Application of bottle cladosporium LC3 in crop growth promotion and bacterial wilt resistance

Abstract

The invention relates to the technical field of microorganisms, in particular to application of Cladosporium LC3 in crop growth promotion and bacterial wilt resistance, the strain LC3 belongs to Cladosporium and Cladosporium immonda species, the strain is separated from Guangxi sugarcane rhizosphere soil, experimental evaluation screening finds that the strain has a good growth promotion function on tomatoes and fresh ginger, and the strain can be applied to crop growth promotion and bacterial wilt resistance. The yield of ginger is remarkably increased, and the growth vigor of tomato plants is effectively improved; meanwhile, the strain has a good prevention and treatment effect on tomato and ginger bacterial wilt and has good plant bacterial wilt resistance, and screening verification of the strain provides a new fungicide and a raw material for later-period growth of an anti-microbial agent or a biological organic fertilizer.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of Cladosporium LC3 in crop growth promotion and resistance to bacterial wilt. Background Art

[0002] Ralstonia solanacearum is a soil-borne pathogen with a wide host range, especially causing devastating damage to important crops such as tomatoes, peppers, potatoes, and ginger. It has the characteristics of rapid onset, rapid spread, and high mortality, and is considered to be the most destructive pathogen on plants. Ralstonia solanacearum is a soil-dwelling fungus and can be widely spread with agricultural tools, soil, and running water. Therefore, it is difficult to control and causes huge economic losses to agricultural production. At present, there are three main methods for the bacterial wilt caused by Ralstonia solanacearum: physical control, chemical control, and biological control. Physical and chemical control cannot be effectively implemented due to time and space limitations, pesticide residues, environmental pollution, and other problems. Biological control uses biocontrol bacteria as the main body, which not only ensures the prevention effect but also meets the requirements of green environmental protection, and is regarded as an important means of sustainable agricultural development. Therefore, screening biocontrol bacteria for crop bacterial wilt is of great significance to the sustainable and green development of agricultural production.

[0003] At present, the screening of biocontrol bacteria for bacterial wilt is mostly based on antagonism and inhibition. The commercially available biocontrol agents are mainly bacteria and Trichoderma products, with relatively single strains, and it is difficult to exert ideal antagonistic effects on crop bacterial wilt, which has serious physiological subspecies differentiation and is complex and diverse. In addition, affected by the complex soil environment in the field, most biocontrol strains have poor field colonization ability and cannot create a microecological environment that is conducive to the healthy growth of plants but not conducive to the occurrence of diseases, resulting in unstable control effects of biocontrol bacteria in field applications. Therefore, new biocontrol microorganisms are still urgently needed in production.

[0004] Plant endophytic fungi have shown obvious advantages in the field of biocontrol because they can establish a relatively stable symbiotic relationship with host plants. Existing technical research has found that endophytic biocontrol fungi can enter the body of new plants through plant seed germination and seedling treatment, preferentially occupy ecological niches, and can more stably exert their biocontrol functions; there are also reports in the existing technology that endophytic fungi can control soil-borne diseases such as banana wilt, and their control mechanism is mainly to induce a significant improvement in the systemic disease resistance of host plants, but there are few reports on their control of bacterial wilt. Screening for endophytic fungal strains resistant to bacterial wilt can broaden the application scope of this type of fungi and promote their development and utilization. Summary of the invention

[0005] In view of the above, it is necessary to continuously screen out effective endophytic fungi, study their infection effects on the host, enrich the endophytic fungal germplasm resource bank, and provide new germplasm for the manufacture of biocontrol agents.

[0006] To achieve the above-mentioned purpose, the deposit number of the Cladosporium strain Cladophiaphora immunda LC3 of the present application is CGMCC NO.41540; the strain is classified and named as: Cladophiaphora immunda and is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is October 12, 2024.

[0007] The present invention also includes a bacterial agent comprising the Cladosporium phialum Cladophiaphora immunda LC3 strain.

[0008] The present invention also includes the use of the Cladosporium immunda LC3 strain and the bacterial agent in resisting plant bacterial wilt.

[0009] The present invention also includes the use of the Cladosporium phialariae immunda LC3 strain or the bacterial agent in promoting plant growth.

[0010] Furthermore, the plant is: tomato and / or ginger.

[0011] The present invention also includes a method for plant cultivation using the Cladosporium phialaria strain or the bacterial agent, which comprises inoculating a bacterial solution containing the Cladosporium phialaria LC3 into the plant and then carrying out cultivation.

[0012] Furthermore, the inoculation method is: transplanting plant seedlings onto a cultured Cladophiaphora immunda LC3 colony; irrigating the plant roots with a bacterial solution containing Cladophiaphora immunda LC3; or soaking the plant roots in the bacterial solution of Cladophiaphora immunda LC3.

[0013] The present invention has the following beneficial effects:

[0014] The strain LC3 of the present invention belongs to the genus Cladosporium and the species Cladophiaphora immunda. The strain is screened and obtained from a tomato disease field with serious bacterial wilt in Guangxi. Experimental verification shows that the strain has a good growth-promoting function on both tomatoes and ginger, significantly improves the ginger yield, and effectively improves the growth of tomato plants. At the same time, the strain also has a good prevention and control effect on tomato and ginger bacterial wilt, and has a good plant resistance to bacterial wilt. The screening and verification of the strain provides new bacterial agents and raw materials for the later development of growth control fungi or bio-organic fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the morphological identification result of strain LC3.

[0016] Figure 2 This is the phylogenetic tree of strain LC3 constructed based on the ITS sequence.

[0017] Figure 3 This is a graph showing the results of the determination of the symbiotic characteristics of strain LC3 on tomato seedlings, wherein this graph is an experimental graph after 14 days of cultivation, CK is a blank control group not inoculated with strain LC3, and LC3 is an experimental group inoculated with the strain.

[0018] Figure 4 This is a microscopic examination of the colonization structure of strain LC3 at the root of tomato seedlings; the arrows in the figure point to the dark septate hyphae of strain LC3.

[0019] Figure 5 This is a diagram of the survival status of ginger potted plants 55 days after inoculation with pathogens; in the figure, CK is the blank control group without inoculation of strain LC3; LC3 is the experimental group inoculated with the strain.

[0020] Figure 6 The following are the potted control effects of strain LC3 on ginger bacterial wilt; Figure a shows the plant height experimental results, Figure b shows the ginger tiller number experimental results, Figure c shows the survival rate experimental results, Figure d shows the disease index experimental results, and Figure e shows the LC3 control effect experimental results; in the figure, * represents P < 0.05; ** represents P < 0.01.

[0021] Biomaterial Deposit Information

[0022] The deposit information of strain LC3 in this application is:

[0023] ① Cladophiaphora immunda strain LC3, its classification name is: Cladophiaphoraimmunda, its Chinese classification name is: Cladosporium, and its preservation number is CGMCC NO.41540; the strain is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is October 12, 2024. DETAILED DESCRIPTION

[0024] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0025] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0026] The culture medium and formula used in the examples of this application are as follows:

[0027] Potato dextrose agar medium (PDA): 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, sterilized by high pressure steam at 121°C for 30 min.

[0028] Malt extract medium (CMMY): 10.0 g malt extract, 2.0 g yeast extract, 8.5 g corn flour agar, 7.5 g agar, 1000 mL distilled water, sterilized at 121°C with high pressure steam for 30 min. Used to observe the colony morphology of Cladosporium sporeans.

[0029] Oatmeal medium (OMA): MgSO4 7H20 1.0g, KH2PO4 15g, NaNO3 1.0g, oatmeal powder 10g, agar powder 11g, distilled water 1000mL, 121℃ high pressure steam sterilization for 30min. Used to observe the spore structure of Cladosporium strains.

[0030] 1.5% agar medium (WA): 15 g agar, 1000 mL distilled water, sterilized by high pressure steam at 121°C for 30 min. OMA medium for carrying small pieces.

[0031] Example 1

[0032] This example is the classification and identification of strain LC3.

[0033] 1. Cladosporium LC3 was isolated from sugarcane rhizosphere soil in Guangxi.

[0034] 2. Identification of Cladosporium LC3:

[0035] ① Morphological identification: The strain was inoculated on CMMY medium and the colony morphology was observed after 14 days of culture. At the same time, a sterilized coverslip was placed on WA medium, and a 0.5 cm × 0.5 cm OMA medium was placed in the center of the coverslip. At the same time, the LC3 strain was inoculated on OMA medium and covered with another sterilized coverslip. After culturing at 28°C for 2 weeks, it was placed in a 4°C refrigerator to stimulate spore production. After one week, the coverslip was removed and the morphology of hyphae, spore production structure and conidia was observed under an Olympus BX53 microscope.

[0036] like Figure 1As shown, the colony grows at a moderate rate on CMMY medium. After 2 weeks of culture at 28°C, the colony is 28 mm in diameter, gray, tapered, round, and black on the back. The hyphae are transparent to light brown, septate, and 2.3-4.1 μm in diameter. Conidiophores are not significantly different from hyphae, slightly thicker than hyphae, 2.6-5.5 μm in diameter, light brown, 1-8 septa, upright, branched or unbranched. The conidial cells are drop-shaped, 1.5-4.2 μm in diameter, often branched, and the conidia on them will leave obvious tooth-like spore scars after falling off. Conidia are single-celled, terminal, often chain-arranged or head-shaped, translucent to light brown, spherical, elliptical to lemon-shaped, spindle-shaped or teardrop-shaped, smooth, and unseptate. Some spores have a hilum at both ends or one end. Non-spherical spores are 3.2-6.4×2.1-3.6μm in size; spherical spores are 2.2-4.2μm in diameter.

[0037] The morphological characteristics of strain LC3 were basically consistent with the description of Cladophialophora immunda by Badali et al. (2008), so the strain was identified as Cladophialophora immunda.

[0038] ② Molecular identification: After the LC3 strain was activated in PDA medium, the mycelium was scraped and the genomic DNA of the strain was extracted using a fungal DNA kit. The extracted DNA was used as a template and the ITS rRNA sequence was amplified by PCR using the universal fungal primers ITS1 and ITS4. The amplification procedure was as follows: after pre-denaturation at 94℃ for 5min; 35 cycles were performed in the order of denaturation at 94℃ for 40s, annealing at 56℃ for 40s, and extension at 72℃ for 1min60s; and then extended at 72℃ for 10min. The PCR amplification product was detected by 0.8% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequence was uploaded to NCBI, and the approximate genus-level classification status of the strain LC3 was confirmed by BLAST comparison. Then the ITS sequences of the known species model strains of the genus and the representative strains of its closely related species were downloaded from NCBI. The phylogenetic evolutionary tree was constructed by the neighbor-joining method using MEGA 11.0 to determine the taxonomic status of the target strain. The ITS similarity between strains was calculated by NCBI's BLAST comparison.

[0039] The comparison results in NCBI are as follows Figure 2As shown, the strain with the highest ITS sequence similarity to strain LC3 is Cladophialophora immunda FMR20179, with a similarity of 99.7% (578 / 580). In the phylogenetic tree constructed based on the ITS sequences of the type strains of known species of the genus and the representative strains of the closely related species of strain LC3, strain LC3 was clustered in a monophyletic evolutionary branch with 13 representative strains of Cladophialophora immunda, including the type strain, with a support rate of 54%. Among them, strain LC3 and FMR20179 from marine sediments (Guerra-Mateo et al., 2024) and CBS126725 from healthy palm stems (Vicente et al., 2014) formed a small evolutionary branch, while the type strain CBS 834.96 was clustered in another small evolutionary branch with two strains related to environmental pollution with a support rate of 100%, and the remaining strains related to plant pathogens or animals were clustered in a third small evolutionary branch with a support rate of 96%. The ITS similarity between strain LC3 and the Cladophialophora immunda model strain CBS 834.96 was 98.1%, which further proved the reliability of strain LC3 identification as Cladophialophora immunda.

[0040] Example 2

[0041] This example is a method for determining the symbiosis between the strain LC3 of Example 1 and a plant, and the details are as follows:

[0042] The strain was inoculated into OMA medium, with 3 bacterial blocks per dish, and cultured for 7-14 days before use. Referring to the method of Xie Ling (2018), tomato was used as the host plant. After the surface of tomato seeds was sterilized, they were germinated on WA medium. After the seeds germinated into seedlings, they were transplanted to the cultured LC3 colonies. One sterile seedling was transplanted to each colony, with 3 seedlings per dish. The culture dish was placed in a tissue culture bottle and kept at a temperature of 26°C and a light intensity of 180 μmol / m 2 s 2 , and co-cultivated in an incubator with a light duration of 16h / d. The uninoculated treatment was used as the control, with 9 tomato seedlings per replicate and 4 replicates per treatment. After 14 days of co-cultivation, the growth of the host plant was recorded, and the roots of the host plant were washed and divided into 3 samples. One was placed in a 50℃ oven and baked to constant weight, and then the dry weight was weighed; one was re-isolated to determine whether the re-isolated strain was the original inoculated strain; the last was sectioned and stained with lactic acid phenol cotton blue, and the colonization of hyphae in the roots was observed under a microscope.

[0043] The results are as follows Figure 3-Figure 4As shown: Under sterile conditions, LC3 did not show disease symptoms after being co-cultured with tomato seedlings for 14 days. Compared with the uninoculated CK, the plant height was higher, the leaves were flatter and more stretched, the stems had fewer hairs and were darker in color ( Figure 3 ), and its dry weight increased by 15.6%. Through sectioning and lactophenol cotton blue staining, it was observed under a microscope that LC3 hyphae colonized in the epidermal cells of the roots ( Figure 4 ) ; No colonization of endophytic fungal hyphae was observed in the root sections treated with CK. The re-isolated strain was completely consistent with the original inoculated strain LC3 by ITS sequence alignment.

[0044] Example 3

[0045] This example is an experiment on the potted plant biocontrol effect of Cladosporium strain LC3 on tomato bacterial wilt, as follows:

[0046] It was carried out in the greenhouse of the Institute of Plant Protection of Guangxi Academy of Agricultural Sciences. Germination period of tomato seeds: soak in 60℃ warm water for 2h, take out and place in a culture dish (covered with a wet towel on the top and bottom), and germinate for 72h in a light incubator (temperature 28℃, humidity 80℃, no light). Seedling period: select plump seeds with consistent bud length, soak them in LC3 bacterial suspension for 30min, soak them in an equal amount of sterilized water as a control (CK), sow them in seedling trays, inoculate 10mL of bacterial suspension as rooting water for each seedling, and place them on a light culture rack for cultivation (lighting conditions: 16h of light, light intensity 30000lx, open at 6:00 am and close at 22:00 pm). Growth period: After 25 days of seedling cultivation, transplant the seedlings into pots (20.6cm×17.5cm×15cm) filled with 2 / 3 tomato bacterial wilt disease soil (collected from the Lijian Base of Guangxi Academy of Agricultural Sciences) and place them in a greenhouse (temperature 30±5℃, relative humidity 80±5%, natural light). Each replicate has 12 tomato seedlings, and each treatment is repeated 3 times. The disease situation was investigated on the 45th day after transplanting, and the incidence rate, disease index and control effect were calculated. The grading standard of tomato bacterial wilt disease index is as follows: Level 0, no wilting symptoms; Level 1, 1%-25% leaf wilt; Level 2, 26%-50% leaf wilt; Level 3, 51%-75% leaf wilt; Level 4, 76%-100% leaf wilt. Among them, the formulas for incidence rate, disease index and control effect are as follows:

[0047] Incidence rate (%) = (number of diseased plants / total number of plants) × 100;

[0048] Disease index = 100 × ∑ (number of plants at each disease level × representative value of each level) / (total number of plants surveyed × highest representative value);

[0049] The control effect (%) = 100 × (disease index of the control group - disease index of the treatment group) / disease index of the control group.

[0050] The potted plant biocontrol effect on tomato bacterial wilt is shown in Table 1:

[0051] Table 1 Biocontrol effect of strain LC3 on tomato bacterial wilt

[0052]

[0053] As shown in Table 1, under potted conditions, 45 days after the tomato seedlings were transplanted into the bacterial wilt disease soil, the bacterial wilt incidence of the tomato seedlings inoculated with LC3 was significantly lower than that of CK, and the disease index was extremely significantly lower than that of CK, with a prevention efficiency of 46.0%.

[0054] Example 4

[0055] This example is a potted plant biocontrol experiment of Cladosporium strain LC3 against ginger bacterial wilt, as follows:

[0056] The potted experiment was conducted in the greenhouse of the Plant Protection Institute of Guangxi Academy of Agricultural Sciences. Strong and uniformly growing ginger seedlings were selected and treated with a concentration of 5×10 5 CFU / mL LC3 bacterial solution was used to soak the roots for 30 minutes, and the same amount of sterilized water for soaking the roots was used as the control (CK). After transplanting, the remaining bacterial solution or sterilized water was used as rooting water to irrigate the flower pots and placed in a greenhouse (temperature 30±5℃, relative humidity 90±5%, natural light). 35 days after transplanting the ginger seedlings, the pathogen was inoculated. After the stem base of the ginger was pierced with a pin with a diameter of 0.4mm, each plant was inoculated with 100mL of ginger bacterial wilt pathogen (Ralstonia solanacearum) Gg24 bacterial solution. Three plants were planted in each pot, and each replicate had 4 pots of ginger seedlings. Each treatment was repeated 3 times. The disease situation was investigated 10d and 55d after the pathogen was inoculated, and the incidence rate, disease index and prevention effect were calculated. When the disease situation was investigated for the last time, the plant height and tiller number were also investigated.

[0057] The grading standard of ginger bacterial wilt disease index is as follows: 0, the whole plant is disease-free; 1, less than 10% of the leaves turn slightly yellow, and the fleshy stems have no obvious symptoms; 3, 11%-30% of the leaves turn yellow, the leaf margins are slightly curled, and the fleshy stems have water-soaked spots; 5, 31%-50% of the leaves turn yellow, the leaf margins curl, the plants are dwarfed, and the fleshy stems are partially rotten; 7, more than 51% of the leaves turn yellow, the plants wilt, and most of the fleshy stems rot; 9, the plants die, the fleshy stems rot or only the filamentous vascular bundles remain. The calculation formulas for the incidence rate, disease index and prevention effect are the same as the calculation formula for tomato bacterial wilt in Example 3.

[0058] The results are as follows Figure 5-Figure 6As shown in the figure: Under potted conditions, 55 days after inoculation with bacterial wilt pathogen, the early tillers of all treated plants had become diseased and died. Among them, 66.7% of the plants in the LC3 treatment could grow new branches and leaves again, while only 44.4% (survival rate) of the plants in the CK treatment grew new branches and leaves ( Figure 5 ), the survival rate of LC3 was significantly higher than that of CK( Figure 6 c). In the regrown branches and leaves, the plant height of LC3 treatment increased by 61.8% compared with CK, reaching a highly significant difference level ( Figure 6 a); there was no significant difference in tiller number between LC3 and CK ( Figure 6 b). At 10d and 55d after inoculation of pathogens, the disease index of LC3 treatment was significantly lower than that of CK ( Figure 6 d), the protection efficiency was 46.07% and 40.53% respectively ( Figure 6 e).

[0059] The statistical analysis method of the data in Examples 2 to 4 of the present application was performed using Microsoft Excel 2016 and DPS7.05 system. Duncan's new multiple range method in one-way analysis of variance was used for significant difference test (P < 0.05, P < 0.01), and GraphPad Prism 8 was used for plotting. All results are expressed as mean ± standard deviation (SD).

[0060] In summary, the Cladosporium phialium strain LC3 used in the present invention can well coexist with tomatoes and promote the growth of tomatoes and ginger. At the same time, the strain can effectively inhibit the growth of Ralstonia solanacearum, and has a good preventive and therapeutic effect on tomato and ginger bacterial wilt caused by it. It is a strain that has both plant growth promotion and bacterial wilt prevention, and can provide rich germplasm resources for the later development of antimicrobial agents or the preparation of biological organic fertilizers.

[0061] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Cladosporium phialariae immunda LC3 strain, characterized in that The deposit number of the Cladosporium phialariae immunda LC3 strain is CGMCC NO.41540.

2. A bacterial agent comprising the Cladosporium phialariae immunda LC3 strain as claimed in claim 1.

3. Use of the Cladosporium immunda LC3 strain according to claim 1 or the bacterial agent according to claim 2 in resisting plant bacterial wilt.

4. Use of the Cladosporium immunda LC3 strain according to claim 1 or the bacterial agent according to claim 2 in promoting plant growth.

5. The use according to claim 3 or claim 4, characterized in that: The plants are: tomato and / or ginger.

6. A method for plant cultivation using the Cladosporium immunda LC3 strain according to claim 1 or the microbial agent according to claim 2, characterized in that: The method comprises the following steps: inoculating a bacterial solution containing the Cladosporium LC3 into plants, and then cultivating the plants.

7. The method according to claim 6, characterized in that: The inoculation method comprises the following steps: transplanting plant seedlings onto a cultured Cladophiaphora immunda LC3 colony; irrigating plant roots with a bacterial solution containing Cladophiaphora immunda LC3; or soaking plant roots in the bacterial solution of Cladophiaphora immunda LC3.

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