Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers.

Bacillus subtilis strain Ydj3 was applied to sweet peppers to understand the influence of this bacterium on the growth, fruit quality, and rhizosphere microbial composition of sweet pepper. The promotion of seed germination was observed for sweet pepper seeds treated with the Ydj3 strain, indicating that Ydj3 promoted seed germination and daily germination speed (131.5 ± 10.8 seeds/day) compared with the control (73.8 ± 2.5 seeds/day). Strain Ydj3 displayed chemotaxis toward root exudates from sweet pepper and could colonize the roots, which enhanced root hair growth. Following the one-per-month application of strain Ydj3 to sweet pepper grown in a commercial greenhouse, the yield, fruit weight, and vitamin C content significantly increased compared with those of the control. Additionally, the composition of the rhizosphere bacterial community of sweet pepper changed considerably, with the Bacillus genus becoming the most dominant bacterial genus in the treated group. These results suggested that B. subtilis Ydj3 promotes seed germination and enhances fruit quality, particularly the vitamin C content, of sweet pepper. These effects may be partly attributed to the B. subtilis Ydj3 colonization of sweet pepper roots due to Ydj3 chemotaxis toward root exudates, resulting in the modulation of the rhizosphere bacterial community.

Introduction

Sweet pepper (Capsicum annuum L.) belongs to the Capsicum genus and the Solanaceae family and is among the most popular vegetables, has high nutrient contents, and is particularly rich in vitamins A and C. Antioxidant compounds, including water-soluble vitamin C and water-insoluble vitamin A and carotenoids, are abundant in pepper fruits and these vitamins play important roles due to their potent anti-inflammatory properties. Vitamin C has also been shown to act as a disease suppressant in humans. The production area of sweet pepper in Taiwan is approximately 2,498 hectares, which yields 29,221 tons of sweet pepper each year, and the price of sweet pepper fluctuates between 43 and 85 New Taiwan dollars per kg. According to the FAO, more than 70% of the world’s bell peppers are produced in Asia [1]. China is the largest producer of bell peppers, followed by Mexico and Indonesia [2]. The United States is the sixth largest and 363,647 hg/ha chilies and peppers were produced in 2020 [1, 2].

The qualities of pepper fruit are affected by the cultivar [3], agricultural practices [4, 5], fertilizer use [6], macronutrient and micronutrient application [7], disease control strategies, and postharvest conditions [8]. Changes in the soil bacterial community may enhance the suppression of Fusarium wilt disease in pepper [9]. Additionally, root colonization by Bacillus subtilis strain SL-44, which is attracted by the root exudates of pepper, has been reported to promote pepper plant growth [10].

The benefits of plant growth-promoting rhizobacteria (PGPR) on disease suppression and seed germination in pepper have been described previously [11, 12]. Cisternas-Jamet et al. [11] showed that the root inoculation of Bacillus amyloliquefaciens on green bell pepper affected the biochemical composition of fruits, including inducing changes in the calcium, iron, and vitamin C contents, especially when B. amyloliquefaciens was inoculated at the seedbed stage prior to transplantation. Furthermore, Mamphogoro et al. [13] revealed that fresh picked peppers are colonized by different bacterial communities on the fruit surface, and antagonistic bacterial communities that exist on the fruit surface can be found independent of the agronomic strategies employed during their growth. These bacterial communities may be important for peppers established in open fields, protecting them against abiotic and biotic stresses. However, no comprehensive assessment has examined the effects of PGPR treatment from seeds to fruits, and the diversity and dynamics of rhizosphere soil communities for fresh pepper plants grown under different treatment conditions have also not been examined. The objectives of this study were to evaluate the potential effects of the Bacillus subtilis strain Ydj3 on sweet pepper plants, from seeds to fruits, including effects on seed germination, seedling growth, fruit quality, and fruit yield, to reveal the interaction between the bacterial strain Ydj3 and sweet pepper root, and to understand the dynamics of the rhizosphere microbiome in the presence and absence of the Ydj3 strain.

Material and methods

Isolation, identification, and growth conditions of the bacterial strain

Bacillus subtilis strain Ydj3 was isolated from the rhizosphere of dragon Chinese juniper (Juniperus chinensis L. var. kaizuka Hort. ex Endl), in Yongjing Township, Changhua County, Taiwan (23°91’93N, 120°51’33 E), and was assessed for antagonistic activity against Colletotrichum gloeospoioides, Botrytis cinerea, and Phytophthora capsici by dual culture assay [14]. The species identity was characterized based on biochemical and physiological characteristics using the Biolog Gen III microbial identification system (Biolog Inc., CA, USA) and sequence analysis based on 16S rRNA [15] and gyrB sequences [16]. The 16S rRNA sequences of B. subtilis strain Ydj3 were deposited in the GenBank database under accession number MW911495; the gyrB sequences were deposited under accession number MW922029. B. subtilis strain Ydj3 was routinely maintained on Luria-Bertani (LB) agar plates (Difco, Detroit, MI, USA) and was cultured in LB broth (Difco) at 30°C and 150 rpm for 24 h unless otherwise stated. The field research and materials collected were under the agreement by the farm owner and granted permission by the Council of Agriculture, Taiwan.

Seed germination assay and seedling growth

Sweet pepper seeds (Yellow star, Known-You seed, No.SV-030, Kaohsiung, Taiwan) were surface-disinfected in 6% NaClO solution for 5 min and rinsed four times in sterile distilled water. The disinfected seeds were completely dipped in a B. subtilis Ydj3 culture suspension at a final concentration of 1×108 CFU/mL and then transferred into a 128-cell plug tray containing 10% perlite peat soil (Jiffy, Moerdijk, Netherlands) and incubated in a growth chamber at 25°C with a 12-h light/12-h dark cycle. The germination parameters and biomass were evaluated 6 weeks posttreatment. The germination percentage (GP), mean germination time (MGT), germination value (GV), and daily germination speed (DGV) were calculated based on the formulas described by Djavanshir and Pourbeik [17]. Each treatment was performed with three replicates, and each replicate contained 20 seeds.

For the seedling growth assay, sweet pepper seeds (yellow star, Known-You seed) were surface disinfected, as described above. Each germinated seed was transferred into a 3.5-inch-diameter pot containing 10% perlite peat soil (Jiffy) and incubated in a growth chamber at 25°C with a 12-h light/12-h dark cycle. After emergence, each seedling was irrigated with 2 mL of B. subtilis Ydj3 culture broth, once per week for 5 weeks, at a final concentration of 1×106 CFU/mL. Distilled water and uninoculated LB broth were used as controls. Twenty seedlings were used for each treatment, with three replicates. After 40 days of treatment, the plants were harvested, and the dry weights of the aboveground and underground components of the plants were assessed.

Field experiment and growth conditions

Sweet pepper (yellow star) seedlings were grown in a seedbed with 10% perlite peat soil (Jiffy) and then transplanted to a commercial greenhouse in Xinyi Township, Nantou County, Taiwan (23°37’13.2"N, 120°53’00.5"E). The experiment was conducted in a commercial greenhouse. Before transplanting the seedlings, peat soil was sanitized with hot water, and the treatment group was inoculated with B. subtilis Ydj3 culture broth at a final concentration of 1×107 CFU/mL. Tap water was used as a control. Pepper seedlings were transplanted into 10-m-long and 40-cm-wide bed tanks in double rows. Each row was approximately 30 cm apart, with 15 cm between each seedling in the row. Each treatment was performed with three replicates, and each replicate contained approximately 100 plants, which were drip-irrigated and fed liquid fertilizer (Nitrophoska®perfect, EuroChem, Mannheim, Germany) through the irrigation system. Fertilizer management was performed as recommended by The World Vegetable Center (AVRDC).

Fruit quality measurement and vitamin C content

B. subtilis Ydj3 culture broth was applied by root irrigation once per month to sweet pepper plants grown in a commercial greenhouse, and tap water was used as a control as described above. The horticultural traits, including total harvest yield, fruit size, fruit weights, fruit firmness, total seed weight per fruit, total soluble solids (°Brix), and vitamin C content, were recorded. The harvested fruit sizes were classified into large (>200 g/fruit), medium (100–200 g/fruit), and small (<100 g/fruit), based on the weight of each fruit. Total soluble solids were determined using a handheld refractometer (Brix 0–33%, Atago, Master-M, Tokyo, Japan). The vitamin C concentration in sweet pepper determined by measuring ascorbic acid using the ultraviolet (UV) spectroscopy method and 10 μg /mL ascorbic acid was used for standard curve preparation. Briefly, 20 mature pepper fruit samples were collected from plants in each treatment condition, 5 g from each sample was milled with 5 mL of 1% HCl solution, and the milled fruit juice was collected. A 2 mL sample of the milled juice was transferred into a 25-mL tube containing 8 mL of distilled water and shaken gently. Then, 0.2 mL of diluted sample was mixed with 0.4 mL of 10% HCl solution, distilled water was added to a total volume of 10 mL. The absorbance was measured at 243 nm using an ultraviolet–visible (UV–Vis) spectrophotometer (BioRad SmartSpec™ 3000, Bio‐Rad Laboratories, Hercules, California, U.S.A.). All determinations were performed in triplicate, and the results are expressed in μg vitamin C per g fresh fruit weight (μg Vit C/g). Vitamin C content was determined using the following equation: where μ is the vitamin C concentration, as interpolated from the standard curve; v is the total sample volume; v1 is the volume used for spectrophotometer determination; and w is the blended weight of the sample (g).

Microscopy observation and root colonization assay

The colonization assay was performed using a modified method described by Zhang et al. [18]. Sweet pepper seeds were surface-disinfected with 6% NaClO and seeded in 10% perlite peat soil (Jiffy) for 45 days. After removing the peat soil from the roots, the seedlings were transferred into 48 mL of sugar-free, half-strength Hoagland medium No. 2 (Sigma, Darmstadt, Germany). After 2 days of transplantation, 2 mL of B. subtilis Ydj3 culture broth at 1×107 CFU/mL was added to half-strength Hoagland medium No. 2. Distilled water and LB broth were used as controls. For each treatment, five plant roots were observed using optical microscopy and scanning electron microscopy. The plants were incubated in a growth chamber at 100 rpm and 25°C for 24 h. Before microscopic observation, the roots were collected and gently washed twice with sterile water. The sweet pepper roots from plants grown under each treatment condition were observed using an optical microscope (Olympus Fluorescence BX60, Tokyo, Japan) and a scanning electron microscope (SEM, JEO JSM-6330F SEM, Tokyo, Japan). For optical microscope observation, at least ten root hairs from different zones for each treatment were randomly chosen and their lengths were measured. For SEM observations, the roots from plants grown in each treatment were dehydrated as follows: the root samples were gently washed twice with distilled water and completely dehydrated through incubation in an ascending ethanol series: 50%, 70%, 80%, 90%, and 95% for 30 min each and three times in 100% for 30 min each time. The colonization of bacterial cells on the roots was observed using a JEO JSM-6330F SEM at 2.80 kV, and images were obtained. Each sample was observed at 2000X, 3000X and 4000X magnification and at least 3 images were taken for analysis.

For the root colonization assay, a 0.1-g root sample from each treatment was ground with a pestle mill, 0.9 mL of sterile distilled water was added. The suspension was serially diluted and plated on an LB agar plate and cultured at 30°C, 24 h to count the numbers of colonized cells. The Bacillus and other bacteria were differentiated by colony morphology on the LB agar plates, and Bacillus-like colonies were randomly picked for the identification of Bacillus species by 16S RNA sequencing as described above [15].

Collection of root exudates from sweet pepper and chemotaxis assay

Sweet pepper seedlings were prepared as described above. For each treatment, 50 sweet pepper seedlings were used to collect root exudates for use in the chemotaxis assay. After removing the peat soil, the roots were gently washed in sterile distilled water four times. Seedlings with four leaves were transplanted into 50-mL tubes containing 50 mL of sterile, half-strength, sucrose-free Hoagland medium No. 2 (Sigma) at 28°C for 5 days. After 5 days, the roots of the seedlings were washed with sterile distilled water to avoid nutrient contamination and transferred into 50-mL tubes containing 40 mL sterile distilled water in a growth chamber for 24 h (12-h light/12-h dark) at 100 rpm and 25°C. The collected solution (approximately 2000 mL) containing root exudates was filtered through a 0.45-μm membrane filter (Millipore, Darmstadt, Germany), concentrated by a vacuum concentrator (Büchi, vacuum controller B-721, Flawil, Switzerland) to a final volume of 50 mL, and stored at −80°C for further investigation.

A modified capillary assay based on the procedure described by Mazumder et al. [19] was performed for the quantitative measurement of B. subtilis strain Ydj3 chemotaxis in response to root exudates collected from sweet pepper seedlings. Strain Ydj3 was grown in LB broth until it reached the logarithmic growth phase, as determined by an optical density at 600 nm (OD600) of 0.8. The cells were collected by centrifugation, gently washed twice with chemotaxis buffer (20 μM EDTA, 100 mM potassium phosphate, pH 7.0), and resuspended in chemotaxis buffer to an OD600 of 0.8. A disposable 2-cm 25-gauge needle loaded with 100 μL the concentrated root exudate was immersed in a 200-μL pipette tip containing 100 μL of cell suspension. After 30 min of incubation at room temperature, the contents of the needle were transferred into a sterile Eppendorf tube by syringe. The suspension was diluted 103-, 104-, and 105-fold and plated on LB agar plates. Colony-forming units (CFUs) were counted after incubation for 24 h at 30°C. The experiment was repeated three times, and each treatment included three replicates.

Rhizosphere microbiome analysis

For the analysis of the soil microbial community composition for each treatment, untreated and B. subtilis Ydj3-treated rhizosphere soil samples were collected from sweet pepper roots at the beginning of planting and at the end of fruit harvest and stored at −20°C until further analysis. Bulk peat soil samples were collected after hot water sanitization and before transplanting sweet pepper as controls (Ctrl 0). Rhizosphere soil samples without B. subtilis Ydj3 treatment were used as a second control (Ctrl 1). Approximately 5 g of peat soil from each soil sample was subjected to DNA extraction using QIAamp DNA Microbiome Kit (QIAGEN, Germantown, MD, USA), and another 5 g of peat soil was used for the analysis of the total bacterial population by the serial dilution and plating method to count cell numbers.

Microbial identification was performed using amplicon sequencing of 16S rRNA. PCR amplification was performed to amplify the V3–V4 conserved regions of bacterial 16S rRNA gene sequences. The V3 and V4 regions were amplified using a forward primer (5’ CCTACGGRRBGCASCAGKVRVGAAT 3’) and a reverse primer (5’ GGACTACNVGGGTWTCTAATCC 3’). Next-generation sequencing library preparation and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc. (Suzhou, China). DNA samples were quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Amplicons were generated from 30–50 ng DNA using a MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, NJ, USA).

DNA libraries were validated by an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and quantified by a Qubit 2.0 Fluorometer. DNA libraries were multiplexed and loaded onto an Illumina MiSeq instrument, according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2×300 paired-end configuration; image analysis and base calling were conducted using the MiSeq Control Software embedded in the MiSeq instrument.

The QIIME data analysis package was used for 16S rRNA data analysis. Quality filtering of joined sequences was performed, and sequences that did not fulfill the following criteria were discarded: sequence length <200 bp, no ambiguous bases, and mean quality score >20. The retained sequences were compared with the reference database (RDP Gold database) using the UCHIME algorithm to detect chimeric sequences, which were removed. The effective sequences were used in the final analysis. Sequences were grouped into operational taxonomic units (OTUs) using the clustering program VSEARCH (1.9.6) against the Silva 119 database preclustered at 97% sequence identity. The Ribosomal Database Program (RDP) classifier was used to assign taxonomic categories to all OTUs at a confidence threshold of 0.8. The RDP classifier uses the Silva 132 database, which has taxonomic categories predicted to the species level.

Statistical analysis

All data were analyzed by analysis of variance (ANOVA). Each treatment was performed using three replicates, and the results are expressed as the mean values. Significant differences among treatment means were analyzed using the least significant difference (LSD) test at p < 0.05 with IBM SPSS Statistics software, Version 22.0. (Armonk, NY, IBM Corp).

Results

Influence of B. subtilis Ydj3 application on seed germination and seedling growth

To evaluate the effects of B. subtilis Ydj3 application on seed germination and seedling growth, pepper seeds were dipped in B. subtilis Ydj3 broth culture at a final concentration of 108 CFU/mL before transfer to peat soil. Seeds were treated with water and uninoculated LB broth as controls. Treatment with B. subtilis Ydj3 showed the highest GP (91.7 ± 7.2%) compared with the water (66.7 ± 6.8%) and LB broth (66.7 ± 9.0%) treatments (Table 1). The DGV following B. subtilis Ydj3 treatment (131.5 ± 10.8 seeds/day) also increased compared with water (73.8± 2.5 seeds/day) and LB broth (91.4 ± 8.6 seeds/day) treatments (Table 1). The application of B. subtilis Ydj3 broth culture also significantly promoted the daily germination speed (DGV) and shortened the MGT compared with those observed for the control treatments (Table 1). These results suggested that B. subtilis Ydj3 culture broth promoted the seed germination rate and percentage and reduced the germination time for sweet pepper.

Table 1

Effects of B. subtilis Ydj3 colonization on seed germination and seedling growth of sweet peppers.

Treatment	Growth parameters
	Germination percentage (GP) (%)	Mean germination time (MGT) (day)	Daily germination speed (DGV) (seeds/day)	Germination value (GV)	Root dry weight (g/plant)	Shoot dry weight (g/plant)
dH 2 O	66.7±6.8 a	23.3±2.9 ab	73.8±2.5 a	2.9±0.4 a	0.03±0.02 a	0.04±0.03 c
LB broth	66.7+9.0 a	29.3±5.4 b	91.4±8.6 b	2.3±0.5 a	0.02±0.02 a	0.05±0.04 b
Ydj3	91.7±7.2 b	20.5±0.1 a	131.5±10.8c	4.5±0.5 b	0.02±0.02 a	0.11±0.04 a

Means in the same column followed by the same letter are not significantly different at P > 0.05, according to one-way ANOVA and the least significant differences (LSD) test. Treatments: dH2O, distilled water; LB broth, Luria-Bertani broth; Ydj3, B. subtilis Ydj3 culture broths at a final concentration of 1 ×108 CFU/mL.

For measures of seedling growth by irrigation with water, LB broth and B. subtilis Ydj3 culture broth, treatment with B. subtilis Ydj3 showed the highest shoot dry weight per plant (0.11 ± 0.04 g/plant), followed by treatment with LB broth (0.05 ± 0.04 g/plant) and water (0.04 ± 0.03 g/plant; Table 1). The shoot dry weight following B. subtilis Ydj3 treatment was enhanced by 275% compared with that of the water treatment control. However, no significant difference was found for the root dry weight between pepper seedlings treated with B. subtilis Ydj3 (0.02 ± 0.02 g/plant), LB broth (0.02 ± 0.02 g/plant), or water (0.03 ± 0.02 g/plant; Table 1). Our results indicated that the application of B. subtilis Ydj3 once per week to sweet pepper could stimulate shoot seedling growth.

Influence of B. subtilis Ydj3 application on the physicochemical composition of sweet pepper fruits

The effects of B. subtilis Ydj3 application on fruit quality were assessed (Table 2). The results indicated that the total yield of plants treated with B. subtilis Ydj3 (53,903.3 ± 373.9 kg) was significantly higher than that of the control (51,607.7 ± 532.7 kg). The weight of large-sized fruit obtained from plants treated with B. subtilis Ydj3 (247.2 ± 5.1 g/fruit) was significantly (p < 0.05) heavier than that of large-sized fruit obtained from plants treated with water (241.1 ± 5.7 g/fruit); medium-sized fruit from B. subtilis Ydj3-treated plants (164.9 ± 1.6 g/fruit) was also significantly heavier than those from the water control group (157.2 ± 3.2 g/fruit). Although larger quantities of large fruits were collected from plants treated with B. subtilis Ydj3 (89.3 ± 5.9 mean number of large fruits) compared with those collected from control plants (74.0 ± 18.2 mean number of large fruits), the difference was not significant. The differences in numbers of medium- and small-sized fruits, weights of small-sized fruits, total average fruit weight, total seed weight, fruit firmness, and total soluble solids between B. subtilis Ydj3-treated plants and control plants were also not significant. However, sweet peppers collected from plants treated with B. subtilis Ydj3 contained significantly higher vitamin C contents (790.9 ± 17.9 μg/g) than those obtained from untreated control plants (730.8 ± 14.4 μg/g).

Table 2

Effects of B. subtilis Ydj3 colonization on the fruit qualities of sweet peppers.

Physicochemical composition of fruit	Treatment
	Ctrl	Ydj3
Total yield (kg)	51607.7 ± 532.7	53903.3 ± 373.9 *
Mean number of large fruits	74.0 ± 18.2	89.3 ± 5.9
Weight of large fruits (g/fruit)	241.1 ± 5.7	247.2 ± 5.1 *
Mean number of medium fruits	198.7 ± 34.3	180.3 ± 8.5
Weight of medium fruits (g/fruit)	157.2 ± 3.2	164.9 ± 1.6 *
Mean number of small fruits	36.7 ± 9.7	28.7 ± 6.2
Weight of small fruits (g/fruit)	74.1 ± 6.0	80.8 ± 12.0
Mean weight of fruits (g/fruit)	157.5 ± 5.0	164.3 ± 6.2
Total seed weight (g/fruit)	2.4 ± 0.1	2.5 ± 0.1
Firmness [20]	6.4 ± 0.1	6.4 ± 0.1
TSS (°Brix)	5.6 ± 0.1	5.6 ± 0.1
Vitamin C (μg/g)	730.8 ± 14.4	790.9 ± 17.9 *

Means in the same row followed by an asterisk (*) are significantly different at P < 0.05, according to one-way ANOVA and the least significant differences (LSD) test. TSS, total soluble solids.

Influence of B. subtilis Ydj3 application on root hair formation and root colonization

To investigate the effects of B. subtilis Ydj3 application on root hair growth and the root architecture of sweet peppers, the roots were examined by optical microscopy, and pictures were recorded, as shown in Fig 1. Roots treated with B. subtilis Ydj3 presented denser root hairs at apical meristem, elongation zone, and maturation zone than those in the control (Fig 1). Furthermore, the root hairs found in the apical meristem zone and the elongation zone (Fig 1D and 1E, respectively) were longer in the B. subtilis Ydj3-treated group than in the control group (Fig 1A and 1B, respectively). The lengths of root hairs in each treatment were measured using the software (cellSens Standard 1.9) installed in the Olympus Fluorescence BX60 microscope, and results showed that the length of root hair of B. subtilis Ydj3 treatment had was longer than in the Ctrl treatment, with an average length in the apical meristem of 279.4 ± 177.3 μm compared with the Ctrl treatment, with an average length of 61.8 ± 8.8 μm; an average length of 375.9 ± 143.0 μm in the elongation zone, significantly longer than the Ctrl treatment, with an average length of 120.6 ± 63.4 μm; and an average length of 491.4 ± 21.9 μm in the maturation zone, significantly longer than the Ctrl treatment, with an average length of 172.4 ± 17.6 μm. These results suggested that B. subtilis Ydj3 applications enhanced root hair growth and changed the root architecture of sweet pepper.

Fig 1

Root architecture examined by optical microscopy.

(A, B, C) Control; (D, E, F) treatment with B. subtilis Ydj3. (A, D) Apical meristem; (B, E) elongation zone; (C, F) maturation zone of sweet pepper roots. Bar, 100 μm; arrow, root hair.

To reveal the interaction between B. subtilis Ydj3 and the roots of sweet pepper, the colonization capability of strain Ydj3 was assessed by milling 0.1-g root samples, and plating the obtained solution on LB agar plates. The Bacillus and other bacteria were differentiated by colony morphology on the LB agar plate, and the identification of Bacillus species were confirmed by 16S RNA sequencing. The total bacterial population density in roots obtained from plants treated with strain Ydj3 was significantly higher (6.84 ± 0.04 log10 CFU/g) than those detected on the roots of control plants treated with distilled water (5.85 ± 0.22 log10 CFU/g) or LB broth (6.66 ± 0.10 log10 CFU/g). While the bacteria obtained from plants treated with strain Ydj3 were all Bacillus species (data not shown).

Root colonization by B. subtilis Ydj3 was observed by SEM, and the control group revealed no bacterial cells on the root surface (Fig 2A). In contrast, B. subtilis Ydj3 cells were found to colonize the root surface and root hair (Fig 2B and 2C).

Fig 2

Scanning electron microscopy photographs of sweet pepper roots.

(A) Treatment with distilled water as a control (Ctrl); (B, C) treatment with B. subtilis Ydj3. Bar, 10 μm. (A) plant cells were observed; (B) the apex of a root. Arrow head, root hairs; retangular area was enlarged in C; (C) Enlarged image of the retangukar area in B. Arrow head, bacteria.

Chemotaxis to root exudate by B. subtilis Ydj3

The chemotactic effects of root exudates on B. subtilis Ydj3 were assessed using a modified capillary assay. The results showed that strain Ydj3 was chemoattracted by 40-fold concentrated root exudates, resulting in a population density of 4.53 ± 0.08 log10 CFU/mL compared with the distilled water control, which was characterized by a population density of 4.21 ± 0.03 log10 CFU/mL.

Bacterial community composition in soil with and without B. subtilis Ydj3 application to sweet pepper

The analysis of the bacterial communities in the rhizosphere soil of sweet pepper plants treated with and without B. subtilis Ydj3 resulted in 171,368 bacterial reads, consisting of 546 OTUs (97% cutoff). The Q20 values (%) of Ctrl 0, Ctrl 1 and Ydj3 were 91.36%, 90.24% and 91.31%, respectively, and the Q30 values (%) of Ctrl 0, Ctrl 1 and Ydj3 were 87.10%, 85.4% and 86.90%, respectively. A total of 17 distinct bacterial phyla were detected among the samples, including 14 phyla, 130 genera, and 55 species in the peat soil before transplanting (Ctrl 0); 17 phyla, 153 genera, and 73 species in the rhizosphere soil (Ctrl 1); and 16 phyla, 158 genera, and 77 species in the sample with B. subtilis Ydj3 treatment (Ydj3). The most abundant phylum in all treatments was Actinobacteria, which constituted 35.6%, 15.3%, and 14.7% of the bacterial population detected in the Ctrl 0, Ctrl 1, and Ydj3 samples, respectively (Fig 3A). In the Ctrl 0 sample, the other abundant phyla were Proteobacteria (27.9%), Bacteroidetes (16.6%), and Firmicutes (7.1%). In the rhizosphere soil (Ctrl 1), the abundant phyla were Proteobacteria (42.7%), Actinobacteria (15.3%), and Firmicutes (12.0%); in the sample treated with B. subtilis Ydj3 (Ydj3), the abundant phyla were Proteobacteria (35.0%), Firmicutes (22.5%), and Actinobacteria (14.7%; Fig 3A). Except for unclassified genera, Bacillus was the most abundant (16.9%) of the top 17 genera identified in the Ydj3 treatment group and represented 7.0% and 16.9% of the total bacterial populations of Ctrl 0 and Ctrl 1, respectively (Fig 3B). The enriched bacterial genera identified in the pepper rhizosphere between Ctrl 1 and Ydj3 included Nocardioides (1.3% to 1.7%), Bacillus (7.0% to 16.9%), and Gemmatimonas (5.82% to 5.87%).

Fig 3

Composition of the bacterial community in rhizosphere soils with and without B. subtilis Ydj3 treatment.

(A) Relative abundances of most abundant phyla; Ctrl 0, bulk peat soil treated with hot water before sweet pepper transplant; Ctrl 1, rhizosphere soil without B. subtilis Ydj3 treatment, 6 months after sweet pepper transplant; Ydj3, rhizosphere soil with B. subtilis Ydj3 treatment, 6 months after sweet pepper transplant. (B) Relative abundances of the top 17 genera.

Discussion

Microbial seed coating is an efficient technology for the safe and effective delivery of beneficial microbes to plant roots or the rhizosphere. Li and Hu [21] found that exogenous treatments with B. subtilis QM3 applied to wheat seeds increased β-amylase activities and promoted the germination and seedling survival rates [21]. Another study by Tu et al. [22] indicated that the use of B. subtilis SL-13 at 106 CFU/seeds as a microbial seed coating agent could promote seed germination and biomass in cotton [22]. Our results showed that seeds dipped in a B. subtilis Ydj3 cell suspension at 108 CFU/mL promoted the germination of sweet pepper seeds compared with the untreated control. Additionally, the application of B. subtilis Ydj3 broth culture at 108 CFU/mL resulted in a 25% increase in seed germination and a 2.5-fold increase in shoot dry weight compared with the average value of control treatments. Our results suggested that B. subtilis Ydj3 displayed chemotaxis in response to root exudates produced by sweet pepper, was capable of colonizing sweet pepper roots, and induced root hair growth, which may be beneficial for the improved nutrient absorption of sweet pepper plants, resulting in the promotion of plant growth. Whether β-amylase production by B. subtilis Ydj3 is involved in the promotion of seed germination remains to be determined. Additionally, according to Vacheron et al. [23] and Grover et al. [24], understanding and quantifying the impact of PGPR on roots and the whole plant remain challenging. Several studies have indicated that PGPR modifies the root structure, including primary root growth, lateral root density, and root dry weight [24]. However, the root dry weight was not enhanced in our results. This might be because the plants were young and the variation remained nonsignificant.

The rhizosphere plays an important role in plant–microbe interactions. López-Bucio et al. [25] revealed that Bacillus megaterium inoculation inhibited primary root growth followed by increases in the lateral root number, lateral root growth, and root hair length in Arabidopsis thaliana [25]. Their results suggested that plant growth promotion and root architectural alterations induced by B. megaterium may involve auxin- and ethylene-independent mechanisms [25]. Additionally, Vacheron et al. [23] indicated that many PGPR affect root system architecture via modulation of host gormonal balance, including the plant auxin pathway. Bacillus amyloliquefaciens T-5 exhibited a chemotactic response to the malic acids found in root exudates produced by tomato plants, which induced biofilm formation and the colonization of tomato roots by B. amyloliquefaciens T-5 [26]. Similarly, our results indicated that B. subtilis Ydj3 could be chemotactically attracted by root exudates produced by sweet pepper, colonize the root surface, and alter the root architecture by increasing root hair growth. Our unpublished data showed that B. subtilis Ydj3 produces the plant growth regulator indole-3-acetic acid (17 mg/L), which may also contribute to alterations in root architecture and induce plant growth.

Various rhizobacteria are known to promote plant growth, improve plant yields, and alter fruit quality. The inoculation of tomato roots with B. subtilis BEB-l3bs was demonstrated to enhance the marketable yield, fruit weight and length, and the texture of red fruits [27]. Rahman et al. [28] reported improvements in plant growth, yield, various antioxidant contents (carotenoids, flavonoids, phenolics, and total anthocyanins), and total antioxidant activities in strawberry fruits following the application of both B. amyloliquefaciens BChi1 and Paraburkholderia fungorum BRRh-4 compared with nontreated controls [28]. The application of a strain from the genus Phyllobacterium has been reported to significantly increase the vitamin C contents of strawberry [29]. The application of microbial fertilizer products increased the flavonoid and lignin contents in Arabidopsis leaves through the induction of phenylpropanoid pathway genes [30]. In our study, the inoculation of sweet pepper plants with B. subtilis Ydj3 significantly increased the shoot dry weight of seedlings, the total yield, the weights of large- and medium-sized sweet pepper fruits, and the vitamin C contents of the fruits. Whether the observed increase in the vitamin C contents of sweet pepper induced by B. subtilis Ydj3 treatment is associated with any changes in the phenylpropanoid pathway remains to be investigated.

Plant species have been hypothesized to accommodate specific compositions of rhizosphere bacterial communities to optimize growth and prevent infection by pathogens. The use of biofertilizer may shift the composition of the plant microbiome. Sun et al. [31] reported that the application of B. subtilis biofertilizer reduced the abundance of ureC genes and increased the abundance of functional genes (bacterial amoA and comammox amoA) and ammonia-oxidizing bacteria, suggesting that the conversion of fertilizer nitrogen to NH4+-N decreased, and the nitrification process increased. Thus, B. subtilis biofertilizer is considered an effective control strategy for agricultural NH3 emissions and maintaining high crop yields. Additionally, their results indicated that Actinobacteria was the dominant phylum in the soil before fertilization, whereas Proteobacteria became the new dominant phylum during the stable period [31]. Potential PGPR bacteria (Bacillus), nitrobacteria (Nitrospira), and photosynthetic bacteria (Rhodoplanes) were found to be enriched in B. subtilis biofertilizer compared with fertilizer without B. subtilis [31]. Qiao et al. [32] reported a similar finding for a single time point, whereas Sun et al. [31] examined a quantification curve after applying B. subtilis biofertilizer throughout the pak choi growth period. Their findings also indicated that the application of B. subtilis PTS-394 did not result in a permanent effect on the rhizosphere microbial community, suggesting the compatibility of B. subtilis PTS-394 with the environment [32]. Similar to the data presented by Sun et al. [31], the dominant phylum identified in bulk peat soil (Ctrl 0) in our study was Actinobacteria. After a 6-month growth period for sweet pepper, the most abundant phyla shifted to Proteobacteria in the untreated control group and to Firmicutes in the B. subtilis Ydj3-treated group. Additionally, the Bacillus species abundance was enriched in the B. subtilis Ydj3 treatment group compared with the control group.

In conclusion, the application of B. subtilis Ydj3 promoted seed germination, seedling vigor, and fruit production in sweet pepper and enhanced the antioxidant vitamin C contents of sweet pepper fruit. The efficacy of bacterial treatment for plant growth promotion and the enhancement of fruit quality may be partly attributed to the chemotactic response of B. subtilis Ydj3 to sweet pepper root exudates, and the colonization of sweet pepper roots alters the root architecture. Additionally, the application of B. subtilis Ydj3 shifted the bacterial community compositions of the sweet pepper rhizosphere. Our findings suggested that B. subtilis Ydj3 could be used as a bioagent for the sustainable production of high-quality sweet pepper.

19 Nov 2021

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**********

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Reviewer #1: It is a relatively useful article on the study of probiotics "Bacillus subtilis strain Ydj3", and has a positive effect on the sweet pepper industry. The paper needs to be reconsidered after modification. Please see the comments showed as follows:

Line 89: add citation ” assessed for antagonistic activity against Colletotrichum gloeospoioides, Botrytis cinerea, and Phytophthora capsici by dual culture assay.”

Lines 220-223: Supplementary information on where the soil sample was collected, was it collected from the surface?

Fig. 1: Redraw the figure to facilitate understanding of groups and parts. Besides, the length of root hairs needs to be quantified.

Lines 342-348: Combined with the last part of the results, there may be a large number of other bacteria in the root system, so LB is not suitable for counting bacillus.

Lines 349-351: Due to the small sampling area of SCANNING electron microscopy, whether this result is repeated for many times. After all, a large number of bacteria were also detected in the last results (line 347: 5.85 ± 0.22 log10 CFU/g)

Fig. 2: Redraw to help understand groups. In addition, does the bacteria on the root surface caused pathological change?

Lines 364-385:

1. Please add more quality control information about 16s-hiseq.

2. The form of FIG. 3 can only show the average gap between groups, but cannot show the inter-sample gap (standard deviation) within groups. Please add relevant information.

3. Whether similar results of ” antagonistic activity against Colletotrichum gloeospoioides, Botrytis cinerea, and Phytophthora capsici by dual culture assay (in the section materials and methods)” can be obtained through 16s-hiseq.

Reviewer #2: The manuscript describes a comprehensive study on the effects Bacillus subtilis strain Ydj3 on seed germination, growth, fruit quality and rhizosphere microbial composition of sweet pepper. The authors obtained evidences for the promotion of seed germination, shoot biomass, fruit yield, weight and vitamin C content upon inoculation. These effects were partly attributed to chemotaxis of Ydj3 towards root exudates, resulting in the modulation of the bacterial community in rhizosphere. The work is well structured and is presented clearly. Techniques and methodologies are suitable and well described. The results are well presented and analyzed. It is my opinion that this is an important study for this area of research and for regular readers of this journal. I have just a few minor comments and suggestions for corrections:

Line 25 – I don’t think the term “priming” is adequate here. Please rephrase.

Line 108 – Please include the abbreviation of germination value (GV).

Line 118 and 120 – Seedlings are young plants, and so after 40 days of treatment the correct designation should be “plants”.

Line 139 – As described above, correct?

Line 165 – Zhang et al. (2014) is reference [27].

Lines 225-226 – Is this a repetition of the above? (lines 221-222)?

Line 278 – Did you mean germination value (instead of daily germination speed)?

Line 283-284 (Table 1) – Please indicate units for mean germination time, germination value and daily germination speed. Correct the abbreviation of daily germination speed (DGV, not DGP). Are columns “Germination value” and “Daily germination speed” swapped?

Line 304-305 – These are the total fruit yield values, not by group, right? If yes, remove /group.

Line 320 (Table 2) – Total yield: Same as in the previous comment. Large fruits, medium fruits, small fruits: did you mean number of fruits/group (average of three replicates)? Lee et al. is reference [12].

Line 331 – showed that the distribution

Line 338 (Fig 1) – Are the captions of (A,D) and (C,F) swapped?

Line 368 – “A total of 16 distinct bacterial phyla”: Is this correct number of distinct phyla?

Line 404 – 25% increase in seed germination: how was this value obtained and what parameter does it refer to?

Line 405 -… compared with the average value of control treatments

Line 388 (Fig 3 caption) – (A) Relative abundances of most abundant phyla?

Reviewer #3: The manuscript titled “Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers” studies the influence of the Bacillus subtilis strain Ydj3 on the growth, fruit quality, and rhizosphere microbial composition of sweet peppers. Work presents an issue of economic importance due to the use of a bacteria as bioagent which produces high-quality of sweet pepper and deserves to be investigated. The manuscript is short, precise and well written. Authors presents results of original research and the rest of the requirements of the journal are fulfilled.

Below, I list some suggestions for authors:

Introduction

Page 5, line 55: Perhaps the authors can add more up-to-date citations

Material and Methods

-Microscopy observation and root colonization, Page 11 and 12

Please add number of replicates, number of plants per replicate, number of analyzed images.

Results

Page 21, line 338: In the description of figure 1, the letters are wrong, (A, D) is the meristematic zone and (C, F) is the zone of differentiation.

Commonly, the hairy area is called the differentiation zone, perhaps a more in-depth study of the state of differentiation of cells in these areas that present root hairs with treatment with the bacteria should be carried out. Perhaps the meristematic and elongation zones are more apical.

Page 22, line 353: A more detailed description of figure 2 would be desirable, indicating that it is observed in the images.

In image A, cells are observed, in B, the apex of a root (it would be desirable to indicate the bacteria) and in C, the root hairs (it would be desirable to indicate in the image which are the root hairs and which it's bacteria).

Discussion

If possible, explain in more detail the non-variation of the dry weight of the root between treatments (table 1), although the architecture of the root varies, increasing the root hairs in those roots that are treated with the bacteria.

**********

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Reviewer #2: No

Reviewer #3: No

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Submitted filename: renamed_c18b1.docx

Click here for additional data file.

3 Jan 2022

PONE-D-21-26845.

"Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers"

04-January-2022

Dear Dr. Ma and reviewers,

Thank you and the reviewer for the valuable comments to improve the manuscript!

We have addressed you and the reviewer’s comments in black text below, with our responses in blue. Line numbers noted below correspond to the revised manuscript file. We also include a revised manuscript text with track changes in the resubmission. Additionally, the manuscript was edited for proper English language, spelling and grammar by native English speaking editors at American Journal Experts (AJE), and the certificate was also included.

Response to Editor:

“**3. In your Methods section, please provide additional location information, including geographic coordinates of your field collection (for dragon Chinese juniper rhizosphere) if available.**”

We greatly appreciate you and the reviewer to point out what we have missed from the previous review, we had added the geographic information (23°91'93N, 120°51'33 E) of field collection in the description (line 83).

“**4. In your Methods section, please provide additional details regarding the dragon Chinese juniper used in your study, including ensuring you have described the source.**”

We didn’t use the dragon Chinese juniper in our study, we collected the soil from the rhizosphere of the dragon Chinese juniper.

“**5. Thank you for stating the following financial disclosure:

The research was financially supported by the Council of Agriculture, Taiwan [106AS-12.4.1-PI-P1, 110AS-5.4.2-PI-P2], the Ministry of Science and Technology, Taiwan [MOST 110-2321-B-005-006; 109-2321-B-005-022; 109-2313-B-005-032], and the “Innovation and Development Center of Sustainable Agriculture, NCHU” from the Featured Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education in Taiwan. Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Thank you for the instruction of the statement about the financial support. We have put the statement "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." as suggested in the cover letter.

“6. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. “

The statement “The field research and materials collected were under the agreement by the farm owner and granted permission by the Council of Agriculture, Taiwan.” was included in the “Method” section. (Lines 92-93)

Response to Reviewer #1:

“Line 89: add citation ” assessed for antagonistic activity against Colletotrichum gloeospoioides, Botrytis cinerea, and Phytophthora capsici by dual culture assay.”

The reference was cited in the main text (line 85) and included in the reference list [2].

“Lines 220-223: Supplementary information on where the soil sample was collected, was it collected from the surface?”

We collected the soil samples from the roots of sweet pepper. Some of the fibrous root will be cut and removed, and then the soil samples were collected.

The sentence was revised as “For the analysis of the soil microbial community composition for each treatment, untreated and B. subtilis Ydj3-treated rhizosphere soil samples were collected from the sweet pepper roots at the beginning of planting and at the end of fruit harvest and stored at −20 °C until further analysis.” (Lines 213-216)

“Fig. 1: Redraw the figure to facilitate understanding of groups and parts. Besides, the length of root hairs needs to be quantified.”

The figure was labeled as suggested, and the length of the root hairs was quantified and the descriptions were stated in the Material and Methods and Result sections as follows.

In the Material and Methods section, the sentences “For optical microscope observation, at least ten root hairs from different zones for each treatment were randomly chosen and their lengths were measured.” were added (lines 168 to 170).

In the Results section, the sentences “The lengths of root hairs in each treatment was measured using the software (cellSens Standard 1.9) installed in the Olympus Fluorescence BX60 microscope, and results showed that the length of root hair of B. subtilis Ydj3 treatment had was longer than in the Ctrl treatment, with an average length in the apical meristem of 279.4 ± 177.3 µm compared with the Ctrl treatment , with an average length of 61.8 ± 8.8 µm; an average length of 375.9 ± 143.0 µm in the elongation zone, significantly longer than the Ctrl treatment, with an average length of 120.6 ± 63.4 µm; and an average length of 491.4 ± 21.9 µm in the maturation zone, significantly longer than the Ctrl treatment, with an average length of 172.4 ± 17.6 µm.” were added. (lines 318 - 326)

“Lines 342-348: Combined with the last part of the results, there may be a large number of other bacteria in the root system, so LB is not suitable for counting bacillus.”

We agree with the reviewer that there are a large number of bacteria in the root system. The Bacillus and other bacteria were differentiated by colony morphology on the LB agar plates as shown in the following figure and the Bacillus-like colonies were randomly picked for the identification by 16S RNA sequencing. We found that the colonies obtained from the treatment with B. subtilis Ydj3 were all Bacillus species. The material and method and the statement in the result section was revised in the text (lines 180-182 and 336-342, respectively).

(A) (B)

Fig. The colonies obtained from root samples of (A) the control; and (B) the B. subtilis Ydj3 treatment.

“Lines 349-351: Due to the small sampling area of SCANNING electron microscopy, whether this result is repeated for many times. After all, a large number of bacteria were also detected in the last results (line 347: 5.85 ± 0.22 log10 CFU/g)”

As stated in the above reply, the Bacillus and other bacteria were differentiated by colony morphology on the LB agar plates, and the Bacillus-like colonies were randomly picked for the identification of Bacillus species by 16S RNA sequencing.

“Fig. 2: Redraw to help understand groups. In addition, does the bacteria on the root surface caused pathological change?”

We have labeled respective treatment in the figure as suggested. We did not observe pathological effect on the root caused by B. subtilis Ydj3, a plant growth rhizobacterium (PGPR). The change in root architecture- enhancing root hair formation may be partly due to the production of indole-3-acetic acid (17 mg/L) by B. subtilis Ydj3 (our unpublished data and discussed in lines 423-425). Similarly, Richardson et al. (2009) indicated that PGPR could change the root architecture via production of plant hormone.

“Lines 364-385:

1. Please add more quality control information about 16s-hiseq.”

The cutoff value of the bacterial communities in the rhizosphere soil of sweet pepper plants is 97%, and the Effective Tags Q20 (%) and Q30 (%) values of each treatment showed below were all above 85%.

treatment Q20 (%) Q30 (%)

Ctrl 0 91.36 87.10

Ctrl 1 90.24 85.40

Ydj3 91.31 86.90

Additionally, the description of the quality control was also stated in the main text as follows: “The Q20 values (%) of Ctrl 0, Ctrl 1 and Ydj3 were 91.36%, 90.24% and 91.31%, respectively, and the Q30 values (%) of Ctrl 0, Ctrl 1 and Ydj3 were 87.10%, 85.4% and 86.90%, respectively.” (Lines 362 - 365).

“2. The form of FIG. 3 can only show the average gap between groups, but cannot show the inter-sample gap (standard deviation) within groups. Please add relevant information.”

The main focus of the study was to compare the compositions of bacterial community between the control and the B. subtilis Ydj3 treatment, thus the inter-sample gap (standard deviation) within groups were not provided.

“3. Whether similar results of ” antagonistic activity against Colletotrichum gloeospoioides, Botrytis cinerea, and Phytophthora capsici by dual culture assay (in the section materials and methods)” can be obtained through 16s-hiseq.”

Thanks for the suggestion. The effect on the compositions of fungal community by the B. subtilis Ydj3 treatment will be investigated in the future.

Response to Reviewer #2:

“Line 25 – I don’t think the term “priming” is adequate here. Please rephrase.”

The term “ priming” was deleted as suggested. The statement was revised as “The promotion of seed germination was observed for sweet pepper seeds treated with the Ydj3 strain, indicating that Ydj3 promoted seed germination and daily germination speed (131.5 ± 10.8 seeds/day) compared with the control (73.8 ± 2.5 seeds/day).” (Lines 22 - 24)

“Line 108 – Please include the abbreviation of germination value (GV).”

The abbreviation of germination value (GV) was added. (Line 103)

“Line 118 and 120 – Seedlings are young plants, and so after 40 days of treatment the correct designation should be “plants”.

Line 139 – As described above, correct? ”

They were revised as suggested. (Lines 112 and 113)

“Line 165 – Zhang et al. (2014) is reference [27].”

The reference was cited in the main text (Line 156) and the reference list no [32] was revised accordingly.

“Lines 225-226 – Is this a repetition of the above? (lines 221 - 222)?”

The repeated sentence was deleted.

“Line 278 – Did you mean germination value (instead of daily germination speed)?”

Our results indicated that the B. subtilis Ydj3 treatment exhibited higher daily germination speed (DGV) than the control, so that the mean germination value (MGT) was shortened. (Lines 260-266)

“Line 283-284 (Table 1) – Please indicate units for mean germination time, germination value and daily germination speed. Correct the abbreviation of daily germination speed (DGV, not DGP). Are columns “Germination value” and “Daily germination speed” swapped?”

It was revised as suggested and the unit of each term was added in Table 1. (Table 1)

The unit of mean germination time (MGT) is day, and there is no unit of germination value (GV), GV is an index and calculated by the formula cited in reference no [6]. The unit of daily germination speed (DGV) is seeds/day, and the abbreviation was also revised.

“Line 304-305 – These are the total fruit yield values, not by group, right? If yes, remove /group.”

It was revised as suggested and the term “/group” was deleted. (Lines 297 - 298)

“Line 320 (Table 2) – Total yield: Same as in the previous comment. Large fruits, medium fruits, small fruits: did you mean number of fruits/group (average of three replicates)? Lee et al. is reference [12].”

It was revised as suggested. In Table 2, the unit of total yield was kg, the unit of large fruits, medium fruit and small fruits was revised as “numbers of fruits”. The reference was added in Table 2 and the reference list no [16] (Line 305).

“Line 331 – showed that the distribution”

The description was deleted. (Line 313)

“Line 338 (Fig 1) – Are the captions of (A,D) and (C,F) swapped?”

The figure and figure legend were revised as suggested. (A, D) are apical meristem, (C, F) are maturation zone. (Fig 1.) (Lines 330-332)

“Line 368 – “A total of 16 distinct bacterial phyla”: Is this correct number of distinct phyla?”

It was revised as “ A total of 17 distinct bacterial phyla.” (Line 365)

“Line 404 – 25% increase in seed germination: how was this value obtained and what parameter does it refer to?”

The values was obtained as follows: GP (%) of Yjd3 – GP (%) of Ctrl =91.7% - 66.7% = 25%. (Line 398)

“Line 405 -… compared with the average value of control treatments”

The sentence was revised as suggested. (Lines 399-400)

“Line 388 (Fig 3 caption) – (A) Relative abundances of most abundant phyla?”

The Fig 3 caption was revised as suggested. (Line 382)

Response to Reviewer #3:

“Page 5, line 55: Perhaps the authors can add more up-to-date citations”

The reference was updated to the statistical data in 2020 in the main text (Line 52) and the reference list no [8] (Lines 498-499).

“Material and Methods

Microscopy observation and root colonization, Page 11 and 12

Please add number of replicates, number of plants per replicate, number of analyzed images.”

For the assay, each treatment contained 5 plants and at least 3 images were taken at different magnification for each sample.

The statement was revised as the follows accordingly.

“For each treatment, five plant roots were observed using optical microscopy and scanning electron microscopy. “ (Lines 162-163)

“For optical microscope observation, at least ten root hairs from different zones for each treatment were randomly chosen and their lengths were measured.” (Lines 168 – 170)

“Each sample was observed at 2000X, 3000X and 4000X magnification and at least 3 images were taken for analysis.” (Lines 175 - 176)

“Results

Page 21, line 338: In the description of figure 1, the letters are wrong, (A, D) is the meristematic zone and (C, F) is the zone of differentiation.

Commonly, the hairy area is called the differentiation zone, perhaps a more in-depth study of the state of differentiation of cells in these areas that present root hairs with treatment with the bacteria should be carried out. Perhaps the meristematic and elongation zones are more apical.”

The legend of Fig 1 was corrected as suggested. (A,D), apical meristem; (B, E) elongation zone; (C,F) maturation zone. (Lines 330-332)

“Page 22, line 353: A more detailed description of figure 2 would be desirable, indicating that it is observed in the images.

In image A, cells are observed, in B, the apex of a root (it would be desirable to indicate the bacteria) and in C, the root hairs (it would be desirable to indicate in the image which are the root hairs and which it's bacteria).”

It was revised as suggested (Lines 347-350). The revised image was attached with the revised file.

“Discussion

If possible, explain in more detail the non-variation of the dry weight of the root between treatments (table 1), although the architecture of the root varies, increasing the root hairs in those roots that are treated with the bacteria.”

The discussion was added as stated “understanding and quantifying the impact of PGPR on roots and the whole plant remain challenging. Several studies indicated that PGPR modified the root structure including primary root growth, lateral root density, root dry weight, however, the root dry weight was not enhanced in our results. This might be because the plants were young and the variation remained non-significant.” (Lines 406 - 410)

Thank you again for giving us the opportunity to address the question!

Sincerely,

Tzu-Pi

***********************************************************

Tzu-Pi Huang, Ph. D.

Professor, Department of Plant Pathology

Director, Pesticide Residue Analysis Center

National Chung Hsing University

145, Xing-Da Road, Taichung 40227, Taiwan

TEL: 886-4-22840780 ext. 379; 886-4-22859750

FAX: 886-4-22859750

E-mail: tphuang@nchu.edu.tw

***********************************************************

Submitted filename: PONE-D-21-26845 R1 Response to Reviewers.docx

Click here for additional data file.

1 Feb 2022

7 Feb 2022

PONE-D-21-26845.

"Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers"

07-Febuary-2022

Dear Dr. Ma and reviewers,

Thank you and the reviewer for the valuable comments to improve the manuscript!

We have addressed the reviewer’s comments in black text below, with our responses in blue. Line numbers noted below correspond to the revised manuscript file. Additionally, a revised manuscript text with track changes and an unmarked version were included in the resubmission.

“Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.”

We also have reviewed and checked again the reference list and confirmed all the references cited are correct and have not been retracted. The reference cited in the statement “…fertilizer use [11], macronutrient and…” was changed to “…fertilizer use [10], macronutrient and…” (line 54).

Response to Reviewer #2:

“**1. Section "Fruit quality and vitamin C content" (Lines 130-132): Field experiment and growth conditions were described in the previous section. Therefore, "as described above" should be added at the end of the first sentence of this section, or it seems to refer to another experiment.**”

We greatly appreciate you and the reviewer to point out what we have missed from the previous review, we had added the description “as described above” at the end of the sentence (line 132-133).

“**2. Table 1. The position of the columns DGV and GV has been changed, but the contents are as in the previous version and are not in agreement with the text (lines 263-264). Please correct.**”

We had revised the data in the text respectively to Table 1 (line 263-264).

“**3. Table 2. The text in the first column should be unified. I suggest using "Number of large fruits" or "Mean number of large fruits" instead of "Large fruits (mean numbers of fruits)", and "Weight of large fruits" instead of "Large fruit weight". The same applies to medium fruits and small fruits. This will make Table 2 easier to read. **”

It was revised as suggested. (Lines 298-299 and line 307 Table 2)

“**4. Lines 314-316 – Suggestion: “Roots treated with B. subtilis Ydj3 presented denser root hairs at apical meristem, elongation zone, and maturation zone than those in the control (Fig 1). **”

It was revised as suggested (lines 316-317).

Response to Reviewer #3:

“**In any case, I do not agree with the concept that there are root hairs in all root zones (apical, elongation and differentiation/maturation), according to what is incorporated in the new version of the work (lines 318-326).

Root hairs are found in the zone of differentiation/maturation only. Perhaps the authors will find bibliography that supports their findings. **”

Thank you again for giving us the opportunity to address the question. Root hairs are cylindrical extensions of root epidermal cells that are important for acquisition of nutrients, microbe interactions, and plant anchorage. As descripted by Grierson et al. in the Arabidopsis Book (2014 (12); doi: 10.1199/tab.0172), root hair development is regulated by auxin and ethylene, especially by auxin homeostasis. Increased auxin or ethylene signaling resulted in moving the initiation site of root hair growth to a more apical position and increasing the amount of elongation during root hair tip growth. Decreased auxin or ethylene signaling showed the opposite effects. Additionally, as stated in the discussion section of our manuscript, Vacheron et al. indicated that many PGPR affect root system architecture via modulation of host gormonal balance, including the plant auxin pathway (lines 418-420). In our study, we observed the formation of root hairs initiated at apical zone, however, the mechanisms underlying the interaction between the B. subtilis Ydj3 treatment and the root hairs growth of sweet peppers remain to be investigated in the future.

Thank you again for giving us the opportunity to address the questions!

Sincerely,

Tzu-Pi

***********************************************************

Tzu-Pi Huang, Ph. D.

Professor, Department of Plant Pathology

Director, Pesticide Residue Analysis Center

National Chung Hsing University

145, Xing-Da Road, Taichung 40227, Taiwan

TEL: 886-4-22840780 ext. 379; 886-4-22859750

FAX: 886-4-22859750

E-mail: tphuang@nchu.edu.tw

***********************************************************

Submitted filename: PONE-D-21-26845 R2 Response to Reviewers.docx

Click here for additional data file.

8 Feb 2022

Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers

PONE-D-21-26845R2

Dear Dr. Huang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Ying Ma, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10 Feb 2022

PONE-D-21-26845R2

Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers

Dear Dr. Huang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

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on behalf of

Dr. Ying Ma

Academic Editor

PLOS ONE