The pepper cultivar Capsicum chinense is a local germplasm of the domesticated species, cultivated exclusively in Hainan Island, China (Hu et al., 2020). C. chinense is widely utilized for preparing canned sauce, a favorite commercial product among tourists. The increased production of C. chinense in Hainan has led to numerous plant disease outbreaks. Notably, five plant viral diseases, including the chilli veinal mottle virus (ChiVMV, Potyvirus), pepper veinal mottle virus (PVMV, Potyvirus), chilli ringspot virus (ChiRSV, Potyvirus), tobacco mosaic virus (TMV, Tobamovirus), and cucumber mosaic virus (CMV, Cucumovirus), are prevalent in the Hainan province fields, causing considerable economic losses over the last decade. PVMV was initially identified in Eastern Ghana in 1971 (Brunt and Kenten, 1971) and belongs to the genus Potyvirus, family Potyviridae, sharing characteristics with other potyviruses (Alegbejo and Abo, 2002; Ha et al., 2008; Matsumoto et al., 2016). PVMV infects a variety of hosts, such as pepper, tomato, tobacco, eggplant, petunia, Solanum nigrum L., S. integrifolim Poir., Datura metel, D. stramonium, Physalis angulata, and P. micrantha (Brunt et al., 1978; Givord, 1982; Atiri and Ligan, 1986; Alegbejo, 1999). Liang et al. (2015) reported a PVMV infection incidence of 74.07% in Wenchang and Wanning, Hainan, which significantly impacts pepper production and fruit quality (Fajinmi and Odebode, 2010). Despite its prevalence, effective pesticides and chemical treatments against PVMV are limited, necessitating the development of novel antiviral agents and resistant varieties.

Currently, effectively controlling the virus once a plant has been infected remains a challenge for farmers. The primary methods for virus control involve cultivating resistant varieties, developing and utilizing natural products, and using chemicals and synthetic compounds. The potential concept of chemically mediated plant virus control relies on compounds that activate the plant immune system. When locally infected with a necrotizing pathogen or non-pathogen, plants often develop long-lasting, broad-spectrum “immunity” against subsequent infection (Kunz et al., 1997). Benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) is a synthetic compound capable of inducing disease resistance in several dicotyledonous and monocotyledonous plant species (Friedrich et al., 1996). Friedrich et al. (1996) found that BTH cannot cause the accumulation of salicylic acid (SA) but can induce disease resistance and nahG gene expression, thus activating the SAR signal transduction pathway at the site or downstream of SA accumulation. These results demonstrate the disease control mechanism of BTH by which it activates SAR. Dufulin is a novel antiviral agent that is highly effective against plant viruses and is widely used to prevent and control viral diseases in tobacco and rice in China (Song et al., 2009). Chen et al. (2012) found that HrBP1 is a target protein of dufulin, which can also activate the SA signaling pathway to induce host plants to generate antiviral responses.

However, in recent decades, plant growth-promoting rhizobacteria (PGPRs) have been shown to interact symbiotically and synergistically and effectively colonize the rhizosphere (Ongena and Jacques, 2008). They represent a mutually helpful plant–microbe interaction. Plant growth is enhanced by PGPR through antibiosis, the induction of systemic resistance, and competitive multiplication. The most crucial biocontrol trait of PGPR is their ability to trigger an immune reaction in plant tissues, leading to a systemically expressed resistance state that renders the host less susceptible to subsequent infection (induced systemic resistance, ISR) (Mariuotto and Ongena, 2015; Pršić and Ongena, 2020). PGPR can be beneficial to plants and can perform the same function as chemical fertilizers, pesticides, and elicitors do. Bacillus is an important and well-characterized model organism of plant growth-promoting rhizobacteria. Bacillus subtilis belongs to a new class of MAMPs. It can effectively inhibit plant activity and ISR, which act as elicitors of plant immunity (Boutrot and Zipfel, 2017; Radhakrishnan et al., 2017). Bacillus also produces various metabolites such as lipopeptides, hydrolytic enzymes, and bacterial volatile compounds (BVCs) (Cazorla et al., 2007; Wang et al., 2018; Rajaofera et al., 2019; Jin et al., 2020a, b). Notably, Bacillus spp. synthesize antibiotic lipopeptides, including surfactin, iturin, and fengycin (Hashem et al., 2019). Fengycin and surfactin can interact with plant cells as bacterial determinants to turn on an immune response through the stimulation of the induced systemic resistance phenomenon. García-Gutiérrez et al. (2013) showed that ISR is activated by Bacillus spp. through the induction of the synthesis of jasmonic acid, ethylene, and NPR1 regulatory genes in plants. Some studies have indicated that Bacillus spp. produce secondary metabolites, such as lipopeptides, which can stimulate and initiate the activities of key enzymes of the oxylipin pathway in tomatoes (Blée, 2002). The phenolic or phenylpropanoid metabolic pathway is also well known to be stimulated concomitantly by the activation of plant defense reactions (Dixon et al., 2002). Ongena et al. (2005) found that when potato tuber cells were treated with fengycins produced by Bacillus, the accumulation of plant phenolics was involved in or derived from phenylpropanoid metabolism. Jayaraj et al. (2004) reported that phenylalanine ammonia-lyase, peroxidase, and de novo protein synthesis in plants was activated; these enzymes were produced when Bacillus was applied to plants. Furthermore, Bacillus and BVCs exhibit potent antiviral activities against various plant viruses, including cucumber mosaic cucumovirus (CMV), tomato mottle virus (TMV), pepper mottle virus (PepMoV), tomato yellow leaf curl virus (TYLCV), and tomato spotted wilt virus (TSWV) (Ongena and Jacques, 2008; Kong et al., 2018). In addition, Wu et al. (2017) found that B. amyloliquefaciens FZB42 strains produce two types of cyclodipeptides that can induce resistance against TMV infection in N. benthamiana by activating the SA-mediated plant defense pathway. Bacillus spp. are promising candidates for broad-spectrum antiviral therapy (Ongena and Jacques, 2008). However, despite significant advances made over the past few decades in understanding the regulation of hormonal modulation by PGPR and the induction of acquired systemic resistance in plants (Pieterse et al., 2012, 2014; Köhl et al., 2019), we are still far from having a clear picture of the intricate immune-related molecular events and resistance pathways induced by biocontrol microorganisms.

In a previous study, the cyclic lipopeptide C₁₅ surfactin, isolated from Bacillus velezensis HN-2, was shown to induce systemic resistance to pathogens in plants, thereby contributing to their biocontrol activity. Here we present a novel approach wherein Bacillus velezensis HN-2 is capable of eliciting an immune response in plant tissues, resulting in systemic resistance against PVMV and exhibiting strong colonization. This suggests potential for future field applications in managing plant viral diseases.

PVMV stands as a prominent plant viral ailment in Hainan province’s fields, causing substantial economic losses since its recent emergence. Limited effective pesticides and chemical treatments exist to combat PVMV infection. Previous studies have shown that Bacillus spp. can promote plant growth, and its metabolites can significantly inhibit the effects of plant pathogens, which are commonly used in plant disease management (Fira et al., 2018; Abdelkhalek et al., 2020). These metabolites offer a wide range of secondary compounds that may stimulate plant ISR while impeding pathogen growth (Fira et al., 2018; Vurukonda et al., 2018). Although ISR using Bacillus has shown promise against various plant viruses, such as TMV, CMV, and PVY (Murphy et al., 2000, 2003; Wang et al., 2009), its efficacy against PVMV remains unexplored. In this study, we assessed the antiviral properties of Bacillus velezensis HN-2 and its chemical counterparts. Our study revealed that treatment with B. velezensis strain HN-2 reduced the PVMV infection rates, symptoms, and disease severity in Capsicum chinense, correlating with reduced virus quantities in the leaves. These findings underscore the potent control efficacy of B. velezensis HN-2 against PVMV infection in C. chinense, further affirming its role as a plant growth-promoting rhizobacterium capable of inducing plant-induced systemic resistance against PVMV by eliciting JA production.

Benzothiadiazole and dufulin are exogenous chemical small molecule plant disease activators that lack antimicrobial activity in vitro but induce the accumulation of reactive oxygen and phenolics, enhance defense enzyme activity, induce disease-course-related protein expression in plants, activate the SA signaling pathway, and prompt SAR antiviral immune mechanisms to improve the inhibition of viral infection (Chen et al., 2012; Song et al., 2014; Zhou and Wang, 2018; Huang et al., 2020; Wang et al., 2021). While chemical elicitors effectively manage viral diseases, they often result in significant growth penalties in many cases (Heil and Baldwin, 2002). Previous studies by Azami-Sardooei et al. (2013) observed no changes in plant size or yield of tomatoes treated with BTH relative to that in the controls, except for low-level leaf stunting and slight leaf scorching at high concentrations (1,000 mg L^-1). However, in cucumbers and beans, 100 mg L^-1 BTH treatment reduced the plant size, growth rate, and flower and fruit numbers. Spychalski et al. (2021) demonstrated that BTH-mediated induction of SAR may alter plant resource allocation, leading to a growth–immunity trade-off and potentially reducing yield. Furthermore, Yu et al. (2021) found that dufulin exposure affected the fatty acid transport involved in carnitine formation, altering free fatty acid concentrations and Tubifex’s oxidative damage response. Moreover, dufulin may act on the urea cycle by inhibiting ASL, causing urea cycle disorder. However, the long-term toxic effects of BTH and dufulin on plants and the environment remain unclear. Conversely, B. velezensis is recognized as an important biocontrol strain capable of triggering disease resistance across various plant species while being environment friendly, as it completely degrades in soil without residue (Ye et al., 2018).

Recent studies have emphasized that B. velezensis can induce plant disease resistance through various cluster genes involved in the synthesis of non-ribosomal and ribosomal secondary metabolites, volatile compounds, and cyclic lipopeptides with antimicrobial properties; these genes also act as stimulators of ISR (Ye et al., 2018; Borriss et al., 2019). A previous study revealed the potent inhibitory effect of B. velezensis strain PEA1 against F. oxysporum and CMV infections, suggesting its potential as a biocontrol agent (Abdelkhalek et al., 2020). Vinodkumar et al. (2018) reported the effective reduction of TSV incidence by B. amyloliquefaciens (VB7) under field and glasshouse conditions. Similarly, Lee and Ryu (2016) observed no reduction in fruit yield with Bacillus amyloliquefaciens strain 5B6 treatment compared to that in water-treated control plants. However, BTH treatment consistently reduced fruit yield in pepper. Moreover, few studies have directly compared the efficacy of biological controls and chemical inducers of plant immunity (such as dufulin and BTH) against plant viruses. In the present study, we selected BTH and dufulin as positive controls and revealed that the primary mechanism of B. velezensis HN-2 resistance to PVMV is the induction of systemic resistance. Under greenhouse conditions, soil and plant root irrigation with B. velezensis strain HN-2-GFP, combined with foliar application of benzothiadiazole and dufulin 48 h before viral inoculation, significantly (P < 0.05) reduced the disease symptoms and PVMV accumulation levels in treated tobacco plants compared to those in the PVMV-, BTH-, or dufulin-treated plants at 7 dpi. Visualization of green fluorescent signals in the inoculated leaves treated with B. velezensis HN-2 revealed much lower intensities at 5 and 7 dpi compared to those in negative and positive controls. This reduction was accompanied by mosaic patterns and severe shrivel symptoms in the negative and positive controls, while no evident symptoms were observed in the mock or B. velezensis HN-2 extract-treated tobacco plants.

In DAB staining, 3,3-diaminobenzidine was used to visually detect H₂O₂, as ROS generation is often associated with plant cell death (Zhu et al., 2013). Oxidized DAB precipitates as a brown color at the peroxidase site and is observable via light microscopy (Hans et al., 1997; Liu et al., 2014; Zhu et al., 2016). Furthermore, with increasing processing time, the leaves of plants treated with PVMV, BTH, and dufulin gradually darkened. By the 7th day, the PVMV-treated leaves exhibited substantial browning, whereas those treated with BTH and dufulin displayed dark brown precipitates covering approximately two-thirds of the leaf area. Conversely, HN-2-treated leaves showed the least browning compared to the negative and positive controls, with water-treated leaves appearing almost transparent. These DAB staining results were consistent with the results of green fluorescence analysis. Western blot analysis of young leaves at 15 dpi also showed similar results. These results strongly suggest that B. velezensis HN-2 not only exerts a strong antiviral effect but also exhibits sustained efficacy. This may be attributed to the B. velezensis HN-2-mediated induction of plant ISR, conferring robust resilience in N. benthamiana against PVMV-GFP infection and sustaining its antiviral effects over time.

Plants exhibit specific resistance mechanisms when faced with adverse conditions. HR refers to programmed cell death triggered by an exaggerated response at the infection site. Various reports have indicated that HR causes rapid death of plant cells, which can effectively limit pathogen growth at the infection site, thereby preventing further spread to the surrounding healthy tissues and activating plant resistance responses (Guo et al., 2020; Wang et al., 2021). Viral infections can induce a burst of ROS in host plants, resulting in oxidative stress (Yang et al., 2018). Notably, H₂O₂, a type of ROS, serves as an early mediator of plant resistance (Wu et al., 2015). Accordingly, the rapid generation of ROS is a preliminary process by which plants respond to pathogen challenges, with H₂O₂ being capable of initiating cell death during the HR response (Delledonne et al., 2001). Plant cells defend themselves against the oxidative damage caused by ROS through the production of antioxidant enzymes, such as SOD, POD, CAT, MDA, and PAL, which play important roles in plant defense against pathogens and counteract viral infection (Zhang et al., 2016; Lv et al., 2020). Guo et al. (2019) found that following viral infection, POD, PPO, and PAL activities increased more rapidly in the leaves of Ba13-treated plants than in those of the controls. The antiviral activity of Bacillus velezensis PEA1 induces systemic resistance to CMV, with significantly elevated transcriptional levels of PAL, CHS, HQT, PR-1, and POD (Abdelkhalek et al., 2020). Lv et al. (2020) reported that an increase in SOD, POD, CAT, and MDA activities leads to the activation of the plant immune system to combat TMV infection. Similarly, our study revealed that HN-2 treatment reduced PVMV-induced oxidative damage by increasing SOD, POD, and CAT activities compared to those in the blank and negative controls, thereby achieving antiviral effects. These results suggest that SOD, POD, and CAT eliminate excess peroxides, thereby preventing oxidative damage. In contrast, the activity of MDA after HN-2 treatment was lower than that of the blank and negative controls, with no significant difference in activity observed from 1 to 5 dpi following pHNu-GFP inoculation. Following stress exposure, the upregulation of CAT can minimize oxidative stress, which plays a critical role in preventing oxidative damage and protecting plant cells from the oxidative damage caused by ROS (Blackman and Hardham, 2008; Abdelkhalek et al., 2021). In our experiments, the expression of Cat1 remained consistent across all treatments at 1 and 3 dpi, with rapid accumulation observed in the HN-2-treated plants at 5 dpi, aligning with changes in CAT enzyme activities, demonstrating the efficacy of B. velezensis HN-2 in protecting cells from ROS-induced damage, and inducing long-lasting systemic resistance in N. benthamiana following primary inoculation.

SAR and ISR are two major forms of plant defense, wherein the plant hormones SA, JA, ABA, and ET play key roles in regulating the signaling networks associated with plant defense against pathogens. SA is essential for SAR activation in tissues distal to the infection site, whereas jasmonate and ethylene are required for ISR (Zhu et al., 2016). Abdelkhalek et al. (2020) demonstrated that SAR induced B. velezensis PEA1 against CMV-activated gene expression and enzyme activity related to systemic resistance while inhibiting infection. ISR is recognized as an effective biological control method for inducing plant defense against a broad range of pathogens (Nie et al., 2017). JA serves as a regulatory factor of ISR (Zhou and Wang, 2018); previous studies have shown that B. amyloliquefaciens Ba13 can enhance plant resistance against TYLCV disease by directly inducing systemic resistance and increasing the number of beneficial microbes in the rhizosphere (Guo et al., 2019). In the present study, the simultaneous determination of JA and SA revealed that the SA content under the HN-2 treatment gradually increased, reaching a peak at 3 dpi, and subsequently decreased. At 5 dpi, the blank control and BTH-treated groups had 1.33- and 1.37-folds higher SA contents than the HN-2-treated group, respectively. Meanwhile, the expression of JA rapidly increased after 3 dpi and peaked at 5 dpi in the HN-2-treated plants; it was 7.81-, 2.22-, and 4.29-folds higher than that in the blank control and BTH- and dufulin-treated groups, respectively. These results indicate that B. velezensis HN-2 treatment significantly elevated JA expression, potentially activating resistance-related gene expression and defense enzyme activity to enhance plant-induced systemic resistance, thereby aiding in the control of PVMV infection.

Rbohs are key enzymes responsible for ROS production in response to hormonal and environmental signals in plants and play crucial roles in plant growth, development, and stress responses (Suzuki et al., 2011; Sun et al., 2015). ROS levels are elevated by Rboh enzymes in response to multiple biotic and abiotic stresses (He et al., 2017; Liu et al., 2017; Zhai et al., 2018; Chapman et al., 2019). In the present study, the expression of Rboh under the HN-2 treatment increased by 1.89-, 1.18-, and 1.34-folds at 1, 3, and 5 dpi, respectively, compared to that in the blank control group but remained lower than that in the BTH- and dufulin-treated groups. These results suggest that B. velezensis strain HN-2 may potentially maintain Rboh in a low-expression state to restrict ROS production and consequently reduce PVMV-induced oxidative damage. PAL is a key regulatory enzyme of the phenylpropanoid pathway and is involved in the production of polyphenolic compounds (Guo et al., 2020). POD and PAL play important roles in plant defenses against pathogens. Wang et al. (2012) observed that tobacco plants with excessive expression of the PAL enzyme exhibited strong resistance to TMV infection, whereas plants with inhibited enzyme activity were more susceptible to TMV. Interestingly, in the present study, PAL expression was significantly increased under the HN-2 treatment, peaking at 24 h before reducing to near-normal levels at 3 dpi, and subsequently increased by 25.58-, 15.33-, and 6.47-fold compared with BTH- and dufulin-treated controls at 5 dpi, respectively. Conversely, POD activity was lowest at 1 dpi and increased rapidly at 3 dpi, which indicated that PAL expression was induced in advance following virus infection and continued to be induced at 5 dpi, thereby enabling sustained POD activity after 3 dpi. At 5 dpi, the HN-2 treatment demonstrated the highest level at 1,660.31 unit mg^-1 FW, indicating 1.15-, 1.16-, and 1.57-fold increase in activity compared to the BTH treatment, dufulin treatment, and blank control, respectively. This increase in POD activity may activate the plant immune system to combat PVMV infection.

PR proteins are produced in plants during pathogen attacks and constitute vital components of the plant defense mechanism (Sels et al., 2008). Numerous studies have shown that PGPRs boost plant health by improving defense against various pathogens, often associated with JA and ET pathway induction (Beneduzi et al., 2012; Pieterse et al., 2014; Rahman et al., 2015). In tobacco, ISR triggered by non-pathogen PGPR strains is accompanied by the upregulation of PR genes encoding pathogenesis-related proteins, as evidenced by RT-PCR analysis results indicating an increased expression of PR genes upon treatment with B. velezensis (Wang et al., 2009). SAR, demonstrated across many plant species, involves the accumulation of PR proteins (PR-1, PR-2, and PR-5) and SA as its molecular basis (Gao et al., 2014). Niu et al. (2011) found that ISR simultaneously activates the SA- and JA/ET-dependent signaling pathways, leading to the induced expressions of PR1, PR2, PR5, and PDF1.2. NPR1 acts as a regulator of SAR and ISR, mediating the SA–JA crosstalk (Pieterse et al., 2014). The NPR1 (nonexpresser of PR genes) protein serves as a key regulator in transmitting SA and JA/ethylene signals, thus triggering acquired resistance responses (Spoel et al., 2003). Previous studies suggest that the induction of PR1 gene synthesis may be associated with increased NPR1 gene expression and conformational changes in the NPR1 protein (Mou et al., 2003). Nie et al. (2017) showed that root-drench application of Bacillus cereus AR156 significantly reduced disease incidence by activating ISR through a mechanism dependent on NPR1, leading to the expression of downstream defense-related genes, such as PR1, PR2, PR5, and PDF1.2, and activation of cellular defense responses. Similarly, inoculation with B. amyloliquefaciens Ba13 improved the defense ability of tomato plants against TYLCV infection by activating the expression of genes and enzymes related to systemic resistance in tomato—for example, by increasing the expression of the resistance-related genes PR1, PR2, and PR3 (Guo et al., 2019). The expression of PR-1b, PR3, PR5, and NPR1 gradually increased over time in the leaves of HN-2-treated N. benthamiana after PVMV inoculation; we quantitatively analyzed the gene expression at 1, 3, and 5 dpi. Consistent with previous findings, B. velezensis HN-2 treatment significantly increased the transcriptional levels of NPR1, PR-1b, PR3, and PR5, with PR3 and PR5 showing the most significant upregulation compared to those in the blank control. Our experimental results indicated that B. velezensis HN-2 treatment significantly increased PR gene expression and activated plant-induced systemic resistance, thus highlighting the antiviral properties of this strain. From an agronomic perspective, the ISR triggered by PGPR is interesting, given its long-lasting and broad-spectrum protection without growth costs and its minimal potential to promote pathogen resistance (Köhl et al., 2019). In a previous study, Arabidopsis plants were root-drenched with Bacillus cereus AR156 at 5×10⁸ CFU mL^-1, with 10 inoculated plants for each genotype. The results showed that AR156 only colonized the roots and significantly reduced disease incidence through the activation of ISR (Nie et al., 2017). Guo et al. (2019) watered tomato plants with a Bacillus amyloliquefaciens Ba13 suspension at 10⁶ CFU mL^-1; after 3 weeks, post-bacterial inoculation performed viral infection, wherein 65 replicates were sampled for systemic resistance-related assays and virus quantification. It was found that the number and distribution of rhizosphere-dominant bacteria were changed by Ba13 application and can enhance plant resistance against TYLCV disease through the direct induction of systemic resistance. To elucidate the relationship between the colonization of B. velezensis HN-2 in C. chinense roots and its role in plant virus resistance, a total of 600 plants were subjected to root drenches with B. velezensis strain HN-2 at 10⁶ CFU g^-1 under greenhouse cultivation conditions. The results demonstrated that B. velezensis HN-2 rapidly colonized the rhizosphere within 72 h, and with the extension of treatment time, HN-2 mainly colonized the root interior after 5 dpi. Subsequently, the number of colonies in C. chinense root interior increased by fivefold compared to that in the rhizosphere. Western blotting of PVMV-GFP protein accumulation after 60 days of inoculation showed that high levels of GFP protein were detected only in pHNu-GFP treatment, while when treated with benzothiadiazole, dufulin, and B. velezensis HN-2, GFP protein was almost undetectable in plants. Simultaneously, at 60 days after PVMV-GFP inoculation, a comparative experiment was conducted on the antiviral effect of B. velezensis HN-2 on C. chinense plants grown in soil that was irrigated under field conditions. We observed that the growth of C. chinense treated with HN-2 was significantly superior to that of PVMV-infected plants treated with BTH and dufulin, resembling that of the blank control at 180 dpi in the field.

In a word, the data presented in this study indicate that B. velezensis HN-2 functions as a PGPR, rapidly colonizing C. chinense roots and activating the expression of genes and enzymes associated with systemic resistance. When plants were infected with PVMV and treated with B. velezensis HN-2, the JA signaling pathway was activated, leading to increased JA content and the simultaneous activation of the NPR1-dependent mechanism, resulting in the significant upregulation of PR-1b, PR3, and PR5. Moreover, cellular defense responses, including CAT, POD, Rboh, PAL, and CAT1 activities, exhibited a more rapid and significant increase. Furthermore, the increased expression of JA pathway genes suggests the activation of ISR under Bacillus velezensis HN-2 treatment. However, there was no significant increase in SA content, indicating that systemic acquired resistance (SAR) pathway was not induced by this treatment ( Figure 8 ). In conclusion, B. velezensis HN-2 can effectively induce plant ISR-mediated defense responses and enhances plants’ immunity (including C. chinense) by activating the JA signaling pathway, thereby mitigating symptoms caused by PVMV and safeguarding the plants against pathogenic infections.

In summary, Bacillus velezensis HN-2 is a new antiviral agent against plant viruses and plays significant roles in ISR by activating gene expression and enzyme activity related to systemic resistance, thus inhibiting infection. The strain HN-2 may be considered a promising source of plant growth promotion, with highly effective antiviral substances for plant protection and for development against PVMV diseases. This study provides theoretical basis for the green prevention and control of plant virus diseases.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

PJ: Formal analysis, Funding acquisition, Supervision, Writing – review & editing. ZX: Data curation, Investigation, Writing – original draft. YW: Data curation, Investigation, Writing – original draft. YS: Data curation, Methodology, Writing – original draft. XP: Data curation, Investigation, Writing – original draft. JW: Data curation, Investigation, Writing – original draft. WL: Formal analysis, Resources, Writing – review & editing. WM: Supervision, Writing – review & editing.