Application of compound microbial agent in prevention and treatment of citrus huanglongbing

Abstract

The invention belongs to the technical field of microbial fermentation and plant disease prevention and treatment, and particularly relates to application of a compound microbial agent to prevention and treatment of citrus huanglongbing. According to the present invention, the photosynthetic bacteria such as Rhodobacter sphaeroides A13 and the Bacillus velezensis XZT106 are mixed to obtain the microbial agent, such that the significant effect of the microbial agent on the prevention and control of the citrus huanglongbing is found, the leaf yellowing rate is reduced to 6.67%, and the red nose occurrence rate is reduced to 11.11%.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and its plant disease control technology, specifically relating to the application of a compound microbial agent in the control of citrus Huanglongbing (HLB). Background Technology

[0002] Citrus fruits belong to the genus *Citrus* of the family Rutaceae and are one of the world's most important economic crops. The main citrus-producing areas globally are distributed in warm and humid regions north and south of 35°S and 35°N latitude. Citrus has been cultivated in China for over 4,000 years. Since the beginning of the 21st century, after more than 20 years of rapid development, the cultivated area of ​​citrus in China has reached approximately 2.67 million hectares. 2 With a production exceeding 45 million tons, China ranks first in the world in both cultivation area and output. The citrus industry has become one of the pillar industries for promoting rural revitalization in South China.

[0003] The pathogen of Huanglongbing resides in the phloem of citrus trees and is mainly spread by citrus psyllids. Huanglongbing not only causes citrus trees to decline in vigor and growth stunts, but also causes symptoms such as yellowing and mottling of leaves, smaller fruit, and uneven coloring, significantly reducing citrus yield and quality. Diseased young trees generally die within 2-3 years, while mature trees die within 5-8 years.

[0004] The control techniques for citrus Huanglongbing are divided into: (1) Physical control: Physical control mainly uses physical methods such as electricity, light, heat, force, temperature and humidity; (2) Chemical control: At present, domestic and foreign researchers have conducted research on the chemical control of citrus Huanglongbing from different angles, from traditional antibiotics to the latest nanomaterials. Chemical control is one of the most effective means of controlling citrus Huanglongbing, including nanomaterials, plant immune inducers, antibiotics and antimicrobial peptides. (3) Biological control: Biological control is a technology that uses beneficial organisms or microorganisms and their metabolites to resist invasive species and effectively control crop diseases and pests. This control technology is to irrigate the roots of infected plants or spray biocontrol agents. The agents compete with pathogens for nutrients, thereby inhibiting the growth and reproduction of pathogens. CN109306334 A discloses a strain of Bacillus belye L1-21 and its application in controlling citrus Huanglongbing. However, there are not many types of microorganisms that can control citrus Huanglongbing and the effect is not very significant. Summary of the Invention

[0005] In order to obtain a more effective fungicide for controlling citrus Huanglongbing (HLB), the present invention discloses the following technical solution:

[0006] This invention discloses a microbial inoculant containing a photosynthetic bacterium, Rhodobacter sphaeroides A13, which has an accession number of CCTCC No: M 2018605 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China, on September 7, 2018.

[0007] Preferably, the microbial agent further comprises Bacillus velezensis XZT106, which was deposited at the China Center for Type Culture Collection on March 18, 2025, with accession number CCTCC M 2025518.

[0008] Preferably, the concentrations of the photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 are 0.5-4.8 × 10⁻⁶, respectively. 6 cfu / mL and 0.7-5.6×10 9 cfu / mL.

[0009] Preferably, the concentrations of the photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 are 1.0 × 10⁻⁶ respectively. 6 cfu / mL and 5.2×10 9 cfu / mL.

[0010] This invention discloses a method for preparing microbial inoculants by fermentation, the fermentation method comprising the following steps:

[0011] (1) Preparation of photosynthetic bacteria Rhodobacter sphaeroides A13 microbial inoculum:

[0012] The Rhodobacter sphaeroides A13 strain of photosynthetic bacteria as described in claim 1 was prepared through activation, seed culture, and production culture. The bacterial count of the Rhodobacter sphaeroides A13 strain was 5.6-6.8 × 10⁻⁶. 6 The specific preparation method for cfu / mL includes the following steps:

[0013] S1 and A13 strain activation: The preserved strain of Rhodobacter sphaeroides A13, a photosynthetic bacterium, was cultured using the double-layer plate method until red single colonies appeared. The culture medium used was a photosynthetic bacteria solid medium with the following formula: peptone: 5-7 g / L, beef extract: 1.5-2.0 g / L, yeast extract: 1.5-2.0 g / L, K₂HPO₄: 4.8-6.0 g / L, KH₂PO₄: 1.3-1.6 g / L, trace metal element stock solution 1.0 ml / L, agar powder 20 g / L, pH 7.0.

[0014] Formula for trace metal element storage solution: Accurately weigh EDTA: 2.5g, ZnSO4: 6.13g, MgSO4: 0.78g, CuSO4: 0.25g, CoCl2: 0.08g, FeSO4·7H2O: 7.0g and dissolve in 1L of distilled water;

[0015] S2 and A13 strain seed culture: The red single colonies cultured in step S1 were inoculated into 120mL serum bottles and cultured in photosynthetic bacteria liquid medium to the logarithmic growth phase under the conditions of 35-38℃, 4500-5000Lx light, and pH 7.0 to obtain bacterial suspension; the formula of the photosynthetic bacteria liquid medium is as follows: peptone: 5-7g / L, beef extract: 1.5-2.0g / L, YE: 1.5-2.0g / L, K2HPO4: 4.8-6.0g / L, KH2PO4: 1.3-1.6g / L, trace metal element stock solution 1.0ml / L, pH 7.0.

[0016] Formula for trace metal element storage solution: Accurately weigh EDTA: 2.5g, ZnSO4: 6.13g, MgSO4: 0.78g, CuSO4: 0.25g, CoCl2: 0.08g, FeSO4·7H2O: 7.0g and dissolve in 1L of distilled water;

[0017] S3 and A13 strain production culture: The bacterial solution obtained after seed culture in step S2 is inoculated into a production bottle and cultured in a photosynthetic bacteria strain production medium. The composition of the photosynthetic bacteria strain production medium is the same as that of the above-mentioned photosynthetic bacteria liquid medium. The inoculation amount of the bacterial solution is 5-10% of the total volume of the production medium. Under the conditions of temperature of 35-38℃, light conditions of 4500-5000Lx, and pH value of 7.0, the microbial agent of photosynthetic bacteria Rhodophyton floccosum A13 strain is obtained after culturing to the logarithmic growth phase.

[0018] (2) Preparation of Bacillus velezensis XZT106 microbial inoculant:

[0019] (1) Activation of strain XZT106: Bacillus velezensis XZT106 was inoculated onto LB solid medium and cultured at 30-35℃ for 1-2 days to obtain the activated strain;

[0020] LB solid medium (g / L): tryptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 8-12 g / L, agar powder 14-16 g / L, dissolved in water, and finally adjusted to 1000 mL, pH 7.0±0.2, autoclaved (121℃, 30 min).

[0021] (2) XZT106 seed culture: The activated Bacillus velezensis XZT106 was inoculated into LB liquid medium and cultured in a shaker at 160-200 rpm for 8-10 hours at 28-32℃ to obtain Bacillus velezensis XZT106 seed culture;

[0022] The LB liquid culture medium consisted of 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium chloride, 40-60 mmol / L zinc sulfate, 1000-1200 nmol / L PQQ, pH 7.0 ± 0.2, and was cultured under high pressure (121℃ for 30 min).

[0023] (3) XZT106 fermentation: Bacillus velezensis XZT106 seed liquid was inoculated into the fermentation medium of the fermenter at an inoculation rate of 1-5% of the total volume of the fermentation medium. The culture was carried out in a shaker at 190-220 rpm for 36-48 hours at 28-32℃ to obtain a microbial agent containing Bacillus velezensis XZT106.

[0024] The fermentation medium is formulated as follows: 18-25 g / L soybean meal, 14-16 g / L glucose, 8-12 g / L magnesium sulfate, 40-60 mmol / L zinc sulfate, 15-20 g / L peptone, 10-15 g / L yeast extract, 8-12 g / L sodium chloride, 8-12 g / L potassium dihydrogen phosphate, and 1000-1200 nmol / L PQQ. The pH of the fermenter is 7.0 ± 0.2.

[0025] (3) The microbial inoculant is obtained by mixing the photosynthetic bacteria Rhodobacter sphaeroides A13 microbial inoculant with the Bacillus velezensis XZT106 microbial inoculant;

[0026] The fermentation medium is formulated as follows: 18-25 g / L soybean meal, 14-16 g / L glucose, 8-12 g / L magnesium sulfate, 40-60 mmol / L zinc sulfate, 15-20 g / L peptone, 10-15 g / L yeast extract, 8-12 g / L sodium chloride, 8-12 g / L potassium dihydrogen phosphate, and 1000-1200 nmol / L PQQ. The pH of the fermenter is 7.0 ± 0.2.

[0027] Preferably, the concentrations of the photosynthetic bacteria Rhodobacter phaeroides A13 and Bacillus velezensis XZT106 in the microbial agent in step (3) are 0.5-4.8 × 10⁻⁶ respectively. 6 cfu / mL and 0.7-5.6×10 9 cfu / mL.

[0028] Preferably, the concentrations of the photosynthetic bacteria Rhodobacter phaeroides A13 and Bacillus velezensis XZT106 in the microbial agent in step (3) are 1.0 × 10⁻⁶ respectively. 6 cfu / mL and 5.2×10 9 cfu / mL.

[0029] Preferably, the Bacillus velezensis XZT106 microbial agent contains the following components: 2-oxoadipic acid, L-4,5-dihydroorotic acid, eicosapentaenoic acid, undecanoic acid, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, 5-phenylvaleric acid, L-pyroglutamic acid, and mucoacid.

[0030] Preferably, the mass fraction of 2-oxoadipic acid, L-4,5-dihydroorotic acid, docosapentaenoic acid, undecanoic acid, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, 5-phenylvaleric acid, L-pyroglutamic acid, and mucoacid in the Bacillus velezensis XZT106 microbial agent is 0.05-0.35%; the mass ratio of 2-oxoadipic acid, L-4,5-dihydroorotic acid, docosapentaenoic acid, undecanoic acid, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, 5-phenylvaleric acid, L-pyroglutamic acid, and mucoacid is 4-6:1-3:2-8:1-8:12-15:7-16:2-4:5-9.

[0031] The application of a microbial agent containing photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 in the control of citrus Huanglongbing (HLB) is described. The specific method of application is as follows: the microbial agent is applied to the roots of citrus trees by drenching and spraying during the spring, summer and autumn shoots.

[0032] A microbial agent containing photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 is used in the preparation of products for the prevention and control of citrus Huanglongbing (HLB). The specific method of application is as follows: the microbial agent is applied to the roots of citrus trees by drenching and spraying during the spring, summer and autumn shoots.

[0033] The beneficial effects of this invention are:

[0034] This invention mixes photosynthetic bacteria Rhodobacter sphaeroides A13 with Bacillus velezensis XZT106 to obtain a microbial inoculant, which was found to have a significant effect on the control of citrus Huanglongbing (HLB), reducing the leaf yellowing rate to 6.67% and the incidence of red-nosed fruit to 11.11%. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] Example 1: Fermentation of a combined bacterial Rhodotorula glutinis A13 microbial inoculant

[0037] (1) The photosynthetic bacterium Rhodotorula glutinis strain A13, whose accession number at the China Center for Type Culture Collection is CCTCC No: M2018605, the accession date is September 7, 2018, the accession institution is located at Wuhan University, China, and has been published in application CN110205263A.

[0038] (2) Biocontrol agents for photosynthetic bacteria Rhodophyton floccosum strain A13

[0039] The Rhodobacter sphaeroides A13 strain of photosynthetic bacteria was prepared through activation, seed culture, and production culture. The bacterial count of the Rhodobacter sphaeroides A13 strain was 5.6-6.8 × 10⁻⁶. 6 The specific preparation method for cfu / mL includes the following steps:

[0040] S1 and A13 strain activation: The preserved strain of Rhodobacter sphaeroides A13, a photosynthetic bacterium, was cultured using the double-layer plate culture method until red single colonies appeared. The culture medium used was a photosynthetic bacteria solid medium with the following formula: peptone: 5-7 g / L, beef extract: 1.5-2.0 g / L, YE: 1.5-2.0 g / L, K2HPO4: 4.8-6.0 g / L, KH2PO4: 1.3-1.6 g / L, trace metal element stock solution 1.0 ml / L, agar powder 20 g / L, and pH 7.0.

[0041] Formula for trace metal element storage solution: Accurately weigh EDTA: 2.5g, ZnSO4: 6.13g, MgSO4: 0.78g, CuSO4: 0.25g, CoCl2: 0.08g, FeSO4·7H2O: 7.0g and dissolve in 1L of distilled water;

[0042] S2 and A13 strain seed culture: The red single colonies cultured in step S1 were inoculated into 120mL serum bottles and cultured in photosynthetic bacteria liquid medium to the logarithmic growth phase under the conditions of 35-38℃, 4500-5000Lx light, and pH 7.0 to obtain bacterial suspension. The formula of photosynthetic bacteria liquid medium is as follows: peptone: 5-7g / L, beef extract: 1.5-2.0g / L, YE: 1.5-2.0g / L, K2HPO4: 4.8-6.0g / L, KH2PO4: 1.3-1.6g / L, trace metal element stock solution 1.0ml / L, pH 7.0.

[0043] Formula for trace metal element storage solution: Accurately weigh EDTA: 2.5g, ZnSO4: 6.13g, MgSO4: 0.78g, CuSO4: 0.25g, CoCl2: 0.08g, FeSO4·7H2O: 7.0g and dissolve in 1L of distilled water;

[0044] S3 and A13 strain production culture: The bacterial solution obtained after seed culture in step S2 is inoculated into a production bottle and cultured using a photosynthetic bacteria strain production culture medium. The composition of the photosynthetic bacteria strain production culture medium is the same as that of the above-mentioned photosynthetic bacteria liquid culture medium. The inoculation amount of the bacterial solution is 5-10% of the total volume of the production culture medium. Under the conditions of temperature of 35-38℃, light conditions of 4500-5000Lx, and pH value of 7.0, the microbial agent of photosynthetic bacteria Rhodophyton floccosum A13 strain is obtained after culturing to the logarithmic growth phase.

[0045] Example 2: Identification, purification, and preservation of Bacillus velezensis strain XZT106

[0046] (1) Identification

[0047] The 16S rRNA gene of Bacillus velezensis XZT106 of this invention was amplified, and the PCR product was sequenced. The resulting gene sequence (nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO.1) was compared and analyzed by NCBI. The comparison results showed that the strain is 100% related to Bacillus velezensis, and it was named Bacillus velezensis XZT106. This invention provides a strain of Bacillus velezensis XZT106, which was deposited at the China Center for Type Culture Collection (CCTCC) on March 18, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC M 2025518.

[0048] SEQ ID NO.1

[0049]

[0050] Example 3: Preparation of Bacillus velezensis XZT106 microbial inoculant

[0051] The specific preparation process is as follows:

[0052] (1) Activation of strain XZT106: Bacillus velezensis XZT106 was inoculated onto LB solid medium and cultured at 30-35℃ for 1-2 days to obtain activated Bacillus velezensis XZT106 strain.

[0053] LB solid medium (g / L): tryptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 8-12 g / L, agar powder 14-16 g / L, dissolved in water, and finally adjusted to 1000 mL, pH 7.0±0.2, autoclaved (121℃, 30 min).

[0054] (2) Seed culture of XZT106: The activated Bacillus velezensis XZT106 was inoculated into LB liquid medium and cultured in a shaker at 160-200 rpm for 8-10 hours at 28-32℃ to obtain Bacillus velezensis XZT106 seed culture;

[0055] The LB liquid culture medium (g / L) consisted of: 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium chloride, 40-60 mmol / L zinc sulfate, 1000-1200 nmol / L PQQ, pH 7.0 ± 0.2, and was autoclaved (121℃, 30 min).

[0056] (3) XZT106 fermentation: Bacillus velezensis XZT106 seed culture was inoculated into the fermentation medium in a fermenter at an inoculation rate of 1-5% of the total fermentation medium volume. The culture was then incubated at 28-32℃ and 190-220 rpm for 36-48 hours on a shaker to obtain a microbial agent containing Bacillus velezensis XZT106 (colony concentration of 5.6-7.3 × 10⁻⁶). 9 (cfu / ml).

[0057] The fermentation medium is formulated as follows: 18-25 g / L soybean meal, 14-16 g / L glucose, 8-12 g / L magnesium sulfate, 40-60 mmol / L zinc sulfate, 15-20 g / L peptone, 10-15 g / L yeast extract, 8-12 g / L sodium chloride, 8-12 g / L potassium dihydrogen phosphate, and 1000-1200 nmol / L PQQ. The pH of the fermenter is 7.0 ± 0.2.

[0058] Example 4: Preparation of a mixed bacterial agent of Bacillus velezensis XZT106 and Rhodobacter sphaeroides A13 (a photosynthetic bacterium)

[0059] The photosynthetic bacterium Rhodophyton floccosum A13 microbial inoculum (with a colony count of 6.0 × 10⁻⁶) from Example 2 was used. 6 The CFU / mL concentration was compared with that of Bacillus belyeis XZT106 microbial inoculum in Example 3 (colony count 6.2 × 10⁻⁶). 9 Mix (cfu / mL).

[0060] Mixed bacterial agent 1: The volume ratio of photosynthetic bacteria Rhodococcus coccidioides A13 microbial agent to Bacillus belyssus XZT106 microbial agent is 1:1. The concentrations of Rhodococcus coccidioides A13 and Bacillus belyssus XZT106 in the mixed bacterial agent are 3.0 × 10⁻⁶. 6 cfu / mL and 3.1×10 9 cfu / mL.

[0061] Mixed bacterial agent 2: The volume ratio of photosynthetic bacteria Rhodococcus coccidioides A13 microbial agent to Bacillus belyssus XZT106 microbial agent is 1:5. The concentrations of Rhodococcus coccidioides A13 and Bacillus belyssus XZT106 in the mixed bacterial agent are 1.0 × 10⁻⁶. 6 cfu / mL and 5.2×10 9 cfu / mL.

[0062] Mixed bacterial agent 3: The volume ratio of photosynthetic bacteria Rhodococcus coccidioides A13 microbial agent to Bacillus belyssus XZT106 microbial agent is 1:10. The concentrations of Rhodococcus coccidioides A13 and Bacillus belyssus XZT106 in the mixed bacterial agent are 5.5 × 10⁻⁶. 5 cfu / mL and 5.6×10 9 cfu / mL.

[0063] Mixed bacterial agent 4: The volume ratio of photosynthetic bacteria Rhodococcus coccidioides A13 microbial agent to Bacillus belyssus XZT106 microbial agent is 8:1. The concentrations of Rhodococcus coccidioides A13 and Bacillus belyssus XZT106 in the mixed bacterial agent are 5.3 × 10⁻⁶. 5 cfu / mL and 6.9×10 8 cfu / mL.

[0064] Mixed bacterial agent 5: The volume ratio of photosynthetic bacteria Rhodococcus coccidioides A13 microbial agent to Bacillus belyssus XZT106 microbial agent is 4:1. The concentrations of Rhodococcus coccidioides A13 and Bacillus belyssus XZT106 in the mixed bacterial agent are 4.8 × 10⁻⁶. 6 cfu / mL and 1.2×10 9 cfu / mL.

[0065] Example 5: Application of a mixed inoculum of Bacillus velezensis XZT106 and Rhodobacter sphaeroides A13 in the control of Huanglongbing (HLB).

[0066] Seven experimental plots were selected, with 45 six-year-old citrus trees infected with Huanglongbing (HLB) in each plot. The Ct value of HLB bacteria on each citrus tree was determined by real-time quantitative PCR. The specific measurement method was in accordance with the People's Republic of China National Standard GB / T28062-2011. All samples infected with HLB were found to contain *Citrus citrus HLB*.

[0067] Treatment 1: Dilute the mixed bacterial agent 1 in Example 4 with water 100 times, take 5L and mix it with 100g of humic acid fertilizer, and then irrigate the roots. Then take 8L of compound amino acid fertilizer and spray it on the front and back of the citrus leaves. Treat once each in spring, summer and autumn when the citrus leaves are emerging, and then treat again after 10 days.

[0068] Treatment 2: Dilute the mixed bacterial agent 2 in Example 4 with water 100 times, take 5L and mix it with 100g of humic acid fertilizer, and then irrigate the roots. Then take 8L of compound amino acid fertilizer and spray it on the front and back of the citrus leaves. Treat once each in spring, summer and autumn when the citrus leaves are emerging, and then treat again after 10 days.

[0069] Treatment 3: Dilute the mixed bacterial agent 3 in Example 4 with water 100 times, take 5L and mix it with 100g of humic acid fertilizer, and then irrigate the roots. Then take 8L of compound amino acid fertilizer and spray it on the front and back of the citrus leaves. Treat once each in spring, summer and autumn when the citrus leaves are emerging, and then treat again after 10 days.

[0070] Treatment 4: Dilute the mixed bacterial agent 4 in Example 4 with water 100 times, take 5L and mix it with 100g of humic acid fertilizer, and then irrigate the roots. Then take 8L of compound amino acid fertilizer and spray it on the front and back of the citrus leaves. Treat once each in spring, summer and autumn when the citrus leaves are emerging, and then treat again after 10 days.

[0071] Treatment 5: Dilute the mixed bacterial agent 5 from Example 4 with water 100 times, take 5L and mix it with 100g of humic acid fertilizer, then apply it to the roots. Then take 8L of compound amino acid fertilizer and spray it on both sides of the citrus leaves. Apply it once each in spring, summer and autumn when the citrus leaves are emerging, and then treat it again after 10 days.

[0072] Treatment 6: Mix 5L of water with 100g of humic acid fertilizer and apply as a root irrigation. Then, mix 8L of water with amino acid fertilizer and spray it on both sides of the citrus leaves. Apply the treatment once each in spring, summer and autumn when the citrus leaves are emerging, and then repeat the treatment after 10 days.

[0073] Treatment 7: Mix 5L of water with 15g of tetracycline and drench the roots. Then mix 8L of water with 25g of tetracycline and spray the front and back of the citrus leaves. Treat the citrus leaves once each in spring, summer and autumn when the new shoots emerge, and then repeat the treatment after 10 days.

[0074] Treatment 8: Dilute the Bacillus vesiculosus XZT106 microbial agent from Example 3 with water 100 times, take 5L and mix it with 100g of humic acid fertilizer, and then irrigate the roots. Then take 8L of compound amino acid fertilizer and spray it on both sides of the citrus leaves. Treat once each in spring, summer and autumn when the citrus leaves are emerging, and then treat again after 10 days.

[0075] Treatment 9: Dilute the photosynthetic bacteria Rhodobulbus globosum A13 microbial agent from Example 4 with water 100 times, take 5L and mix it with 100g of humic acid fertilizer, and then irrigate the roots. Then take 8L of compound amino acid fertilizer and spray it on both sides of the citrus leaves. Treat once each in spring, summer and autumn when the citrus leaves are emerging, and then treat again after 10 days.

[0076] After the autumn shoot treatment, the yellowing of citrus leaves and the number of red-nosed fruits were counted. The results on the citrus trees are shown in Table 1.

[0077] Table 2 Results of each treatment

[0078]

[0079]

[0080] The data in Table 1 show that mixing Bacillus velezensis XZT106 with the photosynthetic bacterium Rhodobacter sphaeroides A13 has a synergistic effect in controlling citrus Huanglongbing (HLB), reducing the leaf yellowing rate to 6.67% and the incidence of red-nosed fruit to 11.11%.

Claims

1. A microbial inoculant, characterized in that, The microbial inoculant contains the photosynthetic bacterium Rhodobacter sphaeroides A13, which has the accession number CCTCC No: M 2018605 at the China Center for Type Culture Collection on September 7, 2018. The depository is located at Wuhan University, China.

2. The microbial agent according to claim 1, characterized in that, The microbial agent also contains Bacillus velezensis XZT106, which was deposited at the China Center for Type Culture Collection on March 18, 2025, with accession number CCTCC M 2025518.

3. The microbial agent according to claim 2, characterized in that, The concentrations of the photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 were 0.5-4.8 × 10⁻⁶, respectively. 6 cfu / mL and 0.7-5.6×10 9 cfu / mL.

4. The microbial agent according to claim 3, characterized in that, The concentrations of the photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 were 1.0 × 10⁻⁶. 6 cfu / mL and 5.2×10 9 cfu / mL.

5. A method for preparing the microbial inoculant according to claims 2-4 by fermentation, characterized in that, The fermentation method includes the following steps: (1) Preparation of photosynthetic bacteria Rhodobacter sphaeroides A13 microbial inoculum: S1 and A13 strain activation: The preserved strain of Rhodobacter sphaeroides A13, a photosynthetic bacterium, was cultured using the double-layer plate method until red single colonies appeared. The culture medium used was a photosynthetic bacteria solid medium with the following formula: peptone: 5-7 g / L, beef extract: 1.5-2.0 g / L, yeast extract: 1.5-2.0 g / L, K₂HPO₄: 4.8-6.0 g / L, KH₂PO₄: 1.3-1.6 g / L, trace metal element stock solution 1.0 ml / L, agar powder 20 g / L, pH 7.

0. The formula for the trace metal element storage solution is as follows: Accurately weigh EDTA: 2.5g, ZnSO4: 6.13g, MgSO4: 0.78g, CuSO4: 0.25g, CoCl2: 0.08g, FeSO4·7H2O: Dissolve 7.0g in 1L of distilled water; S2 and A13 strain seed culture: The red single colonies cultured in step S1 were inoculated into 120mL serum bottles and cultured in photosynthetic bacteria liquid medium to the logarithmic growth phase under the conditions of 35-38℃, 4500-5000Lx light, and pH 7.0 to obtain bacterial suspension; the formula of the photosynthetic bacteria liquid medium is as follows: peptone: 5-7g / L, beef extract: 1.5-2.0g / L, YE: 1.5-2.0g / L, K2HPO4: 4.8-6.0g / L, KH2PO4: 1.3-1.6g / L, trace metal element stock solution 1.0ml / L, pH 7.0; Trace metal element storage solution formula: Accurately weigh EDTA: 2.5g, ZnSO4: 6.13g, MgSO4: 0.78g, CuSO4: 0.25g, CoCl2: 0.08g, FeSO4·7H2O: Dissolve 7.0g in 1L of distilled water; S3 and A13 strain production culture: The bacterial solution obtained after seed culture in step S2 is inoculated into a production bottle and cultured in a photosynthetic bacteria strain production medium. The composition of the photosynthetic bacteria strain production medium is the same as that of the above-mentioned photosynthetic bacteria liquid medium. The inoculation amount of the bacterial solution is 5-10% of the total volume of the production medium. Under the conditions of temperature of 35-38℃, light conditions of 4500-5000Lx, and pH value of 7.0, the microbial agent of photosynthetic bacteria Rhodophyton floccosum A13 strain is obtained after culturing to the logarithmic growth phase. (2) Preparation of Bacillus velezensis XZT106 microbial inoculant: (1) Activation of strain XZT106: Bacillus velezensis XZT106 was inoculated onto LB solid medium and cultured at 30-35℃ for 1-2 days to obtain activated Bacillus velezensis XZT106 strain; The LB solid medium (g / L) consists of: 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium chloride, and 14-16 g / L agar powder. Dissolve in water and bring the final volume to 1000 mL. The pH is 7.0 ± 0.

2. Sterilize. (2) XZT106 seed culture: The activated Bacillus velezensis XZT106 was inoculated into LB liquid medium and cultured in a shaker at 160-200 rpm for 8-10 hours at 28-32℃ to obtain Bacillus velezensis XZT106 seed culture; The LB liquid culture medium consisted of 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium chloride, 40-60 mmol / L zinc sulfate, 1000-1200 nmol / L PQQ, pH 7.0 ± 0.2, and was sterilized. (3) XZT106 fermentation: Bacillus velezensis XZT106 seed liquid was inoculated into the fermentation medium of the fermenter at an inoculation rate of 1-5% of the total volume of the fermentation medium. The culture was carried out in a shaker at 190-220 rpm for 36-48 hours at 28-32℃ to obtain a microbial agent containing Bacillus velezensis XZT106. The fermentation medium is formulated as follows: 18-25 g / L soybean meal, 14-16 g / L glucose, 8-12 g / L magnesium sulfate, 40-60 mmol / L zinc sulfate, 15-20 g / L peptone, 10-15 g / L yeast extract, 8-12 g / L sodium chloride, 8-12 g / L potassium dihydrogen phosphate, and 1000-1200 nmol / L PQQ. The pH of the fermenter is 7.0 ± 0.

2. (3) The microbial inoculant is obtained by mixing the photosynthetic bacteria Rhodobacter sphaeroides A13 microbial inoculant with the Bacillus velezensis XZT106 microbial inoculant; The fermentation medium is formulated as follows: 18-25 g / L soybean meal, 14-16 g / L glucose, 8-12 g / L magnesium sulfate, 40-60 mmol / L zinc sulfate, 15-20 g / L peptone, 10-15 g / L yeast extract, 8-12 g / L sodium chloride, 8-12 g / L potassium dihydrogen phosphate, and 1000-1200 nmol / L PQQ. The pH of the fermenter is 7.0 ± 0.

2.

6. The fermentation method according to claim 5, characterized in that the concentrations of the photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 in the microbial agent in step (3) are 0.5-4.8 × 10⁻⁶ respectively. 6 cfu / mL and 0.7-5.6×10 9 cfu / mL.

7. The fermentation method according to claim 6, characterized in that the concentrations of the photosynthetic bacteria Rhodobacter sphaeroides A13 and Bacillus velezensis XZT106 in the microbial agent in step (3) are 1.0 × 10⁻⁶ and 1.0 × 10⁻⁶, respectively. 6 cfu / mL and 5.2×10 9 cfu / mL.

8. The fermentation method according to claim 5, characterized in that, The Bacillus velezensis XZT106 microbial inoculant contains the following components: 2-oxoadipic acid, L-4,5-dihydroorotic acid, eicosapentaenoic acid, undecanoic acid, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, 5-phenylvaleric acid, L-pyroglutamic acid, and mucoacid.

9. The fermentation method according to claim 8, characterized in that, The mass fraction of 2-oxoadipic acid, L-4,5-dihydroorotic acid, docosapentaenoic acid, undecanoic acid, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, 5-phenylvaleric acid, L-pyroglutamic acid, and mucoacid in the Bacillus velezensis XZT106 microbial agent is 0.05-0.35%; the mass ratio of 2-oxoadipic acid, L-4,5-dihydroorotic acid, docosapentaenoic acid, undecanoic acid, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, 5-phenylvaleric acid, L-pyroglutamic acid, and mucoacid is 4-6:1-3:2-8:1-8:12-15:7-16:2-4:5-9.

10. The application of the microbial inoculant according to any one of claims 1-4 or the microbial inoculant prepared by the fermentation method according to any one of claims 5-9 in the prevention and control of citrus Huanglongbing.

11. The application according to claim 10, characterized in that, The specific method of application is as follows: the microbial agent is applied to the roots of citrus trees by irrigation and spraying during the spring, summer and autumn shoots.

12. The microbial agent according to any one of claims 1-4 or the microbial agent prepared by the fermentation method according to any one of claims 5-9 is used in the preparation of products for the prevention and control of citrus Huanglongbing.

13. The application according to claim 12, characterized in that, The specific method of application is as follows: the microbial agent is applied to the roots of citrus trees by irrigation and spraying during the spring, summer and autumn shoots.

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