Strain of European gloeosporium with hyperparasitism effect on European gloeosporium and application of strain of European gloeosporium

Abstract

The invention discloses an oosporium stephanianum strain with a hyperparasitism effect on puccinia coffee and application of the oosporium stephanianum strain with the hyperparasitism effect on puccinia coffee. The name of the hyperparasitic bacterium is Ovicillium atteureum HY87, the Ovicillium atteureum is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.41897. The invention further discloses a preparation method of the hyperparasitic bacterium. The Escherichia coli HY87 has an antibacterial effect on the Pucidium caffei. The invention also discloses an application of the oosporium edubia HY87 in the aspect of preventing and treating the coffee rust disease.

Description

Technical Field

[0001] The invention belongs to the field of microbial applications, and particularly relates to a strain of Oosporium attenuata having a heavy parasitic effect on coffee rust fungus and an application thereof. Background Art

[0002] Coffee is a perennial plant of the genus Coffea in the family Rubiaceae (Flora of China, Chinese Academy of Sciences, 1999). It is one of the world's three major beverage crops and a key commodity driving international trade and economic development, generating income for over 125 million people. Commercially, it is the second most traded commodity after petroleum (Gichuru et al., 2021). Coffee has been shown to boost metabolism, stimulate alertness, sober up, improve digestion, and reduce the risk of various diseases (Crous-Bou et al., 2022; Herqutanto et al., 2024; Kong et al., 2024; Westerterp-Plantenga et al., 2006). However, coffee leaf rust (CLR) seriously affects coffee production (Salazar-Navarro et al., 2024).

[0003] Although coffee rust can be reduced through physical control measures such as improved coffee plantation management, appropriate fertilization, and pruning (Zhang Hongbo et al., 2011), this approach is time-consuming, labor-intensive, and difficult to implement on a large scale. Planting rust-resistant coffee varieties is a relatively cost-effective control measure (Sera et al., 2022), but resistant varieties often lose their resistance after a few years due to pathogen mutation. Fungicide application can also be used to control coffee rust (Aristizábal and Johnson, 2022); however, long-term application may lead to pathogen resistance, requiring increased dosages and potentially increasing the risk of environmental pollution and pesticide residues (Araaf et al., 2024; Martinho et al., 2020; Nicolopoulou-Stamati et al., 2016). With increasing global attention to environmental protection and sustainable agriculture, green control technologies have become an important development direction in disease prevention and control (Xu et al., 2024).

[0004] Live cell preparations made from hyperparasitic bacteria offer numerous advantages. These preparations are inherently nontoxic, leave no residual residue in the environment, and are harmless to non-target microorganisms. They exhibit good environmental compatibility and offer a long-lasting effect (Qiao et al., 2002), making them potentially useful for controlling plant diseases, particularly rust. In 2024, Luiz et al. found that fungi potentially antagonistic to coffee rust (Hemileia vastatrix) on Arabica coffee in Hawaii include Simplicilium, Akanthomyces, Cladosporium, Fusarium, and Clonostachys (Luiz et al., 2024). Therefore, identifying and identifying highly effective hyperparasitic strains in target areas for biocontrol is an effective approach to controlling coffee rust. Summary of the Invention

[0005] The invention provides an Ovicillium attenuatum HY87 strain that heavily parasitizes coffee rust fungus and an application thereof.

[0006] The Ovicillium attenuatum of the present invention was isolated from a typical coffee leaf rust sample with heavy parasites collected from a Pu'er coffee plantation in Yunnan Province and identified as Ovicillium attenuatum. It was deposited in the China General Microbiological Culture Collection Center (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​April 21, 2025, with a deposit number of CGMCC No. 41897. After culturing on PDA medium for 14 days, the colony size of Ovicillium attenuatum was 77.33 mm, with white fuzzy hairs, short and sparse hyphae, growing from the center outward in a linear shape, and with neat edges ( Figure 1 A), pale yellow on the back ( Figure 1 B).

[0007] The Ovicillium attenuatum HY87 strain of the present invention has a significant inhibitory effect on Hemileia vastatrix. Therefore, the present invention provides the strain and its metabolites for biological control of coffee rust.

[0008] The present invention has the following good effects: after 96 hours, the germination rate of coffee rust fungus spores treated with the HY87 spore suspension was 11.33%, while the germination rate of untreated coffee rust fungus uredia spores was 61.67%. This shows that the HY87 spore suspension has an inhibition rate of 81.63% on coffee rust fungus uredia germination (Table 1).

[0009] After 16 days of cross-inoculation of leaf cakes of susceptible varieties, the results showed that only the leaf cakes inoculated with coffee rust urediospores showed urediospore piles ( Figure 5 A), while the coffee leaves in the four treatments of inoculation with strain HY87 alone, inoculation with both hyperparasite and rust, inoculation with rust 72 hours before hyperparasite strain HY87, and inoculation with hyperparasite first and coffee rust 72 hours later did not develop chlorotic spots after invasion by coffee uredia ( Figure 5 B-E). This indicates that strain HY87 is not only non-pathogenic to coffee but also has an inhibitory effect on the invasion of coffee leaf rust spores.

[0010] qPCR was used to detect the concentration of C. coffee rust in the cross-inoculated leaf cakes. The results showed that C. coffee rust DNA was only detected from the three leaf cakes inoculated with rust, DNA HV-1, HV-2, and HV-3, with average Ct values ​​of 17.427, 17.638, and 19.680, respectively, and copy numbers of 1.78×10 8 , 1.51×10 8 , 3.32×10 7 However, no C. coffee rust was detected in the DNA of the three leaf cakes inoculated with both C. coffee rust and the heavy parasite HY87. Figure 6 ). This showed that the HY87 strain inhibited the invasion of C. coffee rust.

[0011] In vitro lesion control tests showed that the yellow powdery uredia on the lesions of the control coffee rust fungus grew normally ( Figure 7 A). However, 96 hours after inoculation of the coffee rust lesions with the strain HY87, most of the original yellow powdery coffee rust uredia on the lesions disappeared, and were replaced by clearly visible white mycelium of the hyperparasite ( Figure 7 B). This indicates that strain HY87 can parasitize the uredia of the rust fungus and interfere with its growth and reproduction. Scanning electron microscopy (SEM) observations showed that the uredia on the rust lesions that were not inoculated with the heavy fungus grew and reproduced normally, and uredia with a hunchback shape and short spines on the back were observed ( Figure 7 C). However, the summer spores on the surface of coffee lesions treated with the HY87 strain showed concave and collapsed shapes, and the spore structure had been severely damaged ( Figure 7D) The strain Ovicillium attenuatum HY87 was confirmed to have a significant inhibitory effect on coffee rust, suggesting it has the potential to control coffee rust. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The colony morphology of strain HY87 in Example 1 on PDA medium (cultured for 14 days); Figure 1 Middle (A) is the positive morphology of strain HY87 PDA culture, Figure 1 Middle (B) is the morphological image of the back of PDA culture.

[0013] Figure 2 This is the conidia morphology of strain HY87 in Example 1.

[0014] Figure 3 This is a scanning electron micrograph of strain HY87 in Example 1.

[0015] Figure 4 Phylogenetic identification of strain HY87 and related strains in Example 2; Figure 4 (A) is the phylogenetic tree of ITS gene sequences, and (B) is the phylogenetic tree of LSU gene sequences.

[0016] Figure 5 This is a leaf pie chart of strain HY87 in Example 4 16 days after cross-inoculation with coffee rust; Figure 5 In the middle, Hv, inoculated with coffee rust spores; Hy, inoculated with HY87; Hv0+Hy0, inoculated with coffee rust and HY87 at the same time; Hv0+Hy 72 , first inoculate rust fungus, then inoculate HY87 72h later; Hv0+Hv 72 HY87 was inoculated first, and then rust fungus was inoculated 72 hours later.

[0017] Figure 6 This is the effect of the coffee rust qPCR detection strain HY87 on the biomass change of coffee rust in Example 5.

[0018] Figure 7 The indoor control effect of the strain HY87 in Example 5 on coffee rust; A, CK indicates no treatment; B, indicates inoculation of the strain HY87 on a pile of coffee rust spores; C, scanning electron microscopic morphology of the untreated rust fungus; D, scanning electron microscopic morphology 96 hours after inoculation with the strain HY87 bacterial solution.

[0019] Biomaterial Deposit

[0020] Name: Ovicillium attenuatum HY87;

[0021] Classification: Ovicillium attenuatum

[0022] Deposit date: April 21, 2025;

[0023] Depository: General Microbiology Center, China Culture Collection Administration (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing);

[0024] Deposit number: CGMCC No.41897. DETAILED DESCRIPTION

[0025] Example 1. Acquisition of Ovicillium attenuatum HY87

[0026] Typical coffee rust samples infested with hyperparasitic fungi were collected from a Pu'er coffee plantation in Yunnan. Bringing these samples back to the laboratory, sterile forceps were used to remove white mycelium from the uredia of diseased leaf lesions. These were then inoculated onto potato dextrose agar (PDA) medium. The inoculated plates were placed in a 28°C incubator. After three days of incubation, bacterial masses were excised from the edge of the white colonies and transferred to fresh PDA medium. Purification was performed in a 28°C incubator, and the samples were numbered and stored.

[0027] After culturing HY87 in PDA medium for 14 days, the colony size was 77.3 3mm, with white fuzzy hairs, short and sparse hyphae, growing from the center to the outside in a linear shape, with neat edges and a light yellow back. Figure 1 ). Under an optical microscope (10×40), the spores are transparent, short elliptical or ovate, and the size is (2.3~4.2μm)×(1.9~2.4μm) ( Figure 2 ), SEM (×3000) showed that the spores were oval or ovate, and the spore surface was relatively smooth ( Figure 3 ).

[0028] Example 2 Molecular Identification of Ovicillium attenuatum HY87

[0029] DNA from the purified strain HY87 obtained in Example 1 was extracted from the hyperparasitic mycelium using a fungal DNA extraction kit (OMEGA, Beijing, China) according to the manufacturer's instructions and stored at -20°C. PCR amplification was performed using universal fungal primers ITS1 / ITS4 (White et al., 1990) and LROR (Rehner and Samuels, 1994) / LR5 (Moriya et al., 2005). The PCR reaction system (25 μL) consisted of: 2.5 μL 10x Ex Taq Buffer; 2 μL dNTP Mix; 1 μL each of forward and reverse primers; 0.2 μL Ex Taq enzyme (2 U); 17.3 μL ddH₂O; and 1 μL DNA template. The PCR amplification protocol was as follows: 94°C pre-denaturation for 4 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 35 cycles, and 10 min extension at 72°C, followed by storage at 4°C. PCR amplification products were ligated using the pMDTM18-T Vector Cloning Kit and transformed into competent Escherichia coli Trans 5a cells for cloning. Positive clones were identified and sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. Sequences of each gene fragment were analyzed for homology using the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) to preliminarily identify their species. Sequences of homologous strains and foreign strains were downloaded, and a phylogenetic tree was constructed using the neighbor method (1000 bootstraps) using MEGA11.0 software.

[0030] The DNA of strain HY87 was amplified using universal primers for the ITS and LSU genes, and 532bp and 920bp nucleotide fragments were obtained by sequencing, respectively. The ITS sequence is shown as sequence 1 in the sequence listing, and the LSU sequence is shown as sequence 2 in the sequence listing. The ITS and LSU gene clustering tree showed that strain HY87 and Ovicillium attenuatum CBS 399.86 were both located on the same evolutionary branch ( Figure 4 ). Combining morphological and molecular characteristics, strain HY87 was identified as Ovicillium attenuatum.

[0031] The strain, named Ovicillium attenuatum HY87, was deposited in the China General Microbiological Culture Collection Center (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with a deposit date of April 21, 2025, and a deposit number of CGMCC No. 41897.

[0032] Example 3 Effect of Inhibiting Spore Germination of Coffee Camelus spores

[0033] Fresh uredia spores of C. coffeei were collected from coffee rust-susceptible varieties at the Ruili coffee base in Yunnan and placed in waterproof capsules. Fresh uredia spores of C. coffeei and spores of strain HY87 were prepared in the laboratory with sterile water to a concentration of 1 mg / mL and 1×10 6 Spore suspension (number of spores / mL). A 2 cm × 2 cm × 0.3 cm 15‰ water agar block was placed on a glass slide for the germination inhibition test of C. coffeae rust spores: ① 20 μL of the C. coffeae rust spore suspension and 20 μL of sterile water were vortexed and spread evenly on the water agar block; ② 20 μL of the HY87 spore suspension and 20 μL of the C. coffeae rust spore suspension were vortexed and spread evenly on the water agar block. Each treatment was repeated three times. The glass slide was placed in a Petri dish and incubated at 26-28°C for 96 h. The number of germinated spores was recorded under an optical microscope (NiKon, NI / E, Japan). The inhibition rate was calculated according to the method (Mudyiwa et al., 2017). The criterion for judging spore germination was that the length of the germ tube was greater than half the diameter of the spore. The experiment on the inhibition of spore germination of C. coffeei showed that after 96 hours, the germination rate of C. coffeei spores treated with HY87 spore suspension was 11.33%, while the germination rate of untreated C. coffeei spores was 61.67%. This indicates that the inhibition rate of HY87 spore suspension on the germination of C. coffeei spores reached 81.63% (Table 1).

[0034] Table 1. Inhibitory effect of HY87 spore suspension on spore germination of coffee rust

[0035]

[0036] Example 4 Inhibitory effect of strain HY87 on the infection process of coffee rust

[0037] Three PDA cakes (5 mm) of the purified strain HY87 obtained in Example 1 were inoculated into 150 mL of liquid culture medium (10 g glucose, 3 g yeast powder, 1000 mL purified water, sterilized at 121°C for 20 min) and cultured at 28°C with shaking until the spore concentration was about 1 × 10 6 / mL. Fresh summer spores of coffee rust fungus were collected and prepared into a 1mg / mL spore suspension with sterile water. Healthy susceptible coffee leaves of Matari were collected, leaf cakes were made with a 2cm diameter puncher, and washed with sterile water. The cross-inoculation experiment was divided into 5 groups: Hv, inoculated with 25μL of summer spore suspension of coffee rust fungus; Hy, inoculated with 25μL of spore suspension of strain HY87; Hv0+Hy0, inoculated with 25μL of summer spore suspension of coffee rust fungus and HY87 spore suspension at the same time; Hv0+Hy 72, first inoculate 25 μL of coffee rust uredus spore suspension, and then inoculate 25 μL of HY87 spore suspension 72 hours later; Hy0+Hv 72 25 μL of HY87 spore suspension was first inoculated, and 72 hours later, 25 μL of uredospore suspension of C. coffeae was inoculated. Each experiment was repeated five times. After inoculation, the plants were incubated in a 24°C artificial climate chamber (MMM Climacell 707, Germany) at 100% humidity and darkness for 24 hours. After 24 hours, the cycle was adjusted to 12 hours of light / 12 hours of darkness (Silva et al., 2012). After 16 days of incubation, the disease progression of each treatment was observed.

[0038] The cross-inoculation leaf cake experiment showed that only the coffee leaf cake inoculated with coffee rust uredia grew uredia piles ( Figure 5 A), while the susceptible coffee leaves inoculated with strain HY87 alone, strain HY87 and rust of C. coffeae simultaneously, strain HY87 first inoculated with rust of C. coffeae 72 hours later and then with strain HY87, and strain HY87 first inoculated with rust of C. coffeae 72 hours later did not show visible chlorotic spots after invasion by coffee uredia spores ( Figure 5 B-E). This indicates that strain HY87 is not only non-pathogenic to host coffee plants, but also has an inhibitory effect on the infection of spores of the coffee leaf rust pathogen, C. coffeae.

[0039] Example 5 Effect of strain HY87 on biomass changes of coffee rust fungus

[0040] Using the coffee rust qPCR detection system, 16 days after cross-inoculation, the Hv group, Hy0+Hv 72 Three leaf cakes were randomly selected from the group to extract DNA, and the content of coffee rust in the leaf cakes was quantitatively detected using the qPCR quantitative detection method. The test results showed that the coffee rust DNA was detected in the three leaf cakes HV-1, HV-2, and HV-3 that were only inoculated with rust, with average Ct values ​​of 17.427, 17.638, and 19.680, respectively, and the copy number was 1.78×10 8 , 1.51×10 8 , 3.32×10 7 However, the coffee rust fungus was not detected in the DNA of the three leaf cakes inoculated with the heavy parasite 72 hours before the rust fungus. Figure 6 ).

[0041] Example 6 Growth inhibition of naturally occurring coffee rust by strain HY87

[0042] During the coffee rust season, leaves with coffee rust lesions of severity 5-6 were selected from coffee plantations in Dehong and Ruili, Yunnan Province [Ministry of Agriculture and Rural Affairs. (2016). Standards for the Description and Evaluation of Tropical Crop Germplasm Resources. Institute of Spice and Beverages, Chinese Academy of Tropical Agricultural Sciences, Dehong Institute of Tropical Agricultural Sciences, Yunnan Province]. Leaf cakes with lesions of relatively uniform size were punched using a 2 cm hole punch and subjected to an antibacterial test against hyperparasitic fungi. Each lesion on the leaf cake was inoculated with 25 μL of strain HY87. An equal number of lesions were not inoculated with strain HY87 as a control. Each experiment was repeated 10 times. After inoculation with HY87, the leaves were incubated in an artificial climate chamber (MMM Climacell 707, Germany) at 100% humidity and 24°C under 12 h light / 12 h dark conditions for 96 h. The experimental results were analyzed, and the inhibition of strain HY87 against rust fungi was observed using a scanning electron microscope.

[0043] In vitro lesion control tests showed that the yellow powdery uredia on the control coffee rust lesions grew normally ( Figure 7 A). However, 96 hours after inoculation of the coffee rust lesions with the strain HY87, the original yellow powdery uredus of the coffee rust fungus on the lesions almost completely disappeared, and were replaced by clearly visible white mycelium of the heavy parasite ( Figure 7 B). This indicates that HY87 can parasitize the uredia of the rust fungus and interfere with its growth and reproduction. Scanning electron microscopy (SEM) observations showed that the uredia on the lesions that were not inoculated with the heavy fungus grew and reproduced normally, and uredia with a hunchback shape and short spines on the back were observed ( Figure 7 C). However, after the strain HY87 was parasitized, the summer spores at the coffee lesions were severely damaged. Most of the summer spores of the rust fungus were wrapped by the spores and hyphae of the heavy parasite, and even caused their structures to be sunken and damaged, and they could not develop normally. Figure 7 D) Therefore, strain HY87 has a good effect in controlling coffee rust caused by Puccinia camelus.

[0044] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific, they are intended to be illustrative rather than restrictive of the present invention. Those skilled in the art will readily appreciate that numerous modifications, variations, and improvements can be made without departing from the spirit and scope of the appended claims, all of which fall within the scope of protection of the present invention.

Claims

1. A strain of Oosporium attenuata characterized by: The classification name is Ovicillium attenuatum, and the name is Ovicillium attenuatum HY87. The strain has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No.41897.

2. Use of the Ovicillium attenuatum HY87 according to claim 1 in the preparation of a biocontrol agent, microbial fertilizer or biopesticide for inhibiting coffee rust.

3. Use of Ovicillium attenuatum HY87 according to claim 1 in inhibiting coffee rust.

4. A biocontrol agent for inhibiting coffee rust, wherein the active ingredient is the Ovicillium attenuatum HY87 according to claim 1.

5. A microbial fertilizer for inhibiting coffee rust, comprising the Ovicillium attenuatum HY87 according to claim 1.

6. A biopesticide for inhibiting coffee rust, the active ingredient of which comprises the Ovicillium attenuatum HY87 according to claim 1.

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