Cladosporium sp. Csfl2 and application of cladosporium sp. Csfl2 in clubroot of tumorous stem mustard

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to cladosporium sp. Csfl2 and application of the cladosporium sp. Csfl2 to clubroot of tumorous stem mustard. The cladosporium sp. Is preserved in Institute of Microbiology, Chinese Academy of Sciences on April 14, 2025, the preservation number is CGMCC No.41914, and the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The invention provides Cladosporidium sp. Csfl2, and in-depth study finds that the Cladosporidium sp. Csfl2 can inhibit the occurrence of the clubroot of tumorous stem mustard and has a biocontrol effect on the clubroot of tumorous stem mustard.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a type of spore-bearing bacterium, Csfl2, and its application in clubroot disease of stem mustard. Background Technology

[0002] Clubroot disease of cruciferous plants is a worldwide soil-borne plant disease caused by *Platycotyle brassicae*. Plasmodiophora brassicae Clubroot fungi can infect approximately 3,700 species from 388 genera of cruciferous plants, including major crops such as Chinese cabbage, rapeseed, radish, turnip, and kale. （索欢, 陈龙正, 徐海, 等. 十字花科根肿病研究进展[J]. 安徽农业科学, 2015, 43(14): 115-117+126.） Plasmodium fungi infect the roots of plants, causing tumors of varying sizes to form. This affects the absorption of water and nutrients by the roots, and in severe cases, can lead to the wilting or even death of the entire plant, impacting the production of cruciferous crops in my country. （蒋欢, 彭玉梅, 闫玉芳, 等. 榨菜根肿病生防细菌的筛选、鉴定及评价[J]. 植物保护, 2018, 44(02): 104-110） Clubroot fungus overwinters and oversummers as dormant spores in the soil or diseased plant debris. It is highly resistant to adverse environments and can generally survive for more than 4 years. It has multiple transmission routes; the pathogen can be carried and spread to disease-free areas via seedlings, soil, irrigation water, and farm tools. （邱家德. 芸苔根肿菌致病特性及防治[J]. 农业科技与信息, 2016, (05): 87-88） Because the clubroot pathogen can be spread through seeds and survive for a long time in the soil, the disease has spread rapidly in my country. （谭博元. 油菜根肿病微生物调控防治研究[D]. 沈阳农业大学, 2018） .

[0003] Currently, the control of clubroot disease in cruciferous plants can be broadly categorized into breeding highly resistant varieties, soil improvement, agricultural control, chemical control, and biological control. （贾华生, 朱学松, 何平, 等. 十字花科蔬菜根肿病综合防治技术[J]. 农业科技通讯, 2005, (08): 38-39.） Biological control, as an important means of green pest control, has a relatively small impact on the environment and food safety, and has the advantages of being highly efficient, environmentally friendly, and harmless to humans and animals. （林洪波. 蔬菜病虫害识别与综合防治技术[J]. 安徽农学通报, 2024, 30(23): 31-34.） Previous studies have shown that various beneficial bacteria exist in the soil, which can significantly inhibit the occurrence of clubroot disease, such as Bacillus subtilis XF-1 (…). Bacillus subtilis ) （贾媛, 孙睿揆, 吴毅歆, 等. 枯草芽孢杆菌XF-1提取物对11种植物病原菌的抑制作用[J]. 安徽农业大学学报, 2011, 38(05): 753-756.） Trichoderma ( Trichoderma spp.) （杨力凡. 深绿木霉Trichoderma atroviride生物菌肥的研制及对油菜菌核病、根肿病的生物防治[D]. 四川农业大学, 2010.） Streptomyces ( Streptomyces platensis ) （刘秀秀. 根肿菌生防菌株的筛选及应用[D]. 华中农业大学, 2023） , Lysobacteria ( Lysobacter (spp.) 刘秀秀. 根肿菌生防菌株的筛选及应用[D]. 华中农业大学, 2023） etc., but no Cladosporium species were found. Cladosporium Reports on the prevention and treatment of clubroot disease.

[0004] Cladosporium ( Cladosporium Fungi are a type of abundant fungi, widely distributed in terrestrial and marine environments, such as higher plants, insects, rhizosphere soil, marine sediments, sponges, algae, cnidarians, and seawater. （夏辰曦, 刘昕明, 彭亮, 等. 珊瑚礁泥砂真菌多样性及真菌Cladosporium sp.GXIMD02067天然产物分离与鉴定[J]. 微生物学通报, 2023, 50(11): 4 784-4795）The structural types of chemical components in the secondary metabolites of the genus fungi are various, and the biological activities are outstanding, including alkaloids, polyketides, macrolides, steroids and terpenes and other compound types, most of which have antibacterial, antiviral and cytotoxic biological activities （王梦芹, 帖青清, 黄晓雯, 等. 北部湾珊瑚共附生真菌Cladosporium sp. SCSIO41206次级代谢产物研究[J / OL]. 广西科学, 2025, 1-10） The fungus has no patent application on the resistance to root rot of cruciferous plants, and only two patent publications: one is the fungus DLSH-DA-1 of the genus Cladosporium, which has excellent salt tolerance, especially high-concentration sulfate tolerance. It has high-efficiency COD (chemical oxygen demand) removal capacity in high-salt environment, and is suitable for removal of COD and other environmental harmful substances in high-salt wastewater, especially high-sulfate wastewater (CN202111439942.6); the other is a low-temperature straw degrading fungus NJAU-M3a1 and its application, which can effectively degrade corn straw under low-temperature conditions and promote crop growth, and has important agricultural and environmental protection values (CN202410726294.X).

[0005] After our in-depth research, we cultivate a cladosporium and name it Csfl2, i.e. Cladosporium sp. Csfl2, and explore its biocontrol effect on the root rot of stem tumor mustard, which can inhibit the occurrence of the root rot of stem tumor mustard, and therefore we propose a cladosporium Csfl2 and its application in the root rot of stem tumor mustard. SUMMARY

[0006] The purpose of the present application is to provide a cladosporium Csfl2 and its application in the root rot of stem tumor mustard, which is used for inhibiting the occurrence of the root rot of stem tumor mustard and has a biocontrol effect on the root rot of stem tumor mustard.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: A cladosporium fungus Cladosporium sp. Csfl2, which is preserved in the Institute of Microbiology, Chinese Academy of Sciences on April 14, 2025, with the preservation number of CGMCC No. 41914 and the preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0008] The nucleotide sequence of the cladosporium fungus Cladosporium sp. Csfl2 is shown as SEQ ID No. 1.

[0009] The application of the cladosporium fungus Cladosporium sp. Csfl2 in the prevention and treatment of the root rot of stem tumor mustard.

[0010] The application method is: After the stem tumor mustard seedlings grow for 3 weeks, 1x10 8 mL of the root rot fungus spore suspension is inoculated, and 10 mL of the root rot fungus spore suspension is irrigated for each plant. 24 h after inoculation, the roots were irrigated with the same way with the suspension of Clavibacter Cladosporium sp. Csfl2, 10 mL per plant; Then, the roots were irrigated again at 7 d and 14 d after inoculation, respectively, for a total of 3 times.

[0011] The active bacteria suspension of Clavibacter Cladosporium sp. Csfl2 was used.

[0012] The present application has at least the following beneficial effects: The present application provides a Clavibacter Cladosporium sp. Csfl2, which is found to be able to inhibit the occurrence of root tumor disease of Stemmatum, and has a biocontrol effect on the root tumor disease of Stemmatum. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0014] Figure 1 Figure is a colony chart of Csfl2; a-front, b-back; c-mycelia and spore morphology; Figure 2 Figure is a phylogenetic tree of strain Csfl2 constructed based on ITS sequence; Note: the tree is constructed by Maximum Likelihood method. The numbers on the branch nodes represent the Bootstrap support rate based on 1000 times of repetition (only values ≥50% are shown); Figure 3 Figure is the influence of Csfl2 treatment on the incidence and disease index of root tumor disease of Stemmatum; Figure 4 Figure is the influence of Csfl2 treatment on the incidence (a) and disease index (b) of root tumor disease of Stemmatum; Note: * and *** in the figure indicate significant differences when P<0.05 and 0.001 in T test of two groups of data; Figure 5 Figure is the influence of different treatments on the occurrence of root tumor disease of Stemmatum; (a~d) are the galls phenotype of Stemmatum under different treatments, respectively, root gall treatment (CK), Csfl2 treatment (treatment A), Csfl2 culture solution treatment (treatment B), and Csfl2 bacterial body treatment (treatment C); (e) and (f) are the incidence and disease index, and different lowercase letters indicate P<0.05 level difference (single factor ANOVA, LSD); (a) in the figure indicates 1 cm. Figure 6 Root phenotypes of *Plasmodiophora stylosa* under different treatments; (a) Uninoculated water treatment (water), (b) *Plasmodiophora stylosa* treatment (CK), (c) Csfl2 culture medium treatment (treatment D), (e) Csfl2 cell treatment (treatment E), (d) Csfl2 culture medium 10× treatment (treatment F), (f) Csfl2 cell treatment 10× treatment (treatment G); (a) The scale bar represents 1 cm; Figure 7 The severity of clubroot disease in different treatments of stem mustard; (a) and (b) represent the incidence and severity indices of clubroot disease in stem mustard. Different lowercase letters indicate significant differences at the P < 0.05 level (one-way ANOVA, LSD). CK, treatment D, treatment E, treatment F, and treatment G are references. Figure 6 Notes. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] Example 1: This example discloses a test method for Csfl2. 1.1 Isolation, purification, and identification of Csfl2 strain Microorganisms in the rhizosphere soil of *Musa parasitica* were isolated using a soil dilution separation method. 1.0 g of soil sample was weighed and placed in an Erlenmeyer flask containing 99 mL of sterile water. The flask was shaken at 37°C and 200 r / min for 20 min to prepare a soil suspension. This suspension was then serially diluted 10-fold to prepare 10... -3 10 -4 10 -5Soil suspension, each take 100 μL respectively coated in high's first medium, each concentration repeated 3 times, 27℃ constant temperature incubator culture 3-5 d, and then using the tip of the hypha separation method for purification. Fresh plant genomic DNA column extraction kit (Kofler) was used to extract microbial DNA, and the ITS sequence of fungi was amplified by PCR. The PCR system included 15 μL PCR2x mix, ITS1 primer (5'-CCGTAGGTGAACCTGCGG-3') 1 μL, ITS4 primer (5'-TCCTCGCTTATTGATATGC-3') 1 μL, DNA template 1 μL, and ddH2O 12 μL. The PCR reaction program was 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min. The PCR product was detected by 1% agarose gel electrophoresis and sent to Huada Gene for sequencing. The obtained sequence was submitted to NCBI for comparison and identification.

[0017] 1.2 Csfl2 effect evaluation on clubroot Mix the peat soil and vermiculite in a volume ratio of 1:3, sterilize at 121℃, and then divide into seedling pots (9 cm long x 9 cm wide x 11 cm high) for use. The seeds of Fuzha No. 2 were sterilized with 75% alcohol for 1 min, then washed with sterile water for 2 times, treated with 50% 84 disinfectant for 5 min, and then washed with sterile water for 5 times (1 min each time), and then sowed in the above treated seedling pots.

[0018] Preparation of Agrobacterium tumefaciens suspension: Take the galls out of the-20℃ refrigerator, rinse with tap water, cut into small pieces, and put into a tissue grinder. Add sterile water at a mass-volume ratio of 1:1, homogenize, and filter with 8 layers of gauze to obtain the Agrobacterium tumefaciens suspension. The concentration of Agrobacterium tumefaciens was calculated by using a hemocytometer, and diluted with sterile water to a final concentration of 1×10 8 individuals / mL.

[0019] Preparation of different formulations of Csfl2: First, the Csfl2 strain was activated by transferring to potato sucrose solid (PDA) medium and incubated at 28°C for about one week; then the newly grown mycelium at the edge was scraped and inoculated into potato sucrose liquid (PD) medium (350 mL), and incubated at 28°C for one week; the homogenate was Csfl2 suspension (treatment A) using a tissue homogenizer. After one week of water culture, the mycelium and culture solution were separated by centrifugation at 6000 rpm for 30 min, and Csfl2 culture solution (treatment B) was obtained; the mycelium (about 5 g) was homogenized in sterile water (350 mL) to obtain the mycelium suspension (treatment C). The Csfl2 mycelium was scraped and inoculated into potato sucrose liquid (PD) medium (350 mL), and incubated at 28°C, 170 rpm for one week; then the mycelium and culture solution were separated by centrifugation at 6000 rpm for 30 min, and the shaken Csfl2 culture solution (treatment D) was obtained, which was diluted 10 times to obtain treatment F; the mycelium (about 10 g) was homogenized in sterile water (350 mL) to obtain the mycelium suspension (treatment E), which was diluted 10 times to obtain treatment G.

[0020] Treatment and investigation: After the seedlings of A. thaliana grew for 3 weeks, they were inoculated with 1 × 10 8 spores / mL of the resting spore suspension of P. brassicae, and 10 mL of the suspension was poured into the root of each plant. At 24 h after inoculation, the plants were poured with 10 mL of the Csfl2 suspension (A, B, C, D, E, F, G) in the same way; then the pouring treatment was repeated once again at 7 d and 14 d after inoculation, respectively, for a total of 3 times. The incidence of the clubroot disease was investigated at 22 d after inoculation. The incidence of the clubroot disease was graded as 0-4 (Agarwal et al., 2011), i.e., 0 = no disease, no swollen root; 1 = very small swollen root, mainly on lateral roots, no swollen root on main root; 2 = small swollen root, mainly on main root, a small amount on lateral roots; 3 = medium-sized swollen root, on both main and lateral roots, which may affect the growth of the plant; 4 = severe swollen root on both main and lateral roots, which affects the growth of the plant.

[0021] The data were recorded and statistically analyzed using Excel and IBM SPSS Statistics 27 software.

[0022] Incidence rate (%) = (diseased plant / total plant) × 100%.

[0023] Disease index = (n0×0 + n1×1 + n2×2 + n3×3 + n4×4) × 100 / (N×4), where n0 to n4 are the number of plants at each level, 0-4 is the grade of the clubroot disease, and N is the total number of plants investigated.

[0024] Control efficiency (%) = (control disease index - treatment disease index) × 100% / control disease index 1.3 Experimental result case Experimental result 1: Plasmodiophora brassicae treatment (CK) and Csfl2 treatment (treatment A), 10 seedlings of T. caerulescens per treatment.

[0025] Experimental result 2: Plasmodiophora brassicae treatment (CK) and Csfl2 treatment (treatment A), 3 repeats per treatment, 10 seedlings of T. caerulescens per repeat.

[0026] Experimental result 3: Plasmodiophora brassicae treatment (CK), Csfl2 treatment (treatment A), Csfl2 culture solution treatment (treatment B) and Csfl2 mycelium treatment (treatment C). 3 repeats per treatment, 10 seedlings of T. caerulescens per repeat.

[0027] Experimental result 4: Water treatment (water), Plasmodiophora brassicae treatment (CK), shake culture Csfl2 culture solution treatment (treatment D), shake culture Csfl2 mycelium treatment (treatment E), shake culture Csfl2 culture solution 10-fold dilution treatment (treatment F) and shake culture Csfl2 mycelium 10-fold dilution treatment (treatment G). 3 repeats per treatment, 10 seedlings of T. caerulescens per repeat.

[0028] Example 2, this example is used to disclose specific research results: 2.1 Strain information The morphology of the Csfl2 strain obtained by isolation and purification is as shown in Figure 1 grown at 28°C for 14 days on PDA medium, with a surface of velvet, brownish, wrinkled and black pigment production; hyphae with septa, light brown, about 2.5-6.3 μm directly; conidiophores straight, unbranched; conidia oval, gray, about (3.7-8.3 μm) x (2.3-4.0 μm) in size, produced in a chain-like arrangement towards the top, forming branched spore chains.

[0029] The ITS sequence of the Csfl2 strain was amplified by ITS1 and ITS4 universal primers, and the sequence information obtained by sequencing was: SEQ ID No. 1: GGTCTACCACCGGGATGTTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTCGCTTGGTATTGGGCAACGCGGTCCGCCGCGTGCCTCAAATCGACCGGCTGGGTCTTCTGTCCCCTAAGCGTTGTGGAAACTATTCGCTAAAGGGTGCTCGGGAGGCTACGCCGTAAAACAAACCCATTTCTAAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGGCGGGAGGAA See Figure 2 the phylogenetic tree of strain Csfl2 based on ITS sequence, the above sequence is identified as Fungi, Ascomycota, Dothideomycetes, Cladosporiaceae, Cladosporium by NCBI sequence alignment. Cladosporium The evolutionary analysis result based on ITS rRNA gene sequence shows that Csfl2 is clustered with Cladosporium pseudotenuissimum strain ZJ12E01, but the Bootstap value is lower than 50%.

[0030] 2.2 The prevention effect of Csfl2 strain on root tumor disease of tumorous mustard The experimental result 1: the preliminary screening result of greenhouse test ( Figure 3 ) shows that the incidence of the control group (CK) is 100%, and the disease index is 60.00; the incidence of Csfl2 treatment (treatment A) is 50%, and the disease index is 25.00, with a prevention effect of 58.33%.

[0031] The experimental result 2: the greenhouse test result ( Figure 4) showed that the incidence rate and disease index of CK were 82.59% and 42.22, respectively; the incidence rate and disease index of Csfl2 (treatment A) were 20% and 8.33, respectively, and the control efficiency was 80.27%.

[0032] Experimental result 3: results of greenhouse test Figure 5 ) showed that the incidence rate and disease index of CK were 90.00% and 49.17, respectively; the incidence rate and disease index of Csfl2 (treatment A) were 10.00% and 4.17, respectively, and the control efficiency was 91.52%; the incidence rate and disease index of Csfl2 culture solution (treatment B) were 24.00% and 11.33, respectively, and the control efficiency was 76.96%; the incidence rate and disease index of Csfl2 cell suspension (treatment C) were 63.33% and 30.83, respectively, and the control efficiency was 37.30%. The difference significance analysis showed that the incidence rate and disease index of treatment A and treatment B were significantly lower than those of CK and treatment C, and the control efficiency of treatment C was the weakest.

[0033] Experimental result 4: results of greenhouse test Figure 6 and Figure 7 ) showed that the incidence rate and disease index of CK were 86.67% and 46.67, respectively; the incidence rate and disease index of Csfl2 culture solution (treatment D) were 70.00% and 35.00, respectively, and the control efficiency was 25.01%; the incidence rate and disease index of Csfl2 cell suspension (treatment E) were 96.67% and 46.83, respectively, and there was no control efficiency; the incidence rate and disease index of 10-fold diluted Csfl2 culture solution (treatment F) were 73.33% and 33.33, respectively, and the control efficiency was 28.58%, which was not significantly different from that of undiluted Csfl2 culture solution; the incidence rate and disease index of 10-fold diluted Csfl2 cell suspension (treatment G) were 100.00% and 53.33, respectively, and there was no control efficiency, which was not significantly different from that of CK and treatment C.

[0034] The above results of greenhouse test showed that the control efficiency of Csfl2 (treatment A) on the root rot of T. caeruleum was 58.33-91.52%; the control efficiency of Csfl2 culture solution without shaking (treatment B) was 76.96%; the control efficiencies of Csfl2 culture solution (treatment D) and 10-fold diluted solution (treatment F) were 25.01% and 28.58%, respectively; the control efficiency of Csfl2 cell suspension without shaking (treatment C) was 37.30%; and the control efficiencies of Csfl2 cell suspension (treatment E) and 10-fold diluted solution (treatment G) were 0.

[0035] Therefore, it can be known that the Csfl2 can inhibit the occurrence of the root tumor disease of stem tumor mustard, and has the biological control effect on the root tumor disease of stem tumor mustard.

[0036] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection required by the present application is defined by the appended claims and their equivalents.

Claims

1. A type of Cladosporium Cladosporium sp. Csfl2, characterized in that, The Cladosporium was preserved in the Institute of Microbiology, Chinese Academy of Sciences on April 14, 2025, with a preservation number of CGMCC No. 41914 and a preservation address of No. 3, Xibeiyilu, Beijing Chaoyang District.

2. A Cladosporium sp. according to claim 1 Cladosporium sp. Csf 12, characterized in that, the branch of the fungus Cladosporium The nucleotide sequence of the Csfl2 of the branch of the fungus is shown as SEQ ID No.

1.

3. The Cladosporium sp. of claim 1 Cladosporium Use of the Cladosporium sp. Csf 12 in the control of clubroot disease in Brassica napus.

4. Use according to claim 3, characterized in that, The application method is: After 3 weeks of growth, seedlings were inoculated with 1 x 10 8 spores / mL of A. brassicae, 10 mL per plant. The roots were irrigated with the same way 24 h after inoculation with 10 mL of suspension of Trichoderma harzianum per plant Cladosporium sp. Csfl2 per plant. Then, the root irrigation treatment is carried out again at 7 d and 14 d after inoculation, respectively, for a total of 3 times.

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