Utilizing plant growth promoting rhizobacteria (PGPR) to combat pathogens and enhance crop production is an environmentally friendly and sustainable approach. The biocontrol activity of PGPR depends on their ability to colonize plant roots and synthesize antimicrobial compounds that inhibit pathogens. However, the regulatory mechanisms underlying these processes remain unclear. In this study, we isolated and characterized Bacillus velezensis isolate 118, a soil isolate that exhibits potent biocontrol activity against Fusarium wilt of banana. Deletion of sigX, an extracytoplasmic function (ECF) sigma factor previously implicated in controlling biofilm architecture in B. subtilis, reduced biocontrol efficacy. The B. velezensis 118 sigXmutant displayed reduced biofilm formation but had only a minor defect in swarming motility and a negligible impact on lipopeptide production. These findings highlight the importance of regulatory processes important for root colonization in the effectiveness of Bacillus spp. as biocontrol agents against phytopathogens.
Article
The extracytoplasmic sigma factor SigX supports biofilm formation and increases biocontrol efficacy in Bacillus velezensis 118
https://doi.org/10.21203/rs.3.rs-5005592/v1
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With the rise in global population and dramatic changes in climate, ensuring food security has become an increasingly pressing issue1. This has led to heightened pressure to meet growing food demand, resulting in extensive use of chemical pesticides and fertilizers to boost agricultural productivity2. However, the massive application of agrochemicals can disrupt soil salinity levels and leave toxic residues in agricultural produce, leading to the frequent occurrence of soil-borne diseases. Soil amendment with plant growth promoting rhizobacteria (PGPR) is widely regarded as a promising and sustainable strategy for crop disease management3. Numerous strains from the Bacillus genus have figured prominently in efforts to use PGPR in agricultural settings4,5.
Fusarium wilt, caused by the soil-borne fungus F. oxysporum f. sp. cubense (Foc), and Bacterial wilt, caused by Ralstonia solanacearum (Rs), rank among the most devastating plant diseases6,7. In the rhizosphere of plants, soil-borne pathogens coexist with beneficial microorganisms such as plant growth-promoting rhizobacteria (PGPR)8. PGPR can suppress soil-borne pathogens through competition for essential nutrients and the production of antagonistic compounds9.
B. subtilis and B. velezensis represent a growing class of plant growth promoting rhizobacteria (PGPR) widely recognized for their biocontrol activity against plant diseases10,11. These bacteria form biofilms on plant roots and produce bioactive compounds that competitively exclude pathogens12. The model organism of the B. velezensis species is strain FZB42 (formerly B. amyloquifaciens FZB42)13. Functional information related to this species is available through the AmyloWiki database14. This and numerous other related Bacilli have been isolated from soils and shown to be effective biocontrol agents in a variety of settings.
Biocontrol activity has been shown to depend on the ability of the bacteria amended to the soil to colonize plant roots and produce potent antimicrobial compounds that inhibit the growth of bacterial and plant pathogens. Prior studies have defined the transcriptional responses of Bacilli to root exudates15,16, demonstrated the essential role of motility and biofilm formation in root colonization17–19, and identified the specific antimicrobial secondary metabolites that mediate disease suppression10,20–22.
The extracytoplasmic function (ECF) sigma factors serve as important transcriptional regulators in the Bacilli, responding to diverse stresses and impacting biofilm formation23–25. B. subtilis encodes seven ECF sigma factors (σM, σW, σX σV. σylaC, σZ and σY). Deletion of four sigma factors (σV, σY, σZ, and σylaC) resulted in only a minor reduction in biofilm formation, whereas deleting the remaining three (σM, σW, and σX) led to a modest decrease23. However, deletion of all seven sigma factors exhibited the most significant decrease in biofilm formation23. B. velezensis FZB42 possesses five ECF (σM, σW, σX σV and σylaC) sigma factors26, but the specific functions remain unclear.
B. velezensis FZB42 was isolated from plant-pathogen-infested soil from a sugar beet field in Brandenburg, Germany27. Products based on this strain include RhizoVital® (ABiTEP, GmbH, Berlin, Germany) and Taegro® 2 (Novozymes), and many related species have also been commercialized28. Prior work has demonstrated that the successful application of PGPR benefits from the use of indigenous isolates adapted to the ambient temperature and soil types. For example, isolates indigenous to the Vietnamese highlands were effective in presented disease in that environment29, and cold-adapted Bacilli from the Qinghai-Tibetan Plateau were effective in promoting growth of winter wheat30.
Here, we isolate and characterize B. velezensis 118, a strain indigenous to Guangzhou, a city in South China with a south Asian tropical monsoon oceanic climate and latisolic red soils. This isolate exhibits robust biocontrol activity against the banana fungal pathogen Foc and the bacterial wilt pathogen Rs. We further demonstrate that the ECF sigma factor SigX is required for effective biocontrol against both Foc and Rs under natural environmental conditions, likely through its contribution to biofilm formation. This study highlights the role of SigX in enhancing biocontrol efficacy of B. velezensis.
Isolation and identification of a Bacillus isolate that exhibits strong antagonistic activity against Foc.
To isolate PGPR that can be effective in a sub-tropical climate zone, we collected rhizosphere soil from healthy banana plants at a local farm in Guangzhou, a city in South China. More than 60 Bacillus strains were isolated. The isolate designated as strain 118 (HB19118) exhibited the strongest inhibitory effect against the banana fungal pathogen F. oxysporum f. sp. cubense (Foc) as assayed using a spot-on-lawn assay on Potato-Dextrose-Agar (PDA) plates (Fig. 1A). Compared to unexposed Foc, exposure to strain 118 resulted in altered fungal morphology with swollen and distended Foc spores (Fig. 1B) and hyphae (Fig. 1C), particularly when isolated proximal to the zone of growth inhibition. Phylogenetic analysis based on a partial sequence of rpoB revealed that strain 118 (henceforth B. velezensis 118) clusters with the well characterized B. velezensis FZB4213 and B. velezensis SQR915,16 isolates.
To further evaluate the antagonistic capability of B. velezensis 118, we conducted a plate confrontation assay against several common fungal plant pathogens. B. velezensis 118 significantly inhibited the growth of Magnaporthe oryzae, Peronophythora litchii, Rhizoctorzia solani, Fusarium oxysporum f.sp. cucumerinum (Fig. S1).
B. velenzensis 118 is an effective biocontrol agent for banana Fusarium wilt
To test whether B. velenzensis 118 could effectively control banana Fusarium wilt, we carried out pot experiments under greenhouse settings using micropropagated Cavendish banana seedlings of the ‘Brazilian’ variety that are susceptible to Foc. Following treatment with B. velezensis 118, the wilt incidence (WI) was reduced by nearly two-fold relative to the untreated control plants (CK2), which exhibited a DI of 89% (Fig. 2A and 2E). Consistent with this disease suppression, plants treated with both B. velezensis 118 and Foc displayed a restoration of leaf number (Fig. 2B), plant height (Fig. 2C), and plant biomass (Fig. 2D) to levels significantly higher than CK2 (Foc-exposed plants). Bacillus biocontrol agents can both protect plants against pathogens and in some cases improve growth by production of plant hormones31,32. In this case, the plants in the 118-treated group (Foc + 118) were comparable to the healthy, uninfected control plants (CK1), with no obvious growth stimulation. Furthermore, treatment of B. velezensis 118 led to restoration of the rhizosphere microbial community, with increased levels of bacteria and actinomycetes, and a significant reduction in fungi, relative to the Foc-treated plants (Fig. S2). Together, these results demonstrate that B. velezensis 118 effectively suppresses banana Fusarium wilt and also facilitates the restoration of soil microbial ecological balance, thereby mitigating the damage caused by Foc infection.
Deletion of sigX impairs biofilm development in B. velenzensis and B. subtilis.
The ability of PGPR to reduce the impact of phytopathogen is associated with a strong potential for biofilm formation, which contributes to efficient colonization of the root surface33,34. Assays for biofilm formation are well established for Bacillus isolates, and include analysis of the complex morphology of colonies growing on agar plates35,36 and of the pellicles that form at the interface between a nutrient medium and the air in static cultures37,38. We evaluated the biofilm formation capability of B. velezensis 118 by monitoring pellicle mass and found that this isolate exhibited even higher biofilm production than other biofilm-producing isolates such as B. velezensis FZB42, Y620, F720, and B. subtilis 3610 (NCBI 3610) (Fig. S3).
The regulatory pathways involved in biofilm formation have been investigated in detail in B. subtilis 361036,39–41. Since the transition from planktonic growth to a biofilm is a major life-style transition, we hypothesized that alternative sigma factors might play a role in this process. B. subtilis 3610 strains encode seven sigma factors of the extracytoplasmic function (ECF) subfamily that are important in helping cells adapt to new environments25,42. Initial studies revealed that a triple mutant lacking three of these sigma factors (sigM sigW sigX) were defective in colony morphology and pellicle formation43. Further analysis revealed that sigX mutants are defective in biofilm formation44. SigX controls the expression of Abh, a positive regulator of biofilm formation35. Although there have been some studies of the pathways regulating biofilm formation in B. velezensis FZB4245 and related species, the roles of ECF sigma factors are not yet known.
B. velezensis encodes five ECF sigma factors: σM, σX, σW, σV, and σYlaC 26. To assess the role of each σ factor to biofilm formation, we constructed deletion mutants of each ECF sigma factor in B. velezensis 118. We then examined the impact of these deletions on pellicle formation at the air-liquid interface in liquid culture. B. velezensis 118 WT exhibited densely packed, uniformly structured pellicle with characteristic wrinkled patterns. Four single mutants (sigM::mls, sigW::cat, sigV::kan, and ylaC::cat) displayed comparable levels of biofilm development. However, the sigX::mls mutant showed a much thinner and disorganized pellicle structure with less wrinkling and large gaps (Fig. 3A). We then monitored the colony morphology on solid agar plates. Compared to the wild-type B. velezensis 118, three mutants (sigM::mls, sigW::cat, and sigV::kan) showed similarly structured biofilms with rugged edges. In contrast, the ylaC::cat mutant displayed a wide and dispersed halo at the margin, and the sigX::mls mutant exhibited a disrupted structure with less uniformity and potential central degradation (Fig. 3B).
To further evaluate the effects of sigX deletion on biofilm development, we monitored pellicle formation and colony morphology over five days. The sigX::mls mutant failed to develop a mature and complex pellicle structure by day 5 compared to WT (Fig. 4A). While WT developed well-defined colonies with a rugged and intricate pattern over time, sigX::mls colonies remained smaller and less organized, with parts of the pellicle petal structure missing even after 5 days (Fig. 4B). Additionally, the sigX::mls mutant produced significantly lower biofilm mass across all time points (Fig. 4C).
Next, we compared the effects of the B. velezensis sigX deletion with those observed in the more genetically tractable strain B. subtilis NCIB3610. Consistent with prior studies44, the B. subtilis sigX null exhibited disrupted pellicle formation and less structured colonies compared to B. subtilis 3610 WT. This phenotype can be restored by complementation in trans (Fig. 4D). Together, these data illustrate the pivotal role of SigX in maintaining biofilm structure and integrity in both B. subtilis and B. velezensis. Consistent with the known correlation between biofilm formation and efficiency of root colonization18, we note that the B. subtilis 3610 sigX mutant strain was compromised in its ability to colonize Arabidopsis thaliana roots, particularly at early time points (Fig. S4). In contrast with these effects on root colonization, sigX mutants in both B. subtilis 3610 and B. velezensis 118 had only minor defects in swimming and swarming motility (Fig. S5), and no differences were noted in the production of lipopeptides in B. velezensis 118 (Fig. S6).
The role of SigX in enhancing the biocontrol efficacy of B. velezensis 118
To evaluate the potential involvement of sigX in the biocontrol efficacy (BE) of B. velezensis 118, we monitored disease progression of banana and tomato plants exposed to the fungal pathogen Foc and the bacterial pathogen Rs, respectively (Fig. 5). In the absence of B. velezensis 118 treatment, the DI of banana plants exposed to Foc reached 66% at 21 d after transplanting. Treatment of WT B. velezensis 118 significantly reduced the DI to 23% (Fig. 5A-B), achieving a biocontrol efficacy of 65% against Foc. However, deletion of sigX led to a significantly reduced biocontrol efficacy against Foc, with a DI of 38% (Fig. 5A-B), resulting in a biocontrol efficacy of 42% for the sigX::mls mutant against Foc.
In tomato bacterial wilt pot experiments, deletion of sigX also resulted in decreased biocontrol efficacy against Rs. Thirteen days after exposure to Rs, the DI of tomato plants treated with wild-type B. velezensis 118 was 51%, whereas those treated with the sigX::mls mutant had a DI of 69% (Fig. 5C-D). In contrast, the DI in plants not treated with B. velezensis 118 was 93% (Fig. 5C-D). Notably, sigX contributed to a 23% enhancement in biocontrol efficacy against Rs. These results underscore the significant role of sigX in the biocontrol efficacy of B. velezensis.
B. velezensis 118 is a rhizosphere soil isolate found near cultivated banana plants in Guangzhou, China. This isolated was selected based on screening for strong anti-fungal activity against Foc when assayed on plates (Fig. 1). We further confirmed that strain B. velezensis 118 reduces wilt incidence as assayed on banana seedlings exposed to Foc (Fig. 2). The activity of this isolate is not specific to this disease, and it also reduced the incidence of bacterial tomato wilt (Fig. 5).
The ability of Bacillus spp. to function as PGPR and to suppress plant disease relies on numerous traits. These include the ability to efficiently colonize plant roots and to produce potent antimicrobials, including many lipopeptides. Efficient root colonization is correlated with the ability to form robust biofilms, which can be conveniently assayed by monitoring pellicle formation at the medium-air interface and colony morphology on solid media. Consistent with its strong biocontrol activity, B. velezensis 118 forms well-developed biofilms (Fig. 3, Fig. 4, Fig. S3), and produces numerous lipopeptides, including surfactin, iturin, and fengycin (Fig. S6).
Previous work has suggested that regulators of the extracytoplasmic function sigma factor family can be important in biofilm formation43,44. However, there is little information about their possible role in biocontrol by B. velezensis spp. By screening mutant derivatives of B. velezensis 118 for effects on biofilm formation we determined that only sigX has a notable reduction in pellicle formation (Fig. 3), although not as strong as that seen in the model organism B. subtilis 3610 (Fig. 4). This reduction in biofilm formation was correlated with reduced efficacy of disease suppression by B. velezensis 118 for both banana wilt and bacterial tomato wilt (Fig. 5). Since the role of sigX in biofilm formation is best understood in B. subtilis 361044, we tested the effect of this mutation on root colonization using an Arabidopsis thaliana model. Indeed, root colonization in this mutant derivative was slowed relative to wild-type. These results provide further evidence that a strong capacity for biofilm formation is correlated with root colonization, which is one likely explanation for the importance of sigX in the context of biocontrol.
Although the biological roles of ECF sigma factors have been extensively studied in B. subtilis 168 and 3610 strains, their contributions in B. velezensis are still poorly understood. The results here suggest that sigX is part of the extended regulatory network important for biocontrol, consistent with the regulatory map for biofilm formation that has emerged from studies in B. subtilis41.
Isolation of antagonistic Bacillus species
Samples were collected from the rhizosphere soil of banana plants in Guangzhou, China (23˚ 08’ N 113˚ 16’ E). Bacillus spp. were isolated as reported previously46. Briefly, the soil samples (10 g each) were shaken in 90 ml of sterilized water for 30 min, heated for 30 min at 80°C, serially diluted, and then spread over lysogeny broth (LB) plates. Single bacterial colonies were streaked onto fresh LB plates after 48 h of incubation at 30°C. Frozen stocks of the purified colonies were prepared using 15% glycerol and kept at -80°C for further study.
Growth measurements
The growth kinetics experiments were conducted as described previously20. Briefly, two µl aliquots (OD600 ~ 0.4) were inoculated to 200 µl of LB and LBGM medium in a Bioscreen 100-well microtiter plate. Growth was measured spectrophotometrically (OD600) every 15 min for 24 h using a Bioscreen C incubator (Growth Curves USA, Piscataway, NJ) with continuous shaking at 37°C or 30°C as indicated. Data shown are representative growth curves and experiments were performed with three biological replicates.
Antifungal activity test
We performed two assays, a plate confrontation assay and a spot-on-lawn assay, to test activity of the isolates (118) and derived mutants against common fungal pathogens including Fusarium oxysporum f.sp. cubense 4 strain XJZ2 (Foc4, GenBank accession number JX090598) Magnaporthe oryzae B157 (GenBank accession number AXDJ01000000)47, Peronophythora litchii Shs3 (GenBank accession number PCFV01000001)17, Rhizoctorzia solani AG1-IA GD-118 (GenBank accession number KB317696 AFRT01000000), and Fusarium oxysporum f.sp. cucumerinum (isolated from cucumerium rhizosphere, no accession number available yet).
The plate confrontation assay was conducted as described previously20. Briefly, the four fungi except Peronophythora oryzae B157 were cultivated on potato-dextrose-agar plates (PDA, 20% potato infusion, 2% dextrose, and 1.5% agar), while Peronophythora oryzae B157 was cultivated on carrot juice agar plates (CA, 20% carrot and 1.5% agar) at 30°C. After 5 days of incubation, a 5-mm-diameter block of mycelium agar containing fungi (Rhizoctorzia solani AG1-IA GD-118, Fusarium oxysporum f.sp. cucumerinum and Foc4) was cut and placed at the center of a fresh PDA plate. After one day of incubation, a 5-mm-diameter well was created 2.5 cm away from the center of each plate, and 50 µl of Bacillus spp. cells (OD600 ~ 0.4; 118) grown in LB medium was added into each well. Additionally, the mycelium agar of the slow-growing fungi (Magnaporthe oryzae B157 and Peronophythora litchii Shs3), was cut and placed 2.5 cm away from the center of a fresh PDA or CA plate. After one day of incubation, a 5-mm-diameter well was made in the center of each plate, and 50 µl of Bacillus spp. cells (OD600 ~ 0.4; 118) grown in LB medium was added into each well. Antifungal activity was evaluated by measuring the diameter of the inhibition zone (the distance between the mycelium and the bacterial colony) after 7 days of incubation at 30°C.
The spot-on-lawn assay was conducted as described previously20. This assay is more sensitive compared to the plate confrontation assay and requires only one day of incubation instead of seven days, fungal hyphae of Foc4 were streaked and inoculated with 5 ml of PDA broth. After two days of incubation at 30°C with shaking at 180 rpm, 50 µl of the fungal culture was re-inoculated into 5 ml of fresh PDA broth and incubated for additional 12 h. The culture was then filtered using a cheesecloth to remove hyphae, and 50 µl of the resulting spore suspension was mixed with 4 ml of 0.7% soft PDA agar and poured directly onto a PDA plate (1.5% agar). After drying the plates for 50 min, a 5-mm-diameter well was created in the center of each plate, and 50 µl of Bacillus spp. cells (strain 118, or sigX mutants derived from 118), grown in LB medium to OD600 ~ 0.4, was added into each well. Antifungal activity was evaluated by measuring the diameter of the inhibition zone (in mm) after 24 h of incubation at 30°C. The experiments were performed at least three times.
Antibacterial activity test
The antibacterial activity of the isolates and their derived mutants against R. solanacearum GMI1000 was evaluated using an optimized spot-on-lawn assay as described previously20. Two hundred microliter of R. solanacearum cell culture (OD600 ~ 0.4), grown in Casamino Acid-Peptone-Glucose (CPG, 0.1% peptone, 0.01% casamino acids, 0.05% glucose), was mixed with 4 ml of 0.7% CPG soft agar and directly poured onto a CPG plate (1.5% agar). After drying the plates for 50 min, a 5-mm-diameter well was made in the center of each plate, and 50 µl of each Bacillus strain (OD600 ~ 0.4) grown in LB medium was added into each well. Antagonistic activity was evaluated by the size of the inhibition zone after 24 h incubation at 30oC. The experiments were performed at least three times.
Construction of mutants
In initial studies, we determined that 118 is sensitive to spectinomycin (spc; 100 µg ml− 1), kanamycin (kan; 15 µg ml− 1), chloramphenicol (cat; 10 µg ml− 1), tetracycline (tet; 5 µg ml− 1), and macrolide lincosamide-streptogramin B (mls; contains 1 µg ml− 1 erythromycin and 25 µg ml− 1 lincomycin) antibiotics. The sigM::mls, sigX::mls, sigW::cat, sigV::kan and ylaC::cat single mutants in 118 background were generated by replacing the coding region with an antibiotic resistance cassette using long flanking homology PCR (LFH-PCR) followed by DNA transformation as previously described (Mascher et al., 2003) (Table S1). The sigX::erm mutant in NCIB3610 background was also constructed using sigX::erm from the Bacillus Knockout Erythromycin (BKE) collection (28189581). The sigX Pspac sigX complemented strain in NCIB3610 background was constructed by vector pPL8248 using PCR products from B. velezensis 118 chromosomal DNA. pPL82 contains a chloramphenicol resistance cassette, a multiple cloning site downstream of the Pspac(hy) promoter and the lacI gene between the upstream and downstream fragments of the amyE gene. All the constructs were confirmed by Sanger sequencing.
Swarming and swimming motility assays
Swimming and swarming motility of 118, NCIB3610, and their derived mutants were tested using standard protocols was conducted as described previously20 with minor modification. LBGM plates containing 0.7% (for swarming) or 0.3% agar (for swimming) were dried in a laminar flow hood for 30 min and then 5 µl of LB precultures (OD600 ~ 0.4) were spotted on the center of each plate. The plates were then dried for another 15 min and incubated overnight at 37°C.
Biofilm formation assay
The biofilm formation assays were conducted as described previously20. For colony morphology analysis, 3 µL of LB precultures (OD600 ~ 0.4) were spotted onto LBGM agar plates, which had been dried for 30 min in a laminar airflow hood prior to spotting. The plates were then incubated at 30°C for up to 5 d. To monitor pellicle formation, 10 µL of LB precultures (OD600 ~ 0.4) were inoculated into 2.5 ml of LBGM medium in a 24 well plate and incubated at 30°C for up to 5 d. The pellicle was harvested from the well using a 1 ml pipette tip, placing it into a 1 ml centrifuge tube, and then dried under vacuum for 1 h prior to weighing.
Plant pot experiments
The biocontrol efficacy of B.velezens 118 against banana Fusarium wilt was determined under greenhouse conditions using a similar protocol was conducted as described previously20. Micropropagated Cavendish banana seedling ‘Brazilian’, the F. oxysporum (Foc) susceptible variety, were used for the pot experiments. Each pot contained 1.5 kg soil (pH 4.7, organic matter 22.7 g kg− 1, total nitrogen 0.79 g kg− 1, alkaline hydrolysis nitrogen 118.4 mg kg− 1, available phosphorus 1.61 mg kg− 1, and available potassium 18.6 mg kg− 1). Two control groups were included as follows: CK1 (no Foc), banana seedlings with four or five leaves and approximately 20 cm in height were directly implanted into pots; CK2 (Foc, GenBank accession number JX090598), prior to planting into pots, the roots of the banana seedlings were immersed in Foc (ཞ106cfu ml− 1) for 30 min. In the treated group (Foc + 118), the roots of the banana seedlings were firstly immersed in the Foc suspension for 30 min, then planted into pots. Two days later, the plants were watered with 50 ml of a cell suspension of 118 (OD600 ~ 1.0) around the roots, resulting in a final concentration of ཞ106 cells per gram of soil. The wilt severity index (WSI) was recorded using the following index49: 1 = healthy, 2 = slight chlorosis and wilting with no petiole buckling, 3 = moderate chlorosis and wilting with some petiole buckling and/or splitting of leaf bases, 4 = severe chlorosis, wilting, petiole buckling and dwarfing of the newly emerged leaf, and 5 = dead. Wilt incidence (WI) of the banana plants was monitored every 3 days after transplantation. Wilt incidence (WI) of the banana plants was monitored every 3 days after transplantation. The experiments were conducted at least three times with 30 banana plants per groups. To evaluate the contribution of sigX in biocontrol efficacy against banana Fusarium wilt, four groups were included: CK1 (No Foc), CK2 (Foc), Foc + 118, and Foc + sigX.
The biocontrol efficacy of 118 and its derived sigX::mls mutant against tomato bacterial wilt was determined under greenhouse conditions using a similar protocol as described previously20. Four treatments were included as follows: control 1 (CK1, no Rs), control 2 (CK2, only inoculated with Rs), WT (Rs + 118), and sigX (Rs + sigX::mls mutant). Tomato seeds (Lycopersicon esculentum Miller) were surface-sterilized by immersion in 70% ethanol for 30 s, followed by 5% sodium hypochloride for 15 min, and then washed three times with sterile water for 15 min each time. The sterilized seeds were planted in pots containing non-sterile local soil, and the pots were then placed in an artificial climate chamber (PQX-450R-22HM). After one month, the tomato seedlings were transplanted into new pots containing 1.5 kg of the non-sterile local soil. Seven days after transplanting, the soil used for 118 or sigX::mls treatment was drenched with 50 ml of bacterial suspension of 118 or sigX mutant strain (ཞ106 cells per g of soil). The bacterial suspension was prepared using cell pellets harvested from 50 ml of LB cell culture (OD600 ~ 1.0) by centrifugation. Two days later, each pot was drenched with 30 ml of R. solanacearum cell suspension, which was obtained from 30 ml of CPG cell culture (OD600 ~ 1.0) by centrifugation and resuspension in sterile water, resulting in ཞ107 cells per g of soil. The pots were then placed back into the artificial climate chamber, set to a 16-hour day/8-hour night cycle with temperatures of 30°C during the day and 28°C at night, and relative humidity ranging from 65–80%. Each treatment group included 24 tomato plants with three replicates. The wilt incidence (WI) was calculated on the 30th day after transplanting. The wilt severity index (WSI) was recorded as follows: 0 = no wilt symptoms, 1 = wilt symptoms on 1–25% of the leaves, 2 = wilt symptoms on 26–50% of the leaves, 3 = wilt symptoms on 51–75% of the leaves, and 4 = wilt symptoms on more than 76% of the leaves.
Wilt incidence and biocontrol efficacy of the two pot experiments tomato bacterial wilt and banana Fusarium wilt were calculated according to the following formula49,50:
Root colonization assay
To investigate the contribution of sigX to root colonization, root colonization experiments were conducted as described previously51. Briefly, Arabiposis seeds were surface sterilized with 75% ethanol followed by 0.3% sodium hypochlorite (vol/vol) and germinated on 0.5X MS (Murashige and Skoog) agar plates containing 0.7% agar, 0.05% glucose, the following macronutrients in mM: MgCl2, 3.0; (NH4)2SO4, 0.25; Ca(NO3)2, 1.0; KCl, 2.0; CaCl2, 2.75; KH2PO4, 0.18; and the following micronutrients in µM: H3BO3, 5.0; MnSO4, 1.0; CuSO4, 0.05; ZnSO4, 0.2; Na2MoO4, 0.02; CoCl2, 0.001. pH 5.4). After 6 days of incubation in a plant growth chamber at 25°C under a 16 h light /8 h dark cycle, seedlings were ready for use. One µl of LB preculture (OD600 0.4) and one seedling were added into 200 µL LBGM in 96-well plates. There were incubated at 25oC for 3 h, followed by shaking at 100 rpm in a greenhouse at 25°C for 0, 12, 24, or 48 h. Roots were then washed using 0.1m PBS buffer and imaged by a Leica SP5 confocal scanning laser microscopy (CSLM) with excitation at 488 nm and emission at 509 nm.
Cell recovery counting
The cell recovery counting assay was performed as described previously51. After being washed four times in sterile water, the preprocessed roots (one-cm root segments) were placed in an Eppendorf tube containing 1 mL of sterile water. After adding two glass beads to each tube, each sample was vortexed for 5 minutes. The resulting suspension was serially diluted with distilled water, and 100 µL of the cell suspension from dilutions (10− 1, 10− 2, or 10− 3,10− 4) was plated onto LB agar plates. The plates were then incubated for 7 hours at 37°C. Colony forming units (CFU) per mm root were calculated. The experiment was repeated three times with ten root samples per replicate.
Lipopeptide (LP) extraction from the isolated strains
To understand the impact of pathogen presence on LP production of Bacillus isolates, LPs were extracted from the inhibition zone as described previously20 (Fig. S6C). Two hundred µl of R. solanacearum cell culture (OD600 ~ 0.4) in CPG medium was combined with 4 ml of 0.7% CPG soft agar, mixed thoroughly by vortexing, and then poured onto a CPG plate containing 1.5% agar. Plates were dried for 50 min and a 5-mm-diameter well was made at the center of each plate, and 50 µl of Bacillus isolates (118 or NCIB3610 or their mutant) grown in LB medium (OD600 ~ 0.4) was added into each well. After the inhibition zone became evident with 24 h incubation at 30°C, a 300 mg agar sample was harvested from the inhibition zone, mixed with 1 ml of 1:1 acetonitrile/water mixture, and sonicated for 30 s, and then subjected to centrifugation and filtration. The supernatant was collected from acetonitrile/water extract. LPs were also extracted from the control plates using the same procedure, but without R. solanacearum in the top soft agar layer. Agar sample (300 mg) was collected around the well (2–4 mm).
Identification and quantification of LPs by UPLC–MS
The acetonitrile/water extracts were analyzed by reverse phase Ultra-Performance Liquid Chromatography coupled with a triple quadrupole MS (UPLC-MS) as described previously20 (Waters, Acquity, XEVO-TQD). The identification of lipopeptide compounds was achieved using their mass-to-charge ratio (m/z), and their quantification was performed using standard curves derived from commercial LP standards (Sigma-Aldrich, USA). The column temperature was maintained at 40°C and a gradient elution with (A) acetonitrile (containing 0.1% formic acid) and (B) water (containing 0.1% formic acid) was used. The gradient program was used as follows: 0–0.5 min, 40% A; 0.5–3.5 min, 40–80% A; 3.5–4.0 min, 80% A; 4.0–6.0 min, 80–95% A; 6.0–7.0 min, 95–98% A. The flow rate was set at 0.4 ml min− 1. The Electrospray Ionization (ESI) source was set in positive ionization mode with a capillary voltage of 3.26 kV, and the source temperature was maintained at 150°C. The nitrogen flow rate was 600 L h− 1 and the argon flow rate was 50 L h− 1.
Declaration of Interests
Yanfei Cai is listed as an inventor on Chinese patent # ZL201910391310.3 owned by South China Agricultural University.
Competing Interests
Yanfei Cai is listed as an inventor on Chinese patent # ZL201910391310.3 owned by South China Agricultural University.
Author Contribution
Y.C., H.P., and J.D.H conceived and designed the experiments, Y.C. and H.T. conducted the experiments, Y.C., H.T., and H.P. analyzed the data, Y.C., H.P., and J.D.H wrote the paper with input from all authors.
Acknowledgement
The authors thank Dr. Pete Chandrangsu for advice during these studies. This work was supported by National Institutes of Health grant R35GM122461 awarded to JDH and R00 AI168483 to HP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. YC was supported by the China Scholarship Council during her time in the Helmann lab. This work was also supported by grants from the National Natural Science Foundation of China to YC (41471214 and 41977035).
Data Availability
Data Availability StatementAll relevant data are within the manuscript and its Supplementary Information.
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Competing interest reported. Yanfei Cai is listed as an inventor on Chinese patent # ZL201910391310.3 owned by South China Agricultural University.