Controlling Tomato Fusarium Wilt Disease through Bacillus thuringiensis-Mediated Defense Primining

1. Background

Tomato (Solanum lycopersicum) is considered one of the world’s most important crops due to its high levels of production and consumption. In 2018, the global area of the cultivated tomato was ca. 4.8 million hectares with a production of more than 180 million tons ( 1 ). Also, tomato is a model plant to investigate plant-pathogen interactions ( 2 ). Fusarium wilt is recognized as one of the most destructive diseases affecting tomato plants. The causal fungus, Fusarium oxysporum f. sp. lycopersici (Fol), is ranked as the fifth important plant pathogenic fungus, resulting in a yield loss of ca. 14% ( 3 ). The hyphae of the vascular fungus penetrate tomato plants through wounds or directly via the root cortex and develop in intra- as well as inter-cellular manners in root tissue to reach and colonize vascular vessels ( 4 ). To restrict fungal colonization, the plant reacts through various defensive responses, including the production of substances like gum and tylose ( 5 ). However, these reactions lead to vascular occlusion, endangered water transfer and wilt symptoms ( 6 ). The mode of parasitism of the fungus is controversial. While some researchers believe it is necrotrophic ( 7 ), others know it as a hemibiotrophic parasite ( 8 , 9 ). Gordon ( 10 ) categorized the isolates of F. oxysporum into necrotrophic and biotrophic groups based on the death or survival of host cells near the infection site. The defensive mechanisms of tomato plants against Fol require the recognition of the pathogen through cell surface receptors that recognize pathogen-associated molecular patterns (PAMPs) and induce microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) ( 11 ).

Because of the soil-born nature of the fungus, its capability to colonize vascular tissues of infected plants and the high saprobic competitiveness, the control of the disease is difficult. Furthermore, the high rate of genetic mutation and the development of new, more invasive physiological races of Fusarium species lead to the inefficacy of host resistance. While chemical control has already lost its reputation due to the unwanted impacts of chemicals on the environment and soil microbiology, genetic diversity can also lead to fungicide resistance ( 12 ). So, it seems that the integrated management of the disease is the only choice to reduce yield loss. Besides the host plant resistance, biological control is a fundamental part of most integrated disease management programs ( 13 ).

B. thuringiensis (Bt) Berliner is known as the most abundantly applied Bacillus species throughout the world ( 14 ), and its importance as an effective biological control agent in the integrated management of plant diseases and pests has recently been reviewed ( 13 ). In vitro studies have shown the potential of some Bt isolates in the effective and long-lasting inhibition of Fol ( 15 ). previously, we reported that the Bt strain IBRC-M11096 alone induced the expression of jasmonic acid/ethylene (JA/ET) and salicylic acid (SA)-related marker genes in tomato cultivar Early Urbana prior to Fol inoculation ( 16 ). Furthermore, the study on the effect of the same Bt strain on the transcription of antioxidant genes indicated the increase of superoxide dismutase (SOD) as well as glutathione-S-transferase (GST) genes accompanied by the reduced rate of hydrogen peroxide (H₂O₂) in tomato cultivar Falat C.H. plants challenged with Fol ( 17 ).

2. Objective

The potential of the Bt strain IBRC-M11096 in inducing defensive pathways in tomato plants against Fol was investigated. Also, the effects of the bacterial strain on fusarium wilt severity and plant growth indices under pot conditions were studied.

3. Materials and Methods

3.1. Plant Material

The tomato seeds of the cultivar Falat C.H. were superficially sterilized by soaking in a 2% aqueous solution of sodium hypochlorite for 5 minutes, followed by rinsing twice with sterile deionized water. The seeds were then planted in a culture tray containing pre-autoclaved cocopeat and perlite (in a ratio of 3: 1). When the seedlings reached the 4-leaf stage, they were transferred into 1-liter plastic vases containing sterilized vermiculite and soil (in a 1:1 ratio, W/W). The vases were kept at 25 °C under photoperiod conditions of 16 h light: 8 h dark in a growth chamber.

3.2. Preparation of Bacterial Suspension

The bacterial strain IBRC-M11096 of Bt was purchased from the National Genetic Engineering Institute in Tehran. The bacterial suspension was prepared following the method used by Lacy and Lukezic ( 18 ): a 50 µL bacterial aliquot was transferred into a nutrient broth (1.5%) medium in a 1000 mL flask. The culture was incubated at 25 °C in a shaker-incubator adjusted to a velocity of 150 rounds per minute (rpm) for 48 h. The bacterial suspension was incubated until its optical density reached 0.9, measured at a 600 nm wavelength (almost equal to 10⁹ colony forming units (CFU. mL^-1)); then, it was diluted to a volumetric ratio of 1: 100 (V/V) to achieve a final bacterial cell density of 10⁷ CFU. mL ^-1. The suspension was immediately applied.

3.3. Preparation of Fungal Suspension

The fungus Fol was obtained from the fungal collection at the Department of Plant Protection, Agricultural Sciences and Natural Resources University of Khuzestan. The fungus was cultured on corn meal agar (CMA) plates and incubated at 25 °C under 16 h light: 8 h dark photoperiod conditions for 14 days. The fungal spore suspension (10⁶ spores. mL ^-1) was prepared following the methodology previously described ( 19 ): Briefly, spores (microconidia) were gently collected from 14-day-old culture plates using a sterile loop. The harvested spores were suspended in a 30 mL sterile aqueous solution containing 0.1% Tween 80. The suspension was agitated by vortexing to distribute the spores evenly. Finally, the concentration of microconidia was measured using a hemocytometry lamella (Neubauer Improved, HBG, Germany) under a light microscope (Olympus, Japan) and adjusted to 10⁶ spores. mL ^-1.

3.4. Treatments

Seven days after transferring the seedlings to the plastic vases, experimental treatments, including untreated control, bacterial treatment (T_Bt), fungal treatment (T_Fol), and a combination of both bacterial and fungal treatment (T_Bt+Fol) were performed on 4-5 leaf stage tomato seedlings. Bacterial treatment (T_Bt) involved adding a 30 mL volume of Bt suspension (10⁷ CFU. mL^-1) to the soil inside each pot. The treatment with the pathogenic fungus, Fol (T_Fol), was performed by adding a 20 mL volume of microconidial suspension (10⁶ spores. mL^-1). In the treatment including both microorganisms (T_Bt+Fol), the microconidial suspension of the pathogenic fungus (Fol) was added to each vase’s soil 48 h after the amendment of the bacterial suspension, following the method mentioned above. The effect of bacterial pre-treatment on the induction of defensive genes was evaluated by investigating the transcriptional rates of the selected genes 3, 24, 48, and 72 h after treatment with Fol conidial suspension using the qRT-PCR technique, and the results were compared with those from the plants only treated with the pathogenic Fol conidial suspension.

3.5. Preparation of Foliar Samples

Samples were collected from the middle leaves of seedlings at the 4-5 leaf stage. Three biological replicates were conducted for each temporal treatment, with only one seedling per replicate. The foliar samples were wrapped in pieces of aluminum foil sheet, rapidly frozen in liquid nitrogen, and immediately stored at -80 °C until the next steps.

3.6. RNA Extraction, cDNA Synthesis, and Designing Primers for qRT-PCR

RNA extraction was made using a RNA extraction kit (Access No. # S1020, manufactured in Dena Zist Asia, Mashhad, Iran) following the manufacturer’s recommendations. The extracted RNA samples were quantified by their optical absorbance determined with a Nanodrop apparatus (Spectrophotometer 2000c, Thermo Scientific, USA). Electrophoresis of the RNA on 1% agarose gel was performed for the qualitative analysis of the RNA integrity. The first stranded cDNA was synthesized using a cDNA synthesis kit (Access No. # E6300S, New England BioLabs, England) and Oligo dT primer following the manufacturer’s guidelines. To study changes in the transcription rate of plant defensive pathway genes (COI1, PR1, NPR1, and PIN2), primers were designed using the online software Primer Quest freely available at IDT website (www.idtdna.com). The synthesis of the designed primers (Table 1) was carried out by Takapoozist Company, Tehran, Iran.

Quantitative real time-PCR (qRT-PCR) was performed using Master Mix SYBR Green (High ROX) kit (Cat. No. M3003S) and gene-specific primers and Step One Plus® Real-Time PCR System (ABI Company, USA). The protocol recommended by the kit manufacturer NEB Co., was followed. The thermal cycle’s conditions applied were as follows: one cycle pre-denaturation of 10 min at 95 °C; 40 cycles of denaturation at 95 °C for 15 s; annealing at 60 °C for 15 s, and extension at 72 °C for 20 s. Three seedlings were used as biological replicates, while two technical replicates (cDNA samples) were considered for each biological replicate. The relative expression level (fold change) of target genes in T_Bt+Fol relative to T_Fol was calculated by the 2^-ΔΔCT comparative method ( 20 ). Lycopersicum elongation factor-1 alpha (LOC544055) has been reported as a reference (internal) gene in tomato plants ( 21 ). The data acquired from various samples were first normalized using the difference in cycle threshold (Ct) values against the internal gene LOC544055. Then, the relative expression rate of target genes was calculated using the Relative Expression Software Tool (REST) software ( 22 ).

3.7. Determination of Growth Indices

The experiment was performed based on a completely randomized design with four treatments (control, T_Bt, T_Fol, and T_Bt+Fol), each with 4 replicates. The measurement of growth indices was conducted 40 days after treatment with the pathogen. The weights of fresh shoots and roots were measured using a laboratory balance and recorded. To determine dry weights, the samples were kept in an oven at 72 °C until their dry weights no longer changed after 72 h. The experimental data were analyzed using SAS software (Version 9.1), and the means of treatments were compared using Duncan’s multiple-range test.

3.8. Evaluation of Disease Severity

To investigate the progression of the disease over time, the percentage of leaves displaying symptoms at the whole plant level was monitored every three days, starting from the 10^th day post-inoculation (dpi) and extending for 30 days, as described previously ( 23 ). For this, the number of symptomatic leaves of 4-leaf stage seedlings was recorded on the 10^th, 13^th, 16^th, 19^th, 22^nd, 25^th, 28^th, 31^th, 34^th, 37^th, and 40^th days after fungal treatment. Three seedlings were considered for each treatment (T_Bt, and T_Bt+Fol). The experiment was designed as a random complete block. Disease progress curves were drawn by plotting the disease incidence percentage versus time, and their regression lines were derived using Excel software. The AUDPCs of both treatments were calculated following the published methodology ( 24 ). The ANOVA analyses of total AUDPCs and interval AUDPCs data were performed using IBM^® SPSS^® Statistics 26 software (IBM Corp., USA).

4. Results

4.1. Effect of Bt on Growth Indices of Tomato Plant

Except for root fresh weight, the biological treatments of tomato seedlings led to highly significant differences in shoot fresh weight, shoot dry weight and root dry weight. Bt could significantly increase shoot fresh and dry weights compared to untreated control and Fol-treated plants (Fig. 1A, 1B). Also, compared to the plants treated with Fol (T_Fol), the dry weight of root significantly increased in the plants only treated with Bt (T_Bt) and also in Bt-primed plants challenged with Fol (T_Bt+Fol) (Fig. 1C).

Figure 1.A) Shoot fresh weight, B) shoot dry weight and C) root dry weight in various treatments. The columns with only a common letter are not statistically of significant difference. The mean of treatments has been compared by Duncan’s multiple-range test (P≤ 0.05). Each mean is the average of four repeats.

4.2. Transcriptional Responses of Plant Defense Hormo-nal Signaling Pathway Genes

The study on the transcription rate of plant defense hormonal signaling pathway genes indicated that compared to the plants only treated with Fol, the transcription rate of the NPR1 gene significantly increased in the Bt-primed plants subsequently treated with Fol in the third hour after fungal treatment. Although no significant change was found in the transcriptional rate of the NPR1 gene in the T_Bt+Fol plants in the 24^th h after treatment with Fol compared to T_Fol plants at that time point, a significant 12.2-fold increase of NPR1 gene transcription was found 48 h after Fol treatment. 72 h after treatment with Fol, no significant difference was detected in the transcription of the NPR1 gene between T_Bt+Fol plants and T_Fol plants. A 7.821-fold increase in the COI1 gene transcription occurred in T_Bt+Fol plants compared to T_Fol plants 3 h after treatment with Fol. 48 h after treatment with Fol, the COI1 gene transcription attained its maximal rate, 16.7 folds that of its transcription rate in T_Fol plants. The transcription rate of the COI1 gene in T_Bt+Fol plants was significantly higher 72 h after treatment with Fol (11.75 times higher than that in T_Fol plants). A significant increase in PIN2 gene expression was found in T_Bt+Fol plants up to 48 h after the subsequent treatment with Fol. At this time point, the transcription rate of the gene reached its maximal rate, which was 38.5 times higher than that observed in T_Fol plants. The transcription rate of the PR1 gene in T_Bt+Fol plants was significantly higher than in T_Fol plants at all time points, attaining its highest rate, 66.13 folds, 48 h after treatment with Fol (Fig. 2).

Figure 2. The Effect of pre-treatment of tomato with Bacillus thuringiensis on the relative transcriptional rate of NPR1, COI1, PIN2, and PR1 genes in different time points after treatment with the pathogenic fungus Fusarium oxysporum f. sp. Lycopersici. ns: not significant, *: significantly different (P≤ 0.05), and **: highly significant different (P≤ 0.01) from that in the plants only treated with the fungus.

4.3. Disease Severity

Study on the AUDPC in the T_Fol as well as T_Bt+Fol plants indicated that pre-treatment with Bt led to a highly significant reduction of fusarium wilt disease in T_Bt+Fol plants compared to T_Fol plants (F_{1, 64} = 50.681***; Fig. 3).

Figure 3. Comparison of area under disease progress curves (AUDPCs) calculated for tomato (cv. Falat C.H.) plants primed and non-primed with Bacillus thuringiensis (Bt) IBRC-M11096 and challenged by Fusarium oxysporum f. sp. lycopersici (Fol). The mean of treatments has been compared by Duncan’s multiple-range test (P≤ 0.01). Each mean is the average of three repeats.

Highly significant differences were found in the disease incidence percentage of both treatments in the time points after treatment with Fol (F_{21, 44} = 16.101***). These differences were also well reflected in the AUDPC between time intervals (F_{21, 44} = 16.628***). Also, the comparison of linear regressions of disease progress curves of the bacterial primed and Fol-treated plants at different time points was illustrated in Figure 4.

Figure 4. Effect of Bacillus thuringiensis (Bt) IBRC-M11096- based priming on fusarium wilt disease progress in tomatoes (cv. Falat C.H.) after inoculation with the causal pathogenic fungus, Fusarium oxysporum f. sp. lycopersici (Fol). The trendlines of both disease progress curves (DPCs) have been demonstrated.

5. Discussion

The ability of certain rhizobacteria to enhance plant growth through their interactions with plants and other PGPR (plant growth promoting rhizobacteria) has been formerly reported ( 25 ). The Bt strain, RZ2MS9, was able to improve the growth of maize and soybean under greenhouse conditions, significantly increasing their dry weights. These activities were attributed to atmospheric nitrogen fixation and the production of auxin, phosphate, and siderophores ( 26 ). The strain could endophytically colonize maize roots and leaves, and when co-inoculated with Azospirillum brasilense Ab-V5, it respectively increased maize root and shoot dry weights by approximately 50% and 80% compared to untreated controls ( 27 ). In the present study, the Bt strain significantly increased root and shoot dry weights and raised shoot fresh weight in Bt-primed tomato plants compared to those only treated with the pathogenic fungus, Fol.

Plant hormones act as crucial regulators of plant defensive responses that regulate various aspects of plant immunity against pathogens. The signaling role of JA/ET against necrotrophic pathogens is well known ( 28 - 30 ). To survey the involvement of the JA in the protection mediated by Bt against Fol in tomato, we monitored the transcript level of JA signaling marker genes in plants treated with Bt after Fol challenge. F-box protein coronative insensitive1 (COI1) is a JA receptor, the primary regulator of defensive responses in rhizobacteria-mediated induced systemic resistance (ISR) ( 31 ). Furthermore, the proteinase inhibitor gene is crucial for JA-dependent signaling and serves as a marker gene for the related pathway in the Fol/tomato pathosystem ( 32 ). We showed that the transcription of COI1 significantly increased in the plants pre-treated with Bt at all time points after treatment with the fungus. Furthermore, the level of PIN2 gene (coding for a proteinase II inhibitor) transcription attained its highest rate in the plants pre-treated with Bt 48 h after the fungal treatment. Similarly, Pazarlar et al. ( 33 ) showed that the JA/ET-related marker genes, such as proteinase inhibitor I (I-1) and ethylene receptor4 (ETR4), were up-regulated following the Fol challenge in Bacillus cereus EC9 – treated plants, indicating that EC9 stimulated defense response by priming the JA/ET signaling pathway in tomato-Fol interaction.

Although the efficacy of the external application of SA in the induction of PR1 and its contribution to the development of defensive responses has been illustrated, some studies indicate that the accumulation of internal SA inhibits the development of defensive responses against necrotrophic pathogens (such as Fo). In contrast, it induces an efficient defensive response against biotrophic pathogens ( 28 , 34 ). For instance, some investigations have shown that the necrotrophic pathogen takes advantage of the SA signaling pathway to strengthen the disease development in tomato ( 35 ). However, some reports indicate the effect of SA in the defensive responses against necrotrophy. Despite the proven role of JA and ET in the immune responses of plants against Fo, the role of SA in immune responses is complicated and seemingly dependent on the nature of the host and pathogen ( 36 , 37 ). Hence, we aimed to investigate whether the SA signaling is also implicated in boosting plant immunity by Bt against Fol. This was assessed using the qRT-PCR to measure the expression level of the SA signaling marker genes. As one of the positive regulators of PR gene expression and a SA receptor, the nonexpressor of pathogenesis-related 1 (NPR1) protein plays a vital role in the induction of systemic acquired resistance (SAR) ( 38 ). We observed that Bt pre-treatment elevated the transcript level of NPR1 and PR1 following the Fol challenge; it can be speculated that the SA signaling also functions in Bt-stimulated immune response against Fol in tomato. Former studies have indicated that ISR is more dependent on JA/ET signaling pathway and is regulated through a pathway other than that of SA ( 39 ). However, some PGPR strains induce ISR through a SA-dependent pathway, exactly like pathogen-induced SAR (reviewed in 40). Multiple reports, mentioned subsequently, illustrated that certain PGPR strains could trigger host ISR through the synergistic activation of both JA/ET and SA signaling pathways. Bacillus subtilis MBI600 activated JA and SA signaling pathways simultaneously in tomato plants to control soil-borne pathogens ( 41 ). Similarly, B. cereus AR156 can trigger immune responses against Pseudomonas syringae pv. tomato DC3000 via ISR stimulated by both the JA/ET and SA signaling pathways ( 42 ). It has also been shown that both the SA and JA signaling were involved in endophyte-mediated resistance (EMR) against Fol in tomato plants ( 43 ). In this study, considering increased growth indices and reduced AUDPC obtained with T_Bt+Fol plants, it appears that Bt stimulates an immune response against Fol in tomato plants, possibly by priming both the JA and SA signaling pathways. Such a biological treatment with double effects is preferred to the treatments made only with chemical plant resistance inducers, where the induced resistance is acquired at the cost of reduced crop yield ( 44 ).

Considering the soil-borne nature of fusarium wilt disease, eliminating the potential inoculum of soil-borne pathogens is regarded as one of the most effective methods for disease control ( 45 ). A look at the disease progress curves of both treatments as well as their regression lines, indicates that y-intercept (the point where the regression line meets the axis of disease severity percentage, “y”) in T_Bt+Fol stands lower than T_Fol, indicating the effect of Bt on reducing the pathogenic fungus primary inoculum in tomato soil (Fig. 4). Despite these promising results, the greater slope of the regression line of the disease severity in T_Bt+Fol plants (Fig. 4) illustrates that the disease severity can even precede that in T_Fol plants over time if the conditions are conducive for disease development. Such a prediction does not seem improbable because the tomato plants reach their highest susceptibility in the fruit maturity step under field conditions. These findings indicated that such an approach may not be sufficiently effective in eradicating the whole soil-borne inoculum of the fungus. Even the volatile compounds used in soil fumigation have not led to the complete eradication of the pathogens in the treated soil and have exhibited only short-term effects ( 46 ). Thus, the survived pathogen inoculum can infect the host, and if the plant is not resistant or the applied control method leads to changes in plant defensive hormonal pathways that favor the pathogen, it can lead to a severe disease in the infected plant. Hence, the final confirmation of the practical profitability of the studied Bt strain still requires field studies, where tomato plants are tested in their whole life span, and the effect of Bt-priming can be studied during fruiting stages till crop harvesting.

6. Conclusion

The findings of this research showed that the expression of defensive genes in the Bt-primed plants was detectable from 3 h after plant treatment with the pathogenic fungus (Fol) and continued at least till 48 h or 72 h after fungal treatment, indicating the induction of plant defensive genes of both hormonal pathways, JA as well as SA, following bacterial pre-treatment. We also found that B. thuringiensis acts as PGPR, positively affecting plant shoot and root growth indices, and reduced AUDPC in Bt-primed tomato plants, challenged with Fol (T_Bt+Fol plants). However, since tomato plants become most susceptible during the fruit maturity stage, conducting field experiments is necessary to obtain convincing results.

Acknowledgements

The present research is based on the M.Sc. thesis of the first author performed in the Department of Plant Production and Genetics, Faculty of Agriculture, Agri-cultural Sciences and Natural Resources University of Khuzestan. The research was financially supported by Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Khuzestan, Iran.

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