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Article

Peanut Growth Promotion and Biocontrol of Blight by Sclerotium rolfsii with Rhizosphere Bacterium, Bacillus siamensis YB-1632

by
Yinghang Chang
1,†,
Qianqian Dong
2,3,4,†,
Limei Zhang
1,
Paul H. Goodwin
5,
Wen Xu
2,3,4,
Mingcong Xia
2,3,4,
Jie Zhang
2,3,4,
Runhong Sun
2,3,4,
Chao Wu
2,3,4,
Kun Wu
1,
Shuxia Xu
1,* and
Lirong Yang
2,3,4,*
1
College of Life Science, Henan Agricultural University, Zhengzhou 450046, China
2
Henan Agricultural Microbiology Innovation Center, Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
3
Henan Biopesticide Engineering Research Center, Zhengzhou 450002, China
4
Henan Agricultural Microbiology Innovation Center, Zhengzhou 450002, China
5
School of Environment Sciences, University of Guelph, Guelph, ON N1G2W1, Canada
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(3), 568; https://doi.org/10.3390/agronomy15030568
Submission received: 15 January 2025 / Revised: 10 February 2025 / Accepted: 24 February 2025 / Published: 25 February 2025
(This article belongs to the Section Pest and Disease Management)
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Abstract

:
A total of 34 strains of bacteria were isolated from peanut rhizosphere soil, and all showed some in vitro inhibition of the pathogen Sclerotium rolfsii in co-culture. Strain YB-1632 produced the highest level of inhibition and also produced relatively high levels of biofilm in culture. Cell-free culture extracts and volatiles from it were also inhibitory to S. rolfsii. Based on 16S rDNA, gyrA, and gyrB sequences, it was identified as Bacillus siamensis. In the greenhouse, seed treatment resulted in a level of control of peanut sclerotium blight (PSB) comparable to that of a standard fungicide seed treatment. In addition to its antifungal activity, YB-1632 could induce disease resistance in peanut seedlings based on increasing peanut defense enzyme activities and gene expression. The priming of defense gene expression against a necrotrophic pathogen is consistent with Induced Systemic Resistance (ISR). In addition, YB-1632 produced enzyme activities in culture associated with root colonization and plant growth promotion. In the greenhouse, it increased peanut seedling growth, indicating the YB-1632 is a plant growth-promoting rhizobacterium (PGPR). In summary, YB-1632 is a promising novel PSB biocontrol agent and PGPR of peanut.

1. Introduction

Peanut (Arachis hypogaea) is the fourth largest oil crop in the world and is widely used in the food industry [1]. China is the world’s largest producer of peanuts with more than twice the production of the second largest producer, India, which is followed by Nigeria and the USA [2]. A major factor limiting peanut production is plant diseases, of which over 50 have been described to be caused by different fungi, bacteria, nematodes, and viruses [3]. One of the most serious diseases is peanut sclerotium blight (PSB), also known as Southern blight, which reduces yield and quality of peanuts, causing major economic losses [4,5,6,7,8]. PSB is caused by Sclerotium rolfsii, a wide host range, soil-borne fungal phytopathogen that can survive long periods in soil by forming sclerotia [9]. The fungus infects peanuts near the soil line rotting roots, stem pegs, and pods, with symptoms of yellowing and wilting followed by browning and eventually plant death [10]. During PSB, creamy-colored hyphae cover the lower stems, which may spread onto the surrounding soil with dark brown sclerotia embedded in the hyphae. The aggressiveness of S. rolfsii to peanut has been related to more carbohydrate-active enzymes and differences in putative effector and secondary metabolite synthetic genes [11].
At present, the main control measure against PSB is the use of fungicides [12], but disease resistance has also been investigated [13]. There have also been a number of studies using biological control as an environmentally friendly alternative. Some examples of PSB biocontrol are the application to seeds of Pseudomonas fluorescens isolated from peanut soil that reduced PSB losses by 99% [14], and the application to seeds of Streptomyces sp. isolated from peanut rhizosphere soil, which reduced PSB losses by 64–67% [15]. Bacillus spp. are also promising biological control agents of S. rolfsii, acting through different mechanisms. For example, Bacillus velezensis from pig slurry induced resistance against S. rolfsii [8], Bacillus pumulis from peanut rhizosphere had direct antifungal activity against S. rolfsii [16], and Bacillus subtilis from tomato rhizosphere altered peanut seed fatty acid composition associated with tolerance to S. rolfsii [17]. Despite their potential, the number of Bacillus isolates tested thus far against S. rolfsii is limited, and more isolates should be obtained and tested for PSB control efficacy and their mode of action.
Bacillus siamensis strains were reported to be effective biocontrol agents of fungal root rot phytopathogens as well as promoters of plant growth acting through a variety of mechanisms. Examples of strains with biocontrol activity are an isolate from the inter-rhizosphere soil of soybean that showed antifungal activity against Fusarium oxysporum and Phytophthora sojae, causing their hyphae to expand, deform, and create vesicles, inhibiting growth by 82.96% and 82.44%, respectively [18], and an isolate from cauliflower leaves that induced resistance in rice against Magnaporthe oryzae [19]. Examples of B. siamensis strains as plant growth promotion are a strain from chickpea roots that promoted chickpea growth by solubilizing P, fixing N2, and producing indole acetic acid (IAA) [20]; a strain from wheat rhizosphere that promoted wheat growth associated with the production of IAA, beta-1,3-glucanase, siderophores, amylase, cellulase, and protease [21]; and a strain from banana rhizosphere that promoted Arabidopsis growth related to the secretion of volatiles [22]. Thus far, however, there are no reports of B. siamnensis controlling PSB or promoting peanut growth.
The goal of this study was to isolate bacteria from the rhizosphere soil of peanuts with PSB, screen them for inhibition of S. rolfsii in vitro, and then test the most promising isolate in planta for reduced PSB and peanut growth promotion. To determine the mode of action for biocontrol, various enzyme activities were examined in vitro, and the expression level of peanut disease defense response genes were studied in planta. Potential modes of action for plant growth promotion examined were the production of IAA, siderophores, and other secondary metabolites.

2. Materials and Methods

2.1. Soil Sampling and Microbial Isolates

Peanut plants were collected from Zhumadian (32°36′22″ N latitude 114°23′35″ E longitude), Henan, China in July 2020, from the field, and rhizosphere soil on their roots was gently collected with a soft brush after removing large pieces of soil from the roots. The soil was subsequently passed through a sieve with an aperture of 0.9 mm and stored at 4 °C [23]. The soil type of the peanut plants was sandy loam, and a field was selected for sampling where no pesticides were applied during growth. Dilution plating on Luria–Bertani (LB) agar was performed as described by Q. Dong et al. [21]. After 24 h, single colonies were transferred to fresh LB medium, and pure cultures were stored at −80 °C at the Bacterial Biocontrol Strain Collection of the Henan Academy of Agricultural Sciences. For Sclerotium rolfsii strain ZY-009, isolated from diseased peanut stems in 2020 and identified by cultural characteristics, its virulence to peanut and Internal Transcribed Spacer sequence analysis was obtained from the Plant Pathogen Culture Collection of the Plant Protection Institute (Henan Academy of Agricultural Sciences, Zhengzhou, China).

2.2. Screening Soil Bacteria for In Vitro Antifungal Activity

After growth of S. rolfsii strain ZY-009 on Potato Dextrose Agar (PDA) for 5 days at 28 °C, a 6 mm diameter plug with mycelium was transferred to the center of a 90 mm diameter fresh PDA plate, and 0.001 mL of a solution of 108 CFU/mL of each rhizosphere soil bacterial isolate was placed on the PDA 2.5 cm from S. rolfsii strain ZY-009. After 5 days at 28 °C, the zone of inhibition was measured between the bacterial and fungal colonies with a dial caliper (Deli, Shanghai, China) [24]. Three replicates were performed.

2.3. Biofilm Formation

The five isolates showing the greatest in vitro antifungal activity were grown in 100 mL LB broth overnight at 37 °C and 180 rpm. The broths were diluted to OD600 = 0.040 (±0.005) with sterile Yeast Extract Peptone Dextrose (YEPD) broth and added to 6-well sterile microtiter plates (CCP06-006, Bioland, Huzhou, China) with 2.5 mL per well. After stationary incubation at 28 °C for 72 h, the biofilm was removed with toothpick and transferred to a 0.45 μm mixed cellulose ester (MCE) filter membrane (Millipore, Billerica, MA, USA). The filter was dried at 45 °C for 48 h, and the dry weight was measured [25].

2.4. Antifungal Activity of Bacterial Cell-Free Culture Filtrate

After 3 days in LB broth at 37 °C and 180 rpm, the broth was centrifuged for 20 min at 10,000× g at 4 °C. The supernatant was removed and passed through a 0.22 μm filter to obtain a cell-free culture filtrate. The filtrate was added to melted PDA at 50–55 °C, at 0%, 10%, 15%, 20%, and then the mixture was poured into Petri plates at 15 mL each. After solidification, a 6 mm diameter agar plug with mycelium of S. rolfsii strain ZY-009 was transferred to the center of each plate and incubated at 28 °C for 5 days. Colony diameter was measured, and the percent inhibition was calculated based on the following formula: Percent inhibition = (Colony diameter on PDA without cell-free culture filtrate − Colony diameter on PDA with cell-free culture filtrate)/Colony diameter on PDA without cell-free culture filtrate × 100% [26]. Three replications of each treatment were performed.

2.5. Antifungal Activity of Bacterial Volatiles

Bacteria were grown overnight in LB broth at 37 °C and 180 rpm, and 0.1 mL was spread onto LB agar in a Petri plate, and then a 6 mm diameter agar plug with mycelium of S. rolfsii strain ZY-009 hyphae was placed in the center of the PDA in another Petri plate. The bottoms of the two Petri plates were then sealed together with Parafilm (Amcor, Zurich, Switzerland) in a face-to-face format with the Petri plate containing S. rolfsii strain ZY-009 on top. Petri plates with S. rolfsii strain ZY-009 on PDA and lids coated with non-inoculated LB agar were used as the control. After incubation at 28 °C for 5 days, the diameter of the fungal colony was measured, and inhibition of fungal growth was calculated by comparing the diameter to that of the control [27]. Three replications of each treatment were performed.

2.6. Biocontrol of PSB and Growth Promotion of Peanut by YB-1632

Peanut (Arachis hypogaea Yuhua 22) seeds were incubated in 75% alcohol (Guoyao, Shanghai, China) for 1 min, and then washed 3 times with dsH2O. The surface disinfected seeds were soaked in dsH2O for 8 h and incubated on moist sterile filter paper for 1–2 days. Seeds with buds were placed in pots with 200 g sterile peat–vermiculite–volcanic gravel mixture (2:1:2) per pot and grown in a greenhouse at 28 °C with 12 h light/12 h dark (Lighting equipment is CHNT, model KG316T-D, ZhengTai, Leqing, China). Seed treatment with YB-1632 was carried out by adding mixing 500 g soaked seeds with 5 mL of 108 cells/mL of YB-1632 and then gently stirring the seeds by hand for 5 min to ensure that they had come in relatively uniform contact with the bacterial solution. Seed treatments with chemical fungicide were conducted with the same procedure, except using 5 mL 1 mg/mL oxadiazol (Guoguang, Chengdu, China). Inoculation of S. rolfsii was performed as per Li et al. [7], using a 6 mm PDA plug containing S. rolfsii mycelium placed on the peanut stem at the junction of the root, and the plug was attached to the stem by a sterile toothpick that also created a wound site. The wound site was then moistened with sterile dsH2O. Ther treatments were as follows: peanut seeds treated with dsH2O (CK), peanut seeds treated with B. siamensis YB-1632 (YB), peanut seeds treated with dsH2O and then 7 days later seedlings inoculated with S. rolfsii (SR), peanut seeds treated with B. siamensis YB-1632 and then 7 days later seedlings inoculated with S. rolfsii (SR + YB), and peanut seeds treated with oxadiazol and then 7 days later seedlings inoculated with S. rolfsii (SR + oxadiazol). Ten pots containing two peanut seeds each were used for each treatment group. A total of 20 plants were used for measurements, and the experiment was repeated three times.
Root length, plant height, root fresh weight, and total fresh weight were determined with a ruler, dial calipers, and analytical balance at 28 days after seeding. Percent PSB incidence was determined by measuring the ratio of diseased plants to total plants. PSB severity (%) was determined by measuring the ratio of lesion width to peanut stem width, with the following scale of 0 = healthy plant without any root rot symptoms (no infection); 1 = mild infection with 1–25% of the root surface area infected; 2 = medium infection with 26–50% of the root surface area infected; 3 = strong infection with 51–75% of the root surface area infected; 4 = severe infection with 76–99% of the root surface area infected or a dead plant [28].
Relative PSB control (%) = (PSB severity of SR − PSB severity of treatment)/PSB severity of SR.

2.7. Identification of YB-1632

Genomic DNA of YB-1632 was extracted using the MiniBEST Bacterial Genomic DNA Extraction Kit Ver. 3.0 (Takara, Beijing, China). The 1400 bp 16S rDNA sequence, 2000 bp gyrA sequence and 1900 bp gyrB sequence were amplified from the YB-1632 genomic DNA using primers 27F and 1492R [29], gyrA-F and gyrA-R, gyrB-F, and gyrB-R and the amplicons were sequenced (Tsingke Biotechnology, Beijing, China). The sequences of 16S rDNA, gyrA, and gyrB were used as queries with blastn against the NCBI nr database, respectively. In addition, 19 other 16S rDNA sequences, 14 other gyrA sequences, and 16 other gyrB sequences were selected for the construction of the trees using the neighbor-joining method with 1000 bootstrap iterations [24,30].

2.8. Putative Growth-Promoting and Biocontrol Factors of YB-1632

YB-1632 was inoculated into fresh LB broth for overnight incubation at 37 °C, 180 rpm. Then, the concentration of the bacterial solution was diluted to OD600 = 1.0. Phosphatase [31], protease, pectinase, and siderophore activities were determined by adding 0.001 mL of the diluted bacterial solution to the center of a Petri plate containing phosphorus [31], skim milk [32], pectin [33], or Chrome Azurol S blue [34] agar, respectively. The plates were then incubated at 28 °C for 5, 1, 5, and 2 days, respectively, and then the production of transparent zone was observed [21]. β-glucanase, amylase, and cellulase activities were measured by adding 0.001 mL of diluted bacterial solution to the center of a Petri plate containing β-glucan [35], starch agar [36], or cellulase assay [35] agar, respectively, and incubating at 28 °C for 2, 1, and 5 days, respectively. The appearance of hyaline circles was detected following staining β-glucan agar with 0.1% Congo red [35]. Then, starch agar were stained with 10% iodine [36], and β-glucan agar and cellulase assay agar was stained with 0.1% Congo red [35]. IAA was detected by inoculating the diluted bacterial solution into NB broth containing 2.5 mM L-tryptophan and incubating at 37 °C at 180 rpm for 24 h. After centrifugation for 10 min at 10,000× g, the supernatant was mixed with Salkowski reagent, and then observed for a pink color at 30 min incubation in the dark [21,37].

2.9. Peanut Enzymatic Activities and MDA Levels

At 28 days post planting, 0.1 g of whole peanut seedlings were ground in a mortar and pestle, and then 1 mL extraction reagent from the corresponding assay kit for enzyme activity (BC0200 for catalase (CAT), BC5165 for superoxide dismutase (SOD), BC0025 for malondialaehyde (MDA), BC0095 for peroxidase (POD), and BC0325 for lipoxygenase (LOX), Solarbio, Beijing, China)) was added to each sample. The solution was centrifuged at 8000× g for 10 min at 4 °C. The enzyme activities were detected and calculated according to the instructions of the manufacturer.

2.10. Peanut Gene Expression

Stems (0.1 g) of 28-day-old peanut seedlings were macerated in a mortar and pestle with liquid nitrogen. Total RNA was extracted from the macerate with RNAiso Plus (Takara, Beijing, China) following the instructions of the manufacturer. The RNA was stored at −80 °C. After electrophoresis and visual examination of the RNA in 1% (w/v) TAE agarose gel to ensure that the RNA was not degraded, 1 g of total RNA was reverse-transcribed using the All-in-One First-Strand Synthesis MasterMix (Lablead, Beijing, China) to prepare cDNA. Real-time quantitative PCR (RT-qPCR) was performed using a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Singapore) with 10 μL reactions containing TB Green Premix Ex Taq II (Takara, Beijing, China) and the primers listed in Supplementary Table S1. The transcript level of each target gene (pathogenesis-related proteins PR-4A (PR-4A), pathogenesis-related protein PR10 (PR10), class IV chitinase (PR-2), thaumatin-like protein (PR-5), polygalacturonase-inhibiting protein (pgip2), and glutathione-S-transferase (GST)) was determined relative to that of the β-actin gene and calculated as 2−ΔΔCt [38].

2.11. Statistical Analysis

Three biological replicates were performed for all assays. Statistical analyses were performed using IBM SPSS Statistics 27 with Shapiro–Wilk test for normality, Levene test for chi-square, and Tukey’s and Tamhane tests used for significance analyses (p < 0.05).

3. Results

3.1. Screening and Identification of YB-1632

A total of 34 strains of bacteria were isolated from the rhizosphere soil of peanut fields with PSB. Screening for the inhibition of S. rolfsii in vitro revealed that the largest zone of inhibition was with ZY-7 (5.50 ± 0.24 mm), which was not significantly greater than the next largest inhibition zone created by ZY-30 (Table 1). This was followed by the sizes of the inhibition zones of ZY-10, ZY-23, and ZY-17. Among the five strains producing the largest inhibition zones, biofilm formation, an indicator of potential root colonization was significantly greater for ZY-7 in culture (Figure 1).
Isolate ZY-7 is stored in the Bacterial Biocontrol Strain Collection of the Henan Academy of Agricultural Sciences and is cataloged as YB-1632. A comparison of the 16S rDNA sequence to that of six Bacillus spp. showed that YB-1632 was most similar to several B. siamensis strains (Figure 2A). Similar results were obtained with a sequence comparison of gyrA (Figure 2B) and gyrB (Figure 2C). Thus, YB-1632 was considered to be a strain of B. siamensis.

3.2. In Vitro Activities

Strain YB-1632 in pure culture produced the putative antifungal and plant growth-promoting enzyme activities of beta-glucanase, protease, amylase, pectinase, and cellulose, as well as the production of IAA and siderophores (Supplementary Figure S1). However, it did not have the ability to solubilize phosphorus.
Cell-free culture filtrate prepared from YB-1632 grown in LB, Landy, or YEPD broth inhibited the growth of S. rolfsii mycelium on PDA, but at 15 and 20%, the filtrate of inoculated LB was significantly higher than that of YEPD and Landy (Figure 3A). The inhibition of S. rolfsii growth was highest at 96.56% with 20% cell-free filtrate from LB broth. The inhibition of S. rolfsii growth occurred at 63.72% and 41.09% with 15% and 10% cell-free filtrate from LB broth, respectively. In contrast, cell-free filtrates from YEPD broth and Landy broth inhibited S. rolfsii by up to 50.97% and 63.11%, respectively. YB-1632 grown on the LB agar on the up Petri plates resulted in the inhibition of fungal growth on PDA in the bottom of Petri plates, indicating that YB-1632 can produce fungal inhibitory volatile substances (Figure 3B).

3.3. In Planta Biocontrol and Plant Growth Promotion Activities

The SR treatment resulted in high levels of PSB symptoms by 14 days post inoculation with a PSB incidence of 93.33%, and a PSB severity of 65.85% (Figure 4 and Table 2). However, the SR + YB treatment resulted in significantly lower PSB incidence and severity, which was not significantly different from that achieved treating seeds with the SR + oxadiazol treatment. The relative PSB control was also not significantly different between the SR + YB and SR + oxadiazol treatments.
The treatment of seeds with B. siamensis YB-1632 at the time of planting resulted in significantly lower PSB incidence and severity comparable to that achieved by treating seeds with the fungicide, oxadiazol. The relative PSB control was also very similar between the seed treatment with B. siamensis YB-1632 and the seed treatment with oxadiazol at 89%.
The YB treatment significantly increased root length, plant height, root fresh weight, and fresh weight of peanut compared to CK (Figure 4 and Table 3). In contrast, the SR treatment significantly reduced all those parameters except root length compared to CK. The SR + YB treatment resulted in higher plant height and total fresh weight than the CK or SR treatments, and the root fresh weight and total fresh weight were similar to those of the YB treatment. The SR + oxadiazol treatment yielded root fresh weight and total fresh similar to those of CK, and plant height was significantly higher than that of the other treatments, except for YB.
In contrast, seedling inoculation with S. rolfsii significantly reduced all those parameters compared to the control, demonstrating the damage caused by the phytopathogen. The combination of B. siamensis YB-1632 seed treatment and S. rolfsii seedling inoculation resulted in root lengths, root fresh weights, and total fresh weights greater than the control, phytopathogen inoculation alone, and the oxadiazol treatment, but not as great as with the YB-1632 seed treatment alone. Plant height was somewhat different in that the combination of YB-1632 and S. rolfsii resulted in a significant increase compared to the phytopathogen alone, but plant height was significantly lower than with the YB-1632 treatment alone and the oxadiazol treatment, which were not significantly different from each other.

3.4. Plant Enzymatic Activities and MDA Content

At 28 days after seed treatment, the seedling enzyme activities of CAT, SOD, and POD were significantly higher with YB than the CK treatment, whereas LOX activity was significantly lower with YB than CK treatment (Table 4). CAT and POD activities were not significantly different between the SR and YB treatments, but SOD and LOX activities were lower with SR than the YB treatment, although not significantly different from the SR and CK treatments. The SR + YB treatment resulted in CAT, SOD, POD, and LOX activities that were significantly higher compared to the CK, YB, and SR treatments. The SR + oxadiazol treatment resulted in CAT and POD activities not significantly different from the SR + YB treatment, but significantly lower than the SR + YB treatment for SOD and LOX activities.
MDA levels were not significantly different between the CK and YB treatments, both of which were significantly lower than those with the SR treatment. MDA levels with the SR + YB and SR + oxadiazole treatments were not significantly different from each other but were significantly higher than those with the CK and YB treatments.

3.5. Plant Gene Expression

The expression levels of PR-4A (Figure 5A), class IV chitinase (Figure 5B), and pgip2 (Figure 5C) were similar in their response to the treatments with expressions not significantly different between the CK, YB, and SR treatments. However, while the expression of gluathtione-S-transferase (Figure 5D) and PR-10 (Figure 5E) were not significantly different between the CK and YB treatments, it was significantly higher than with the SR treatment. The expression of all the tested genes with the SR + YB treatment was significantly higher than with the CK, YB, and SR treatments, except for PR-10 between the SR + YB and SR treatments.

4. Discussion

Bacillus siamensis is emerging as a promising biocontrol and plant growth-promoting agent [18,39,40,41]. Thus far, there is one report of B. siamnesis from peanut soil as a biocontrol agent, which reduced the incidence of collar rot of peanut caused by Aspergillus niger by 53.6–60.8%, and increased the yield by 20.5–22.7% compared to non-treated plants in field trails [42]. There is also a report of an endophytic isolate of B. siamensis obtained from healthy roots of peanut that had peanut growth promotion activity increasing stem root lengths by more than 2.5-fold under salt stress [43]. This study shows that other isolates of B. siamensis can act as effective agents for plant disease biocontrol and plant growth promotion.
One mode of action of biocontrol agents, such as Bacillus spp., is the direct inhibition of fungal growth [44]. Among all the isolates tested, B. siamensis YB-1632 showed the strongest inhibition of S. rolfsii in vitro, demonstrating that it expresses relatively strongly at least one of the mechanisms for biocontrol. One mechanism of antimicrobial activity for Bacillus spp. is the production of lipopeptides, such as surfactin, fengycin, and iturin, which inhibit fungi by binding to membranes, causing them to be disrupted [45]. Another mechanism is the production of antimicrobial enzymes, such as beta-glucanases, that can digest beta-glucan, which is the second most abundant polysaccharide in fungal phytopathogen cell wall after chitin [46], and chitinases that can lyse fungal cells by digesting chitin in fungal cell walls [47]. Another antimicrobial mechanism is the production of antimicrobial volatiles, such as benzothiazole produced by Bacillus subtilis CF-3 that produced shrunken, twisted, and ruptured hyphae of Monilinia fructicola [48] and 2-methylbutanoic acid and 3-methylbutanoic acid produced by Bacillus siamensis LZ88 that caused hyphae of Alternaria alternata to deform and collapse [49]. It appears that B. siamensis YB-1632 has several of these mechanisms.
Among the isolates with the strongest inhibition of S. rolfsii in vitro, B. siamensis YB-1632 had the greatest biofilm formation, making it a promising candidate for future study. A bacterial biofilm is a surface attached community of bacteria surrounded by an extracellular matrix of proteins, carbohydrates, and/or extracellular DNA, that results in traits distinct from those of free-living cells [50]. Biofilm should be considered in developing successful biocontrol and plant growth-promoting agents as it can be involved in space and nutrient competition, antibiosis, plant hormone-like activity, and the bacterial biofilm may constitute a physical barrier that hinders the infection of host root phytopathogens, and also increases the root’s ability to retain water and resist abiotic stress [51,52]. The relatively high levels of biofilm production by B. siamensis YB-1632 further indicates that it has the potential to be developed into an effective biocontrol and plant growth-promoting agent.
Aside from their role in direct antimicrobial activity mentioned above, extracellular enzymes of Bacillus spp. can contribute in other ways to biocontrol and plant growth promotion activity. The examination of B. siamensis YB-1632 showed that it produced extracellular beta-glucanase, protease, amylase, pectinase, and cellulase in culture but not phosphatase. The secretion of beta-glucanase could help in the promotion of plant growth as it can enhance plant cell expansion [53]; protease can increase plant protein turnover, accelerating amino acid recycling and degradation of non-functional proteins [54], as well as deactivate virulence factors like phytopathogen-secreted hydrolytic enzymes [55]; and amylase can promote seed germination by digesting starch, increasing the release of sugars for growth and development [56]. Thus, B. siamensis YB-1632 also has several of these potential mechanisms, which could help explain its ability to provide plant disease biocontrol and growth promotion.
Siderophores are also important for rhizobacteria to help provide nutrition to plants and defend against root pathogens [57]. Siderophores were detected from B. siamensis YB-1632. Siderophores are low-molecular-weight iron chelators able to bind iron with high affinity that are divided into three groups: hydroxamates, catecholates, and carboxylates [58]. They can inhibit plant pathogenic fungi by reducing Fe availability and can promote plant growth by providing Fe to plants under low Fe conditions [59]. Siderophore production may contribute to B. siamensis YB-1632, reducing root infections by S. rolfsii and promoting the growth of peanut seedlings.
Seed treatment with B. siamensis YB-1632 reduced PSB incidence and severity, resulting in disease control of 89%, which was not significantly different than that of a traditional fungicide treatment with oxadiazol. Other biocontrol agents of PSB have shown 67% control of Sclerotinia minor with Coniothyrium minitans [60], 65% control of S. rolfsii with Bacillus pumilus LX11 [16], and 62.6–70.8% control of S. rolfsii with Bacillus velezensis LHSB1 [61]. Compared to these strains, B. siamensis YB-1632 was more effective for the biocontrol of PSB.
In addition to having antimicrobial activity to control plant diseases, Bacillus spp. can also control plant diseases by inducing disease resistance in the host, such as B. siamensis B-612 increasing plant defense enzyme activities like peroxidase, associated with increased resistance to rice blast [19]. In this study, host catalase and peroxidase, superoxide dismutase, and lipoxygenase were measured, as they can act to inhibit and reduce damage caused by free radicals in cells and are often increased with diseases as the plant attempts to prevent damage caused by oxidative substances [62,63,64]. Infection by S. rolfsii triggered catalase, superoxide dismutase, and peroxidase activities that may have been related to oxidative stress and defense activation, but B. siamensis YB-1632 treatment also triggered these without pathogen infection, potentially providing a better antioxidative response of the plant at the start of infection. The combination of S. rolfsii and B. siamensis YB-1632 resulted in the highest catalase, superoxide dismutase, and peroxidase activities compared to other treatments, indicating a stronger response of seedlings treated with B. siamensis YB-1632, potentially contributing to reduced disease. Lipoxygenase was different in that B. siamensis YB-1632 treatment alone lowered its activity and infection by S. rolfsii did not significantly increase it compared to the control. However, it was like the other plant enzyme activities measured in that the highest levels were observed with the combination of S. rolfsii and B. siamensis YB-1632, once again indicating induced disease resistance with the B. siamensis YB-1632 treatment.
Although activities of antioxidative enzymes were triggered, it is important to determine if oxidative damage is occurring in the plant. MDA is a measurement of lipid peroxidation in plants [65]. MDA levels often increase with infections because reactive oxygen species (ROS) are produced in large quantities during plant disease, but biocontrol agents can lower this by triggering mechanisms like antioxidant enzymes to remove ROS [66]. In this study, MDA levels were not affected by YB-1632 alone but were increased with S. rolfsii infection. B. siamensis YB-1632-treated plants had less MDA than non-treated plants with S. rolfsii infection compared to the control. The combination of S. rolfsii and B. siamensis YB-1632 resulted in significantly lower MDA levels than S. rolfsii infection alone. This is consistent with B. siamensis YB-1632 antioxidative mechanisms, such as catalase and superoxide dismutase activities, to reduce phytopathogen-induced membrane damage, which could contribute to increased host resistance.
Induced host resistance by Bacillus spp. was also related to triggering the expression of host defense genes like those for pathogenesis-related proteins [19]. Plant disease biocontrol Bacillus spp. are typically associated with Induced Systemic Resistance (ISR) [67]. ISR is primarily active against necrotrophic phytopathogens and mostly involves priming of defense-related gene expression, dependent on jasmonic acid and ethylene signaling, but independent of salicylic acid [68]. In this study, PR-4A, class IV chitinase, pgip2, gluathtione-S-transferase, and PR-10 gene expression were measured following the different treatments. PR-4A genes in plants encode class I and II type chitinases that catalyze the hydrolysis of chitin, which is a major component of fungal cell walls [69]. Class IV chitinase genes encode a type of chitinase to hydrolyze beta-glycosidic linkage [70]. Pgip2 genes encode polygalacturonase-inhibiting proteins, which are found in plant cells walls that bind and inhibit the activity of fungal polygalacturonases, increasing oligogalacturonides in the apoplast that can elicit plant defense responses [71]. GST genes encode glutathione S-transferase, acting to reduce oxidative stress during infections [72]. Plant PR10 genes encode proteins with ribonuclease, papain inhibitory, phenolic oxidate coupling, antimicrobial activities [73]. In this study, PR-4A, class IV chitinase, and pgip2 gene expression were all not significantly affected by S. rolfsii or B. siamensis YB-1632 alone, but B. siamensis YB-1632 primed the plant to increase its expression several-fold during S. rolfsii infection, which could be contributing to greater resistance. While GST and PR10 expression were also not induced by B. siamensis YB-1632, they were triggered by S. rolfsii infection. Only GST expression was significantly further induced in S. rolfsii-infected seedlings treated with B. siamensis YB-1632, indicating priming of its expression to infection. The evidence for priming (i.e., no change in expression with the PGPR but stronger expression after infection) of PR-4A, class IV chitinase, pgip2, and GST is consistent with ISR, indicating that one potential biocontrol mechanism of B. siamensis YB-1632 against PSB is ISR.

5. Conclusions

In this study, a new strain of B. siamensis, YB-1632, was examined with strong in vitro antagonism to S. rolfsii, biofilm formation ability, control of PSB, and plant growth promotion. While many Bacillus spp. have been studied as biocontrol and plant growth-promoting agents, there are much fewer reports of B. siamensis compared to others like B. subtilis and B. amyloliquefaciens, which were developed into commercial products [74,75,76]. B. siamensis YB-1632 is a promising commercial product as it can be a seed treatment, providing disease control levels comparable to fungicide seed treatment. As peanut is attacked by more than 50 phytopathogens [77], YB-1632 may provide control of other diseases of peanuts as well, which would make it more practical by controlling multiple diseases. Formulations for commercial applications are important of the success of biocontrol agents as they need to be mass-produced and have a prolonged shelf life along with other traits [78]. Future work should examine formulations of B. siamensis YB-1632, such as B. siamensis MW586052, where including an emulsifier and osmotic protectant improved its antifungal and plant growth-promoting activities [40]. Overall, B. siamensis YB-1632 is a promising biocontrol and growth-promoting agent of peanut.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15030568/s1. Figure S1. Plant growth promotion and biocentrol traits of strain YB-1632. Tests for production of (A) IAA, (B) siderophore, (C) β-glucanase, (D) protease, (E) amylase, (F) pectinase and (G) cellulase. Activity for IAA is indicated by pink color, siderophore by a yellow color, β-glucanase by a clear zone, protease by a clear zone, amylase by a clear zone, pectinase by a clear zone and cellulase by a clear zone in the agar. Table S1. Primers for target and the reference genes examined in this study. The selection of genes and the design of primers were referred to Jogi et al. [38]. Table S2. Results of the normality test for inhibition of S.rolfsii by the Shapiro-Wilk. Table S3. Results of the normality test for biofilm dry weight by the Shapiro-Wilk. Table S4. Results of the normality test for inhibition of the growth of S.rolfsii on PDA with cell-free culture filtrate of YB1632 from LB, Landy and YEPD by the Shapiro-Wilk. Table S5. Results of significance of difference analysis of the growth of S.rolfsii on PDA with cell-free culture filtrate of YB1632 from LB, Landy and YEPD by Tamhane’s test. Table S6. Results of the normality test for PSB incidence by the Shapiro-Wilk. Table S7. Results of significance of difference analysis of PSB incidence by Tamhane’s test. Table S8. Results of the normality test for PSB severity by the Shapiro-Wilk. Table S9. Results of the normality test for Root length by the Shapiro-Wilk. Table S10. Results of significance of difference analysis of Root length by Tamhane’s test. Table S11. Results of the normality test for Plant height by the Shapiro-Wilk. Table S12. Results of the normality test for Root fresh weight by the Shapiro-Wilk. Table S13. Results of significance of difference analysis of Root fresh weight by Tamhane’s test. Table S14. Results of the normality test for Total fresh weight by the Shapiro-Wilk. Table S15. Results of significance of difference analysis of Total fresh weight by Tamhane’s test. Table S16. Results of the normality test for activity of CAT by the Shapiro-Wilk. Table S17. Results of the normality test for activity of SOD by the Shapiro-Wilk. Table S18. Results of the normality test for activity of POD by the Shapiro-Wilk. Table S19. Results of the normality test for activity of LOX by the Shapiro-Wilk. Table S20. Results of the normality test for activity of MDA by the Shapiro-Wilk.

Author Contributions

Conceptualization, L.Y., K.W., S.X. and Y.C.; methodology, Y.C., Q.D. and L.Z.; validation, Q.D. and W.X.; formal analysis, Y.C., P.H.G. and L.Z.; investigation, Y.C., Q.D. and L.Z.; resources, M.X., J.Z. and K.W.; data curation Y.C., W.X. and L.Y.; writing—original draft preparation: Y.C.; writing—review and editing, Y.C. and P.H.G.; visualization, Y.C.; supervision, R.S. and C.W.; funding acquisition, L.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Henan Provincial Science and Technology Major Project (221100110100), the Central Plains Science and Technology Innovation leader Project (234200510010), and the Special Project for Science and Technology Innovation Team of Henan Academy of Agricultural Sciences (2023TD15).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

The authors would like to thank Shimin Zhang (Henan Agricultural University), Jiran Zhang (Henan Agricultural University), and Tian Tian (Henan Agricultural University) for comments on the manuscript. In addition, we are grateful to Qingxiang Liu, for his valuable suggestions.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The biofilm dry weight produced by the isolates ZY-7, ZY-10, ZY-17, ZY-23, and ZY-30. A 2.5 mL of bacterial suspension (OD600 = 0.040) was inoculated into a 6-well plate, incubated at 28 °C for 48 h, and then the dry weight of the biofilm was determined. Three replicates were performed, and the means are shown with bars indicating one standard deviation (SD). The different letters on the columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test. The normality test as well as the chi-square test are shown in Table S3.
Figure 1. The biofilm dry weight produced by the isolates ZY-7, ZY-10, ZY-17, ZY-23, and ZY-30. A 2.5 mL of bacterial suspension (OD600 = 0.040) was inoculated into a 6-well plate, incubated at 28 °C for 48 h, and then the dry weight of the biofilm was determined. Three replicates were performed, and the means are shown with bars indicating one standard deviation (SD). The different letters on the columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test. The normality test as well as the chi-square test are shown in Table S3.
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Figure 2. Relatedness of YB-1632 to other Bacillus species. Phylogenetic tree based on (A) 16S rDNA, (B) gyrA, and (C) gyrB. The numbers in the figure represent Bootstrap value.
Figure 2. Relatedness of YB-1632 to other Bacillus species. Phylogenetic tree based on (A) 16S rDNA, (B) gyrA, and (C) gyrB. The numbers in the figure represent Bootstrap value.
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Figure 3. Inhibition of S. rolfsii strain ZY-009 by cell-free culture filtrate and volatiles produced by B. siamensis YB-1632. (A) Inhibition rates of S. rolfsii on PDA by cell-free culture filtrate of YB-1632 made with LB, Landy, and YEPD broths. (B) Growth of S. rolfsii on PDA with Petri plate coated without (left) or with (right) YB-1632. Means in Figure 2A are from three replicates and error bars represent one standard deviation (SD). Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test and Tamhane test. Normality test as well as the chi-square test are shown in Tables S4 and S5.
Figure 3. Inhibition of S. rolfsii strain ZY-009 by cell-free culture filtrate and volatiles produced by B. siamensis YB-1632. (A) Inhibition rates of S. rolfsii on PDA by cell-free culture filtrate of YB-1632 made with LB, Landy, and YEPD broths. (B) Growth of S. rolfsii on PDA with Petri plate coated without (left) or with (right) YB-1632. Means in Figure 2A are from three replicates and error bars represent one standard deviation (SD). Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test and Tamhane test. Normality test as well as the chi-square test are shown in Tables S4 and S5.
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Figure 4. Effect of seed inoculation with B. siamensis YB-1632 on 28-day-old peanut seedlings with and without stem inoculation with S. rolfsii strain ZY-009. CK represents peanut seeds grown in greenhouse and only treated with dsH2O. YB indicates peanut seeds only treated with B. siamensis YB-1632. SR indicates peanut seeds treated with dsH2O and seedling stems inoculated 7 days later with S. rolfsii. SR + YB indicates peanut seeds treated with B. siamensis YB-1632 and seedlings inoculated 7 days later with S. rolfsii. SR + oxadiazol indicates peanut seeds treated with fungicide, oxadiazol, and seedling stems inoculated 7 days later with S. rolfsii.
Figure 4. Effect of seed inoculation with B. siamensis YB-1632 on 28-day-old peanut seedlings with and without stem inoculation with S. rolfsii strain ZY-009. CK represents peanut seeds grown in greenhouse and only treated with dsH2O. YB indicates peanut seeds only treated with B. siamensis YB-1632. SR indicates peanut seeds treated with dsH2O and seedling stems inoculated 7 days later with S. rolfsii. SR + YB indicates peanut seeds treated with B. siamensis YB-1632 and seedlings inoculated 7 days later with S. rolfsii. SR + oxadiazol indicates peanut seeds treated with fungicide, oxadiazol, and seedling stems inoculated 7 days later with S. rolfsii.
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Figure 5. qRT-PCR of defense-related genes in 28 day-old peanut seedlings. RNA was extracted from roots and qRT-PCR analysis. Target genes were PR-4A (A), class IV chitinase (B), pgip2 (C), GST (D), and PR10 (E). Transcript level of each target genes was determined relative to that of actin elongation factor 1 and calculated as 2(−ΔΔCt). Primers for each target and normalization gene are shown in Table S1. CK represents peanut seeds grown in greenhouse and only treated with dsH2O. YB indicates peanut seeds only treated with B. siamensis YB-1632. SR indicates peanut seeds treated with dsH2O and seedling stems inoculated 7 days later with S. rolfsii. SR + YB indicates peanut seeds treated with B. siamensis YB-1632 and seedlings inoculated 7 days later with S. rolfsii. SR + oxadiazol indicates peanut seeds treated with the fungicide, oxadiazol, and seedling stems inoculated 7 days later with S. rolfsii. Means ± standard deviation (SD) from three replicates shown. Means for treatments with different letters in each column are significantly different (p < 0.05) using one-way ANOVA with Tukey’s test.
Figure 5. qRT-PCR of defense-related genes in 28 day-old peanut seedlings. RNA was extracted from roots and qRT-PCR analysis. Target genes were PR-4A (A), class IV chitinase (B), pgip2 (C), GST (D), and PR10 (E). Transcript level of each target genes was determined relative to that of actin elongation factor 1 and calculated as 2(−ΔΔCt). Primers for each target and normalization gene are shown in Table S1. CK represents peanut seeds grown in greenhouse and only treated with dsH2O. YB indicates peanut seeds only treated with B. siamensis YB-1632. SR indicates peanut seeds treated with dsH2O and seedling stems inoculated 7 days later with S. rolfsii. SR + YB indicates peanut seeds treated with B. siamensis YB-1632 and seedlings inoculated 7 days later with S. rolfsii. SR + oxadiazol indicates peanut seeds treated with the fungicide, oxadiazol, and seedling stems inoculated 7 days later with S. rolfsii. Means ± standard deviation (SD) from three replicates shown. Means for treatments with different letters in each column are significantly different (p < 0.05) using one-way ANOVA with Tukey’s test.
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Table 1. In vitro inhibition of S. rolfsii strain ZY-009 when co-cultured with peanut rhizosphere soil bacterial isolates on PDA. Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test. Normality test as well as the chi-square test are shown in Table S2.
Table 1. In vitro inhibition of S. rolfsii strain ZY-009 when co-cultured with peanut rhizosphere soil bacterial isolates on PDA. Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test. Normality test as well as the chi-square test are shown in Table S2.
Bacterial IsolateWidth (mm)Bacterial IsolateWidth (mm)
ZY-151.13 ± 0.13 qZY-222.70 ± 0.20 i
ZY-141.21 ± 0.15 qZY-272.73 ± 0.15 i
ZY-251.23 ± 0.23 pqZY-92.77 ± 0.21 i
ZY-81.32 ± 0.22 opqZY-13.23 ± 0.15 h
ZY-51.38 ± 0.25 nopqZY-43.24 ± 0.24 h
ZY-281.57 ± 0.15 mnopZY-203.27 ± 0.21 h
ZY-31.58 ± 0.26 mnoZY-213.57 ± 0.15 gh
ZY-341.63 ± 0.06 mnoZY-293.77 ± 0.06 g
ZY-161.70 ± 0.18 mnZY-243.87 ± 0.15 g
ZY-261.73 ± 0.15 lmZY-114.18 ± 0.19 f
ZY-332.03 ± 0.15 klZY-24.24 ± 0.25 f
ZY-312.07 ± 0.21 kZY-184.30 ± 0.10 ef
ZY-122.12 ± 0.24 kZY-174.57 ± 0.15 de
ZY-62.23 ± 0.24 jkZY-234.87 ± 0.25 cd
ZY-192.30 ± 0.20 jkZY-105.05 ± 0.23 bc
ZY-322.50 ± 0.10 ijZY-305.27 ± 0.21 ab
ZY-132.68 ± 0.13 iZY-75.50 ± 0.24 a
Table 2. Effect of B. siamensis YB-1632 on Peanut Stem Blight (PSB) caused by S. rolfsii. CK represents peanut seeds grown in greenhouse and only treated with dsH2O. YB indicates peanut seeds only treated with B. siamensis YB-1632. SR indicates peanut seeds treated with dsH2O and seedling stems inoculated 7 days later with S. rolfsii. SR + YB indicates peanut seeds treated with B. siamensis YB-1632 and seedlings inoculated 7 days later with S. rolfsii. SR + oxadiazol indicates peanut seeds treated with fungicide, oxadiazol, and seedling stems inoculated 7 days later with S. rolfsii. Different letters on columns indicate significant difference (p < 0.05) using Tamhane test and Tukey’s test. Normality test as well as the chi-square test are shown in Table S8.
Table 2. Effect of B. siamensis YB-1632 on Peanut Stem Blight (PSB) caused by S. rolfsii. CK represents peanut seeds grown in greenhouse and only treated with dsH2O. YB indicates peanut seeds only treated with B. siamensis YB-1632. SR indicates peanut seeds treated with dsH2O and seedling stems inoculated 7 days later with S. rolfsii. SR + YB indicates peanut seeds treated with B. siamensis YB-1632 and seedlings inoculated 7 days later with S. rolfsii. SR + oxadiazol indicates peanut seeds treated with fungicide, oxadiazol, and seedling stems inoculated 7 days later with S. rolfsii. Different letters on columns indicate significant difference (p < 0.05) using Tamhane test and Tukey’s test. Normality test as well as the chi-square test are shown in Table S8.
PSB Incidence (%)PSB Severity (%)Relative PSB Control (%)
CK
YB
SR93.33 ± 2.89 a65.85 ± 1.16 a
SR + YB11.67 ± 2.89 b6.79 ± 0.95 b89.65 ± 0.81
SR + oxadiazol13.33 ± 2.89 b6.75 ± 0.99 b89.79 ± 0.13
Table 3. Effect of B. siamensis YB-1632, S. rolfsii strain ZY-009, or oxadiazol on growth of 28-day-old peanut seedlings. Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test and Tamhane test. Normality test as well as chi-square test are shown in Tables S9–S15.
Table 3. Effect of B. siamensis YB-1632, S. rolfsii strain ZY-009, or oxadiazol on growth of 28-day-old peanut seedlings. Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test and Tamhane test. Normality test as well as chi-square test are shown in Tables S9–S15.
Root Length (cm)Plant Height (cm)Root Fresh Weight (g)Total Fresh Weight (g)
CK11.04 ± 1.62 a14.00 ± 1.80 c1.06 ± 0.45 a2.92 ± 0.69 b
YB11.99 ± 3.11 a16.33 ± 1.64 a1.41 ± 0.69 a3.69 ± 0.61 a
SR10.64 ± 1.70 a6.71 ± 1.80 d0.39 ± 0.22 b1.14 ± 0.41 c
SR + YB11.57 ± 1.40 a15.18 ± 1.73 b1.20 ± 0.58 a3.61 ± 0.87 a
SR + oxadiazol11.36 ± 2.47 a16.26 ± 1.61 a1.10 ± 0.43 a3.16 ± 0.19 b
Table 4. Activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), lipoxygenase (LOX), and MDA content in peanut seedlings at 28 days post planting. Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test. Normality test as well as chi-square test are shown in Tables S16–S20.
Table 4. Activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), lipoxygenase (LOX), and MDA content in peanut seedlings at 28 days post planting. Different letters on columns indicate significant difference (p < 0.05) using one-way ANOVA with Tukey’s test. Normality test as well as chi-square test are shown in Tables S16–S20.
CAT (U/g)SOD (U/g)POD (U/g)LOX (U/g)MDA (nmol/g)
CK136.629 ± 11.019 c18.026 ± 1.286 d8659.460 ± 581.372 c7480.759 ± 1918.007 bc48.146 ± 3.495 c
YB225.28 ± 10.826 b35.333 ± 1.513 b13,505.147 ± 556.389 b963.902 ± 126.205 d43.715 ± 4.689 c
SR182.104 ± 33.304 b22.628 ± 2.145 cd12,580.234 ± 1473.519 b10,158.530 ± 1390.438 b86.930 ± 3.738 a
SR + YB369.412 ± 81.563 a43.747 ± 3.713 a16,285.935 ± 1045.415 a13,688.035 ± 1020.885 a64.083 ± 2.550 b
SR + oxadiazol307.315 ± 33.457 a28.257 ± 5.170 c14,786.548 ± 1877.844 ab5136.207 ± 631.574 c64.252 ± 3.435 b
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MDPI and ACS Style

Chang, Y.; Dong, Q.; Zhang, L.; Goodwin, P.H.; Xu, W.; Xia, M.; Zhang, J.; Sun, R.; Wu, C.; Wu, K.; et al. Peanut Growth Promotion and Biocontrol of Blight by Sclerotium rolfsii with Rhizosphere Bacterium, Bacillus siamensis YB-1632. Agronomy 2025, 15, 568. https://doi.org/10.3390/agronomy15030568

AMA Style

Chang Y, Dong Q, Zhang L, Goodwin PH, Xu W, Xia M, Zhang J, Sun R, Wu C, Wu K, et al. Peanut Growth Promotion and Biocontrol of Blight by Sclerotium rolfsii with Rhizosphere Bacterium, Bacillus siamensis YB-1632. Agronomy. 2025; 15(3):568. https://doi.org/10.3390/agronomy15030568

Chicago/Turabian Style

Chang, Yinghang, Qianqian Dong, Limei Zhang, Paul H. Goodwin, Wen Xu, Mingcong Xia, Jie Zhang, Runhong Sun, Chao Wu, Kun Wu, and et al. 2025. "Peanut Growth Promotion and Biocontrol of Blight by Sclerotium rolfsii with Rhizosphere Bacterium, Bacillus siamensis YB-1632" Agronomy 15, no. 3: 568. https://doi.org/10.3390/agronomy15030568

APA Style

Chang, Y., Dong, Q., Zhang, L., Goodwin, P. H., Xu, W., Xia, M., Zhang, J., Sun, R., Wu, C., Wu, K., Xu, S., & Yang, L. (2025). Peanut Growth Promotion and Biocontrol of Blight by Sclerotium rolfsii with Rhizosphere Bacterium, Bacillus siamensis YB-1632. Agronomy, 15(3), 568. https://doi.org/10.3390/agronomy15030568

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