Ovulinia tenuis strain with hyperparasitic effect on hemileia vastatrix and application thereof

Abstract

The application discloses a strain of Ovicillium attenuatum with hyperparasitic action on Hemileia coffeicola and application thereof. The hyperparasitic fungus is named Ovicillium attenuatum HY87, and the Ovicillium attenuatum has been preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.41897. The Ovicillium attenuatum HY87 has a bacteriostatic effect on the Hemileia coffeicola. The application further discloses application of the Ovicillium attenuatum HY87 in prevention and treatment of coffee rust.

Description

Technical Field

[0001] This invention belongs to the field of microbial applications, specifically relating to a strain of Oomycetes acuminata that has a hyperparasitic effect on Camelopardalis coffeeensis and its application. Background Technology

[0002] Coffee is a plant of the Rubiaceae family ( Rubiaceae Caffeine ( ) Coffea Coffee is a perennial plant (Flora of China Committee, 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 largest traded commodity after oil (Gichuru et al., 2021). Coffee has effects such as promoting metabolism, stimulating energy, sobering up, aiding digestion, and reducing 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) severely impacts coffee growth (Salazar-Navarro et al., 2024). It has been reported that coffee rust causes significant economic losses in over 50 coffee-growing countries, resulting in annual losses of $1 billion to $2 billion (Gichuru et al., 2021). From 2012 to 2017, the CLR caused an estimated $3 billion loss to coffee production across Latin America (Pham et al., 2019). In peak years, coffee leaf drop can reach over 50%, resulting in yield reductions of 30% to 50% (Fu et al., 2024).

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

[0004] Live cell preparations made using hyperparasitic bacteria have many advantages. These preparations are non-toxic, leave no residue in the environment after application, do not harm non-target microorganisms, have good environmental compatibility, and can achieve a relatively long-lasting disease-preventing effect (Qiao Hongping et al., 2002). They have potential application value in the control of plant diseases, especially rust. A 2024 survey by Luizd et al. found that rust may be present in Arabica coffee grown in Hawaii. Hemileia vastatrix Fungi with antagonistic effects include Simplicillium , Akanthomyces , Cladosporium , Fusarium and Clonostachys (Luiz et al., 2024). Therefore, identifying and utilizing highly efficient hyperparasitic strains within the target area for biocontrol is one of the effective methods for controlling coffee rust. Summary of the Invention

[0005] This invention provides a strain of Oomycetes that heavily parasitizes Camelopardalis laciniata (a type of rust fungus). Ovicillium attenuatum HY87 strain and its application.

[0006] Oomycetes in this invention ( Ovicillium attenuatum It was isolated from typical coffee leaf rust disease samples with hyperparasitic fungi collected from coffee plantations in Pu'er, Yunnan, and identified as *Oomycetes zeylanica*. Ovicillium attenuatumThe fungus *Oomycetes* was deposited on April 21, 2025, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 41897. Ovicillium attenuatum After 14 days of cultivation on PDA medium, the colony size was 77.33 mm, white and fluffy, with short and sparse hyphae that grew linearly from the center outwards and had neat edges. Figure 1 A) The back is light yellow ( Figure 1 B).

[0007] The present invention is based on Oomycetes ( Ovicillium attenuatum HY87 strain against *Camelus coffeeensis* rust ( Hemileia vastatrix It has a significant inhibitory effect. Therefore, the present invention provides the strain and its metabolites for the biological control of coffee rust.

[0008] The present invention has the following advantages: after 96 hours, the germination rate of *Camponotus coffeeensis* spores treated with spore suspension of strain HY87 was 11.33%, while the germination rate of untreated *Camponotus coffeeensis* urediniospores was 61.67%. This indicates that the spore suspension of strain HY87 inhibited the germination of *Camponotus coffeeensis* urediniospores by 81.63% (Table 1).

[0009] Sixteen days after cross-inoculation of leaf cakes from susceptible coffee rust varieties, the experimental results showed that only coffee leaf cakes inoculated with coffee rust fungus urediniospores developed uredinia (uredinia). Figure 5 A), while coffee leaves treated with only strain HY87, simultaneously inoculated with both parasitic and rust fungi, inoculated with rust fungi 72 h before inoculating with parasitic strain HY87, and inoculated with parasitic fungi 72 h before inoculating with coffee rust fungi, did not show chlorotic spots after coffee urediniospore invasion. 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 rust spores.

[0010] qPCR was used to detect the content of *Camelus spp.* rust in cross-inoculated leaf cakes. The results showed that *Camelus spp.* DNA was detected only in the DNA samples from three leaf cakes inoculated with the rust (HV-1, HV-2, and HV-3). The average concentration of *Camelus spp.* rust in cross-inoculated leaf cakes was [missing data]. Ct The values ​​were 17.427, 17.638, and 19.680, respectively, and the copy numbers were 1.78 × 10⁻⁶. 8 1.51×10 8 3.32×10 7 However, no *Camelopardalis coffeeensis* was detected in the DNA of any of the three leaf cakes simultaneously inoculated with *Camelopardalis coffeeensis* and the parasitic fungus HY87. Figure 6 This indicates that strain HY87 inhibited the invasion of *Camponotus coffeeensis*.

[0011] Indoor lesion control experiments showed that the yellow powdery urediniospores on the control *Camelopardalis camelopardalis* lesions grew normally. Figure 7 A). However, 96 hours after inoculating coffee rust lesions with strain HY87, most of the original yellow powdery coffee rust uredinia on the lesions disappeared, replaced by clearly visible white mycelium of the hyperparasite (A). Figure 7 B). This indicates that strain HY87 can parasitize the urediniospores of *Russula cambogia* and interfere with their growth and reproduction. Scanning electron microscopy (SEM) observation showed that urediniospores grew and reproduced normally on uninoculated rust lesions, and humped-back urediniospores with short spines on their backs were visible. Figure 7 C). The urediniospores on the surface of coffee lesions treated with HY87 bacterial solution showed signs of depression and collapse, indicating severe damage to the spore structure. Figure 7 D). Confirmed Oomycetes ( Ovicillium attenuatum HY87 has a significant inhibitory effect on coffee rust fungus and has the potential to control coffee rust disease. Attached Figure Description

[0012] Figure 1 The colony morphology of strain HY87 in Example 1 on PDA medium (cultured for 14 days); Figure 1 Image A shows the frontal morphological view of strain HY87 PDA during culture. Figure 1 Image B shows the morphology of the PDA cultured on the back.

[0013] Figure 2 The image shows the morphology of conidia of strain HY87 in Example 1.

[0014] Figure 3 This is a scanning electron microscope image of strain HY87 from Example 1.

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

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

[0017] Figure 6 This example illustrates the effect of strain HY87, a qPCR assay for coffee rust, on changes in coffee rust biomass.

[0018] Figure 7 The indoor control effect of strain HY87 in Example 5 on coffee rust; A, CK represent no treatment; B, represents inoculation of strain HY87 on coffee rust spore piles; C, scanning electron microscope morphology of untreated rust fungus; D, scanning electron microscope morphology of HY87 bacterial suspension 96 h after inoculation.

[0019] Preservation of biological materials Name: Ovicillium attenuatum HY87; Classification and naming: Ovicillium attenuatum Date of preservation: April 21, 2025; Preservation institution: China General Microbiological Culture Collection Center (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing); Accession number: CGMCC No.41897. Detailed Implementation

[0020] Example 1: Obtaining Ovicillium attenuatum HY87 Typical coffee rust disease samples with hyperparasitic fungi were collected from coffee plantations in Pu'er, Yunnan. These samples were brought back to the laboratory, where, using sterile forceps under a sterile operating table, white mycelium was extracted from the uredinia of diseased leaves and inoculated onto potato dextrose agar (PDA) medium. The inoculated petri dishes were then placed in a 28°C incubator. After 3 days of incubation, mycelial blocks were cut from the edges of the white colonies and transferred to fresh PDA medium. These blocks were then purified and cultured at 28°C, and the samples were numbered and stored.

[0021] After 14 days of cultivation on PDA medium, strain HY87 produced colonies measuring 77.33 mm in size. The colonies were white and fluffy, with short, sparse hyphae that grew linearly from the center outwards, with neat edges and a pale yellow underside. Figure 1 Under an optical microscope (10×40), the spores are transparent, short-elliptical or oval, and measure (2.3 ~ 4.2 µm) × (1.9 ~ 2.4 µm). Figure 2 Under a scanning electron microscope (×3000), the spores appear elliptical or oval, and the spore surface is relatively smooth. Figure 3 ).

[0022] Example 2 Oomycetes acutigera ( Ovicillium attenuatum Molecular identification of HY87 The purified strain HY87 obtained in Example 1 was used to extract DNA from the reparasitic mycelium using a fungal DNA extraction kit (OMEGA, Beijing, China) according to the manufacturer's instructions. The extracted DNA was 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 volume was 25 μL: 10 x Ex Taq Buffer 2.5 μL; dNTP Mix 2 μL; forward primer and reverse primer 1 μL each; Ex Taq Enzyme (2 U) 0.2 μL; ddH2O 17.3 μL; DNA template 1 μL. PCR amplification program: 94℃ pre-deformation for 4 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, 72℃ extension for 10 min, storage at 4℃. The PCR amplification products were ligated using the pMDTM18-T Vector Cloning Kit, and the ligation products were transformed into E. coli competent cells. Trans 5a Cloning was performed on positive clones, which were then sent to Beijing BGI Genomics Co., Ltd. for sequencing. The sequenced gene fragments were compared for homology in the NCBI database to preliminarily identify their species. Homologous strain sequences and exogenous strains were downloaded, and a phylogenetic tree was constructed using the MEGA 11.0 software's proximity method (1000 bootstraps).

[0023] use ITS , LSU DNA from strain HY87 was amplified using universal primers, and 532 bp and 920 bp nucleotide fragments were obtained by sequencing. The ITS sequence is shown in Sequence 1 of the sequence listing, and the LSU sequence is shown in Sequence 2. Clustering phylogenetic trees of the ITS and LSU genes show that strain HY87 is related to… Ovicillium attenuatum All CBS strains 399.86 belong to the same evolutionary branch. Figure 4 Based on morphological and molecular characteristics, strain HY87 was identified as... Ovicillium attenuatum .

[0024] This strain was named Oomycetes acutigera ( Ovicillium attenuatum HY87, deposited at the China General Microbiological Culture Collection Center (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), deposited on April 21, 2025, with accession number CGMCC No. 41897.

[0025] Example 3: Inhibition effect on spore germination of *Camelus coffeeensis* Fresh urediniospores of *Camelus coffee rust* were collected from coffee rust-susceptible varieties at the Ruili coffee base in Yunnan Province and encapsulated in waterproof capsules. In the laboratory, the fresh *Camelus coffee rust* urediniospores and strain HY87 spores were prepared into concentrations of 1 mg / mL and 1 × 10⁻⁶ spores, respectively, using sterile water. 6 Spore suspension / mL. A 2 cm × 2 cm × 0.3 cm block of 15‰ water agar was placed on a glass slide for the germination inhibition test of *Camponotus coffee* spores: ① 20 μL of *Camponotus coffee* spore suspension and 20 μL of sterile water were vortexed and then spread evenly on the water agar block; ② 20 μL of HY87 spore suspension and 20 μL of *Camponotus coffee* spore suspension were vortexed and then spread evenly on the water agar block; each treatment was repeated 3 times. The glass slides were placed in petri dishes and incubated at 26-28℃ for 96 h. The number of germinating 100 rust spores was observed and recorded under an optical microscope (NiKon, NI / E, Japan), and the inhibition rate was calculated according to the method (Mudyiwa et al., 2017). The criterion for judging the germination of *Camponotus coffee* spores was: a germ tube length greater than 1 / 2 of the rust spore diameter was considered germination. The germination inhibition experiment of *Camponotus coffeeensis* spores showed that after 96 h, the germination rate of *Camponotus coffeeensis* spores treated with HY87 spore suspension was 11.33%, while the germination rate of untreated *Camponotus coffeeensis* urediniospores was 61.67%. This indicates that the HY87 spore suspension inhibited the germination of *Camponotus coffeeensis* urediniospores by 81.63% (Table 1).

[0026] Table 1. Inhibitory effect of HY87 spore suspension on spore germination of coffee rust fungus.

[0027] Example 4: Inhibitory effect of strain HY87 on the infection process of coffee rust fungus. Three 5 mm mycelial blocks of the purified strain HY87 PDA obtained in Example 1 were inoculated into 150 mL of liquid culture medium (10 g glucose, 3 g yeast extract, 1000 mL purified water, sterilized at 121°C for 20 min) and cultured at 28°C with shaking until the spore concentration was approximately 1 × 10⁻⁶. 6Fresh urediniospores of *Camelus spp.* were collected and prepared into a spore suspension of 1 mg / mL using sterile water. Leaves of the healthy, susceptible *Matari* coffee variety were collected, and leaf cakes were prepared using a 2 cm diameter perforator and washed with sterile water. Cross-inoculation experiments were conducted in 5 groups: Hv, inoculated with 25 μL of *Camelus spp.* urediniospore suspension; Hy, inoculated with 25 μL of HY87 spore suspension; Hv0+Hy0, inoculated simultaneously with 25 μL each of *Camelus spp.* urediniospore suspension and HY87 spore suspension; Hv0+Hy... 72 First, inoculate with 25 μL of urediniospore suspension of *Camelus coffeeensis*, and then inoculate with 25 μL of HY87 spore suspension 72 h later; Hy0+Hv 72 First, inoculate with 25 μL of HY87 spore suspension, and then 25 μL of *Camelopardalis coffeeensis* urediniospore suspension after 72 h. Each experiment was repeated 5 times. After inoculation with the rust fungus, the fungus was cultured in the dark for 24 h in a climate chamber (MMM Climacell 707, Germany) at 100% humidity and 24°C. After 24 h, the light / dark cycle was adjusted to 12 h light / 12 h dark (Silva et al., 2012). After 16 days of culture, the disease incidence of each treatment was observed.

[0028] The cross-inoculation leaf cake experiment showed that only coffee leaf cakes inoculated with coffee rust fungus urediniospores produced rust fungus uredinia ( rust fungus urediniospores). Figure 5 A), however, in four treatments—inoculation with strain HY87 only, inoculation with both strain HY87 and *Camellia spp.*, inoculation with *Camellia spp.* 72 hours prior followed by inoculation with the heavily parasitic strain HY87, and inoculation with the heavily parasitic strain HY87 followed by inoculation with *Camellia spp.* 72 hours prior—no visible chlorotic spots were observed on coffee leaves after invasion by coffee urediniospores. Figure 5 (B~E). This indicates that strain HY87 is not only non-pathogenic to the host coffee, but also has an inhibitory effect on the spore infection of *Camponotus coffeeensis*, the pathogen of coffee leaf rust.

[0029] Example 5: Effect of strain HY87 on biomass changes of *Camelopardalis coffeeensis* Using the qPCR detection system for *Camelopardalis coffeeensis*, 16 days after cross-inoculation, samples were collected from the Hv group and the Hy0+Hv group, respectively. 72 DNA was extracted from three randomly selected leaf cakes in the group, and the content of *Camelopardalis coffeeensis* rust in the leaf cakes was quantitatively detected using qPCR. The results showed that *Camelopardalis coffeeensis* DNA was detected in the DNA from the three leaf cakes (HV-1, HV-2, and HV-3) inoculated only with rust, with an average concentration of... Ct The values ​​were 17.427, 17.638, and 19.680, respectively, and the copy numbers were 1.78 × 10⁻⁶. 8 1.51×10 83.32×10 7 However, no *Camelopardalis coffeeensis* was detected in the DNA of three leaf cakes inoculated with *Camelopardalis coffeeensis* 72 hours after inoculation with *Camelopardalis coffeeensis*. Figure 6 ).

[0030] Example 6: Growth inhibition of naturally occurring coffee rust by strain HY87 During the coffee rust season, diseased leaves with rust lesion severity of 5-6 were selected from coffee plantations in Dehong and Ruili, Yunnan Province [Tropical Crop Germplasm Resource Description and Evaluation Standard for Coffee. Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, and Dehong Tropical Agricultural Research Institute, Yunnan Province]. Leaf cakes with relatively uniform lesion size were prepared using a 2 cm punch and subjected to a hyperparasitic fungal inhibition test indoors. Each leaf cake lesion was inoculated with 25 μL of bacterial suspension of strain HY87. A control group with the same number of lesions not inoculated with strain HY87 was used. Each experiment was repeated 10 times. After inoculation with HY87, the leaves were cultured in a 100% humidity, 24℃ artificial climate chamber (MMM Climacell 707, Germany) for 96 h under 12 h light / 12 h dark conditions. The experimental results were observed, and the inhibitory effect of strain HY87 on *Camponotus spp.* was observed using scanning electron microscopy.

[0031] Indoor in vitro lesion control experiment showed that the yellow powdery urediniospores on the control coffee rust lesions grew normally. Figure 7 A). However, 96 hours after inoculating coffee rust lesions with strain HY87, the original yellow powdery uredinia of *Camponotus coffeeensis* on the lesions almost completely disappeared, replaced by clearly visible white mycelium of the hyperparasitic fungus growing on the lesions. Figure 7 B). This indicates that HY87 can parasitize the urediniospores of *Russula cambogia* and interfere with their growth and reproduction. Scanning electron microscopy (SEM) observation showed that urediniospores grew and reproduced normally on lesions not inoculated with the superparasite, and humped-back urediniospores with short spines on their backs were visible. Figure 7 C). After parasitizing coffee lesions with HY87 bacterial suspension, the urediniospores were severely damaged. Most rust urediniospores were encased by hyperparasitic spores and hyphae, even leading to structural depression and damage, preventing normal development. Figure 7 D). Therefore, strain HY87 has a good effect in controlling coffee rust caused by Camelospermia ulmoides.

[0032] The embodiments described above are merely illustrative of several implementations of the present invention, and are described in detail, but are not intended to limit the scope of the invention. Those skilled in the art will recognize that many modifications, variations, and improvements can be made without departing from the spirit and scope defined by the appended claims, and these all fall within the protection scope of the present invention.

Claims

1. A strain of Oomycetes ( Ovicillium attenuatum ), characterized in that, Its name is Ovicillium attenuatum HY87 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41897.

2. The use of Oomycetes zedoaria according to claim 1 in the preparation of biocontrol agents, microbial fertilizers, or biological pesticides for inhibiting coffee rust.

3. The use of Oomycetes zeylans as described in claim 1 in the suppression of coffee rust.

4. A biocontrol agent for inhibiting coffee rust, characterized in that, Its active ingredient is Oomycetes acuminata as described in claim 1.

5. A microbial fertilizer for inhibiting coffee rust, characterized in that, Contains Oomycetes as described in claim 1.

6. A biological pesticide for inhibiting coffee rust, characterized in that, Its active ingredient includes Oomycetes acuminata as described in claim 1.

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