Rice (Oryza sativa L.) is one of the world’s major food crops and also serves as a staple food for more than half of the world’s population (Zeng et al., 2017; Huang et al., 2021). During its life cycle, rice can be attacked by a variety of microbial pathogens, thus causing huge economic losses to agricultural production while restricting the healthy and stable development of the rice industry. In particular, rice blast has a devastating effect on rice production. Rice blast is a worldwide rice disease caused by the ascomycete fungus (Magnaporthe oryzae). The disease affects rice all year round, producing lesions on leaves, leaf necks, culms, culm nodes, spike nodes and spike necks. These lesions cause leaf blast, neck blast, and spike blast and the development of spots. The color and shape of the spots vary depending on environmental conditions and the developmental stage of the plant (Law et al., 2017).

Rice fungus is a filamentous ascomycete fungus that exists in two hybrid types: MAT-1 and MAT-2. This fungus can form sexual substrates called ascospores within 21 days with flask-shaped mycelium carrying a large number of ascospores (Talbot, 2003; Ryder and Talbot, 2015). Rice blast begins when asexual spores land on the surface of rice leaves and attach themselves to the cuticle by releasing an adhesive in the apical spacing of the spores (Hamer et al., 1988). The disease causing process can be divided into the following five steps: conidia formation, budding, attachment cell formation, infestation peg differentiation, mycelial infestation expansion, and disease spot formation (Zhang et al., 2016; Osés-Ruiz et al., 2017; Gupta et al., 2021).

At present, the main control methods for rice blast are divided into three strategies: the selection of resistant rice varieties, chemical control, and biological control. Screening disease-resistant varieties can effectively improve the ability of rice to resist external disturbances, reduce the occurrence of disease, and promote the healthy growth of rice. However, the genetic complexity and diversity of the rice fungus itself leads to differences in its pathogenicity; furthermore, rice can exhibit differences in resistance (Ou, 1980). Furthermore, the fungi responsible for plague are prone to mutation which enables them to overcome the resistance of rice by evolutionary processes (Oerke, 1996). Chemical control is an important means of prevention and control because it is rapid and inexpensive; mizoram, paddy strain, tebuconazole, and tricyclozole are common chemical pesticides (Kunova et al., 2013). However, the annual application of a large number of pesticides has led to an increase in the resistance of rice to plague bacteria. Thus, the resistance of rice is becoming weaker; this, combined with environmental pollution, has caused socio-economic losses, and could eventually lead to the poisoning of humans and animals, and increased mortality.

With the increasing concern for sustainable agricultural development, biopesticides are highly valued for their environmental friendliness, low resistance, low harm to natural enemies of pests, and low risk to human and animal health (Zhang, 2020). Biopesticides involve organisms, or their secondary metabolites, producing antagonistic inhibitory effects on diseases (Samada and Tambunan, 2020). According to their source, biopesticides can be divided into plant-derived pesticides and microbial-derived pesticides. Research has shown that Bacillus can secrete a variety of hydrolytic enzymes, including lipase, protease, and amylase, because of its strong resistance to adversity, thus leading to high levels of resistance to temperature, ultraviolet radiation, and other factors (Fravel, 2005). Moreover, this bacterium has a wide spectrum of inhibitory properties, rapid reproduction, low production costs, a good safety profile, and a wide variety of advantageous properties for biological control. The main species of Bacillus used in biological control research are Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliq uefaciens, and Bacillus polymyxa (Wang et al., 2019; Roy et al., 2021).

In this study, an endophytic strain, named UTF-33, with strong inhibitory effects on rice fungus, was isolated and identified from the leaves of acid mold. The strain was identified by morphological, biochemical, and physiological characterization, and confirmed by 16S rRNA sequence analysis. The drug sensitivity profile of this bacterium was investigated and the effects of its filtrate fermentation solution on the defense mechanism of rice were investigated. The crude extraction of filtered fermentation broth with n-butanol was tested for its inhibitory effects on conidial germination, attachment cell formation, mycelial growth, cell permeability, and the infestation process of Fusarium oxysporum. In addition, the effect of antagonistic bacteria on the biological control of rice blast was evaluated in field trials. The detection of active expression genes provided further directions and methods for subsequent extraction and purification. Collectively, these studies confirmed the potential value of strain UTF-33 for development as a biopesticide.

As the global population increases, agriculture and natural resources are facing unprecedented demands. In order to meet the needs of the world’s future food security and sustainable development, there is a need to increase food production while effectively reducing environmental hazards (Foley et al., 2011; Zeng et al., 2017). To alleviate the problem of rice yield reduction caused by rice plague, many chemicals have been used as pesticides and fungicides over recent years. However, many of these have been gradually replaced due to the environmental pollution and drug residues they cause; thus increasing interest in the use of biopesticides. Microbial pesticide control effect, production of raw materials and active ingredients are natural products, safe, non-toxic residue-free, to ensure sustainable development (Starnes et al., 1993; Fravel, 2005). Bacillus, as one of the most important microbial pesticides, exerts inhibitory effects through mechanisms such as competitive action, antagonism, and the induction of plant resistance (Bacon et al., 2001; Durairaj et al., 2017; Hasan et al., 2020; Diabankana et al., 2021).

In this study, an endophytic strain of the antagonistic bacterium UTF-33 was extracted from the plant acid mold, a perennial herb that is rich in vitamin A, vitamin C, and oxalic acid; this herb is often used as a seasoning for cooking. The plant itself is edible, and it can be inferred that its endophytic bacteria have no toxic effects on human body. The antagonistic bacteria UTF-33 was identified as Bacillus mojavensis UTF-33 by physiological and biochemical indicators and by molecular biology. A review of published information on Bacillus mojavensis species showed that this bacterium is non-toxic and non-pathogenic; thus, the endophyte can be guaranteed in terms of crop safety and suitable to be developed as biopesticides (Fanaei, 2018; Camele et al., 2019; Mnif et al., 2021). Further analysis found that Bacillus mojavensis UTF-33 was sensitive to most of the antibiotics tested, thus indicating that it was not a drug-resistant microorganism, a characteristic consistent with the use of biocontrol bacteria.

The plant’s innate immune system consists of two immune responses: PTI (PAMP-triggered immunity) and ETI (effector-triggered immunity) (Li et al., 2015). PTI is mainly induced by the stimulation of pathogen-associated molecular patterns on the surface of pathogenic microorganisms, thus leading to non-specific defense responses in plants, including the production of reactive oxygen species ROS, MAPK activation, and SA (salicylic acid) production. ETI is triggered by the recognition of effector proteins produced by pathogenic microorganisms by the plant’s disease-resistant R proteins, including the production of resistant SAR (systemic acquired resistance) and the activation of various protein kinase cascades (Feys and Parker, 2000). The defense mechanism in rice is specifically divided into three expression pathways: those related to disease course, those related to signal transduction, and those related to the encoding of transcription factors (Yuan et al., 2007; Pandey and Somssich, 2009; Mahdavi et al., 2012; Liu et al., 2016).

After treatment with the fermentation products of Bacillus mojavensis UTF-33, PR1a and PR5 (genes related to the course of disease) reached maximal expression levels at 72 h. The genes associated with the titin signaling receptors (OsCEBiP and OsLYP4 and OsLYP6) also reached maximal levels during the same period. OsCEBiP is known to play a key role in the perception (Kaku et al., 2006) and transduction of titin oligosaccharide inducers and OsLYP4 and OsLYP6 encoding proteins that are capable of sensing PGN (peptidoglycan) and fungal titin as bifunctional PRs (pathogenesis-related proteins) (Liu et al., 2012). The genes encoding the OsEREBP, OsWRKY53 and OsWRKY89 transcription factors were upregulated significantly at 24 h. The expression of the salicylic acid signaling receptor pathway and the OsMAPK6 signaling pathway (OsNH1) also reached maximal levels at this time; this is consistent with the timing and characteristics of the rice autoimmune response when analyzed together with the H₂O₂ accumulation results. It was evident that the immune response of the treated rice was concentrated in the early and mid-late stages, with the initial defense against infestation in the early stage; this was due to pathogenic infection stimulating PRS and up-regulating the expression of the fungal titin recognition pathway. The mid-late stage mainly targeted the formation of the infestation pegs, mediating the internal synthesis of SA, and upregulating the expression in concert with multiple transcription factor-related pathways to complete the immune response. In recent studies (Chaiharn et al., 2020; Rong et al., 2020; de Sousa et al., 2021) identified Bacillus safensis, Streptomyces spp. and Trichoderma sp.as potentially being useful for the control of rice blast. These studies mainly focused on the antagonistic effects of microbial pesticides and explored the direct inhibitory effects of metabolites on rice blast. However, we do not know whether these pesticides enhance the autoimmune capacity of rice or activate the response of relevant immune mechanism pathways. However, Bacillus mojavensis UTF-33 can indirectly fight against rice fungus infestation by promoting the upregulation of the defense mechanism response in rice itself.

In addition, fermentation products were extracted with n-butanol to obtain crude extracts. We found that these crude extracts had a significant effect in delaying and inhibiting the germination of rice fever conidia and the formation of attachment cells; furthermore, some of the treated conidia showed distortion after 12 h. Most of the mycelium that could germinate also swelled and broke and could not form a normal attachment cell structure. This indicates that these extracts can directly inhibit the developmental growth process of rice fever conidia, as described previously by Chen et al. who treated conidia using the fermentation product of Bacillus velezensis (Chen et al., 2021); however, the inhibitory effect of Bacillus mojavensis UTF-33 was clearly more effective. Considering both the control effect of biopesticides and the economic effect at the same cost, Bacillus mojavensis UTF-33 is more advantageous.

Functional gene amplification experiments were also conducted; we found that Bacillus mojavensis UTF-33 expressed genes required for the synthesis of various lipopeptides, including bioA, bmyB and fenB, ituD, srfAA. fenB, ituD, and sfp. Synthesis products are useful as Bacillus extracts are often used in antifungal studies and have recognized potential for use in biocontrol (Zhang et al., 2017; Li et al., 2021; Rasiya and Sebastian, 2021). For example, B. velezensis 9D-6 produces surfactins C14 and C15 to antagonize fungal pathogens such as Fusarium oxysporum, Gibberella zeae, Pyrenochaeta terrestris (Grady et al., 2019) and Bacillus subtilis WL-2 antagonizes potato blight by producing Iturin A (Wang et al., 2020). In a previous study, Xiao and Hanif purified fengycin from Bacillus subtilis Z-14 and Bacillus amyloliquefaciens FZB42 to destroy the structure of Gaeumannomyces graminis var. tritici, and Fusarium graminearum to achieve a suppressive effect (Hanif et al., 2019; Xiao et al., 2021). In contrast, Bacillus mojavensis UTF-33 has the potential to synthesize a variety of lipopeptides and is presumed to have direct antagonistic effects on a variety of plant pathogens. It is necessary to further purify and investigate the metabolic activity of a variety of antibacterial active substances to investigate their mechanisms of action and broaden the scope of application.

In the field trials, the control effect of Bacillus mojavensis UTF-33 as a biopesticide was further evaluated. Leaf disease and spot statistics of the fermented filtrate spray treatment were significantly lower than those of the water and LB treatment groups, and were similar to those of the carbendazim treatment group. Analysis showed that Bacillus mojavensis UTF-33 also exhibited excellent bacterial inhibition activity in practical use and could be a beneficial tool for controlling rice blast in the field.

In recent years, Snook et al., Bacon et al., and Hinton et al. used maize as experimental material to study the antagonistic mechanism of Bacillus mojavensis against Fusarium verticillioides. They also used the metabolites of this species to reduce the lesions of maize stalk seedlings, and developed it as a biopesticide for disease control in maize (Snook et al., 2009; Bacon and Hinton, 2011a,b). Hanen et al., Ayed et al., and Samiha et al. used Bacillus mojavensis to extract lipopeptides such as surface activator and eugenol (Ayed et al., 2014; Hanen et al., 2014; Samiha et al., 2014), and also purified the protease synthesized by their metabolism (Beg and Gupta, 2003), or optimizing the production process (Beg et al., 2004; Sepahy et al., 2011). The present literature relating to the biocontrol of crops is relatively homogeneous. This is the first study focusing on the antagonistic effect of Bacillus mojavensis on rice diseases. Our findings illustrate the direct inhibitory effect of Bacillus mojavensis UTF-33 on rice blast and the indirect induction of the rice defense gene expression mechanism. These data confirm the potential of Bacillus mojavensis as a biopesticide and advance the progress of research on the disease suppression mechanisms in plant endophytes.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

ZX, RC, YZ, and YY contributed to conception and design of the study. YZ, YY, LZ, and ZZ organized the database. YZ, JZ, XG, and JY performed the statistical analysis. YZ wrote the first draft of the manuscript. YY, YH, ZH, and LL wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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