Characterization and Biocontrol Efficacy of Bacillus velezensis GYUN-1190 against Apple Bitter Rot

Article information

Plant Pathol J. 2024;40(6):681-695

Publication date (electronic) : 2024 December 1

doi : https://doi.org/10.5423/PPJ.OA.05.2024.0076

Hyeonjin Park ¹, Younmi Lee ¹, Kotnala Balaraju ², Jungyeon Kim ¹, Yongho Jeon ¹^,

¹Department of Plant Medicals, Andong National University, Andong 36729, Korea

²Agricultural Science and Technology Research Institute, Andong National University, Andong 36729, Korea

^*Corresponding author. Phone) +82-54-820-5507, FAX) +82-54-820-6320, E-mail) yongbac@andong.ac.kr

Handling Editor: Hun Kim

Received 2024 May 9; Revised 2024 November 10; Accepted 2024 November 15.

Abstract

The application of synthetic fungicides has resulted in environmental pollution and adverse effects on non-target species. To reduce the use of agrochemicals, crop disease management requires microbial biological control agents. Bacillus-related genera produce secondary metabolites to control fungal pathogens. Bacillus velezensis GYUN-1190, isolated from soil, showed antagonistic activity against Colletotrichum fructicola, the apple anthracnose pathogen. Volatile organic compounds and culture filtrate (CF) from GYUN-1190 inhibited C. fructicola growth in vitro, by 80.9% and 30.25%, respectively. The CF of GYUN-1190 inhibited pathogen spore germination more than cell suspensions at 10⁸ cfu/ml. Furthermore, GYUN-1190 CF is effective in inhibiting C. fructicola mycelial growth in vitro, and it suppresses apple fruit bitter rot more effectively than GYUN-1190 cell suspensions and pyraclostrobin in planta. The mycelial growth of C. fructicola was completely inhibited 48 h after immersion into the CF, in compared with positive controls and GYUN-1190 cell suspensions. The genetic mechanism underlying the biocontrol features of GYUN-1190 was defined using its whole-genome sequence, which was closely compared to similar strains. It consisted of 4,240,653 bp with 45.9% GC content, with 4,142 coding sequences, 87 tRNA, and 28 rRNA genes. The genomic investigation found 14 putative secondary metabolite biosynthetic gene clusters. The investigation suggests that B. velezensis GYUN-1190 might be more effective than chemical fungicides and could address its potential as a biological control agent.

Keywords: anthracnose; Bacillus velezensis; biocontrol; Colletotrichum; growth inhibition

Colletotrichum is a genus of considerable global importance due to its reputation for inducing a wide range of diseases in crop plants (Zakaria, 2021). Anthracnose disease is particularly detrimental to these plants (Hyde et al., 2009). This genus of pathogens infects a wide variety of crops, including apples, when exposed to high temperatures and precipitation (Heo et al., 2024). As a consequence, there is a significant decrease in yields both prior to and following the harvest season (Salotti and Rossi, 2022).

Apple (Malus domestica) is vulnerable to a diverse array of diseases, with bitter rot being the most prevalent, primarily due to the presence of Colletotrichum species (Khodadadi et al., 2020). Significant economic losses are incurred as a consequence of the disease’s impact on apple fruits both pre- and post-harvest (Chen et al., 2022; Nodet et al., 2019). Colletotrichum, a genus of fungi that spans the globe, comprises more than 189 species that are distributed across tropical and temperate regions (Khodadadi et al., 2020). Colletotrichum species infect a wide range of host plants, causing diseases such as anthracnose and fruit rots (Dean et al., 2012), as well as several fruit crops, including apples (Cannon et al., 2012; Kim et al., 2009). The most typical symptoms are a depressed lesion on the fruit’s surface; a conidial layer forms in a round pattern on the surface of the lesion, causing the fruit rot. Furthermore, the cross-section of the fruit shows that it is rotting in a V-shape (Cheon and Jeon, 2015). The fruits become infected through spore germination, appressorium formation, and hyphal penetration of the host’s epidermis. It has been proven that disease symptoms worsen during the rainy season due to the spread of conidia to fresh produce, resulting in secondary infections (Lee et al., 2012; Yan et al., 2018). In order to increase the quantity of fruits for the present demand in the country and to manage apple bitter rot, apple growers around the world, including Korea, employ a large amount of chemical fertilizers (Kai and Adhikari, 2021) and pesticides (Enserink et al., 2013).

In order to manage anthracnose diseases in Korea, synthetic fungicides are currently being applied and utilized (Kim et al., 2021). On the contrary, the ongoing utilization of synthetic fungicides leads to adverse effects and toxicity on both human health and the environment (Fang et al., 2022), resulting in the development of resistance in the pathogens (Jayasinghe and Fernando, 2009). Moreover, the implementation of chemical control measures may induce microbial community imbalances, thereby impeding the functionality of beneficial organisms and potentially fostering the emergence of resistant pathogen strains (Shanmugam and Kanoujia, 2011). Hence, biological control agents (BCA) have surfaced as environmentally sustainable alternative to chemical fungicides for the purpose of managing plant diseases (Kim et al., 2021).

The utilization of BCA has demonstrated considerable potential as a method of controlling a wide range of plant diseases (Chowdhury et al., 2015; Fan et al., 2018; Mao et al., 2020). A considerable amount of research has been devoted to BCAs; however, commercial development has been limited to select few strains, primarily those of Bacillus species (Chowdhury et al., 2015; Fan et al., 2018; Ye et al., 2020). Bacillus spp., are a class of bacteria that are ubiquitous and found in all natural environments, with particular abundance in the rhizosphere and plant roots. Of numerous Bacillus species, it has been documented that the B. velezensis isolate generates a number of secondary metabolites (Kim et al., 2022; Liang et al., 2023) that exhibit activity against a diverse range of phytopathogens (Keshmirshekan et al., 2024). A variety of B. velezensis isolates, including B. velezensis ES2-4 (Palazzini et al., 2016), B. velezensis E68 (Liang et al., 2023), B. velezensis TSA32-1 (Kim et al., 2022), B. velezensis 9D-6 (Grady et al., 2019), produced secondary metabolites, such as surfactin, iturin, fengycin, and bacillibactin.

In addition, Bacillus species have a significant capacity to regulate infections by directly impeding the growth of pathogens via the synthesis of volatiles, metabolites, enzymes, and low molecular weight compounds (Rehman and Leiknes, 2018). According to Weng et al. (2013), their ability to heat-resistant endospores that are robust enough to be stored and transported makes them highly promising candidates for BCAs. Despite this, the implementation of BCAs continues to be difficult due to their frequently inconsistent result in the field (Weng et al., 2013). Soil is the primary source of these bacteria; their capacity to develop spores enables them to endure unfavorable conditions; which can be used to develop effective microbial BCA (Lee et al., 2020). Furthermore, numerous investigations have demonstrated that antagonistic bacteria produce a variety of secondary metabolites that exhibit diverse antibacterial mechanisms against plant pathogens (Han et al., 2015; Zhang et al., 2008). Whole-genome analysis of Bacillus species provides remarkable insight into the biological regulatory mechanisms of plant growth-promoting rhizobacteria and is critical for the uses of these bacteria (Liu et al., 2017). It provides the foundation for understanding plant-microorganism interactions.

In light of these possible benefits, this study aims to (1) determine the in vitro antagonistic activity of B. velezensis GYUN-1190 against Colletotrichum fructicola, the causative agents of apple bitter rot, respectively; (2) evaluate the potential effectiveness of this strain as a BCA against apple bitter rot in planta conditions; and (3) analyze the full genome sequence of GYUN-1190 in order to identify the divergent genomic attributes.

Materials and Methods

In vitro screening of antagonistic bacteria against C. fructicola

The antagonistic bacteria tested were isolated previously from various sources deposited in our laboratory (Supplementary Table 1). Bacillus velezensis strains were subjected to a dual culture plate assay to determine their in vitro antagonistic activity against C. fructicola, an apple bitter rot pathogen. The fungal pathogen was cultured onto potato dextrose agar (PDA) plates (90 mm diameter) at 25°C for 7 days. The bacterial isolates were cultured in tryptic soy agar (TSA) plates at 28°C for 2 days. A mycelial plug was removed from the fully grown PDA plate using a sterile cork borer (5 mm diameter) and inoculated onto PDA medium supplemented with peptone (PDK) on one side 2 cm away from the edge, whereas the bacterium was streaked on the other side. Plates inoculated with fungal disks alone were used as controls. All plates were incubated at 25°C for 10 days. The distance between bacterial cells and the mycelium was measured as the inhibition of mycelial growth. The results are expressed as the percentage inhibition of the growth of fungal mycelia in the presence and absence of bacteria using the following equation. Inhibition of mycelial growth (%) = [(Control – Test/Control)] × 100.

Evaluation of volatile organic compounds from B. velezensis strains on inhibition of fungal pathogens

To determine the volatile organic compounds (VOCs) secreted by six B. velezensis isolates (GYUN-1178, GYUN-1187, GYUN-1190, GYUN-1198, and GYUN-2376) against the growth fungal pathogen C. fructicola, exposure to these six B. velezensis isolates was performed using a overlapping plate assay described by Li et al. (2018). C. fructicola was cultured on PDA plates at 25°C for 7 days. The antagonistic bacterial isolates were cultured on TSA plates for 48 h. Mycelial plugs (5 mm diameter) of fungal pathogen were placed on the center of freshly prepared PDA plates, and bacteria were streaked onto TSA plates. Bacteria-inoculated plates were placed on top of PDA plates inoculated with mycelial plugs of the fungal pathogen C. fructicola. Fungal pathogen-inoculated plates without bacterial inoculation were used as a control group. Colony diameter was measured after incubating the C. fructicola-inoculated plates at 28°C for 7 days. The experiment was performed two times in triplicates.

In vitro evaluation of cell-free culture filtrate from B. velezensis isolates

The antifungal activity of cell-free culture filtrate (CF) derived from six strains of B. velezensis was assessed by culturing the bacterial cells of all six strains for 5 days in tryptic soy broth (TSB) at 28°C under shaking conditions (180 rpm) for 3 days. In order to acquire the CF, the culture broth was centrifuged for 5 min at 13,000 ×g and 4°C. The resulting supernatant was filtered through a membrane filter (0.22 μm). The plates were spread with 100 μl of C. fructicola spore suspensions (10⁵ conidia/ml) on PDK medium, followed by placing a sterile paper disk at four sides of Petri dish impregnated with 10 μl of bacterial CF each. PDK plates spread with TSB were used as untreated controls. Inhibition zones were measured after incubating the plates at 25°C for 2 days. The experiment was performed two times, and each treatment had three replicates.

In vitro inhibition of spore germination of C. fructicola by treatment with GYUN-1190 cell suspension and CF

In order to ascertain the potential inhibitory effect of B. velezensis GYUN-1190 on germination of pathogenic fungal spores, conidia were harvested from 7-day-old cultures maintained at 25°C on PDA using sterile distilled water (SDW). The conidia concentration was adjusted to 10⁵ conidia/ml using a hemocytometer. GYUN-1190 was cultured for 48 h at 28°C on TSA medium. Cell suspensions containing four different concentrations (10⁵–10⁸ cfu/ml) in SDW were prepared. In order to assess the inhibitory effect of secondary metabolites, the CF was also prepared. Conidial germination and appressorium formation of C. fructicola were evaluated on a glass slide surface treated with B. velezensis GYUN-1190 bacterial suspensions or CF using a previously described method (Kwak et al., 2012). Briefly, conidial suspensions (10 μl) were mixed with bacterial suspensions (10 μl) at varying concentrations (10⁵–10⁸ cfu/ml) and placed onto glass slides. Conidia suspensions without bacterial suspensions were considered as a control. Conidial germination and appressorium formation were assessed during incubation at 25°C and at varying times (0, 6, 12, and 24 h) in Petri dishes containing moist paper. The glass slides were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan). The experiments were carried out three times in triplicate. The conidial germination inhibition rate (%) was calculated using the following formula: Conidia germination inhibition rate (%) = (Germination of treated conidia/Germination of control) × 100.

Evaluation of the in vitro inhibitory effect of B. velezensis GYUN-1190 against C. fructicola

The inhibitory effect of B. velezensis GYUN-1190 on C. fructicola growth was verified in vitro. The freshly prepared cell suspension (10⁸ cfu/ml in SDW) or CF of GYUN-1190 were combined with C. fructicola conidia suspensions of (10⁵ conidia/ml) and incubated at 25°C for different duration of time (0, 8, 24, and 48 h). Following that, 100 μl per sample was distributed onto Petri dishes with PDA containing streptomycin. Following 2 days of incubation at 25°C, individual colonies of C. fructicola were counted. The chemical fungicides tebuconazole and pyraclostrobin were used as positive controls. SDW and TSB were used as negative controls.

Evaluation of the efficacy of B. velezensis GYUN-1190 in controlling apple bitter rot

The disease control efficacy of B. velezensis GYUN-1190 against apple bitter rot was tested. Apple fruits (cv. Fuji) of similar size were surface-disinfected with 70% ethanol for 3 min, followed by 1% NaOCl for 3 min, rinsed three times with SDW, and air-dried. Then, four places on the apple fruits were wounded with a sterile needle at a depth of 1–2 mm. The freshly prepared cell suspension (10⁸ cfu/ml) or CF of GYUN-1190 were combined with a spore suspension of C. fructicola (10⁵ conidia/ml) and incubated at 25°C for different duration of time (0, 8, 24, and 48 h). Afterwards, 10 μl of the mixture was treated. Tebuconazole (AI; 25% WP) and pyraclostrobin (AI; 22.9%, EC) diluted using field concentration (Guild line; KCPA, Korea Crop Protection Associate, Seoul, Korea) were served as positive controls. Apples treated with SDW or TSB served as negative controls. Nine apples were used per treatment, and the experiment was performed twice. The disease severity (%) was determined by the disease index (0 to 4; where 0 = no symptoms, 1 = 0–25% with disease lesions ≥ 2 mm; 2 = 26–50% with tissue showing disease lesions ≥ 4 mm; 3 = 51–75% with disease lesions < 8 mm; 4 = 76–100% with sunken lesions and spore production).

The disease severity (%) is calculated using the following formula:

[Sum of all disease index/(Total number of apples×Disease index)]×100.

Inhibition of C. fructicola growth and prevention of apple bitter rot by GYUN-1190 CF and cell suspension

To determine the growth inhibition of fungal pathogen C. fructicola by treatment with cell suspension (10⁸ cfu/ml) and CF of GYUN-1190, the mycelia plugs (5 mm diameter) of C. fructicola cultured on PDA plates for 7 days at 25°C were immersed in 100 ml of cell suspensions or CF of GYUN-1190. SDW and TSB were used as negative controls; while tebuconazole and pyraclostrobin diluted to field concentration were used as positive controls. All treatments were prepared in the same volume (100 ml) and incubated at 25°C for 0, 8, 24, and 48 h to determine their effectiveness against the pathogen at different time points in vitro. At each time point, treatments were mixed with C. fructicola conidia and spread onto PDA medum containing streptomycin (300 μg/ml). After incubation at 25°C for 7 days, the growth of C. fructicola mycelial plugs was measured. To determine the effectiveness of GYUN-1190 treatment in preventing apple bitter rot in planta at different time points, mature apples were surface-disinfected as described earlier, and wounds were artificially created on the surface of the apples using a sterile needle. On each wound of the apple, the mycelial plugs after immersion in different treatments as above for 0, 8, 24, and 48 h, were placed on the wounded sites of apples. The diseased lesion area was measured after incubation at 25°C for 9 days, and the disease severity (%) of apple bitter rot was calculated.

Whole-genome sequencing, clusters of orthologous genes annotation, and secondary metabolites of GYUN-1190

Bacterial gDNA of GYUN-1190 was extracted using a Solg Genomic DNA Prep Kit (Solgent, Daejeon, Korea), following the manufacturer’s instructions. The quantification of the gDNA was performed using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) and agarose gel electrophoresis was employed to validate its integrity. Sequencing libraries were prepared using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, CA, USA) and the BluePippin Size-Selection System (Sage Science, Beverly, MA, USA) in accordance with the manufacturer’s instructions for 20 kb template preparation. In brief, g-tubes (Covaris, Woburn, MA, USA) were used to shear 10 μg of the gDNA to 20 kb. The gDNA was then purified, end-repaired, and ligated with blunt-end SMRTbell adapters. Libraries were quantified and validated using a Qubit 2.0 fluorometer (Invitrogen) and a DNA 12,000 chip (Agilent Technologies, Santa Clara, CA, USA), respectively. Subsequently, the libraries were sequenced using PacBio P6C4 chemistry in an 8-well SMART Cell v3 with the PacBio RSII (Pacific Biosciences).

Statistical analysis

To assess the mean differences between each treatment, analysis of variance (ANOVA) was conducted. For an in-depth evaluation of differences between individual treatments, the least significant difference test was utilized for multiple comparisons. These statistical analyses were carried out using R statistical software (R Foundation for Statistical Computing, Vienna, Austria).

Results

In vitro screening of antagonistic B. velezensis strains

Among the 33 strains of B. velezensis strains, 6 strains (GYUN-1178, GYUN-1187, GYUN-1190, GYUN-1196, GYUN-1198, and GYUN-276) exhibited the strongest inhibition activity against mycelial growth of C. fructicola (Supplementary Table 1, Supplementary Fig. 1). GYUN-1178, GYUN-1187, GYUN-1190, GYUN-1196, GYUN-1198, and GYUN-2376 inhibited by with 60.0%, 60.6%, 62.9%, 60.6%, 58.9%, and 57.7%, respectively (Fig. 1A).

Fig. 1

(A) In vitro antagonistic effect of various Bacillus velezensis strains GYUN-1190 against mycelial growth of Colletotrichum fructicola GYUN-10893 using a dual culture plate assay. (B) Inhibitory effect of volatile organic compounds produced by B. velezensis strains, including strain B. velezensis GYUN-1190 against the growth of C. fructicola GYUN-10893 using the sandwiched-plate assay. One set of plates streaked with the GYUN-1190 on tryptic soy agar were overlapped with pathogen-inoculated plates. The colony diameter was measured 14 days after incubation at 28°C. Bars with the same letters do not differ from each other according to the least significant difference at P < 0.05.

In vitro mycelial growth inhibition effect of VOCs produced by B. velezensis isolates against the growth of C. fructicola

The effect of VOCs produced by six selected strains against the mycelial growth of C. fructicola was tested using a sandwiched-plate assay. The VOCs released by all the B. velezensis isolates were involved inhibiting the growth of fungal pathogenic mycelia to a greater extent when compared to the non-treated control (Fig. 1B). Among six B. velezensis antagonistic isolates tested, the GYUN-1190 strain exhibited the highest mycelial growth inhibition rate with 80.9% compared to the control, the VOCs of all strains were effective in inhibiting the mycelial growth of C. fructicola VOCs from GYUN-1178, GYUN-1187, GYUN-1190, and GYUN-1196 showed mycelial growth inhibition of 74.1%, 73.5%, 81.1%, and 69.4%, respectively.

In vitro antifungal activity of CFs of the B. velezensis strains

The antifungal activity of CF from the six B. velezensis strains against the mycelial growth of C. fructicola was evaluated in vitro (Fig. 2). The CFs of six strains showed turbid zones of similar size (Fig. 2). However, clear zones were different. The clear zone sizes formed by CFs of GYUN-1178, GYUN-1187, GYUN-1190, and GYUN-1196 were 15.0, 9.25, 19.5, 13.75, 12.75, 12.5, and 12.25 in diameter, respectively. Based on this, the GYUN-1190 strain was chosen as a representative isolate for subsequent investigation.

Fig. 2

The inhibitory effect of culture filtrate of GYUN-1190 derived from tryptic soy broth (TSB) against the growth of Colletotrichum fructicola GYUN-10893, causes bitter rot in apples. Plates treated with TSB alone were used as a non-treated control group. The diameter of mycelial growth inhibition area of fungal pathogen on PDK plates was recorded 9 days after incubation at 25°C. Asterisk shows the highest inhibition zone. Each treatment consisted of three replicates (Petri dishes) and the experiment was performed two times. Bars with the same letters do not differ from each other according to the least significant difference at P < 0.05.

Effect of treatment with cell suspensions or CF of GYUN-1190 on the germination of C. fructicola conidia

The effect of GYUN-1190 cell suspension (10⁵ to 10⁸ cfu/ml) or CF on conidial germination of C. fructicola was examined in vitro. GYUN-1190 cell suspension treatment inhibited conidial germination of C. fructicola in a concentration-dependent manner (Fig. 3). CF treatment also completely inhibited conidial germination (Fig. 3). The highest inhibition of spore germination was observed in GYUN-1190 cell suspensions at 10⁸ cfu/ml concentrations, with a 100% inhibition rate, when compared to other concentrations. Conversely, conidia germination rate was inhibited by 19.4%, 23.1%, and 64.5% at 10⁷ cfu/ml after 6, 12, and 24 h of incubation, respectively. In contrast, the spore germination was observed in GYUN-1190 cell suspensions at reduced concentrations (10⁵, 10⁶, and 10⁷ cfu/ml) after 24 h of incubation. Overall, between cell suspensions of GYUN-1190 at 10⁸ cfu/ml concentration and CF were used, 100% of pathogen spore germination was inhibited by CF.

Fig. 3

Effect of cell suspensions and culture filtrate (CF) of Bacillus velezensis GYUN-1190 on conidial germination rate (%) of Colletotrichum fructicola GYUN-10893. Microscopic observation of the fungal spore germination of C. fructicola GYUN-10893 was recorded after treating with cell suspensions of B. velezensis GYUN-1190 at various concentrations (10⁵, 10⁶, 10⁷, and 10⁸ cfu/ml) and CF during the incubation period from 0 to 24 h. Control used sterile distilled water and CF treatment used tryptic soy broth. The experiment was performed three times in triplicates. Bars with the same letters do not differ from each other according to the least significant difference at P < 0.05.

Effect of antagonistic GYUN-1190 bacterial treatment on inhibiting the in vitro fungal growth and control of anthracnose disease in apple fruits in planta

Cell suspensions and CF of antagonistic isolate B. velezensis GYUN-1190 were used to investigate the growth inhibition effect of C. fructicola, the causal agent of apple bitter rot, and its effectiveness in preventing bitter rot of apple fruit in planta. The mycelial growth of C. fructicola was completely inhibited, indicating that GYUN-1190 played an effective role in inhibiting the growth of the pathogen C. fructicola (Fig. 4). On the other hand, the chemical fungicides tebuconazole and pyraclostrobin were less effective than GYUN-1190, with inhibition rates of 88.4 and 10.0%, respectively. We also confirmed the effectiveness of B. velezensis GYUN-1190 cell suspensions in preventing bitter rot on apple fruits in planta (Fig. 5). Treatment with cell suspensions and CF of GYUN-1190 completely prevented the severity of bitter rot in apple fruits. Apples teated with tebuconazole and pyraclostrobin, which were used as positive controls, showed 41.7 and 75.0% disease severity, respectively. The inhibitory effect of fungicides notably diminished in comparison to that of B. velezensis GYUN-1190.

Fig. 4

Effect of bacterial cell suspensions and culture filtrates of strain Bacillus velezensis GYUN-1190 on mycelium growth of the apple bitter rot pathogen Colletotrichum fructicola GYUN-10893 in vitro. Solid medium supplemented with streptomycin was used, and a GYUN-10893 spore suspension concentration of 10⁵ conidia/ml was used. Chemical fungicides (tebuconazole and pyraclostrobin) were used as positive controls, and sterile distilled water (SDW) and tryptic soy broth (TSB) were used as negative controls. Non-treatment control was used two types (control and chemical control; SDW, culture filtrate control; TSB). Bars with the same letters do not differ from each other according to the least significant difference at P < 0.05.

Fig. 5

Effect of treatment with bacterial cell suspensions and culture filtrate of Bacillus velezensis GYUN-1190 on disease suppression of bitter rot caused by Colletotrichum fructicola GYUN-10893 on apples. Apples treated with chemical fungicides (tebuconazole and pyraclostrobin) and another antagonist bacterium (B. subtilis GYUN-2435) were used as positive controls, and sterile distilled water and TSB as negative controls. The experiment was performed two times and each treatment consisted of 12 replicates. The results were compared with a non-treated control 9 days after incubation at 25°C. Bars with the same letters do not differ from each other according to the least significant difference (P < 0.05).

Effect of GYUN-1190 treatment on control of bitter rot of apple caused by C. fructicola

The growth inhibitory effect of C. fructicola causes apple bitter rot was investigated. The experimental findings indicate that the mycelial plugs, which were immersed at each time point, were subsequently re-cultured in a PDA medium supplemented with streptomycin for 7 days. Notably, the treatment group immersed in the CF of GYUN-1190 at 48 h completely inhibited the mycelial growth. Conversely, cell suspension treatment group did not exhibit a significant effect, as evidenced by a mycelial growth inhibition rate of 10.6%. The positive controls, tebuconazole, and pyraclostrobin, inhibited mycelial growth rates by 41.2 and 3%, respectively (Supplementary Fig. 2). In addition, the application of time-deposited mycelial plugs to apple fruits led to a significant reduction of 70.7% in the development apple bitter rot after 48 h, of the treatment with B. velezensis GYUN-1190 cell suspensions. The apple bitter rot was completely inhibited by B. velezensis GYUN-1190 CF and the positive control tebuconazole, whereas pyraclostrobin exhibited no preventive effect (Supplementary Fig. 3).

Whole-genome sequencing, clusters of orthologous genes annotation, and secondary metabolites of GYUN-1190

The complete genome sequence data of the isolate B. velezensis GYUN-1190 was obtained using a PacBio RSII NGS instrument with a sequencing depth of 179.344×. The generated raw data was assembled using the HGAP2 protocol to obtain a FASTA file consisting of a single contig. The genome of strain GYUN-1190 consists of a circular chromosome (contig 1) measuring 4,240,653 bp, with a total of 4,142 predicted protein coding sequences (CDS), 28 rRNA genes, and 87 tRNA genes (Fig. 6A). The genome also has an average G + C content of 45.9% and was annotated using the NCBI Prokaryotic Genome Automated Annotation Pipeline (PGAAP) analysis. In the gray area of the genome map, the first circle represents the CDS of the forward strand, the second circle represents the CDS of the reverse strand, the third circle represents the location of tRNA and rRNA genes, and the fourth circle represents the GC skew used as a reference point: higher values are shown in green and lower values are shown in red. The fifth circle represents the GC ratio metric. Values above the average GC ratio are colored blue, and values below the average are colored yellow. GC skew and GC ratio are displayed at 10 kB intervals.

Fig. 6

Whole-genome map and clusters of orthologous genes (COG) annotation of Bacillus velezensis GYUN-1190. (A) In the gray area of the genome map, the first circle represents the coding sequence (CDS) of the forward strand, the second circle represents the CDS of the reverse strand, the third circle represents the location of tRNA and rRNA genes, and the fourth circle represents the GC skew used as a reference point: higher values are shown in green and lower values are shown in red. (B) Distribution of orthologous genes in the GYUN-1190 and B. velezensis FJAT-52631, NRRL B-41580, YAU B9601-Y2. The Venn diagram shows the summary of unique SNPs from the total genes of the GYUN-1190. This analysis exploits all CDS of the genomes and is not restricted to the core genome. (C) The COG function annotation of GYUN-1190, and distribution of genes in different COG function categories. (D) Analysis of secondary metabolites of B. velezensis GYUN-1190 using the antiSMASH program.

The Venn diagram (Fig. 6B) shows the comparison of the common CDSs between B. velezensis GYUN-1190 and B. velezensis FJAT-52631, NRRL B-41580, and YAU B9601-Y2. Between them, 3,465 genes were found to be identical. We compared all the predicted protein sequences of GYUN-1190 with the protein sequences in the clusters of orthologous genes (COG) database to identify homologous amino acid sequences in the database (Fig. 6C). According to the COG annotation, the 3,623 proteins in GYUN-1190 were categorized into 20 COG families. The largest gene groups were involved in general function prediction (1,185 genes) and amino acid transport and metabolism (285 genes). A total of 88 proteins were involved in secondary metabolite biosynthesis. Furthermore, according to the antibiotic and secondary metabolite analysis of the GYUN-1190 genome, 14 gene clusters were involved in the secondary metabolism of the strain, and 5 gene clusters were involved in cyclic lipopeptides synthesis via non-ribosomal peptides (NRPs): locillomycin, surfactin, bacillaene, fengycin, and bacillibactin. In addition, gene clusters were likely involved in the secondary metabolites of difficidin and macrolactin H with antibacterial activity (Fig. 6D).

Discussion

Anthracnose is a significant disease caused by the Colletotrichum species complex, leading to severe damage to various fruits and vegetables, including apples, and resulting in substantial economic losses in crops (Guo et al., 2022; Víchová et al., 2012). The primary objective of this study was to evaluate the efficacy of a BCA against apple bitter rot (Kim et al., 2021). The management of apple bitter rot caused by Colletotrichum sp. has primarily involved the extensive use of synthetic fungicides such as mancozeb, carbendazim, prochloraz, and Tecto 60 by farmers (Chechi et al., 2019; Sengupta et al., 2020). Nevertheless, the effectiveness of chemical control methods against fungal diseases is generally limited due to the emergence of resistance mechanisms to these fungicides (Cao et al., 2018). Therefore, the utilization of biological control represents a promising alternative approach. B. velezensis, classified as an antagonistic bacterium within the genus Bacillus, possesses the capability of producing an extensive array of antimicrobial compounds as secondary metabolites that are effective against a variety of phytopathogens (Koilybayeva et al., 2023; Romero et al., 2007). In recent times, additional Bacillus species have been identified as having antifungal properties against plant pathognes (Ashwini and Srividya, 2014; Fan et al., 2017). The use of BCAs to control bitter rot in apples, however, has been the subject of limited research. This is the first report on the utilization of the B. velezensis GYUN-1190 as a potential BCA of apple bitter rot, caused by C. fructicola.

The growth of plant pathogens is inhibited by various secondary metabolite such as bacteriocins, antimicrobial peptides, lipopeptides, polyketides, and siderophores, are produced by Bacillus (Caulier et al., 2019; Raaijimakers et al., 2010; Sarwar et al., 2018). These compounds disrupt the life cycle of fungal phytopathogens, inhibiting their growth (Pliego et al., 2011). Of 33 strains examined in our study, six strains have demonstrated an in vitro antagonistic effect against C. fructicola. In recent studies, it has been documented that a number of Bacillus species exhibit display antagonistic properties against a wide range of Colletotrichum species, including C. fructicola (Heo et al., 2024). A similar pattern has been observed regarding the growth of mycelia in vitro in response to VOCs produced by six different Bacillus strains. Nevertheless, GYUN-1190 exhibited a significantly higher level of mycelium inhibition relative to the other five strains. This result is supported by a recent study conducted by Ghazala et al. (2022), which revealed that the VOCs produced from Bacillus mojavensis 14 exhibited inhibitory effects on the growth of multiple fungal phytopathogens. The beneficial effects of VOCs produced by soil microorganisms on plants have been extensively documented (Gao et al., 2017; Poveda 2021). This study investigated a potential alternative for chemical fungicides by demonstrating biological control of the B. velezensis GYUN-1190 isolated from soil against the apple bitter rot pathogen, C. fructicola. The results demonstrated that GYUN-1190 effectively inhibited the mycelial growth and spore germination by CF or VOCs in vitro.

We found that the GYUN-1190 CF had the strongest inhibitory effect on the mycelial growth of C. fructicola. CFs of other Bacillus species have been found to contain antifungal lipopeptide antibiotics and proteins (Goswami and Deka, 2019). A recent report by Xia et al. (2023) demonstrated that the CF derived from Bacillus subtilis YL13 exhibited antagonistic activity against C. fructicola. The inhibitory effect of CF could potentially be attributed to the existence of diverse antifungal compounds (Ehteshamul-Haque and Ghaffar 1993). Similarly, Khan et al. (2018) documented that CFs derived from Bacillus spp. demonstrated noteworthy antifungal properties against Fusarium spp. Conversely, CFs from Bacillus licheniformis MH48 (Jeong et al., 2017) and B. subtilis YL13 (Xia et al., 2023) inhibited the growth and spore germination of C. gloeosporioides and C. fructicola, respectively. In our study, the CF of GYUN-1190 completely inhibited the growth of C. fructicola. On the other hand, pyraclostrobin, the positive control employed in this investigation, did not demonstrate an inhibitory effect on growth of across all conducted experiments. The observed outcomes are consistent with the emergence of fungicide-resistant strains, which was previously documented (Zhang et al., 2014).

Additionally, the effectiveness of B. velezensis as a biocontrol agent against Colletotrichum species, which are exceptionally destructive pathogens that impact a wide range of crops, including apples was investigated in the study. In apples, B. velezensis has been shown to inhibit anthracnose caused by C. gloeosporioides (Kim et al., 2021). Previous research has demonstrated the efficacy of Paenibacillus polymyxa in inhibiting anthracnose of apple in planta through the application of cell suspensions (Kim et al., 2016). Similarly, Kim et al. (2009) documented in a prior study that Paenibacillus spp., produce a variety of secondary metabolites, including antibiotics, siderophores, hydrogen cyanide, and enzymes. A recent report by Yuan et al. (2022) investigated the biocontrol activity and possible mechanism of B. velezensis strain P2-1 against Botryosphaeria dothidea in postharvest apple fruits. A multitude of approaches have been documented to elucidate the biocontrol mechanism through which Bacillus spp. afflict plant diseases (Chen et al., 2020; Fan et al., 2017; Liu et al., 2020). Several species of Bacillus have been observed to inhibit the growth of diverse fungal pathogens. In addition, further investigation is necessary to understand the mechanisms of action and signaling pathways associated with secondary metabolites that target pathogenic fungi.

Additionally, the complete genome sequence of the strain GYUN-1190, comprising 4,240,653 bp of chromosomes is presented in this study. Using whole genome analysis, the genome of B. velezensis GYUN-1190 was compared to those of other species. Gene clusters that are linked to biological control and antibiotic resistance may potentially account for the variations in efficacy and targets of biological control exhibited by B. velezensis GYUN-1190 in comparison to other Bacillus strains. Based on the findings, it can be concluded that the disease prevention genes associated with the synthesis of secondary metabolites are present in each strain. We checked the presence of secondary metabolites in strain GYUN-1190 using antiSMASH, which revealed the presence of 14 secondary metabolites encoding NRPs, including five antimicrobial peptides (surfactin, fengycin, bacillibactin, bacilysin, and subtilin), polyketide antibiotics (bacillaene and difficidin), and a macrolide antibiotic (macrolactin H), all of which were found in contig 1 with 100% similarity. They contribute to the antibiosis-mediated suppression of microorganisms (Wu et al., 2023). Similarly, in a recent report by Ding et al. (2021), genomic analysis of the strain B. velezensis GUMT319 isolated from the rhizosphere of healthy tobacco plants revealed 13 putative gene clusters involved in the biosynthesis of secondary metabolites with potential antimicrobial activities responsible for non-ribosomal peptide synthetases, trans-acyl transferase polyketide synthases (transAT-PKSs), and one encoded type III polyketide synthase.

In conclusion, the strain GYUN-1190, isolated from the soil demonstrated remarkable effectiveness in inhibiting C. fructicola, the causative agent of apple bitter rot. Both cell suspensions and CF of GYUN-1190 exhibited efficacy in impeding the germination of pathogen spores and the development of apple bitter rot. In particular, it was confirmed that GYUN-1190 is an antagonistic bacterium with potent antimicrobial substances that completely inhibit the pathogen when using GYUN-1190 CF. It was determined to be B. velezensis GYUN-1190 through whole genome analysis, and the secondary metabolite gene cluster of GYUN-1190 was also identified. Our findings suggested that GYUN-1190 could be a potentially effective BCA to control phytopathogens in an eco-friendly manner. Subsequent research endeavors will employ proteomics and transcriptomics techniques to investigate the signaling pathways implicated in the detrimental impacts of secondary metabolites, such as fengycin, and bacillomycin, on pathogenic fungi.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This work was supported by a Research Grant of Andong National University.

Electronic Supplementary Materials

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

PPJ-OA-05-2024-0076-Supplementary-Fig-1.pdf

PPJ-OA-05-2024-0076-Supplementary-Fig-2.pdf

PPJ-OA-05-2024-0076-Supplementary-Fig-3.pdf

PPJ-OA-05-2024-0076-Supplementary-Table-1.pdf

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