Bacillus subtilis for preventing and treating pecan dry rot and related applications

Abstract

This invention belongs to the field of forest tree disease control technology, specifically relating to a Bacillus subtilis strain for controlling pecan dry rot and its related applications. The Bacillus subtilis strain is Bacillus subtilis BSLA101, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2019160. This strain exhibits significant antagonistic effects against various pathogens causing pecan dry rot. This invention also provides a microbial agent containing this strain and its application method in controlling pecan dry rot. The constructed agent has advantages such as being environmentally friendly, having stable efficacy, and being less prone to developing resistance, providing a new solution for the green and efficient biological control of pecan dry rot, suitable for development as a biopesticide or for use in ecological agriculture practices.

Description

Technical Field

[0001] This invention belongs to the field of forest tree disease control technology, specifically relating to a Bacillus subtilis strain for controlling walnut dry rot and its related applications. Background Technology

[0002] Bacillus spp. is a non-pathogenic bacterium commonly found in the rhizosphere soil and other environmental environments. It is a Gram-positive bacterium with peritrichous flagella, enabling motility. Bacillus spp. spores exhibit strong resistance to extreme environments such as radiation, heat, dryness, pH fluctuations, and toxic chemicals. It has been reported that Bacillus spp. spores can remain viable for years or even decades in a dormant state. Furthermore, their rapid reproduction rate and simple nutritional requirements make them well-suited for development as a biocontrol resource. Studies have shown that the mechanism of action of Bacillus against plant pathogens is mainly related to secondary metabolites such as lipopeptides. Different strains from different sources often exhibit different antibacterial spectra against pathogens, and may even show different antagonistic activities against the same pathogen.

[0003] Hickory (Carya cathayensis) is a deciduous tree belonging to the genus Carya in the family Juglandaceae, and has high nutritional and economic value. Dry rot is prevalent in young and middle-aged hickory trees. Mild dry rot affects tree growth, causing fruit drop and reduced yield; severely affected hickory trees are on the verge of death, resulting in significant losses. Simply put, hickory dry rot not only causes huge economic losses for forest farmers but also severely dampens their enthusiasm for hickory production and management, which is extremely detrimental to the sustainable development of the industry. In the past, it was believed that hickory dry rot was mainly caused by soil degradation due to acid rain and the overuse of glyphosate and fertilizers, falling into the category of non-infectious plant diseases. However, this view cannot explain why soil degradation only led to an outbreak of hickory dry rot, but not to other diseases in other crops in the same area or other hickory diseases; and to date, no research has provided scientific evidence of a causal relationship between soil degradation and the outbreak of dry rot.

[0004] Our research group has previously demonstrated for the first time that pecan dry rot is caused by *Botryosphaeria dothidea*, rather than other pathogens or causes reported in the past. Botryosphaera sp., belonging to the phylum Ascomycotion, order Dothidelales, family Botryosphaeriaceae, primarily overwinters inside the tree trunk. In early spring, it forms lesions on the trunk, exhibiting a clear transformation process from a living symbiotic organism to a dead pathogen. Spores are then produced on the lesions and released into the air, causing reinfection. The peak spore flow in the forest occurs in May and June, when the pathogen overwinters as mycelium inside the tree. The following spring, under suitable conditions, it breaks through the epidermis, forming ulcers. Small black granules, the conidiophores, form on the lesions. Conidia produced by the conidiophores are spread by wind and rain, entering through wounds and natural openings, forming infected strains, demonstrating a clear pattern of latent infection.

[0005] However, existing technologies report various biological control agents that not only have the drawback of being difficult to directly apply due to large differences in growth environment, but also make it difficult to find control methods that are effective for the same forest crops. Summary of the Invention

[0006] Based on the aforementioned background, this invention attempts to analyze the relationship between pathogens and microbial communities within pecan trees at different stages, isolates and screens different strains of Bacillus with important functions, and obtains strains with good antagonistic effects against *Botrytis cinerea*, for novel biological control of pecan dry rot. The *Bacillus subtilis* BSLA101 provided by this invention was directly isolated from healthy pecan tissue in the field, exhibiting significantly better adaptability and environmental tolerance to pecan trees than exogenously added Bacillus strains isolated from other plant disease samples. Furthermore, experimental results from field pecan orchards show that the BSLA101 fermentation broth provided by this invention has a control efficacy of up to 75.5% against field pecan dry rot. Compared to theoretical experiments in the laboratory, the efficacy results of this invention are closer to actual production. Finally, the antibacterial active substances produced by BSLA101 are more readily soluble in the organic phase ethyl acetate, which effectively reduces impurities and improves extraction efficiency and purity when isolating and purifying these substances from the BSLA101 fermentation broth. The easy solubility in organic solvents allows the active substances to be more easily formulated into different types of preparations, such as emulsifiable concentrates, microemulsions, or suspensions. These formulations can be flexibly adjusted according to control needs, meeting the requirements of different crops and disease control scenarios. Furthermore, the enhanced solubility of the antibacterial active substances in organic solvents may help improve their absorption and utilization efficiency in organisms, enabling good control effects even at lower doses and reducing environmental residue risks.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a Bacillus subtilis strain for preventing and controlling pecan dry rot. The strain is Bacillus subtilis BSLA101, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2019160 and deposit date of March 15, 2019.

[0009] This invention also provides the application of the above-mentioned Bacillus subtilis BSLA101 in the prevention and control of pecan dry rot.

[0010] This invention also provides the application of the above-mentioned Bacillus subtilis BSLA101 in the control of Botryosphaeria dothidea, Botryosphaeria qingyuanensis, Botryosphaeria corticis, Botryosphaeria fabicerciana and Lasiodiplodia theobromae.

[0011] The present invention provides a microbial agent comprising the above-mentioned Bacillus subtilis BSLA101.

[0012] In this invention, the dosage forms of the microbial agent include live bacteria preparations and fermentation broths.

[0013] This invention provides a method for preventing and controlling pecan dry rot, the method being as follows:

[0014] Apply the above-mentioned Bacillus subtilis BSLA101 or the above-mentioned microbial inoculant to the root system of pecan plants.

[0015] Typical field symptoms of pecan dry rot include: Figure 1 As shown.

[0016] Beneficial effects:

[0017] (1) The Bacillus subtilis BSLA101 provided by the present invention was isolated from healthy hickory trees and is an endophytic strain. According to the description of the examples, it has natural adaptability and affinity to hickory trees and can better colonize and function in hickory trees.

[0018] (2) The strain of this invention has a significant inhibitory effect on five major pathogens of pecan dry rot, with an inhibition rate of over 90%, showing broad-spectrum and highly effective disease resistance characteristics.

[0019] (3) The strain of the present invention has shown excellent control effect in actual field application, with a field control efficacy of 75.5% against pecan dry rot, which is significantly higher than other reported Bacillus strains;

[0020] (4) The antibacterial active substances in the fermentation broth of the strain of the present invention are easily soluble in the organic solvent ethyl acetate, which is beneficial for the extraction, purification and formulation development of active ingredients, and provides convenience for industrial application;

[0021] (5) Compared with Bacillus strains isolated from other plant disease samples, the BSLA101 strain provided by this invention is derived from healthy pecan tissue. Its endogenous characteristics make it more capable of colonizing within the pecan tree and more adaptable to the environment, resulting in a more stable and reliable control effect. This provides a new solution for the green control of pecan dry rot.

[0022] Biological Preservation Instructions

[0023] Bacillus subtilis BSLA101 was deposited on March 15, 2019, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M2019160. Attached Figure Description

[0024] Figure 1 These are typical field symptoms of pecan dry rot;

[0025] Figure (A) shows the dark brown ulcerative lesions on the bark in the early stage of the disease; Figure (B) shows the lesions expanding longitudinally and encircling the trunk, leading to bark necrosis; Figure (C) shows the dark brown gelatinous liquid oozing from the infected area; Figure (D) shows the dark brown granular substances of pecan dry rot.

[0026] Figure 2 Relative abundance of microbial species in diseased and healthy samples from different sampling periods;

[0027] Figure (A) shows the species composition of the diseased group, including the composition and dynamic evolution of the microbial community at the family level in diseased samples from January to July (01B, 03B, 05B, 07B); Figure (B) shows the species composition of the healthy control group, including the distribution of the microbial community at the family level in healthy control samples during the same period (01J, 03J, 05J, 07J).

[0028] Figure 3 Comparison of plate culture morphology of endophytic bacteria isolated from hickory trees;

[0029] in Figure 1 A: Bacillus subtilis BSLA101; Figure 1 B: Sphingomonas sanguinis; Figure 1 C: Microbacterium sp. Figure 2 A: Xanthomonas sp. Figure 2 B: Commamonas intermedia; Figure 2 C: Deinococcus sp. Figure 3 A: Acinetobacter calcoaceticus;

[0030] Figure 4 The plate inhibition activity of Bacillus BSLA101 against five pecan rot pathogens was determined.

[0031] Figure 4 The top row shows the control group (CK) inoculated with blank culture medium (NB), and the bottom row shows the Bacillus treatment group;

[0032] Figure 4 The locations in the middle are: blank culture medium NB (top), Bacillus subtilis BSLA101 (right), Bacillus subtilis ZT4-2 (left) and Bacillus amyloliquefaciens WK1 (bottom);

[0033] Figure 5 Determination of the antibacterial activity of Bacillus BSLA101 against five pecan rot pathogens in fermentation broth;

[0034] Figure 5 The top row shows the fermentation broth of Bacillus subtilis BSLA101, the middle row shows the fermentation broth of Bacillus subtilis ZT4-2, and the bottom row shows the blank control CK.

[0035] The figure shows, from left to right, the inhibitory effects of L. theobromae, B. fabicerciana, B. dothidea, B. corticis, and B. qingyuanensis on the pathogens causing pecan dry rot. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0037] Example 1

[0038] Sample collection and analysis and isolation of key endophytic bacteria in healthy pecan trees.

[0039] (1) Sample collection and processing

[0040] Sampling location: Wucun Village, Taiyang Town, Lin'an (119°16′49″E, 30°19′6″N). Our laboratory has conducted long-term monitoring in the Wucun hickory sample plot and has made detailed records of the occurrence of canker disease in various hickory trees in the forest in recent years.

[0041] Sampling time: January (dormant period), March (initial period), May (peak period), July (plateau period).

[0042] Sampling method: Ten hickory trees with similar disease severity (similar number and size of lesions, see attached) were selected from the hickory forest. Figure 1 Representative plants were selected. The existing number of lesions was recorded before sampling. A 20 mm diameter sterile punch was used to sample the boundary tissue between diseased and healthy tissue in the current year's lesions. To prevent cross-contamination, sampling tools from different plants were sterilized with 75% ethanol and flame. The sample size per plant was approximately 0.8 g, and two biological replicates were set up for each group. The obtained samples were mixed and placed into 50 mL sterile centrifuge tubes, labeled as follows: the prefixes 01, 03, 05, and 07 correspond to the sampling months of January, March, May, and July, respectively; the suffix "B" represents diseased samples, and "J" represents healthy control samples. After the collected samples were stored at low temperature on dry ice and returned to the laboratory, they were immediately aliquoted into three portions (8 g each). The following operations were performed on the three samples in parallel: First, the pathogen of dry rot was purified and identified by conventional tissue isolation methods; second, metagenomic total DNA extraction, library construction, and high-throughput sequencing were performed according to standard procedures; finally, endophytic bacteria were directionally isolated by tissue grinding and selective heat treatment. All of the above processes were performed under sterile conditions. This invention isolated five pathogens from pecan dry rot samples: *Botryosphaeria dothidea*, *Botryosphaeria qingyuanensis*, *Botryosphaeria corticis*, *Botryosphaeria fabicerciana*, and *Lasiodiplodia theobromae*.

[0043] (2) Results of relative abundance analysis of metagenomic microbial species

[0044] like Figure 2As shown, the endophytic microbial communities of diseased pecan tissues (01B, 03B, 05B, 07B) and healthy tissues (01J, 03J, 05J, 07J) at different sampling periods exhibited certain differences at the family level. Overall, the two types of samples showed some similarity in dominant microbial composition, but the community structure of healthy tissues was richer and relatively stable, with the main dominant groups including Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Acidobacteria. As the sampling time changed from January to July, the overall community composition of healthy samples remained relatively stable, while the proportions of some microbial groups changed in diseased samples. Combined with the strain isolation results, Bacillus and Sphingomonas could be stably isolated from all healthy pecan tissue samples in January, March, May, and July, and persisted in the microbial community of healthy tissues. These results suggest that the presence of these two types of bacteria may be associated with the natural inhibition of pecan dry rot, indicating that Bacillus and Sphingomonas may play a role in preventing pathogen invasion and have the potential to be used as biocontrol resources for pecan dry rot.

[0045] (3) Isolation and purification of endophytic bacteria

[0046] As shown in Table 1, endophytic bacteria were isolated from the healthy group samples, yielding a total of 3137 strains belonging to 30 genera. Bacillus and Sphingomonas were isolated from all samples. Some of the isolated endophytic bacteria were then cultured on plates for morphological observation. Figure 3 The isolation process employed a tissue homogenization method: 4 g samples from both diseased and unaffected sites were taken, rinsed with running water to remove surface impurities, and then cut into approximately 5 mm × 5 mm tissue blocks. These blocks were then sequentially immersed in 75% ethanol for 30 seconds, rinsed once with sterile water, immersed in 3% sodium hypochlorite for 2 minutes, and rinsed three times with sterile water. Finally, a blank control was inoculated using the rinsing solution to confirm thorough surface disinfection. After homogenization, the disinfected samples were allowed to stand for 1 hour. The homogenate was then inoculated onto NA medium, with each group incubated three times. After 3 days of incubation, single colonies were picked based on colony morphology, streaked, and inoculated onto NB liquid medium for shaking incubation. After confirming no contamination, the samples were mixed with 80% glycerol saline at a 5:2 ratio and frozen at -80℃. Endophytic bacteria were identified using the universal 16S rDNA primers 27F (5'-AGAGTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). The plant endophytic Bacillus strain finally isolated and purified from the healthy group sample was named BSLA101.

[0047] Table 1. Results of isolation of endophytic bacteria from disease-free parts of hickory nuts.

[0048] 1J 3J 5J 7J Bacillus ＋ ＋ ＋ ＋ Sphingomonas ＋ ＋ ＋ ＋ Pseudomonas ＋ ＋ － ＋ Paenibacillus ＋ ＋ ＋ － Allorhizobium ＋ ＋ ＋ － Chryseobacterium ＋ － － ＋ Lysinibacillus ＋ － － ＋ Staphylococcus ＋ － ＋ － Massilia ＋ － － ＋ Microbacterium － ＋ ＋ － Enterobacter － － ＋ ＋ Clostridium － － ＋ ＋ Undibacterium ＋ － － － Xanthomonas ＋ － － － Comamonas ＋ － － － Sphingobium － ＋ － － Deinococcus － ＋ － － Cohnella － ＋ － ＋ Ochrobactrum － ＋ － － Cellulomonas － ＋ － － Mucilaginibacter － ＋ － － Gibbsiella － － ＋ － Erwinia － － ＋ － Pantoea － － ＋ － Mucilaginibacter － － － ＋ Acinetobacter － － － ＋ Exiguobacterium － － － ＋ Serratia － － － ＋ Flavobacterium － － － ＋ Stenotrophomonas － － － ＋

[0049] Note: "+" indicates that the sample was obtained after separation; "-" indicates that the sample was not obtained after separation.

[0050] Example 2

[0051] Morphological identification of Bacillus subtilis BSLA101

[0052] (1) Test methods

[0053] Bacillus BSLA101 isolated from healthy pecan trees was inoculated onto NA medium for resuscitation and culture. A comprehensive identification strategy was employed to systematically identify the strain, including: 1) morphological observation (colon morphology, size, color, transparency, and spore morphology); 2) routine bacterial biochemical tests; and 3) 16S rDNA sequence analysis and phylogenetic analysis.

[0054] (2) Results Analysis

[0055] The BSLA101 strain was derived from a healthy pecan tree. The bacteria are short rod-shaped, with a cell size of approximately 1.1 × 7.6 μm, and are Gram-positive. The colonies formed by this strain are flat and grayish-white, with morphological characteristics consistent with the reported Bacillus type strain. Based on morphological characteristics and routine physiological and biochemical results, strain BSLA101 was classified as Bacillus. 16S rDNA sequence alignment and phylogenetic analysis showed that BSLA101 is most closely related to Bacillus subtilis. The Bacillus subtilis BSLA101 provided in this invention is deposited under the name Bacillus subtilis BSLA101, deposited at the China Center for Type Culture Collection, Wuhan University, China, with accession number M2019161, on March 15, 2019.

[0056] Example 3

[0057] Determination of antibacterial activity of Bacillus subtilis BSLA101

[0058] (1) Screening of plate antagonistic activity of Bacillus subtilis BSLA101 against five pecan dry rot pathogens

[0059] Initial screening was conducted using a confrontation culture method. Bacillus BSLA101, preserved at 4℃, was revived and activated on PDA plates along with the five pathogens of pecan dry rot isolated above (B. dothidea, B. qingyuanensis, B. corticis, B. fabicerciana, and L. theobromae). Bacillus culture was inoculated on the left side of a 9cm diameter petri dish, while mycelial cakes of the five pecan dry rot pathogens were inoculated on the right side of the same dish. A separate culture plate containing the pathogens without biocontrol was used as a control. All petri dishes were incubated in the dark at 25℃ for 4 days. After incubation, relevant growth indicators were measured, and the inhibition rate was calculated. The inhibition rate was calculated using the following formula:

[0060] Radial growth inhibition rate (%) = 100 × (R0 − R1) / R0

[0061] Where R0 is the longest growth radius of the pathogen in the control group, and R1 is the growth radius of the pathogen in the treatment group in the direction corresponding to the central axis.

[0062] (2) Preparation of Bacillus BSLA101 fermentation broth

[0063] Bacillus BSLA101 was activated and cultured on NA (nutrient agar) medium at 37°C with shaking at 200 rpm until the cells reached the logarithmic growth phase. 20 μL of the activated bacterial solution was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of NB (nutrient solution) medium and cultured on a shaker at 30°C and 250 rpm for 2 days. After culturing, the fermentation broth was filtered through two layers of sterile lens paper (11–15 μm) to remove bacterial cells and large particulate impurities, yielding the Bacillus BSLA101 fermentation broth. The fermentation broth was cultured at 28°C for 24 hours, and the OD (oxidative stress) was measured. 600 =3.453.

[0064] (3) Determination of the antibacterial activity of Bacillus BSLA101 fermentation broth against five pecan rot pathogens.

[0065] The BSLA101 fermentation broth was added to melted PDA medium to a final concentration of 1% (v / v). Five strains of dry rot pathogens isolated from pecans were inoculated into the center of PDA plates and incubated at 25°C. A sterile distilled water treatment group served as a blank control. After 3 days of incubation, the colony diameter was measured, and the inhibition rate was calculated using the following formula:

[0066] Inhibition rate (%) = 100 × (control colony diameter - treated colony diameter) / control colony diameter

[0067] like Figure 4 and Figure 5 The results showed that Bacillus BSLA101 had a significant inhibitory effect on all five pecan dry rot pathogens, with an inhibition rate of over 90%, which was significantly higher than that of Bacillus subtilis ZT4-2 and Bacillus amyloliquefaciens WK1, which are currently disclosed in the prior art.

[0068] Example 4

[0069] Evaluation of the field control efficacy of Bacillus BSLA101 fermentation broth against pecan dry rot

[0070] (1) Test methods

[0071] In the hickory forest of Wucun Village, Taiyang Town, Lin'an City, as described in Example 1, the forest was randomly divided into 12 plots for efficacy testing. Four treatments were established: BSLA101, ZT4-2, WK1, and water, with three plots per treatment. The fermentation broth prepared using the method in Example 3 was diluted 200 times and sprayed around the roots of the hickory plants. 5-10 L of the diluted broth was sprayed onto the base of each plant, ensuring the solution completely saturated the bark and penetrated into the rhizosphere soil.

[0072] (2) Results Analysis

[0073] An investigation was conducted 35 days after spraying, and the results are shown in the table below.

[0074] Table 2. Field control efficacy statistics of Bacillus BSLA101

[0075]

[0076] As shown in Table 2, under conventional management conditions, the endophytic Bacillus subtilis BSLA101 isolated from healthy pecan trees by spraying significantly inhibited the occurrence of pecan dry rot, with an average control efficacy of 75.5%, compared to only 11.3% and 27.8% for the control group. In summary, the strain of this invention has a significant inhibitory effect on pecan dry rot pathogens and can effectively prevent the occurrence of the disease.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A Bacillus subtilis strain for controlling pecan dry rot, characterized in that, The strain is Bacillus subtilis ( Bacillus subtilis BSLA101, deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M2019160, on March 15, 2019.

2. The application of Bacillus subtilis BSLA101 as described in claim 1 in the prevention and control of pecan dry rot.

3. The Bacillus subtilis BSLA101 described in claim 1 in the prevention and control of... Botryosphaeria dothidea , Botryosphaeria qingyuanensis , Botryosphaeria corticis , Botryosphaeria fabicerciana and Lasiodiplodia theobromae Application of [the concept / method] in [the context].

4. A microbial inoculant, characterized in that, The microbial agent includes Bacillus subtilis BSLA101 as described in claim 1.

5. The microbial agent according to claim 4, characterized in that, The dosage forms of the microbial agents include live bacterial preparations and fermentation broths.

6. A method for preventing and controlling pecan dry rot, characterized in that, The method is as follows: Applying Bacillus subtilis BSLA101 as described in claim 1 or the microbial agent as described in claim 4 to the root system of pecan plants.

Images