapevine. Two hundred and ninety-three bacteria which were earlier isolated from the grapevine ecosystem were systematically screened in laboratory, glass house and field for plant growth promotion, production of plant growth promoting metabolites and biochemicals associated with ISR and finally for induction of systemic resistance in grapevines.

Materials and methods

Bacteria

Two hundred and ninety-three bacteria which were earlier isolated from the grapevine eco-system (Sawant et al. 2016a) were used for the study. The cultures were maintained on nutrient agar slants (NA, M002, HiMedia) at 4 °C and were retrieved and grown on NA plates as and when required.

Screening for plant growth promotion activity

The plant growth promotion ability of the 293 bacteria was tested using healthy cowpea seeds which were surface sterilized using 4% sodium hypochlorite solution. The seeds were soaked in any one the bacterial suspension for 6 h and then transferred into sterile 2% water agar plates using sterile forceps. Cowpea seeds soaked in sterile distilled water (SDW) were maintained as control. Three replicates each of eight seeds were maintained for each isolate. Plates were incubated at 28 °C for 72 h in incubator (Binder, KBW 450) and the root length was measured. Percent increase in root length in treatments over control was calculated. The test was repeated twice. Twenty-two bacteria which provided more than 15 percent increase in root length were selected for pot studies. These 22 plant growth promoting bacteria (PGPB) were characterized based on morphological features as per Bergy’s manual of determinative bacteriology.

Development of simple liquid formulation

100 ml nutrient broth (NB, M001, HiMedia) contained in 250 ml conical flasks was steam sterilized and inoculated with a loop full of culture taken from a 24 h old growth on NA. The flasks were incubated at 28 ± 2 °C on an orbital shaker at 120 rpm for 72 h. The bacterial cells were harvested by centrifugation at 5000 rpm for 5 min. The pellets were washed twice with SDW and were used immediately or stored at 0 °C (for maximum 7 days) till use. The pellets were re-suspended in SDW, containing two to three drops of 0.05% Tween 80 per litre, and the OD of the suspension was read at 595 nm using UV visible spectrophotometer (Thermo Scientific Evolution 201). The OD was adjusted to 0.3 using SDW, to provide approximately 1 × 108 CFU/ml. In the mixture, each Bacillus isolate was at half concentration to maintain uniformity in concentration of applied inoculum. Freshly prepared formulation was used in all studies.

In vivo screening for ISR in pots

The ability of the selected 22 PGPB to induce ISR in grape against powdery mildew was studied on healthy 2 months old own rooted cuttings of Vitis vinifera cv. Thompson Seedless. The plants were raised in 25 cm diameter pots filled with 7 kg soil: sand: FYM (2:1:1v/v) mixture. The plants in all the three studies were maintained with two shoots trained on bamboo sticks. Plants were maintained with regular cultural operations except that they were not applied with any chemical effective against powdery mildew. At the first prediction of weather based risk period, fifty mL of inoculum was applied as a soil drench to each plant. Plants drenched with water were maintained as control. Five replicates of one plant each were maintained. All treatments were repeated after 15 days. Plants were rated for disease severity 21 days after the date of first treatment. Powdery mildew severity and percent disease index (PDI) was calculated as given earlier (Sawant et al. 2016a)

Analysis of biochemicals associated with disease resistance

Leaves of plants treated with eleven Bacillus spp., DR-38, DR-39, TS-45, DR-92, CS-126, TL-171, DRo-197, CS-212, TP-230, TP-232 and GR-280, which restricted powdery mildew severity to less than 50 PDI as compared to 76.80 PDI in control of were taken for analysis of peroxidase, polyphenol oxidase, total phenols and total proteins. Total phenol content was determined by the Folin- Ciocalteu method (Singleton and Rossi 1965), using gallic acid as the standard. Polyphenol oxidase (E.C. 1.14.18.1) and peroxidase (E.C. 1.11.1.7) activities were measured as per the procedures given by Sadasivam and Manickam (1996) using catechol as substrates. Total protein content was determined by Lowry’s method (Lowry et al. 1951) using bovine serum albumin (BSA) (Sigma) as standard.

In vitro screening for production of growth promoting metabolites

The ability of the eleven Bacillus spp. which restricted powdery mildew severity to less than 50 PDI, to produce growth promoting metabolites was tested qualitatively in vitro. Twenty-four h grown culture of bacteria were used for the studies. Indole acetic acid (IAA) production was detected in sterilized 20 ml nutrient broth containing 2 mg/ml tryptophan following the procedure of Brick et al. (1991). IAA production was indicated by the production of pink colour in the presence of tryptophan after addition of Salkowski reagent. Ammonia (NH3) production was studied in 10 ml peptone water in tubes using Nessler’s reagent as per the procedure given by Cappuccino and Sherman (1992). Ammonia production was indicated by yellow to brown colour development after addition of Nessler’s reagent. Production of hydrogen cyanide (HCN) was studied by the procedure of Lorck (1948) using nutrient agar amended with 4.5% glycine and using Whatman filter paper No. 1 soaked in 2% sodium carbonate and 1% picric acid solution as indicator for HCN production. HCN production was indicated by a change in colour from yellow to dark brown. Siderophore production was tested using Chrome azurol S agar medium (O-CAS assay) as given by Schwyn and Neilands (1987). Phosphate solubilization was detected in Pikovskaya’s agar medium (Gaur 1990). Halo zone around colony indicated phosphate solubilization.

Field evaluation

Grape cultivar, planting distance and trellis architecture

Studies were conducted on highly susceptible Vitis vinifera table grape cultivar Centennial Seedless. Vines were eight year old at the beginning of the trials and were spaced at 300 × 180 cm and trained with four cordons on extended ‘Y’ trellises with 10-11 shoots per cordon. All trials were conducted under regular growing conditions at the research farm of this Centre at Manjari, Pune, Maharashtra.

Field treatments

The four promising Bacillus spp., TS-45, DR-92, TL-171, and TP-232 were evaluated in field for induction of systemic resistance. Each isolate was used alone and in combination with TL-171 which was a good inducer of resistance against downy mildew, another important disease of grapevines (unpublished data). Furthermore the combination of TS-45 and DR-92 the two isolates which produced/induced higher levels of biochemicals in vitro and in vivo was also included. Due to limitation of experimental vines, the other possible combinations were not included. One L Bacillus formulation was applied on each side of the vine in the drip circle as soil drench. The vines were applied with recommended fungicides as foliar sprays whenever high disease risk was predicted. Vines applied with only fungicide sprays were treated as control.

Three trials were conducted during the fruiting growth phase, 2015–16; vegetative growth phase, 2016; and fruiting growth phase, 2016–17. The treatment applications were need based and the intervals were decided on the predicted risk of powdery mildew based on weather data. In the first trial, Bacillus spp. were applied thrice at 15 day intervals from 23 November to 23rd December 2015. In the subsequent two trials, Bacillus spp. were applied five times at weekly intervals from 6th June to 2nd July 2016 and from 15th November 2016 to 14th March 2017, respectively. Trials were conducted in RBD with 4 replicates consisting of three vines per replicate.

Disease observations and calculations

Powdery mildew severity was recorded periodically on 10 leaves per shoot as ratings on a 0–5 scale and PDI was calculated as given earlier (Sawant et al. 2016a). Powdery mildew ratings were also recorded at harvest on four bunches per vine using a 0–9 scale where, 0, no disease; 1, ≤ 10%; 2, 10% ≤ 20%; 3, 20% ≤ 30%; 4, 30% ≤ 40%; 5, 40% < 60%; 6, 60% < 70%; 7, 70% < 80%; 8, 80% < 90%; 9, ≥ 90% area covered with powdery growth (Bennett and Westcott 1982).

Induction of host defense mechanisms by the promising Bacillus spp.

The levels of disease related bio-chemicals were analysed in leaves of Bacillus treated vines during March 2017. The 5th, 6th and 7th leaf from the apex of the shoots were harvested 6 days after last drench. The leaves were shredded, mixed well, and a composite sample was taken for analysis. Total phenols, peroxidase, polyphenol oxidase and total proteins were determined as mentioned above. Chitinase and β-1,3-glucanase activities were measured by the colorimetric method given by Gupta et al. (2013) using colloidal chitin (Sigma 1.0%) and laminarin (Sigma 0.1%). Chitinase and β-1,3-glucanase activities were calculated using the standard curve of N-Acetyl-D-glucosamine and glucose, respectively.

16S rDNA gene analysis of selected promising Bacillus spp

The four promising Bacillus spp. were grown in Nutrient broth (HiMedia MM244) at 120 rpm for 24 h at 28 ± 1 °C in dark and DNA was extracted using the QIAamp DNA mini Kit (Qiagen). Gene portion of 16S rDNA was amplified by PCR using a combination of the universal primers, 27F (5′-AGAGTTTGATCCTGGTCAGAACGCT-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTTCACCCC-3′) which covers the region approximately 1.4 Kb. The PCR reactions were carried out in 50 µl reaction mixture including 1 U Taq polymerase (Merck Bioscience), 2.5 µl of 10X Taq buffer, dNTP at 200 µM, 10 pmol of each primer and 25 ng of DNA. Reactions were carried out in an ABI Gold GeneAmp PCR System programmed for initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, primer extension at 72 °C for 1.5 min and a final extension at 72 °C for 10 min. The obtained PCR products were checked on 1.4% agarose gel (1X TAE) embedded with ethidium bromide. The resulting PCR products were purified and used directly for sequencing (Sci genome, Kochin, India). The resulting forward and reverse sequences were aligned and edited to obtain the consensus sequence.

Phylogenetic analysis of four promising Bacillus spp.

The 16S rDNA sequences were compared to authentic sequences of different bacterial species available at the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) using BLAST search. Isolates showing maximum similarity were retrieved and were used for phylogenetic analysis. Multiple sequence alignment and construction of phylogenetic tree was carried using MEGA 6 program (Tamura et al. 2013). Kimura-2-parameter distance model was used for computation of pairwise evolutionary distances and phylogenetic tree was constructed using Neighbor Joining (NJ) method.

Statistical analysis

The data was analyzed in CRD and RBD with analysis of variance (ANOVA) using SAS (ver. 9.3; SAS Institute Inc., Cary, North Carolina, USA). The percentage data was arcsine-transformed before analysis. Means were compared using Tukey’s Studentized Range Test using SAS system.

Results and discussion

Screening for plant growth promotion activity

Out of 293 bacteria, only twenty-two bacteria showed 15 to 90% increase in root length (Table S1a) and were selected for in vivo studies on induction of systemic resistance against powdery mildew in potted plants. These bacteria were DR-38, DR-39, TS-45, TS- 66, DR-79, DR-92, DR-118, CS-126, DR-131, TL-171, DRo-197, DRo-198, DRo-199, TS-204, TS-205, TS- 212, DR-219, TP-230, TP-232, TR-259, GR-280 and GR-281. All the twenty two bacteria were Gram-positive, rod shaped, spore forming, motile, arranged in chains, and catalase positive indicating that they belonged to the genus Bacillus. The Bacillus species are preferred for agricultural use as they form spores which are heat and desiccation resistant, hence can be formulated easily into stable products for commercial use (Emmert and Handelsman 1999). Remaining two hundred and seventy one bacteria were either less effective, not effective or restricted the root growth and were not considered for further studies. The association of different species of bacteria with plants and their variable growth effects is well documented (Schwachtje et al. 2012) and hence, the selection of efficient isolates from the micro-biota is a necessary prerequisite.

In vivo screening for induction of resistance

After 21 days, the powdery mildew PDI was 76.80 in control plants (Table S1b). The PDI was significantly less than control in all plants treated with the twenty two Bacillus isolates showing that all PGPB isolates were capable of inducing resistance against powdery mildew. Plants treated with eleven of these Bacillus isolates had PDI in the range of 40 to 50%, while the remaining 11 isolates had PDI in the range of 51 to 60%. Different species of Bacillus are known to control plant diseases through induction of systemic resistance (Park et al. 2001; Ahn et al. 2002). The leaves of plants with PDI in the range of 40 to 50% were analysed for biochemicals associated with disease resistance.

Analysis of biochemicals associated with disease resistance

The levels of peroxidase, polyphenol oxidase, total phenols and total proteins were higher in the leaves of plants treated with the Bacillus isolates than that in the leaves of plants treated with fungicide flusilazole and the untreated control (Table 1) showing that the treated plants had higher levels of resistance. Among the isolates, maximum level of peroxidase, polyphenol oxidase, total phenols and total proteins were seen in plants treated with isolate DR-92 and then in plants treated with isolate TS-45. Isolates DR-38, TP-232 and CS-126 also produced good amounts of these biochemicals. Phenolic compounds are antioxidants which provide resistance to the plant against pathogens and an increase in their levels in plants generally indicates enhanced resistance to pathogens (Thakker et al. 2007).

Table 1 The levels of peroxidase, polyphenol oxidase, total phenols and total proteins in leaves of Thompson Seedless plants with soil application of Bacillus strains after powdery mildew infection
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In vitro screening for production of growth promoting metabolites

All Bacillus isolates tested positive for IAA, NH3, HCN and siderophore production, but only three isolates, DR-38, TS-45 and GR-280 tested positive for phosphate solubilization (Table S2). The change in colour of the overlaid medium from blue to purple showed that all the isolates produced catechol-type siderophores. Isolate TS-45 scored higher in production of growth promoting metabolites (Table 3). Isolate TP-232 showed similar activities except phosphate solubilization. The secondary metabolites, IAA, NH3, HCN, siderophores and phosphatase stimulate plant growth directly (Devi and Thakur 2018) and also act as inducers of systemic resistance (Hartmann and Schikora 2012; Lee et al. 2012).

We selected four isolated for field testing. Isolates TS-45, DR-92, TP-232 which rated higher for production of biochemicals associated with disease resistance and growth promoting metabolites were selected. From the remaining 8 isolates, we selected TL-171 which had good ability to control powdery mildew when applied to the foliage. Isolate DR-92 was obtained from Dogridge rhizosphere while TS-45, TL-171 and TP-232 were obtained from Thompson Seedless stem, leaf and petiole, respectively, hence they can be called as plant growth promoting bacteria rather than PGPR in true sense. Earlier Aziz et al. (2016) were also able to induce resistance in grapevines by using endophytic bacteria which was associated with increased phytoalexins production.

Compatibility

Bacterial consortium may be more efficient than individual isolates in stimulating plant growth and inducing systemic resistance. To prepare such a consortium it is necessary to check the compatibility among the isolates. The cross streak culture study showed that all the four promising isolates in our study, TS-45, DR-92, TP-232 and TL-171, did not inhibit each other’s growth and thus were compatible with each other. Earlier, Jetiyanon et al. (2003) reported the broad spectrum protection against plant pathogens in field condition by mixture of PGPR. Similarly, Raupach and Kloepper (1998) also found PGPR consortium more effective than individual bacteria in controlling cucumber pathogens.

Field evaluation

The data on powdery mildew severity on leaves and bunch in the field trials conducted during three seasons from 2015 to 2017 is presented in Table 2.

Table 2 Reduction in severity of powdery mildew on grapes by soil application of Bacillus isolates alone or in combination
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Fruiting growth phase 2015–16

In 2015–16 fruiting growth phase, the  PDI of powdery mildew on leaves of vines in control was  25.26. Soil application of Bacillus isolates, either alone or in mixtures, reduced the powdery mildew severity on leaves as compared to the control. Maximum reduction in disease severity was found in vines treated with strain TP-232, either alone ( 17.90) or in combination with TL-171 ( 16.15), followed by TS-45+DR-92 ( 17.17). On bunch, powdery mildew severity was minimum in vines treated with TS-45+DR-92 (26.68 ), and then in vines treated with strain TP-232, either alone (27.47) or in combination with TL-171 (27.24).

Vegetative growth phase 2016

Similar results were obtained in the subsequent trial during vegetative growth phase, 2016. The  PDI of powdery mildew in the control was 5.21 and all Bacillus treatments reduced powdery mildew severity. Maximum reduction was seen in vines treated with TP-232+TL-171 and TS-45+DR-92, and then in vines treated with TL-171 alone.

Fruiting growth phase 2016–17

In 2016–17 fruiting growth phase, the powdery mildew severity in the control vines was 5.21 PDI on leaves. Soil application of all Bacillus spp., either alone or in mixtures, reduced the powdery mildew severity. Least PDI was observed in vines treated with TP-232+TL-171, and then in vines treated with TS-45+DR-92 and TL-171 alone. On bunches treatment with TP-232+TL-171 was most effective in minimizing severity.

The three seasons data shows that TP-232 in combination with TL-171was more effective in minimizing powdery mildew severity in grapevines followed by the combined application of TS-45+DR-92. The reduction was higher than that obtained in solo treatments with these four isolates. In many studies it was seen that using mixture of two different PGPB provided better control as compared to application of individual PGPB. This enhanced efficacy might be due to the different mechanism of action of each PGPB and their synergism. A synergistic effect was noticed with isolates with higher chitinase production ability in combination with isolates with antibiotic production ability in suppression of rice sheath blight (Sung and Chung 1997). Enhanced ISR effect of binary mixture of two PGPB against the control of grapevine to gray mold was also reported (Aziz et al. 2016).

Induction of host defense mechanisms by the promising Bacillus spp. in field

Chitinase, β-1,3-glucanase activities and total phenols were highest in leaves of vines treated with TP-232+TL-171 (Table 3). Chitinases and glucanases impart protection to plants by digesting fungal cell walls (Franzener et al. 2018). The peroxidase and polyphenol oxidase activities were highest in leaves of vines treated with TS-45+DR-92 and then in TP-232+TL-171. Isolates TS-45 and TP-232 were most effective in increasing root length (> 75%). The enzyme activities and total phenol contents were higher in vines where mixtures of isolates were used as compared to vines where the isolates were used alone. Peroxidases were reported in reduction in powdery mildew severity in melon plants inoculated with B. subtilis (García-Gutiérrez et al. 2013). Accumulation of PR proteins, chitinases and β-1,3 glucanases, were found to be associated with induced resistance against TNV in tobacco (Maurhofer et al. 1994) and against C. falcatum in sugarcane (Viswanathan and Samiyappan 1999).

Table 3 The activities of defense related enzymes and total phenols in leaves of field grown vines of Thompson Seedless plants applied with Bacillus spp.in their drip circle after infection with powdery mildew disease
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16S rDNA gene analysis of selected promising Bacillus spp

BLAST analysis of the 16S rDNA sequences of the four bacterial isolates with the sequences available in the National Centre for Biotechnology Information (NCBI) database showed maximum (99% to 100%) sequence similarity to Bacillus strains. However, all four isolates were identified as different species. In phylogenetic analysis isolate TS-45 grouped with B. subtilis, B. amyloliquefaciens and B. tequilensis species supported with 100% bootstrap value, however, it formed internal clade with isolates of B. subtilis (Fig. 1). Isolate DR-92 formed a clade with B. endophyticus and B. filamentosus with 100% support of bootstrap value. However it was more closely related to B. endophyticus. Isolate TL-171 formed monophyletic clade with B. licheniformis isolates supported with 99% bootstrap value. Isolate TP-232 grouped with B. flexus isolates supported with 98% bootstrap values. Thus isolate TS-45, DR-92, TL-171 and TP-232 were identified as Bacillus subtilis, B. endophyticus, B. licheniformis and B. flexus respectively. The 16S rDNA sequences of these four isolates were deposited in GenBank under the accession number MH337369 (TS-45), MH333286 (DR-92), MH337245 (TL-171), MH333713 (TP-232).

Fig. 1
figure 1

Phylogenic position of TS-45, DR-92, TL-171 and TP-232 inferred by analysis of 16S rRNA sequences. The tree was obtained from analysis by the NJ method using the Kimura (1980) two-parameter technique of the MEGA6 program

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The study reports the potential of Bacillus species in reducing the severity of powdery mildew incidence in vineyards when used solo or in binary consortiums by inducing enhanced production of defense related proteins and bio-chemicals. Soil application of these selected, efficient Bacillus species will considerably reduce the dependence on single-site action fungicides for disease management.