ARD, which is mainly caused by soil-borne plant diseases, has severely restricted the sustainable development of China's apple industry. Given the environmental pollution and pathogen resistance caused by the overuse or misuse of chemical fungicides, there is an urgent need to develop effective and environmentally friendly methods to replace the use of chemical control methods to suppress the spread of this disease(Yin et al., 2017; Wang et al., 2018; Sheng et al., 2020; Xiang et al., 2021). In recent years, the introduction of beneficial microorganisms into soil has been shown to be an attractive alternative for controlling plant diseases, especially endophytic strains with antibacterial effects or strains that promote plant growth; however, these microorganisms have been rarely used to control ARD (Nan et al., 2011; Deketelaere et al., 2017). Cao et al. (2011) found that B. subtilis SQR 9 isolated from a healthy cucumber root in a field with a high incidence of Fusarium wilt disease can control cucumber wilt by colonizing plant roots. An endogenous B. licheniformis XNRB-3 was isolated from the root tissues of healthy fruit trees in orchards where the incidence of ARD was high; this strain could stably colonize the roots of apple seedlings, and it showed high phloridin-degrading activity and multiple PGP properties. It could also significantly inhibit the mycelial growth and spore germination of Fusarium by producing antifungal compounds. Inhibition of spore germination can protect plant roots from fungal infection and is essential for the development of fungal disease during the early stage (Miyara et al., 2010). This reveals that the strain XNRB-3 has the potential to be used as a biological control agent to control ARD.
Some BCAs that can colonize root tissues without causing harmful effects are known as beneficial endophytes (Cao et al., 2011; Santoyo et al., 2016) they exhibit several disease suppression mechanisms and promote plant growth, including preventing vascular pathogens from colonizing ecological niches; producing hydrolytic enzymes, broad-spectrum antibiotics, PGP hormones, and VOCs; and promoting induced systemic resistance (Santoyo et al., 2012; Chowdhury et al., 2015; Eljounaidi et al., 2016; Afzal et al., 2019). Previous studies indicate that many Bacillus spp. have PGP traits such as IAA production, phosphate solubilization, potassium solubilization, nitrogen fixation, ACC deaminase, and biocontrol attributes such as the production of siderophores, hydrolytic enzymes, HCN, and antibiotics (Penrose and Glick, 2003; Senthilkumar et al., 2009; Kumar et al., 2012; Vacheron et al., 2013). The endogenous B. licheniformis XNRB-3 isolated in this study also has several of the aforementioned PGP properties and antagonistic traits. For example, IAA, CTK, and GA can significantly increase the root length, root tip, and branch number of apple seedlings, The production of IAA can also promote the establishment of a symbiotic relationship between plants and arbuscular mycorrhizal (AM) fungi to improve their adaptability to the external environment (Wahyudi et al., 2011; Liao et al., 2015; Afzal et al., 2017); nitrogen fixation, ACC deaminase, and ammonia production can induce plant resistance and promote plant growth (Penrose and Glick, 2003; Senthilkumar et al., 2009); and phosphate and potassium solubilization can effectively increase the absorption of phosphorus and potassium by plants and promote root development (Richardson, 2001), Therefore, adding the strain XNRB-3 under potted and field conditions can improve the ability of plants to resist pathogens and promote the growth of apple plants. Ryu et al. (2003) found that the bacterial volatile 2,3-butanediol can significantly increase the biomass of Arabidopsis thaliana. Similar results were obtained in our study. Strain XNRB-3 produced butanedioic acid, monomethyl ester, and dibutyl phthalate and had a strong growth-promoting effect on the root system of Arabidopsis. This revealed that strain XNRB-3 can promote root elongation and lateral root development by secreting volatile compounds.
Yu et al. (2011) found that the siderophore-producing bacterium B. subtilis CAS15 has a biocontrol effect on Fusarium wilt. The strain XNRB-3 in this study also has a siderophore-producing function, can directly stimulate the biosynthesis of other antibacterial compounds by increasing the availability of minerals to bacteria, inhibit the growth of pathogenic bacteria, and induce the activity of plant root protection enzymes. The increase in root protection enzyme activity indicates that the root system's defense response against pathogens has been initiated (Ramamoorthy et al., 2001; Joseph et al., 2007; Wahyudi et al., 2011). Strain XNRB-3 can also produce enzymes that dissolve fungal cell walls (cellulose, pectinase, β1,3-glucanase, chitosanase, and protease), antifungal compounds (2,4-di-tert-butylphenol and alpha-bisabolol), and low molecular weight metabolites (HCN), which limits the growth of pathogens and protects plants from phytopathogenic fungi. Among them, cellulases and pectinases are important for the intracellular root colonization of PGP bacteria, as these are hydrolytic enzymes with the ability to degrade cellulose/pectin material of the plant cell wall (Verma et al., 2001). This feature helps strain XNRB-3 better colonize the root system and continue to produce substances that are beneficial to plant growth to promote the development of the root system, which enhances the growth of the aboveground parts of plants. The above results indicate that the endophytic bacterium XNRB-3 has high potential to be used as a biofertilizer and biopesticide and could aid the development of a sustainable, safe, and effective agriculture system.
The production and transportation of BCAs are essential for successful biological control under field conditions (Thangavelu et al., 2004). An appropriate carrier can support the survival of BCAs while inhibiting the growth of target pathogens, thereby improving the performance of BCAs for plant disease control (Ling et al., 2010; Wei et al., 2015). The selection of a suitable vector for strain XNRB-3 is thus necessary for the successful application of BCAs (Malusá et al., 2012). A carrier ideally possesses the following properties: high water-holding capacity, ease of processing, free of lump-forming materials, near-sterile or easy to sterilize by autoclaving or by other methods (e.g., gamma irradiation), high pH buffering capacity, low cost, available in adequate amounts, no toxicity, and environmental safety (Stephens and Rask, 2000; Ferreira and Castro, 2005).Smith (1992) found that dry inoculants can be produced using different types of soil materials (peat, coal, clays, and inorganic soil), organic materials (composts, soybean meal, wheat bran, and sawdust), or inert materials (e.g., vermiculite, perlite, kaolin, bentonite, and silicates). In this experiment, dry inoculants in Table S7 were used to optimize the fermentation conditions of strain XNRB-3 using response surface analysis (RSM), which significantly increased the survival rate and shelf life of strain XNRB-3 and complies with the Chinese bio-organic fertilizer production standard stipulating that the functional microorganism content should be greater than 2.0 × 107 CFU g−1 dry formulation after storage for 6 months at room temperature (Emmert and Handelsman, 1999). The raw materials (cow dung compost and wheat straw) in the formula are cheap and easy to obtain, and the fermentation level is high, which provides a good foundation for its large-scale industrial production. We verified the results under field conditions and found that the addition of optimized XNRB-3 bacterial fertilizer can significantly promote the growth of replanted young apple trees and inhibit the growth of Fusarium in the soil. The abundance of Fusarium in the soil was significantly lower after treatment with the optimized XNRB-3 bacterial fertilizer compared with CK1. This ability to promote plant growth might also stem from the ability of XNRB-3 to enhance the soluble mineral nutrient content and produce indole-3-acetic acid (IAA), as well as its multiple PGP properties and antagonistic traits (Pii et al., 2015) .To improve the effectiveness of the application of XNRB-3 bacterial fertilizer, fertilizer application should be repeated 3–4 times during the growing season with an interval of 2–4 weeks each time (Wei et al., 2015).
The optimized strain XNRB-3 fermentation broth can significantly inhibit the mycelial growth and spore germination of Fusarium, and an abnormal structure of the mycelia (mycelia and conidia breakage, deformity, and dissolution) from the edge of the inhibition zone was observed using scanning electron microscopy (SEM) in vitro assays. This antagonism may be caused by the secreted antifungal compounds (lipopeptides, polyketides, and bacteriocins) (Cawoy et al., 2014; Harwood et al., 2018). Among these compounds, lipopeptides (surfactin, iturin, and fengycin families) show potent antimicrobial activity against a wide variety of microorganisms in vitro, especially filamentous fungi (Bonmatin et al., 2003; Hofemeister et al., 2004; Moyne et al., 2001; Frikha-Gargouri et al., 2017). Aside from their antimicrobial activity, lipopeptides are involved in the attachment to plant surfaces, the formation of biofilms, and the induction of resistance against phytopathogens (Hofemeister et al., 2004; Chen et al., 2013). In our study, non-ribosomal peptide synthetase genes involved in the synthesis of antibiotics were detected in strain XNRB-3 using PCR-based assays, and the antibiotics that can be synthesized include Yndj protein, subtilisin, bacillomycin, iturin A, fengycin, and surfactin, which confer broad-spectrum resistance to plant pathogenic fungi. These findings are similar to the results of Cao et al. (2012). The antibiotics (surfactin, fengycin, iturin, bacillomycin, and subtilosin) produced by B. subtilis SQR 9 significantly inhibit the growth of F. oxysporum, Verticillium dahliae, Phytophthora capsici, and Phytophthora nicotianae. The production of lipopeptides substances might also be one of the important reasons why strain XNRB-3 can form a biofilm on the surface of roots. Besson et al. (1990) found that asparagine appeared to be the optimal precursor among the α-amino acids in the peptidic part of iturin, indicating that the production of amino acids also affects the biosynthesis of peptide antibiotics. In this experiment, capillary electrophoresis was used to detect the content of free amino acids during fermentation by strain XNRB-3. The concentration of four free amino acids (aspartic acid, glutamic acid, proline, and tyrosine) in the extracellular matrix was the highest. This finding is consistent with the results of Ren et al. (2012). This indicates that the production of amino acids might be involved in the biosynthesis of peptide antibiotics.
Recently, Bacillus spp. belonging to PGPR have been shown to promote plant growth and mediate the biocontrol of various pathogens (Cao et al., 2011; Cawoy et al., 2014; Chowdhury et al., 2015; Harwood et al., 2018). However, when applied in the field, they are usually unable to impart these beneficial effects, which might be closely related to the formation of bacterial biofilm and their ability to colonize the rhizosphere and/or roots (Compant et al., 2010; Bhattacharyya et al., 2012). Mendis et al. (2018) found that B. firmus I-1582 and B. amyloliquefaciens QST713 can colonize plant roots, promote plant growth, and provide protection against pathogens/pests, such as F. oxysporum, Pythium aphanidermatum, and Rhizoctonia solani. Nan et al. (2011) found that B. subtilis N11 mainly colonizes the roots by forming a biofilm along the elongation zone and differentiation zone of the roots, thereby protecting the plants from fungal infection. Some studies have found that the presence of lipopeptide biosynthetic genes or the co-production of multiple lipopeptides are important for the colonization of Bacillus and the formation of biofilms (Bais et al., 2004; Frikha-Gargouri et al., 2017). There is thus a need to evaluate the colonization ability of strain XNRB-3 in the root system. Given that strain XNRB-3 has a variety of lipopeptide biosynthetic genes and forms a thick biofilm in a static medium, we also evaluated the colonization ability of the root system by dilution-plate counting in a greenhouse and in the field in non-sterile soil (Mendis et al., 2018). Strain XNRB-3 could colonize plant roots, and its fresh weight ranged from 105 to 107cfu/g within 21 days. This is consistent with the results of Hallmann (2001) indicating that strain XNRB-3 can colonize the roots of apple seedlings, which is critical for its ability to become established in the soil environment after applying it in the field. We also found that watering the strain XNRB-3 in soil pre-infected with Fusarium can significantly reduce the abundance of Fusarium in rhizosphere soil. After 5 weeks, the relative control effect was as high as 51%. These findings are consistent with the results of Cao et al. (2011) showing that the pathogen density in the rhizosphere of cucumber seedlings inoculated with Bacillus subtilis SQR9 was significantly reduced. This result indicated that strain XNRB-3 can stably colonize the rhizosphere of apple seedlings and provide protection to plants. Similar results were obtained in the PAS staining test. The roots treated with the fermentation broth of strain XNRB-3 did not show symptoms of Fusarium infection, which demonstrated that strain XNRB-3 can colonize the roots of the plant and grow root epidermis, forming a biofilm that prevents Fusarium infection and improves the resistance of plants to infection (Duan et al., 2021), Similar results were obtained by Benhamou et al. (1998): seed treatment of tomato with the endophytic bacterium B. pumilus SE 34 prevents the entry of the vascular wilt fungus F. oxysporum f. sp. radicis-lycopersici into the vascular stele, and the mycelial growth is restricted to the epidermis and outer root cortex. Infected roots can also produce a large amount of sticky substances and result in the deposition of formed callose and starch granules to form a mechanical barrier that inhibits the invasion of pathogens (Lagopodi et al., 2002; Grunewaldt-Stöcker et al., 2020). A similar structure was also observed in this experiment.
Soil enzyme activity is often used to monitor changes in soil microbial activity and soil fertility because it is involved in all soil biochemical processes (e.g., soil organic matter formation and degradation; C, N, and P cycling; and plant nutrient transformation); it is also sensitive to changes in soil management (Kandeler et al., 2006; Song et al., 2012). Changes in soil attributes (e.g., SOC, TN, TP, AN, and AP concentrations) are also important indicators of changes in soil fertility and long-term ecosystem sustainability (Pan et al., 2013). Therefore, we evaluated soil microbial activity and fertility status by measuring the activity of soil-related enzymes and soil nutrient attributes after adding strain XNRB-3. The application of strain XNRB-3 significantly increased the activity of soil-related enzymes, and it increased after the second year of applying strain XNRB-3. This is consistent with the results of several previous studies. For example, the addition of PGPR can significantly improve the physical and chemical properties of soil and soil enzyme activity (Ren et al., 2019). PGPR strains can induce the production of lytic enzymes by utilizing carbon from the cell wall of microorganisms, thereby increasing soil urease activity (Karthik et al., 2017). The increase in urease activity indicated that the application of XNRB-3 may increase the gross N mineralization rate because urease can catalyze the hydrolysis of urea into CO2 and NH4+ and promote the soil nitrogen cycle (Xu et al., 2015). The increase in invertase activity can promote the conversion of carbohydrates and increase the concentration of soil nutrients (e.g. N, P, K) under the action of microorganisms and improve soil fertility (Han et al., 2017). Phosphorus (P) plays a key role in crop productivity, and its availability depends on P mineralization from soil organic matter. This enzymatic process is performed by a group of phosphatases, such as AP, which provide inorganic P to the soil solution (Krämer, 2000). Strain XNRB-3 can increase AP activity, which increases the availability of soluble P and promotes plant growth. Many studies have shown that soil phosphatase and urease activity is significantly positively correlated with TN, TP, and SOC, and soil pH is significantly negatively correlated with the activity of soil enzymes (Xie et al., 2017; Ren et al., 2019). Soil sucrase activity is significantly positively correlated with soil available nitrogen (Fu et al., 2018). This finding is consistent with the results of this study; after adding strain XNRB-3, changes in the content of N, P, K, and SOC in the soil and soil pH were similar to changes in the activity of soil-related enzymes. The increase in the nutrient concentration in the soil might also be related to the characteristics of strain XNRB-3 such as nitrogen fixation, phosphorus dissolution, and potassium dissolution. Some soil environmental factors (such as soil temperature, pH, soil aggregate, organic matter, nitrogen, phosphorus, and other nutrients) affect the number and activity of microorganisms and thus affects the activity of soil enzymes (Floch et al., 2009; Qi et al., 2016). The increase in CAT activity after the addition of strain XNRB-3 might be closely related to soil microbial activity (Kabiri et al., 2016). This indicates that the application of strain XNRB-3 can increase the number and activity of soil microorganisms by enhancing soil environmental factors and soil enzyme activity and promoting the growth of young apple trees.
Soil microorganisms play an important role in agroecosystems because they participate in nutrient cycling, the decomposition of organic matter, and the development of soil-borne diseases (Lambers et al., 2009 ), The functional diversity and overall activity of microbial communities in soil can reflect soil quality (Islam et al., 2011). Biological methods are some of the main methods used to measure the functional diversity and overall activity of microbial communities for their simple operation, high sensitivity and resolution, and rich data (Dang et al., 2015; Guo et al., 2015). Therefore, biological methods were used in this study to investigate the functional diversity and carbon source utilization of the rhizosphere soil microbial community after treatment with strain XNRB-3, as well as evaluate the safety of its use in the soil environment. The AWCD value of rhizosphere soil was significantly higher after the addition of strain XNRB-3 compared with other treatments; the addition of strain XNRB-3 also significantly enhanced the use of carbon sources such as polymers, carboxylic acids, and amino acids, which might be related to the increase in the number of soil bacteria and actinomycetes after the addition of strain XNRB-3. Biolog GEN III microplate identification revealed that strain XNRB-3 can use a wide range of carbon sources, which permits this strain to grow and reproduce in environments with different nutrient levels (Schutter and Dick, 2001).
Previous studies have shown that the occurrence of ARD is closely related to the structure and diversity of soil microbial communities. Increases in the number of rhizosphere soil pathogens and decreases in the number of beneficial microorganisms are also important factors leading to disease outbreaks (Mazzola and Manici, 2012; Kelderer et al., 2012). The T-RFLP data from this study showed that strain XNRB-3 can significantly alter the structure of the rhizosphere soil fungal and bacterial communities after treatment; compared with the treatment with strain XNRB-3 addition, the community structure observed in CK1 and T1 differed, but the community structure observed in the fumigation treatment was similar. Application of this strain also significantly increased the abundance and diversity of rhizosphere soil bacteria and fungi, reduced the relative abundance of Fusarium, provided a stable and beneficial rhizosphere ecosystem for plants, and promoted plant growth. These results were consistent with the conclusion of Liu (2011), who indicated that adjustment of soil microbial community structure and the abundance of soil microbes can reduce disease caused by continuous soil cropping. The addition of strain XNRB-3 may improve the soil microbial environment by promoting the aggregation of some beneficial microorganisms or the secretion of some VOCs (Romoli et al., 2014; Liu et al., 2021). Soil microbial VOCs have been shown to promote plant growth (Ryu et al.2003) and inhibit bacterial and fungal growth (Fernando et al., 2005). This finding is consistent with the results of our research. The volatile antibacterial compounds in the fermentation broth of strain XNRB-3 can not only strongly inhibit the growth of pathogenic fungi but can also promote root growth. Bacterial VOCs can also decrease soil fungal biomass and increase soil bacterial biomass (Yuan et al.,2017), The same pattern was observed in this study: after the addition of strain XNRB-3, the number of soil bacteria significantly increased, and the number of fungi significantly decreased. Strain XNRB-3 can also reduce the damage of ARD to the replanted young apple trees by improving the soil microbial environment.
Imbalances in soil physical and chemical properties and the allelopathy of root exudates and residues are considered the main causes of soil sickness (Pant et al., 2013; Zhang Y et al., 2010). Yin et al. (2017) found that the roots of apple plants under continuous cropping can secrete the same substances (such as phenolic acid autotoxic substances) for a long time, and these substances significantly affect the composition and distribution of the rhizosphere microflora, increasing the number of pathogenic fungi and inhibiting plant growth (Liu H et al., 2019; Xu et al., 2020). Phenolic substances related to ARD currently known mainly include 2,4-di-tert-butylphenol, vanillic acid, benzoic acid, p-hydroxybenzoic acid, ferulic acid, cinnamic acid, and phloridzin (Ye et al., 2006; Qu and Wang, 2008; Yin et al., 2013, 2017; Chen et al., 2021). Phlorizin is a unique phenolic acid substance of apples that mainly exists in the roots, stems, bark, tender leaves, and fruits of apples. The high concentration of phlorizin can significantly inhibit the growth of apple seedlings and reduce the rate of plant photosynthesis and transpiration (Ehrenkranz et al., 2005; Zhang Y et al., 2010). Ye et al. (2006) found that ROS-induced membrane damage caused by cinnamic acid can facilitate the attack and colonization of the vascular bundle system of roots by pathogenic Fusarium. The use of microbial degradation methods to degrade phenolic acids in the environment is becoming increasingly popular because of its various advantages, including low cost, high degradation efficiency, lack of secondary pollution, and environmental safety (Ma et al., 2017; Wang et al., 2021), Use of the medium containing phloridzin as the sole carbon source revealed that strain XNRB-3 can efficiently degrade phloridzin; this strain can also effectively degrade phloridzin, cinnamic acid, ferulic acid, benzoic acid, and p-hydroxybenzoic acid in soil and culture fluid, thereby promoting the growth of apple seedlings. This finding is similar to the results of Zhang Y et al. (2010), which used a screening medium containing p-coumaric acid as the sole carbon source. Four microbes were isolated from plant soils, and these microbes could effectively degrade ferulic acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde and promote seedling growth. Phloridin is degraded in the soil in two main ways. The first is through the hydrolysis of phloretin into phloroglucinol and p-hydroxyphenylpropionic acid by secreting a phloretin hydrolase, followed by decomposition to phloretin and glucose by β-glucosidase, which is then used by bacteria (Chatterjee et al., 1969), Alternatively, it can be degraded to pyruvic acid by the protocatechuic acid pathway in Pseudomonas (Mohan and Phale, 2017), and pyruvic acid can be converted to acetyl CoA and enter the tricarboxylic acid cycle, which produces organic acids, such as citric acid, succinic acid, malic acid, and oxaloacetic acid (Priefert et al., 2001; Zhang Y et al., 2010), These substances play an important role in promoting the absorption and transportation of certain nutrients and improving the photosynthetic efficiency of plants and the accumulation of nitrogen, phosphorus, and potassium (Liu et al., 2005). Therefore, the method of phloridin degradation in the soil environment by the strain XNRB-3 is thought to be an effective approach for overcoming the obstacles of continuous apple cropping.