1 Introduction

To respond to the increasing food demands of a growing global population under climate change and pesticide limitations, biological pest control is a highly sought-after alternative to conventional pesticides, for health and ecological reasons (Savary et al., 2012, 2019). As a result of injudicious use of chemical pesticides, pesticide resistance and environmental harm have occurred worldwide (Sharma et al., 2019). Further, many developing countries face low crop productivity in agriculture due to plant health issues. These occurrences have resulted in persistent threats to food security and the environment (Singh et al., 2023). The introduction of innovative, cost-effective biocontrol strategies, constitutes an important avenue for improving the economic status of farmers, increasing global food security, reducing environmental impacts, and minimizing health hazards (Birch et al., 2011; Flood, 2010; Savary, 2020; Box 1). In this regard, microbial bio-pesticides are microorganism-based, low-risk, environmentally friendly agents for managing plant pathogens and pest populations. One of the most studied microbial pesticide microorganisms is the entomo-pathogenic, gram-positive, soil bacterium Bacillus thuringiensis (Bt), which is commonly used in agriculture, horticulture, and forestry (Box 1). Bt was identified and characterized as entomo-pathogenic in the early 1900s and has been used worldwide in pest management for many years (Azizoglu et al., 2023). The discovery of Bt in dead insects led to the development of the first commercial pest control product (Bravo et al., 2011). Bt forms spores (Sanahuja et al., 2011), and produces insecticidal crystal proteins (Cry) during the sporulation process (Box 1). Cry proteins effectively kill a wide range of insect species, making them a good alternative for chemical insecticides (Hang et al., 2007). Bt and the Cry proteins it produces are among the earliest recognized commercially available natural insecticides to be used extensively in agriculture (Copping & Menn, 2000; Sanahuja et al., 2011). Recently, a few reports have also provided evidence of Bt controlling various phytopathogenic fungi (Azizoglu, 2019; Yoshida et al., 2019), and promoting plant growth, through several direct or indirect mechanisms (Azizoglu, 2019; Delfim & Dijoo, 2021). Indirect mechanisms by which Bt could inhibit plant pathogens and promote plant growth and development include the production of bacteriocins, autolysins, lactonases, siderophores, β-1,3-glucanase, chitinases, antibiotics, and hydrogen cyanide, and the ability to degrade indole-3-acetic acid (IAA), respectively (Azizoglu et al., 2023). Direct plant growth stimulation may include providing plants with fixed nitrogen, soluble nutrients, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and production of plant hormones such as IAA, gibberellic acid and cytokinins (Delfim & Dijoo, 2021).

Box 1
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Key concepts presented in this work

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Interaction between plants and microorganisms can trigger plant immunity (Gupta & Bar, 2020; Gupta et al., 2021a; Van Wees et al., 2008). Recognition of beneficial microbes by the plant may activate various plant metabolic pathways, leading to faster and stronger responses upon pathogen attack (Harman et al., 2004; Nawrocka & Małolepsza, 2013). As a result of plant tissue colonization by microorganisms, including microbial biocontrol agents, interaction between the host plant and these microbes results in the activation of resistance against pathogens (Meller Harel et al., 2014; Perazzolli et al., 2008; Box 1). At the same time, the robustness of the root system can be promoted, enhancing nutrient uptake and leading to plant growth promotion (Contreras-Cornejo et al., 2013; Macías-Rodríguez et al., 2018). Recent work by our group has demonstrated that the activation of plant immunity can promote plant growth (Leibman-Markus et al., 2023b). Extensive research into biocontrol agents and their interaction with the host plant has unveiled several different mechanisms by which biocontrol agents can suppress plant diseases by direct management of plant pathogens. Among these reported mechanisms are myco-parasitism, antibiosis, and competition, as well as induction or enhancement of systemic plant resistance, either acquired (systemic acquired resistance; SAR) or induced (induced systemic resistance; ISR; Gupta & Bar, 2020). Plants in which SAR and/or ISR have been activated possess a rapid, stronger activation of defence responses following pathogen infection that has been referred to as priming (Conrath et al., 2015). See Box 1 for further details.

Tomato (Solanum lycopersicum L.) is an important vegetable crop in terms of cultivation area, production, commercial use and consumption. It constitutes 72% of the economic value in fresh vegetables produced worldwide (Cammarano et al., 2022). According to the statistics of the Food and Agriculture Organization of the United Nations, FAOSTAT (https://www.fao.org/faostat/en/#data/QCL), since 2018, over 5 million hectares of tomatoes have been cultivated yearly worldwide, mostly in open field, with greater than 180 million tons produced each year, making tomato currently the first vegetable crop worldwide. However, infectious diseases and pests have always been one of the main factors that constrain tomato production (Olowe et al., 2022). Worldwide, the arthropod pests Bemisia tabaci, Tetranychus urticae and Tuta absoluta cause severe damage in tomato cultivation. B. tabaci transmits viral disease (Czosnek & Rubinstein, 1997), while T. absoluta can reduce crop productivity by up to 90% (Batalla-Carrera et al., 2010), with chemical pesticide resistance becoming an increasing problem (Guedes et al., 2019; Roditakis et al., 2018). The two-spotted spider mite infests many crops, causing particular damage to Solanaceae crops such as tomato (Migeon et al., 2009; Weinblum et al., 2021). The necrotrophic grey mould causing fungus B. cinerea, the biotrophic powdery mildew fungi Leveillula taurica and Oidium neolycopersici, and the hemibiotrophic bacterial pathogen Xanthomonas euvesicatoria, are all major problems in worldwide tomato cultivation and cause severe damage in various parts of the plant (An et al., 2019; Elad et al., 2007; Fillinger & Elad, 2016; Jones et al., 2001).

In the present work, to examine whether Bt can effectively control tomato pathogens, we tested the ability of Bt to inhibit fungal pathogens such as the necrotrophic grey mould -causing fungus B. cinerea, the two biotrophic powdery mildew fungi L. taurica and O. neolycopersici, and the bacterial tomato pathogen X. euvesicatoria.

To characterize the mechanism through which Bt acts, we quantified immune system output in the host plant. Bt commercial products are a mixture of spores and crystals. To examine which component of Bt contributes to the suppression of tomato pests and pathogens, we separated the Bt preparations into fractions, composed of either spores or pure crystals, and compared the activity of these fractions to the Bt product activity as a whole.

2 Materials and methods

In depth procedural details of all experimental methods are provided in Annex 1.

2.1 Plant material and growth conditions

Plants of tomato Solanum lycopersicum cultivar 4107 Ram were obtained from a commercial nursery (Hishtil, Ashkelon, Israel) at 30 to 40 days after seeding, and were transplanted into 1-L pots containing a coconut fiber:tuff (7:3 vol.:vol.) potting mixture (Green Mix; Even‐Ari, Ashdod, Israel). Plants were drip-fertigated 2-3 times per day with a 5:3:8 NPK fertilizer solution (irrigation water prepared to have N, P, and K concentrations of 120, 30, and 150 mg L-1), allowing for 25-50% drainage. Plants were trained on bamboo sticks and maintained at 20 to 30 °C. Bean (Phaseolus vulgaris cv "N9059"), Potato (Solanum tuberosum cv "Desiree"), and Rapeseed (Brassica napus cv "Ruhama") were also grown essentially as described for tomato plants.

2.2 Bt and Bt fraction treatments and fraction preparation

The commercial Bt subsp. krustaki (Btk, referred to herein as Bt) was obtained from BioDalia Microbiological Technologies, Israel. Haemocytometer counts indicated an initial concentration of 2×1010 spores ml-1. The whole Bt product, consisting of both spores and crystals, was used for the preparation of the various Bt fractions (Annex 1). The product was applied by spraying or soil drenching, as indicated in each experiment, using a 0.3% v/v preparation, prepared according to the product label instructions. A hand-held sprayer was used to spray 15 mL of Bt preparation on each plant for full coverage, once a week, for 3 consecutive weeks, on a total 15 plants per treatment in 3 separate experiments. For drench application, roots of each plant were drenched with 15 mL of Bt preparation once a week for 3 successive weeks, on a total of 10 plants per treatment, in 2 separate experiments.

For the preparation of the various fractions of Btk, the whole Btk product consisting of both spores and crystals, was used for the separation procedure. We followed the protocol of Pendleton and Morrison (1966) to separate spores from crystals (Annex 1).

2.3 Fungal and bacterial plant inoculations, and disease assessment

Strain BcI16 of Botrytis cinerea (Elad, 2000) was inoculated on tomato, bean, potato, and rapeseed plants, and disease severity (area under the disease severity progression curve, AUDPC) was calculated (Elad et al., 2010; Annex 1). Oidium neolycopersici and Leveillula taurica were collected from young leaves of infected tomato plants and conidia were used for powdery mildew inoculations. Disease severity on leaves was assessed on a 0–100% scale (Elad et al., 2010; Swartzberg et al., 2008; Annex 1). The bacterial leaf spot pathogen Xanthomonas euvesicatoria strain 85‐10 (Xcv) was inoculated on tomato plants according to Kocal et al. (2008). Bacterial populations were determined by plating and counting colonies (Gupta et al., 2020a, b; Annex 1).

2.4 Pest infestation and quantification

The tomato leaf miner Tuta absoluta (Pizarro et al., 2020), the two-spotted spider mite, (TSSM) Tetranychus urticae (Weinblum et al., 2021), and Bemisia tabaci Genadius (Hemiptera: Aleyrodidae) (Pizarro et al., 2020) assays were performed by exposing plants to infested plants under controlled conditions (Annex 1).

2.5 Reactive oxygen species (ROS) measurement and ion leakage measurement

ROS measurements were performed on tomato leaf discs of control and treated plants, 24 h after the final treatment with Bt or Bt fractions, according to (Gupta et al., 2020a, b; Leibman-Markus et al., 2017 ; Anand et al., 2021; Annex 1). Electrolyte leakage measurements were performed according to (Leibman-Markus et al., 2017; Annex 1).

2.6 RNA extraction and qRT‐PCR analysis

Total plant RNA was extracted using Tri-reagent (Sigma‐Aldrich). RNA was converted to first‐strand cDNA using reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instructions. qRT‐PCR was carried out according to the Power SYBR Green Master Mix protocol (Life Technologies, Rockville, MD), using a Rotor‐Gene Q machine (Qiagen, Valencia, CA). For further information see Annex 1. The expression of the following genes was assayed: The SA-responsive PR1a, PR1b and Pi1 (Meller Harel et al., 2014; Pizarro et al., 2020; Vega et al., 2015); and the ET/JA responsive PR1b, ERF1, Pti5 and LoxD (Gu et al., 2002; Mehari et al., 2015; Müller & Munné-Bosch, 2015; Gupta et al., 2021b; Gupta et al., 2022a, b; Leibman-Markus et al., 2023a). The specific primers used and primer pair efficiencies are detailed in Table S1.

2.7 Plant growth and yield measurement of tomato

Growth traits including height, number of nodes per stem, plant yield, number of fruits, length of a selected mature and young leaf (L5, L9), and terminal leaflet (TL) size of a selected mature and young leaf (L5, L9), were measured on the day of the first treatment, and 2-3 weeks later, and are reported as percent change. Average yield and number of fruit were measured at the end of the growing season and are reported in absolute values.

2.8 Insect feeding bioassays for Bt fractions

To evaluate the insecticidal activity per os of the Bt fractions, we assayed their activity against the leaf-feeding larvae of S. litorallis. Hatched unfed first instars were fed with artificial media supplemented with the Bt fractions at 2% v/v for up to 7 days. Survival was assessed at 3 time points. Only "live" fractions demonstrated insecticidal activity. For further details see Annex 1.

2.9 Bt in-vitro activity against B. cinerea

Direct activity of Bt against B. cinerea was examined in vitro using fungal mycelium (Annex 1).

2.10 Statistical analysis

Experimental data are presented as minimum to maximum values in box-plots or as floating bar graphs. All experiments were carried out in at least 3-5 biologically independent repeats. The replicate numbers and p-values obtained are given in the figure captions for each experiment. Differences were deemed non-significant at P>0.05. Gaussian distribution of data sets was verified using the Shapiro-Wilk test. Differences between two groups were tested for statistical significance using a two‐tailed t-test, with Welch's correction where applicable (unequal variances; Derrick & White, 2016). Differences among three groups or more were tested for statistical significance using a one‐way Analysis of variance (ANOVA). Conventional ANOVA was used for groups with equal variances, and Welch's ANOVA for groups with unequal variances. When ANOVA indicated significant differences among groups, differences between group means were tested using post‐hoc tests. Tukey's or Dunnett's tests were used for samples with equal variances when the mean of each sample was compared to the mean of every other sample. Bonferroni's test was applied for samples with equal variances when the mean of each sample was compared to the mean of a control sample. Dunnett's test was employed for samples with unequal variances. All statistical analyses were conducted using GraphPad Prism version 9.5.1., for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com.

3 Results

3.1 Bt confers resistance to tomato arthropod pests and bacterial and fungal pathogens

We examined the effect of the Bt-based commercial product (Bio T Plus, BioDalia) on disease symptoms induced by various tomato pathogens. Plants sprayed with Bt were better at preventing natural infestation of all three examined pests, demonstrating similar reductions in actual pest numbers and the associated tissue damage, in the case of infestation with B. tabaci (Fig. 1A), TSSM (Fig. 1B) and T. absoluta (Fig. 1C). Spray application of Bt resulted in significant fungal disease reduction of up to 60% in tomato plants in the case of all three tested fungi (Fig. 2A–C). Tomato plants challenged with Xcv demonstrated reduced Xcv CFU count in plants treated with Bt, leading to a decrease of about 40% in bacterial CFU as compared with untreated plants, 3 days after inoculation (Fig. 2D). Bt inhibited B. cinerea disease when applied by soil drench (Fig. 2E), to a slightly lesser level than observed with spray application (Fig. 2A).

Fig. 1
figure 1

Pest resistance following Bt treatment. Tomato plants were treated with a Bt suspension (0.3% of the commercial Bt strain) by spraying once a week for 3 weeks. Plants were placed in a naturally infested net-house 3 days after the first treatment. The plants were challenged with Bemisia tabaci whiteflies (A, B), Tetranychus urticae, Two spotted spider mite (TSSM) (C, D), and Tuta absoluta tomato leafminer (E, F). Number of pests (A, C, E), AUPPC (Area under the pest infestation progression curve, individuals*days) (B), and damage severity (D, F) were assessed two weeks after exposure. At least 3 independent experiments were conducted in all cases. Boxplots represent minimum to maximum values and are shown with the inter-quartile ranges (box), medians (line in box), and outer quartile whiskers. Asterisks represent statistically significant infestation rate or damage severity reduction in the indicated treatment over control, in Welch's t-test. A: N = 10, p < 0.0008. B: N = 15, p < 0.0072. C: N = 15, p < 0.025. D: N = 15, p < 0.0038. E, F: N = 25, p < 0.0001. *p < 0.05; **p < 0.01; ****p < 0.0001

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Fig. 2
figure 2

Pathogen resistance following Bt treatment. Tomato plants were treated with Bt (0.3% of the commercial Bt strain) by spraying once a week for 3 weeks (A–D), or by drenching the plant roots with 15 mL of the same Bt suspension (E). The plants were inoculated with fungi and bacteria as indicated, 3 days after the final treatment. The plants were challenged with Botrytis cinerea (A, E), Leveillula taurica (B), Oidium neolycopersici (C), and Xanthomonas euvesicatoria (Xcv) (D). At least 3 independent experiments were conducted in all cases. Boxplots represent minimum to maximum values and are shown with the inter-quartile ranges (box), medians (line in box) and outer quartile whiskers, all points shown. In all cases, asterisks represent statistically significant disease reduction upon Bt treatment, in a two-tailed t-test with Welch's correction. A N = 20. B N = 15. C N = 10. D N = 12. E N = 20. ***p < 0.001, ****p < 0.0001

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3.2 Bt induces tomato immunity

ROS production and ion leakage were compared in plants treated with Bt with control plants. Pre-treatment with Bt increased ROS elicited by both flg‐22 and EIX. (Figs. 3A, B and S1). Bt induced the expression of the pathogenesis‐related proteins PR1a and PR‐1b, Pto‐interacting 5, and Pi‐1, but did not induce the expression of ERF1 (Fig. 3C). Further details on the plant immune system are provided in Box 1.

Fig. 3
figure 3

Plant defence responses following Bt treatment. Tomato plants were treated with a Bt suspension (0.3% of the commercial Bt strain) by spraying once a week for 3 weeks. Defence responses were measured 24 h after the final treatment. A, B ROS production was measured immediately after flg22 (A) or EIX (B) application using the HRP-luminol method. Bars represent the mean ± SE of 3 independent experiments, all points shown. Asterisks represent increased ROS production as compared with the control treatment in a two-tailed t-test with Welch's correction, *p < 0.05, N = 8. C Defence gene expression of the pathogenesis‐related proteins PR1a (Solyc01g106620) and PR1b (Solyc00g174340), Pto‐interacting 5 (Pti‐5, Solyc02g077370), pathogen-induced 1 (PI‐1, Solyc01g097270), and Ethylene-responsive factor 1 (ERF1, Solyc05g051200) was assayed by RT-qPCR following Bt treatments. Relative expression was calculated using the geometric mean between the gene copy number obtained for two reference genes: RPL8 (Solyc10g006580), and EXP (Solyc07g025390), and normalized to the control. Boxplots represent inner quartile ranges (box), outer quartile ranges (whiskers), and median (line in box) of five independent replicates, all points shown. Asterisks represent statistical significance from control treatment in a t-test with Welch's correction comparing each gene (*p-value < 0.05; ns- non-significant)

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3.3 Bt promotes plant growth and yield production

Bt treatment did not affect plant height or mature leaf length (Fig. S2A, C), but promoted the number of nodes, young leaf length, mature and young leaf terminal leaflet (TL) size, and plant yield and No. of fruits (Fig. S2B, D–H).

3.4 Analysis of the ability of different Bt fractions to confer resistance to tomato arthropod pests and microbial pathogens

We observed tomato immune system activation against pests and diseases (Figs. 1, 2, 3 and S1). To examine which fraction of the Bt product causes these effects, we isolated live bacteria from the Bt product, and generated spore and protein fractions (Annex 1). We also generated heat-inactivated preparations of all fractions. B. tabaci was deterred from plants sprayed with Bt spores or the inactivated whole preparation (Fig. 4A), while T. absoluta infection was reduced in plants sprayed with active or inactivated proteins or whole preparation (Fig. 4B, C). In the case of T. absoluta, treatment with the proteins and whole preparation (which contains both the proteins and spores) reduced the number of galleries irrespective of "live", native activity (Fig. 4B). However, the percentage of damage severity was only significantly reduced when the fractions possessed "live", native activity (Fig. 4C).

Fig. 4
figure 4

Plant resistance to pests following treatment with different Bt fractions. Tomato plants were treated with indicated Bt fractions by spraying once a week for 3 weeks. "X" indicates an inactivated fraction. Plants were placed in a naturally infested net house 3 days after the first treatment. The plants were challenged with Bemisia tabaci whiteflies (A), and Tuta absoluta tomato leafminer (B, C). AUPPC (Area under the pest infestation progression curve, individuals*days, A), Average number of pest galleries/ plant, B, and damage severity % (C) were assessed two weeks after exposure. At least 5 independent experiments were conducted in all cases. Boxplots represent minimum to maximum values and are shown with the inter-quartile ranges (box), medians (line in box), and outer quartile whiskers. Asterisks represent statistically significant infestation rate or damage severity reduction in the indicated treatment over control, in a two-tailed t-test with Welch's correction. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ns- non-significant

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Treatment with Bt fractions resulted in a significant fungal disease reduction of up to 75% (Fig. 5A, B). With the exception of the boiled crude preparation that had a slight increase in activity against B. cinerea compared to the other fractions, all fractions inhibited both fungal diseases in a similar manner. Tomato plants challenged with Xcv following Bt fraction treatment also demonstrated reduced Xcv CFU count in the treated plants, with Bt fractions leading to 30-40% decrease in bacterial CFU compared to untreated plants, irrespective of the Bt fraction applied (Fig. 5C). Similar results were achieved when examining the characteristic water-soaked bacterial lesion caused by Xcv infection (Fig. S3).

Fig. 5
figure 5

Pathogen resistance following treatment with different Bt fractions. Tomato plants were treated with indicated Bt fractions by spraying once a week for 3 weeks. "X" indicates an inactivated fraction. The plants were inoculated with fungi and bacteria as indicated, 3 days after the final treatment. The plants were challenged with Botrytis cinerea (A), Leveillula taurica (B), and Xanthomonas euvesicatoria (Xcv) (C). At least 3 independent experiments were conducted in all cases. Boxplots represent the minimum to maximum values and are shown with the inter-quartile ranges (box), medians (line in box) and outer quartile whiskers, all points shown. A, B: letters represent statistically significant differences among samples, and C, Asterisks represent statistically significant disease reduction upon Bt fraction treatment, in Welch's ANOVA with Dunnett's post hoc test. A N = 40, p < 0.032. B N = 5, p < 0.004. C N = 24. *p < 0.05, **p < 0.01

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3.5 Analysis of the ability of different Bt fractions to induce tomato immunity

The similar activity of all Bt fractions, whether native or inactivated, against tomato pathogens, suggests that all Bt fractions share similar, non- component-specific, activity. To confirm this, we examined ROS production and defence gene expression in plants treated with the different Bt fractions, compared to control plants. With the exception of the inactivated proteins in the case of EIX, pre-treatment with all Bt fractions increased ROS elicited by both flg‐22 and EIX (Fig. 6A, B). Ion leakage was promoted only by treatment with the native proteins or whole Bt preparation (Fig. S4).

Fig. 6
figure 6

Reactive oxygen species (ROS) production following treatment with different Bt fractions. Tomato plants were treated with indicated Bt fractions by spraying once a week for 3 weeks. "X" indicates an inactivated fraction. Defence responses were measured 24 h after the final treatment. A, B ROS production was measured immediately after flg22 (A) or EIX (B) application using the HRP-luminol method. Boxplots represent minimum to maximum values and are shown with the inter-quartile-ranges (box), medians (line in box) and outer-quartile whiskers, all points shown. Asterisks represent increased ROS production as compared with control treatment in a two-tailed t-test with Welch's correction, A: N = 30, B: N = 40. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns- non-significant

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We also examined the effect of treatment with different Bt fractions on tomato defence gene expression. SAR-related genes PR1a, PR1b, and PI‐1 were induced primarily by the live spores and whole preparation, while the ISR-related JA/ET genes Pti‐5, ERF1, PR1b, and LoxD were induced by most fractions, irrespective of viability (Fig. 7).

Fig. 7
figure 7

Defence gene expression following treatment with different Bt fractions. Tomato plants were treated with indicated Bt fractions by spraying once a week for 3 weeks. "X" indicates an inactivated fraction. Defence gene expression was measured 24 h after the final treatment. Defence gene expression of the pathogenesis‐related proteins PR1a (Solyc01g106620, A, SAR responsive gene) and PR1b (Solyc00g174340, B, SAR and ISR responsive gene), Pto‐interacting 5 (Pti‐5, Solyc02g077370, C, ISR responsive gene), pathogen induced 1 (Pi‐1, Solyc01g097270, D, SAR responsive gene), Ethylene-responsive factor 1 (ERF1, Solyc05g051200, E, ISR responsive gene), and Tomato lipoxygenase D (LoxD, Solyc03g122340, F, ISR responsive gene) was assayed by RT-qPCR. Relative expression was calculated using the geometric mean between the gene copy number obtained for two reference genes: RPL8 (Solyc10g006580), and EXP (Solyc07g025390), and normalized to the control. Floating bars represent minimum to maximum values, line in box represents median, N = 8. Asterisks represent a statistically significant increase when compared with control treatment, in t-test with Welch's correction comparing each gene (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns- non-significant)

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3.6 Direct insecticidal and pathogen inhibition activity of Bt fractions

The insecticidal activity per os of the Btk fractions was assayed against leaf-feeding larvae of S. litorallis. Only "live" fractions demonstrated insecticidal activity against S. litorallis (Fig. 8A). Direct pathogen inhibition assays were performed with B. cinerea mycelia. All tested fractions inhibited B. cinerea growth in their "live" form (Fig. 8B). The whole Bt preparation also inhibited B. cinerea growth at 1%. In the inactivated fractions, only the spores inhibited B. cinerea growth, possibly due to the release of active proteins that were not sufficiently deactivated in the inactivation process.

Fig. 8
figure 8

Direct activity of Bt fractions against S. litorallis and B. cinerea. Direct activity of Bt fractions was examined in insect bioassays against S. litorallis (A) and B. cinerea (B). "X" indicates inactivated fraction. For S. litorallis, first instar larvae were fed a diet supplemented with each of the Bt fractions for 7 days. 2% v/v of the different Bt fractions as indicated was added to the insect diet. Survival was assessed up to 7 dpi. For B. cinerea, fungal mycelium discs 0.5 cm in diameter were transferred to the center of PDA plates containing 1 or 2% of each fraction as indicated. Growth, expressed as mycelia area, was measured 3 days after co-culturing. Boxplots represent minimum to maximum values and are shown with the inter-quartile-ranges (box), medians (line in box) and outer quartile whiskers, all points shown, N = 6. A: Different letters represent statistically significant differences among samples in a one-way ANOVA with Tukey's post hoc test. B: Asterisks indicate significance from control treatment in Welch's ANOVA with Dunnett's post hoc test, **p < 0.01; ***p < 0.001; ****p < 0.0001; ns- non-significant

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3.7 Growth promoting activity of different Bt fractions

Plants treated with live spores displayed increased height and node growth (Fig. S5A), and plants treated with live spores or "live", native protein demonstrated increased leaf sizes (Fig. S5B), suggesting that, while not necessarily required for disease prevention, the "live" activity of the Bt fractions is required for the observed growth promotion effects.

3.8 Bt confers resistance to B. cinerea in several crop plants

Further to our results in tomato, we examined the ability of Bt to control grey mould incited by B. cinerea in bean, potato, and rapeseed. We found that Bt treatment reduced disease in all crop plants examined (Fig. 9).

Fig. 9
figure 9

B. cinerea resistance following Bt treatment in crop plants. A: Bean (P. vulgaris French Bean, cv "N9059"), B: Potato (S. tuberosum cv "Desiree"), and C: Rapeseed (B. napus cv "Ruhama") plants were treated with Bt (0.3% of the commercial Btk strain) by spraying once a week for 3 weeks. Plants were inoculated with B. cinerea 3 days after the final treatment. At least 2 independent experiments were conducted in all cases. Boxplots represent minimum to maximum values and are shown with the inter-quartile-ranges (box), medians (line in box) and outer quartile whiskers, all points shown. In all cases, asterisks represent statistically significant disease reduction upon Bt treatment, in two-tailed t-test with Welch's correction. A, B: N = 40. C: N = 24. *p < 0.05, ****p < 0.0001

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4 Discussion

We examined the possibility of systemic plant immune system activation and induced resistance by Bt, and found that Bt suppresses a range of plant diseases, as well as infestation with arthropod pests (Figs. 1 and 2). Bt was found to reduce the damage caused by this diversity of plant pests and pathogens (Figs. 1 and 2), and to reduce grey mould caused by B. cinerea in a range of important crop plants (Figs. 2 and 9).

Systemic immunity modification in plants is associated with transcriptional reprogramming, and with increases in plant defence responses and defence gene expression (Gupta & Bar, 2020). These changes in the plant were indeed observed upon the Bt treatment (Figs. 3 and S1). It therefore appears that Bt induces systemic plant immunity. The activity against B. cinerea was also retained when Bt was applied through soil drenching. Along with the enhanced defence responses and defence gene activation (Figs. 3 and S1), this seems to confirm the systemic nature of the Bt activity. The enhancement of plant productivity by Bt (Fig. S2) is also likely to be a result of host priming and promotion of plant hormonal pathways. Recently, our group reported that activation of plant defence also promotes plant growth and development (Leibman-Markus et al., 2023b).

The activity of Bt against fungal and bacterial plant pathogens has been reported in a number of studies (Djenane et al., 2017; Hernández-Huerta et al., 2023). The mechanism for this antifungal and antibacterial activity was previously attributed to several factors produced by Bt, including chitinases, autolysins, AHL-Lactonases, or Zwittermicin A (Hollensteiner et al., 2017; Raddadi et al., 2007), as well as induced plant resistance (Akram et al., 2013; Azizoglu, 2019). Our results confirm that Bt can indeed control fungal and bacterial plant pathogens via host-induced resistance, confirming recent reports of Bt being effective against B. cinerea (Yoshida et al., 2019) and X. euvesicatoria (Hernández-Huerta et al., 2023). To the best of our knowledge, the present report is however, the first showing Bt-induced plant resistance toward powdery mildew diseases (here, L. taurica and O. neolycopersici). In addition to induced resistance, a direct activity of Bt could develop on the tested plant pathogens via active metabolites and enzymes, particularly in the case of foliar spray application. Such direct activity requires further investigation.

To characterize the nature of Bt activity, we produced different Bt fractions containing spores, protein crystals, or both. We examined the effect of treatment with these fractions on plant immunity and disease resistance, and found that all fractions, both viable and inactivated, promoted plant immunity and enhanced disease resistance (Figs. 5, 6 and 7). This suggests that different molecular motifs present in the Bt fractions (possibly microbe-associated molecular patterns, or MAMPs; Newman et al., 2013), may promote effector-triggered immunity. Some MAMPs are known to retain immune-promoting activity despite heat inactivation (Furman-Matarasso et al., 1999; Wang et al., 2023). Treatment with nearly all fractions increased Reactive Oxygen Species (ROS) elicited by both flg‐22 and EIX (Fig. 6A, B). However, ion leakage was promoted only by treatment with the viable proteins or the whole preparation (Fig. S4). This suggests that different immune pathways might be triggered by different Bt fractions. Another possibility is that the Bt proteins and metabolites may be slightly toxic to plant cells, as reflected in the ion leakage measurements. This possible toxicity did not, however, suppress plant growth (Figs. S2 and S5). The hypothesis that different immune pathways are activated by the different Bt fractions is also supported by the fact that the different fractions promoted the expression of different defence genes (Fig. 7). Our results suggest that live spores may promote SAR, while all Bt components irrespective of viability likely promote ISR.

Confirming previous reports, the fractions containing Bt proteins were more active than spores against the considered pests (Fig. 4). As each of the pests represents different feeding behaviour, it allowed us to differentiate to some extent between the per os activity and the induced plant immunity activity of Bt. A clear example of induced plant immunity is in the inhibitory effect against B. tabaci. As whiteflies feed on phloem, Bt and its crystal proteins are not directly effective in controlling all of its life stages (Deist & Bonning, 2016; Mishra et al., 2022). Still, the results obtained here demonstrate that Bt spores possess activity against B. tabaci, possibly via induced immunity. The viable Bt fractions were better performing in controlling pest populations (Fig. 4A, C), though inactivation did not always abolish the observed activity. This could also suggest induced plant immunity conferring resistance toward the pests. Interestingly, the direct activity of Bt fractions against S. litorallis and B. cinerea relied mostly on "live" activity (Fig. 8). Taken together, these results bring forth the notion that plant pathogens could be controlled by Bt in a combinatorial manner, through both host priming and direct activity against the pathogens, possibly via chitinases, as previously reported (Reyes-ramírez et al., 2004).

In the case of pest control, this combined activity is also possible, although inactive Bt fractions, did not reduce pest damage as much as they reduced plant pathogen disease, despite inducing immunity in the plant (Fig. 4). In the S. litorallis bioassay, larvae were directly fed the various fractions by artificial diet. Thus, this assay cannot distinguish direct activity of Bt toward the larvae from plant-induced resistance following Bt treatment. The live spore fraction reduced S. litorallis survival. This could represent the activity of residual proteins in the live spore fraction, however, the live spore fraction showed no significant in planta activity against T. absoluta. Possible explanations are either the different modes of feeding of these two insects; leaf chewing versus leaf mining, or, variation in key gut features important for the full inhibitory activity of Bt and its proteins, which affect its toxicity toward the insect host.

Induction of systemic plant immunity can also activate plant growth and improve yield, as was reported for Trichoderma, and other plant-associated microorganisms (Gupta & Bar, 2020; Leibman-Markus et al., 2023a, b). Bt was reported to promote plant growth (Akram et al., 2013; Azizoglu, 2019), although the reports on this are limited, and growth-promoting activity could differ among different Bt isolates. For some Bt isolates, growth promotion has been reported to be correlated with the ability of the bacterium to produce plant hormones or promote nutrient uptake (Azizoglu, 2019). Here, we found that treatment with Bt enhanced plant growth and productivity (Fig. S2).

Global attention emphasises the development of farming techniques that are not only socio-economically balanced, but also improve plant productivity and protection, in an eco-friendly manner (Arif et al., 2020; Movilla-Pateiro et al., 2021; Riyaz et al., 2022). The shelf-life of plant protection products is where synthetic pesticides have a decisive advantage over biopesticides products. Therefore, the use of inactivated biopesticides that retain pathogen control capabilities could have significant advantages. A simple protocol for Bt product preparation could be distributed to farmers with the product (or even the bacterium itself). This could offer higher value than other microbe-based products, because the product can be improved by the farmer prior to use, and also, used in its "inactivated" form. In the present work, the inactivated fractions reduced fungal and bacterial diseases, and B. tabaci infestation. This suggests that a Bt-based product could be transported across the world without loss of efficacy. Ease of transport and use should result in Bt-based products being price-competitive and having an acceptable shelf-life, both for the retailer and end-user. Our results indicate that Bt-based products could be developed as combined products aimed at pathogen and pest control as well as plant growth promotion. Thus, Bt-based products could be integrated into a combined strategy aimed at increasing plant productivity through both plant protection and plant growth promotion, while at the same time potentially dropping farming costs and food prices as a result. Taken together with the disease-reducing activity we observed for Bt in several important crop plants (Figs. 2 and 9), use of Bt-based products could have significant advantages in the cropping of several different crops in third world farming. Bt-based products are already on the market, and testing them against additional pathogens and in additional farming systems beyond its current labels should be straightforward. Such "real-world" testing is necessary to determine the applicability of our suggested broad uses for this product, and will undoubtedly be conducted in the near future.