Colletotrichum carpus CZ102 and application thereof

Abstract

The invention discloses a colletotrichum carmichaeli CZ102 strain and application thereof, and relates to the technical field of microorganisms. The colletotrichum caringenum CZ102 disclosed by the invention is characterized in that the preservation number is GDMCC 65838. The invention constructs a colletotrichum caryophyllum CZ102 infection model, verifies that the content of an oxidant in the carya illinoensis and the activity of defensive enzyme are remarkably induced after the colletotrichum caryophyllum CZ102 is inoculated, and in-vivo defensive related genes are remarkably induced and expressed in the early stage of infection so as to start a defensive reaction and participate in a disease-resistant process. It is verified that the resistance of different varieties of carya illinoensis to colletotrichum caryophyllum CZ102 is obviously different. The toxicity of eight bactericides to pathogenic bacteria is verified, the medicament for preventing and treating the colletotrichum gloeosporioides is screened in a targeted manner, and a research basis and a scientific basis are provided for later research on an interaction mechanism of the colletotrichum gloeosporioides and carya illinoensis and scientific prevention and treatment of carya illinoensis anthracnose.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a Colletotrichum fructicola CZ102 and its application. Background Art

[0002] Carya illinoinensis (Wangenh.) K. Koch, with the common name "pecan", is a deciduous tree of the genus Carya in the Juglandaceae family, native to the United States and northern Mexico. It is reported that anthracnose pathogens are currently one of the most serious pathogens harming Carya illinoinensis in China, but there are differences in anthracnose fungi on Carya illinoinensis in different regions. For example, some studies have found that Colletotrichum fructicola and C. viniferum are the pathogenic fungi of pecan black spot disease in Jiangsu and Zhejiang regions; some studies have respectively identified the pathogens of pecan black spot disease from Chuzhou, Anhui, Jiande, Zhejiang, and Ji'an, Jiangxi, and found that multiple anthracnose fungi can cause black spot disease, among which the anthracnose fungus causing pecan black spot disease in Chuzhou, Anhui is C. siamense. Some studies have isolated and identified the pathogenic fruits of pecan anthracnose in South Korea, and its pathogen has also been identified as C. siamense. Some studies have identified the pathogens of pecan diseased leaves and fruits in Brazil, and determined that its pathogen is C. nymphaeae.

[0003] Respiratory burst oxidase homolog D (RBOHD), LRR receptor-like protein kinase (LRR-RLK), pathogenicity-related gene transcriptional activator PTI6, glutathione peroxidase (GPX), and pathogenesis-related protein (PR) are all important plant defense-related genes and play important roles in resisting pathogen infection. Currently, in the research on pecan diseases, it mainly focuses on identifying the types of pathogenic bacteria and their biological characteristics, and there are few reports on the impact of pathogen infection on the defense response of pecan.

[0004] In nature, there are significant differences in disease resistance among different plants or different varieties of the same plant, which can generally be divided into: slightly disease-resistant, moderately disease-resistant, highly disease-resistant, or completely immune. Therefore, this natural difference provides valuable genetic resources for disease prevention and control. Currently, many pecan varieties have been introduced in China, among which 'Pawnee' is the main cultivated variety, and other varieties such as 'Shawnee','Mahan', and 'Stuart' are planted as pollinizer trees. Although multiple anthracnose fungi have been reported to cause pecan anthracnose, there are few studies on the resistance differences of pecan varieties to anthracnose fungi. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a Colletotrichum fructicola CZ102. Another technical problem to be solved by the present invention is to provide an application of Colletotrichum fructicola CZ102 for verifying plant defense responses.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A strain of Colletotrichum fructicola CZ102, which is deposited in the Guangdong Microbial Culture Collection Center, with the deposit number: GDMCC65838, the deposit date: January 21, 2025, and the deposit address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0008] Application of Colletotrichum fructicola CZ102 in the preparation of an infection model.

[0009] Application of the Colletotrichum fructicola CZ102 infection model in verifying plant defense responses.

[0010] The plant defense responses include changes in the activities of defense enzymes, the contents of oxidants, and the expression of defense-related genes.

[0011] The defense enzymes include SOD, POD, CAT, and APX.

[0012] The oxidants include H2O2 and O2 - .

[0013] The defense-related genes include RBOHD, LRR-RLK, PTI6, GPX, and PR-10.

[0014] Application of the Colletotrichum fructicola CZ102 infection model in verifying the disease resistance of plants.

[0015] The plants include pecan varieties 'Pawnee', 'Nacono', 'Stuart','Mahan', 'Shawnee', 'Shoshoni', 'Forkert', and 'Yizheng No.10'.

[0016] An agent for controlling Colletotrichum fructicola CZ102 is carbendazim.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1) The present invention identifies pathogens on thin-shell pecan leaves with typical anthracnose symptoms collected from a thin-shell pecan plantation in Dingyuan County, Anhui Province, and screens out the pathogenic bacteria of thin-shell pecan anthracnose, Colletotrichum spp. CZ102, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC 65838, a deposit date of January 21, 2025, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0019] 2) The present invention verifies the changes in the activities of defense enzymes in thin-shelled walnuts after inoculation with Colletotrichum oleraceus CZ102. The results show that after 24h and 72h of inoculation, the activities of H2O2 and O2 - The content was significantly induced (P<0.05); the SOD, POD, CAT and APX enzyme activities in the body increased significantly 24h after inoculation. At 72h after inoculation, the SOD, POD and CAT enzyme activities in the thin-shell pecan were still higher than those at 24h after inoculation, while the APX enzyme activity decreased significantly at this time, and there was no significant difference with the APX content at 0h after inoculation.

[0020] 3) The present invention verifies the relative expression of defense-related genes in thin-shelled pecans after inoculation with fruit spiny spore CZ102. The results show that after inoculation with fruit spiny spore, RBOHD, LRR-RLK, PTI6, and GPX in the leaves of thin-shelled pecans were induced to express at 6 hours after inoculation, and their relative expression levels increased significantly at 24 hours and reached a peak, and then decreased significantly at 36 hours, but were still significantly higher than the level before inoculation; while the relevant expression of the PR-10 gene in the leaves of thin-shelled pecans increased significantly at 6 hours, and continued to increase at 24 hours and 36 hours. The results show that in the early stage of infection by fruit spiny spores, defense-related genes in the thin-shelled pecans were significantly induced to start the defense response and participate in the disease resistance process.

[0021] 4) The present invention verifies the resistance of different varieties of thin-shelled pecans to the fruit-borne thorny spore CZ102. The results show that different varieties of thin-shelled pecans have significant differences in resistance to anthracnose caused by the fruit-borne thorny spore CZ102.

[0022] 5) The present invention verifies the toxicity of 8 fungicides against pathogenic bacteria, and the results show that carbendazim is the fungicide with the best inhibitory effect on CZ102. The agents for the prevention and treatment of anthrax are screened out in a targeted manner, providing a research foundation and scientific basis for the subsequent study of the interaction mechanism between anthrax pathogens and thin-shelled pecans and the scientific prevention and control of anthrax in thin-shelled pecans. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Morphological characteristics diagram of strain CZ102 (scale bar is 50 µm; A is the front view of the colony after 5 days of culture on PDA medium; B is the back view of the colony after 5 days of culture on PDA medium; C is the conidial morphology);

[0024] Figure 2 Diagram of the pathogenicity determination results of strain CZ102 (A is the leaf spot symptom after inoculation on detached leaves; B is the leaf spot symptom after inoculation on living seedlings);

[0025] Figure 3 Phylogenetic tree diagram of Colletotrichum constructed based on ITS, ACT, CHS-1, CAL, GAPDH, and TUB2 gene sequences (representative strains are marked with *; 0.020 represents the genetic distance);

[0026] Figure 4 Diagram of the relative expression levels of defense-related genes in Carya illinoinensis after inoculation with Colletotrichum fructicola. Detailed implementation methods

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well known to those skilled in the art.

[0028] The diseased leaves of the Carya illinoinensis 'Pawnee' variety used in this application were collected from Dingyuan County, Chuzhou City, Anhui Province.

[0029] Example 1

[0030] 1. Isolation and purification of Colletotrichum fructicola strain CZ102

[0031] Using the diseased leaves of the Carya illinoinensis 'Pawnee' variety collected during the field survey as materials, the diseased leaves were washed and dried, and tissues of 3 mm × 3 mm were cut at the junction of diseased and healthy parts. They were disinfected with 75% ethanol for 30 s, 1.5% sodium hypochlorite for 90 s, rinsed 3 times with sterile water, and then the surface excess water was blotted with sterilized filter paper. The diseased tissues were placed on PDA medium and cultured in the dark at 25 °C for 5 days. After the colonies grew, they were cut and transferred to a new PDA plate one by one until only one type of colony grew on the PDA plate.

[0032] Healthy detached leaves of the Carya illinoinensis 'Pawnee' variety and 3-month-old Carya illinoinensis seedlings were selected for the inoculation experiment. 20 μL of the spore suspension of the pathogen (1×10 6They were inoculated into the left sides of the leaves of in vitro Carya illinoinensis and 3-month-old Carya illinoinensis seedlings at 1×10⁶ conidia / mL respectively, and 20 μL of sterile water was inoculated on the right side as a control. The inoculated in vitro leaves were covered with transparent plastic wrap for moisturizing, and the inoculated living seedlings were bagged for moisturizing, and then placed in a greenhouse at 25 °C and 80% humidity. Each group of experiments was repeated 3 times. The disease symptoms of the inoculated materials were observed 7 days after inoculation. After the disease occurred, the diseased leaves were selected to isolate and culture the pathogen again for morphological and molecular biological identification.

[0033] Finally, 23 strains of fungi were isolated from the diseased leaves of the Carya illinoinensis 'Pawnee' variety in the field. Among them, 15 strains of Colletotrichum fungi with the same morphology and consistent ITS sequences were isolated (the isolation rate was 65%). One of them was selected as a representative strain and named CZ102.

[0034] 2. Morphological and biological identification of the pathogen

[0035] After culturing the pathogen on PDA medium at 25 °C in the dark for 7 days, observe the front and back morphologies of the purified colonies. Observe the morphology of the pathogen mycelium, conidia and sporulation structures under an optical microscope, randomly measure and record the sizes of 50 spores, and preliminarily judge the type of fungi.

[0036] The total DNA of the pathogen was extracted by the CTAB method. Six pairs of specific primers reported by predecessors for the identification of Colletotrichum were used to amplify the genes by PCR respectively. The primer sequences are shown in Table 1. The PCR products were detected by 1% agarose gel electrophoresis. After the gene fragments were purified, recovered, ligated to the T vector, and transformed into Escherichia coli competent DH5α, the positive transformants were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The obtained sequences were uploaded to NCBI (National Center for Biotechnology Information) and subjected to homology comparison in the nucleic acid database. Finally, the method of combining multiple genes to construct a phylogenetic tree was used. The maximum likelihood method (Maximum Likelihood, ML) in MEGA 7.0 software was used to construct a phylogenetic tree to determine the type of pathogen at the species level.

[0037] Table 1 Primers for amplifying the target fragment

[0038]

[0039] The results are as Figure 1 shown. At 25 °C, in the initial stage, the front morphology of the CZ102 strain colony on the PDA medium was light gray in the middle and grayish white on the outer edge, and the back morphology was dark gray in the middle and grayish white on the edge. The mycelium was loose and villous; in the later stage, the colony color became darker, the front was dark gray, and the back was black, only the outermost edge was grayish white ( Figure 1A). The fruiting body is orange-yellow. The conidia are single-celled, oblong, transparent, smooth, with blunt ends at both sides, and the size is (12.4~15.8) µm × (4.4~6.0) µm (n = 50) ( Figure 1 B–C). The morphological characteristics are consistent with the previous descriptions of Colletotrichum fructicola.

[0040] The results are as Figure 2 shown. After inoculation for 5–7 d, dark brown circular lesions appeared at the inoculation sites of both detached leaves and pecan seedlings, while there were no obvious changes in the control sites ( Figure 2 A–B), and the leaf spot symptoms were relatively consistent with the natural symptoms. The pathogen was re-isolated from the diseased leaf spot sites after inoculation, and a strain with a similar morphology to CZ102 was obtained, and the ITS sequencing results were the same as those of CZ102. Thus, Koch's postulates were verified. Therefore, this anthracnose fungus is the pathogenic bacterium of pecan anthracnose.

[0041] The ITS, ACT, CHS-1, CAL, GAPDH, and TUB2 genes of the representative strain CZ102 were amplified by PCR and sequenced. The results of BLAST homology alignment showed that these gene sequences had the highest homology with Colletotrichum fructicola in the database. The results are as Figure 3 shown. The results of multi-gene combined phylogenetic tree construction showed that the CZ102 strain clustered on the branch of C. fructicola, and the bootstrap support rate was 96%. Therefore, combining the morphological and molecular biological identification results, the CZ102 strain was identified as Colletotrichum fructicola. It is preserved in the Guangdong Provincial Culture Collection of Microorganisms, with the preservation number: GDMCC 65838, the preservation date: January 21, 2025, and the preservation address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0042] Example 2

[0043] A 5-mm mycelial disc was cut from the edge of the pathogen colony and inoculated on the leaves of 3-month-old pecan seedlings, with the seedlings inoculated with the blank medium as the control (0 h). The seedlings were bagged and kept moist in a greenhouse at 25 °C and 80% humidity.

[0044] 1. Changes in the activities of defensive enzymes in pecan after inoculation with Colletotrichum fructicola CZ102

[0045] Leaves were collected at 0 h, 24 h, and 72 h after inoculation, immediately wrapped with tin foil and quickly frozen in liquid nitrogen, and stored in a -80 °C refrigerator. Kits for detecting superoxide dismutase (SOD) (product number: PMHA4-M96), peroxidase (POD) (product number: PMHA1-M96) produced by Nanjing Mofan Biotechnology Co., Ltd., kits for detecting catalase (CAT) (product number: BC0200), ascorbate peroxidase (APX) (product number: BC020), hydrogen peroxide (H2O2) (product number: BC3590), and superoxide anion (O2 - )produced by Beijing Solarbio Science & Technology Co., Ltd. (product number: BC1290) were used to determine the activities of SOD, POD, CAT, and APX and the contents of H2O2 and O2 - in each sample.

[0046] As shown in Table 2, after 24 h and 72 h of inoculation, the contents of H2O2 and O2 - in Carya illinoinensis were significantly induced (P < 0.05), and the contents were the highest at 72 h.

[0047] As shown in Table 3, the activities of SOD, POD, CAT, and APX in vivo increased significantly at 24 h after inoculation, and were significantly increased by 98.2%, 201.7%, 106.1%, and 137.5% respectively compared with those at 0 h after inoculation; at 72 h after inoculation, the activities of SOD, POD, and CAT in Carya illinoinensis were still higher than those at 24 h after inoculation, while the activity of APX decreased significantly at this time and there was no significant difference from the APX content at 0 h after inoculation.

[0048] Table 2 Changes in the content of reactive oxygen species in Carya illinoinensis after inoculation with Colletotrichum fructicola

[0049]

[0050] Table 3 Changes in the activities of SOD, POD, CAT, and APX in Carya illinoinensis after inoculation with Colletotrichum fructicola

[0051]

[0052] 2. Relative expression levels of defense-related genes in Carya illinoinensis after inoculation with Colletotrichum fructicola CZ102

[0053] Collect Carya illinoinensis leaf tissues at 0 h, 6 h, 24 h, and 36 h after inoculation, quickly freeze them in liquid nitrogen, and store them in an -80 °C refrigerator. Weigh 100 mg of leaf tissues at different inoculation times, use the SteadyPure Plant RNA Extraction Kit to extract total RNA and measure its concentration. After the RNA samples are qualified, take 0.4 μg of total RNA and reverse transcribe the RNA into cDNA using the HiScript III® All-in-one RT SuperMix Perfect for qPCR reverse transcription kit. Select 5 Carya illinoinensis defense-related genes for relative expression detection. Design specific primers using the SIGMA online design software (http: / / www.oligoarchitect.com / LoginServlet) according to the gene nucleic acid sequences in the genome. The primer sequences are shown in Table 4. Using Carya illinoinensis cDNA as the template and the Actin gene as the internal reference, perform real-time fluorescence quantitative PCR.

[0054] The PCR reaction system is as follows: 10 μL of 2×ChamQ SYBR qPCR Master Mix, 0.4 μL of each upstream and downstream primer, 2 μL of template cDNA, and make up to 20 μL with ddH2O.

[0055] The PCR reaction conditions are as follows: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, for 40 cycles. The experiment is repeated 3 times, with 3 biological replicates for each treatment. Using Carya illinoinensis inoculated at 0 h as the control, use the 2 -ΔΔCT algorithm to calculate the relative expression levels of each gene.

[0056] Table 4 Primer information for Carya illinoinensis defense-related genes

[0057]

[0058] The results are as Figure 4 shown. After inoculation with Colletotrichum fructicola, RBOHD, LRR-RLK, PTI6, and GPX in Carya illinoinensis leaves were all induced to express at 6 h after inoculation, and their relative expression levels all increased significantly at 24 h and reached the peak, which were 846.08 times, 21.70 times, 5.94 times, and 7.25 times that before inoculation, respectively. Subsequently, they all decreased significantly at 36 h, but were still significantly higher than the level before inoculation ( Figure 4 A-D). The relative expression level of the PR-10 gene in Carya illinoinensis leaves increased significantly at 6 h and continued to increase at 24 h and 36 h ( Figure 4 E). The results show that in the early stage of Carya illinoinensis infected by Colletotrichum fructicola, defense-related genes in the body are significantly induced to express to initiate the defense response and participate in the disease resistance process.

[0059] 3. Resistance of Different Varieties of Carya illinoinensis to Colletotrichum fructicola CZ102

[0060] On the healthy leaves of 3-year-old seedlings of 8 Carya illinoinensis varieties [‘Pawnee’, Nacono, ‘Stuart’, ‘Mahan’, ‘Shawnee’, ‘Shoshoni’, ‘Forkert’, ‘Yizheng No.10’ respectively], pathogen discs with a diameter of 5 mm were inoculated using the same method as described above. Three repeated experiments were conducted. Seven days after inoculation, the lesion diameters after Colletotrichum fructicola infection of each variety were counted.

[0061] The results are shown in Table 5. ‘Shoshoni’ and ‘Yizheng No.10’ showed almost no disease after inoculation with CZ102, with no significant difference from the control, indicating high disease resistance; small black spots appeared at the inoculation sites of ‘Mahan’, ‘Shawnee’, and ‘Stuart’ after inoculation with CZ102, showing significant differences from the control, indicating moderate disease resistance. The lesion areas at the inoculation sites of ‘Pawnee’, Nacono, and ‘Forkert’ expanded rapidly, forming obvious larger lesions, showing significant differences from the control and ‘Mahan’, ‘Shawnee’, and ‘Stuart’, indicating that these 3 varieties are highly susceptible to Colletotrichum fructicola. The results show that there are significant differences in disease resistance among different varieties of Carya illinoinensis.

[0062] Table 5 Detection of Resistance Differences of Different Carya illinoinensis Varieties to Colletotrichum fructicola

[0063]

[0064] Different lowercase letters in the table indicate significant differences analyzed by Duncan's new multiple range test (P<0.05).

[0065] 4. Virulence of 8 Fungicides against the Pathogen

[0066] Eight kinds of agents were diluted in concentration gradients to prepare PDA medium containing the agents, with 5 replicates for each treatment (see Table 6). Discs with a diameter of 5 mm were inoculated in the center of the agent-containing plates, and PDA plates prepared with sterile water containing the inoculated discs were used as the control. They were placed in an incubator at 25°C for cultivation. When the control colonies grew to cover the entire culture dish, the colony diameters of the pathogen under each agent treatment were measured by the cross method to screen the fungicide with the best inhibitory effect on it.

[0067] Table 6 Tested Agents and Treatment Concentrations

[0068]

[0069] The results are shown in Table 7. Carbendazim had the highest inhibitory activity against CZ102, EC50 was 0.071 mg / L, followed by prochloraz, tebuconazole, pyraclostrobin, and thiophanate-methyl, with EC 50 being 0.394, 1.527, 1.990 mg / L, and 1.965 mg / L, respectively; CZ102 was moderately sensitive to myclobutanil, with EC 50 being 9.442 mg / L; iprodione had relatively low antibacterial activity, with EC 50 being 35.816 mg / L, and mancozeb had the worst inhibitory effect, with EC 50 being 280.106 mg / L. Therefore, the fungicide with the best inhibitory effect on CZ102 was carbendazim.

[0070] Table 7 Toxicity determination of different fungicides on the mycelial growth of Colletotrichum fructicola

[0071]

[0072] EC 50 Less than 5 mg / L indicates high sensitivity; EC 50 Between 5 mg / L and 20 mg / L indicates moderate sensitivity; EC 50 Higher than 20 mg / L indicates insensitivity.

[0073] The above is illustrative rather than restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, variations, or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A Colletotrichum fructicola CZ102, which is deposited in the Guangdong Microbial Culture Collection Center, with the deposit number: GDMCC65838, the deposit date: January 21, 2025, and the deposit address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

2. Use of the Colletotrichum fructicola CZ102 according to claim 1 in the preparation of an infection model.

3. Use of the Colletotrichum fructicola CZ102 infection model in the verification of plant defense responses.

4. The application according to claim 3, characterized in that, The plant defense responses include changes in defense enzyme activities, oxidant contents, and defense-related gene expressions.

5. The application according to claim 4, wherein The defense enzymes include SOD, POD, CAT, and APX.

6. The application according to claim 4, wherein The oxidant includes H2O2 and O2 - .

7. The application according to claim 4, wherein The defense-related genes include RBOHD, LRR-RLK, PTI6, GPX, and PR-10.

8. Use of the Colletotrichum fructicola CZ102 infection model in the verification of plant disease resistance.

9. The application according to claim 8, wherein The plants include pecan varieties "Pawnee", pecan variety nacono, pecan variety "Stuart", pecan variety "Mahan", pecan variety "Shawnee", pecan variety "Shoshoni", pecan variety "Vaughn", and pecan variety "Yizheng 10".

10. A medicament for preventing and treating Colletotrichum fructicola CZ102, characterized in that, Is carbendazim.

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