Lecanicillium szewalskii strain with hyperparasitism effect on puccinia caffei and application of Lecanicillium szewalskii strain

Abstract

The invention discloses a lecanicillium szewalskii strain with a hyperparasitism effect on caffeia spp. And application of the lecanicillium szewalskii strain. The name of the hyper-parasitic bacterium is Lecanicillium sp. HHV, the Lecanicillium sp. Is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the hyper-parasitic bacterium is CGMCC No.41898. The hyper-parasitic bacterium disclosed by the invention has the advantages that the hyper-parasitic bacterium can be used for preparing the hyper-parasitic bacterium; the Lecanicillium slecanii HHV has the advantages that the Lecanicillium slecanii HHV has an antibacterial effect on the puccinia caffei; the invention also discloses an application of the Lecanicillium sp.sp.HHV in the aspect of preventing and treating the coffee rust disease. The Lecanicillium sp.sp.HHV can be used for 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 Lecanicillium spenceae HHV strain having a heavy parasitic effect on coffee rust fungus and an application thereof. Background Art

[0002] Coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix, is the most devastating disease in the coffee industry, resulting in significant yield losses (Wang et al., 2024). Under favorable conditions, CLR can cause severe leaf drop, yield losses exceeding 70%, and even plant death (Aristizábal and Johnson, 2022). Coffee leaf rust has been reported to cause severe economic losses in over 50 coffee-growing countries, costing US$1 to 2 billion annually (Gichuru et al., 2021). Furthermore, outbreaks of CLR in Latin America have caused coffee yield declines of 30% to 90%, with profound impacts on local economies and societies (de Resende et al., 2021). In epidemic years, leaf drop rates can exceed 50%, and yield reductions can reach 30% to 50% (Fu et al., 2024). Catimor 7963, the primary coffee variety cultivated in my country, has gradually lost its rust resistance over many years.

[0003] Although various methods are currently available for controlling coffee rust, including physical and chemical methods and planting rust-resistant varieties (Aristizábal and Johnson, 2022; Consonni et al., 2018; Sera et al., 2022), physical methods are inefficient and labor-intensive, while chemical methods can cause the coffee rust fungus to develop resistance and pollute the environment. Planting rust-resistant varieties can be overtaken by new pathogens within a few years (Araaf et al., 2024; Martinho, 2020; Sera et al., 2022). In contrast, biological control is not only environmentally friendly and prevents the development of resistance, but can also effectively control plant diseases if implemented within the critical control window (Li Jing et al., 2022). The increasing number of coffee rust species makes control more challenging. It can be seen that more effective and safe control measures are needed for the prevention and control of coffee rust, including the use of biological control methods such as antagonistic fungi and bacteria (Gómez-de la Cruz et al., 2024; Thambugala et al., 2020; Santiago-Santiago et al., 2023).

[0004] Hyperparasites are organisms that live on other parasites, utilizing their resources for survival and reproduction. They have potential applications in controlling plant diseases, particularly rust. Hyperparasites are widely present in coffee rust, and developing their biocontrol applications is a key approach to achieving green control. Summary of the Invention

[0005] The invention provides a Lecanicillium spenceae HHV strain that heavily parasitizes coffee rust fungus and applications thereof.

[0006] The Spencer wax scale fungus (Lecanicillium spenceae) HHV in the present invention was isolated from a typical coffee leaf rust sample with heavy parasites collected from a coffee plantation in Ruili, Yunnan, and identified as Spencer wax scale fungus (Lecanicillium spenceae) HHV. 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. 41898. After 14 days of culture on PDA medium, the colony size of Spencer wax scale fungus (Lecanicillium spenceae) HHV was 66.67 mm, with white fuzz, dense hyphae, neat edges, and a fast growth rate ( Figure 1 A), pale yellow on the back ( Figure 1 B).

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

[0008] The present invention has the following good results: after 96 hours, the germination rate of coffee rust fungus spores treated with the HHV spore suspension was 0.67%, while the germination rate of untreated coffee rust fungus uredia spores was 58%. This shows that the HHV spore suspension has an inhibition rate of 98.84% on coffee rust uredia germination (Table 1).

[0009] After 16 days of cross-inoculation of leaf cakes of susceptible rust varieties, the test results showed that ( Figure 5), only coffee leaves inoculated with coffee rust uredia developed chlorotic yellow spots. However, leaves inoculated with the HHV strain alone, the hyperparasitic HHV strain and the rust simultaneously, the rust strain first inoculated 72 hours before the hyperparasitic HHV strain, and the hyperparasitic HHV strain first inoculated 72 hours before the coffee rust strain developed no visible chlorotic spots after the coffee uredia invasion. This indicates that the HHV strain is not only non-pathogenic to coffee but also has an inhibitory effect on the invasion of coffee rust spores.

[0010] The qPCR detection system was used to detect the content of coffee rust in cross-inoculated leaf cakes. The test results showed that the coffee rust DNA was detected from the three leaf cakes inoculated with only the rust fungus DNA HV-1, HV-2, and HV-3, with average Ct values ​​of 17.688, 16.785, and 17.034, respectively, and the copy numbers were 1.41×10 8 , 2.81×10 8 ,2.34×10 8 , while no C. coffee rust was detected in the DNA of the three leaf cakes inoculated with both C. coffee rust and HHV. Figure 6 ). This showed that the HHV 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 HHV, the original yellow powdery coffee rust uredus on the lesions mostly disappeared, and were replaced by clearly visible white mycelium of the hyperparasite ( Figure 7 B). This indicates that strain HHV can parasitize the uredia of H. humeroides 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), while the summer spores on the surface of coffee lesions treated with strain HHV showed concave and collapsed shapes, and the spore structure had been severely damaged ( Figure 7 D) The results confirmed that Lecanicillium spenceae HHV has a significant inhibitory effect on coffee rust fungus, suggesting that it has the potential to control coffee rust. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0013] Figure 2 This is the conidia morphology of the strain HHV in Example 1;

[0014] Figure 3 This is a scanning electron micrograph of the HHV strain in Example 1;

[0015] Figure 4 Phylogenetic identification of strain HHV and related strains in Example 2; Figure 4 A in the middle is the phylogenetic tree of ITS gene sequences; Figure 4 B in the middle is the phylogenetic tree of RPB2 gene sequences.

[0016] Figure 5 The leaf pie chart of the strain HHV in Example 4 after cross-inoculation with coffee rust for 16 days; Hv, inoculated with coffee rust uredia; Hy, inoculated with HHV; Hv0+Hy0, inoculated with coffee rust and HHV at the same time; Hv0+Hy0 72 , first inoculate rust fungus, then inoculate HHV 72h later; Hy0+Hv 72 , first inoculate HHV, and then inoculate rust fungi 72 hours later.

[0017] Figure 6 The effect of strain HHV on the biomass change of coffee rust in Example 5;

[0018] Figure 7 The effect of the strain HHV in Example 6 on indoor prevention and control of coffee rust; A, CK means no treatment; B, HHV means inoculation of strain HHV 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 HHV bacterial solution.

[0019] Biomaterial Deposit

[0020] Name: Lecanicillium spenceae HHV;

[0021] Deposit date: April 21, 2025;

[0022] Depository: General Microbiology Center, China Culture Collection Administration (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing);

[0023] Deposit number: CGMCC No.41898. DETAILED DESCRIPTION

[0024] Example 1 Obtaining Lecanicillium spenceae HHV

[0025] Typical coffee rust samples, infested with a hyperparasitic fungus, 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 rust spores on diseased leaves and lesions. These were then inoculated onto potato dextrose agar (PDA) medium. The inoculated plates were incubated at 28°C. 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 stored with serial numbers.

[0026] After 14 days of culture on PDA medium, the colony size of strain HHV was 66.67 mm, with white fuzzy, dense hyphae, neat edges, fast growth rate, and light yellow back ( Figure 1 ). Under an optical microscope (10×40), the spores are transparent, oval, sickle-shaped or spindle-shaped, and the size is (3.6~8.9μm)×(1.8~2.3μm) ( Figure 2 ), SEM (×3000) showed that the spores were oval, sickle-shaped or spindle-shaped, and the spore surface was relatively smooth ( Figure 3 ).

[0027] Example 2 Molecular Identification of Lecanicillium spenceae HHV

[0028] DNA from the purified HHV strain obtained in Example 1 was extracted from the parasitic 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 RPB2-5F2(F) / RPB2-7CR(R) (O'Donnell et al., 2007). 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, 94°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 1 min, 35 cycles, and 72°C extension for 10 min, 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. Sequence fragments obtained 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 maximum likelihood method (1000 bootstraps) using MEGA 11.0 software.

[0029] The HHV DNA was amplified using universal primers for the ITS and RPB2 genes, and sequencing yielded 596bp and 1021bp nucleotide fragments, respectively. The ITS sequence is shown as Sequence 1 in the sequence listing, and the RPB2 sequence is shown as Sequence 2 in the sequence listing. A homology search of the NCBI database revealed that the ITS and RPB2 sequence fragments were 99.14% and 99.36% similar to Lecanicillium spenceae BRIP 72646a, respectively. Cluster phylogenetic trees revealed that the ITS and RPB2 gene phylogenetic trees revealed that HHV and Lecanicillium spenceae BRIP 72646a strains were located on the same evolutionary branch ( Figure 4 Combining morphological and molecular characteristics, the strain HHV was identified as Lecanicillium spenceae.

[0030] The strain, named Lecanicillium spenceae HHV, 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. 41898.

[0031] Example 3: Inhibitory effect of HHV spore suspension on spore germination of coffee rust fungus

[0032] 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 HHV were prepared in the laboratory with sterile water to a concentration of 1 mg / mL and 1×10 6 Spore suspension (2 cm × 2 cm × 0.3 cm) was placed on a glass slide. A 15‰ water agar block (2 cm × 2 cm × 0.3 cm) was placed on a glass slide for spore germination inhibition testing against C. coffeae rust: ① 20 μL of the C. coffeae rust uredia suspension and 20 μL of sterile water were vortexed and spread evenly on the water agar block. ② 20 μL of the HHV spore suspension and 20 μL of the C. coffeae rust uredia 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 uredia germination was that the length of the germ tube was greater than half the diameter of the spore. The experiment on inhibiting the germination of coffee rust uredia showed that after 96 hours, the germination rate of coffee rust uredia treated with HHV spore suspension was 0.67%, while the germination rate of untreated coffee rust uredia was 58%. This shows that the inhibition rate of HHV spore suspension on coffee rust uredia germination reached 98.84% (Table 1).

[0033] Table 1. Inhibitory effect of HHV spore suspension on spore germination of coffee rust

[0034]

[0035] Example 4: Inhibitory effect of strain HHV on coffee rust infection

[0036] Three cakes of the purified HHV PDA bacterial block (5 mm) 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 HHV; Hv0+Hy0, inoculated with 25μL of spore suspension of coffee rust fungus and HHV spore suspension at the same time; Hv0+Hy 72 , first inoculate 25 μL of coffee rust spore suspension, and then inoculate 25 μL of HHV spore suspension 72 hours later; Hy0+Hv 72 Plants were inoculated with 25 μL of a spore suspension of HHV (H. chinensis) and then, 72 hours later, with 25 μL of a uredospore suspension of C. coffeae. Each experiment was repeated five times. After inoculation, the plants were incubated in a 24°C artificial climate chamber (MMM Climacell 707, Germany) in darkness for 24 hours, 100% humidity, and a 12-hour light / 12-hour dark cycle. After 16 days of incubation, the disease progression of each treatment was observed.

[0037] The cross-inoculation leaf cake test showed that only the coffee leaf cake inoculated with coffee rust uredus spores showed chlorosis spots after 16 days ( Figure 5 A), while the rust-susceptible coffee leaves inoculated with the HHV strain alone, inoculated with both the hyperparasitic and rust fungi, inoculated with the rust fungi 72 hours before the hyperparasitic HHV strain, or inoculated with the hyperparasitic HHV strain first and then the rust fungi 72 hours after the coffee rust appeared no chlorotic spots after the invasion of coffee uredia spores ( Figure 5 B-E). This indicates that strain HHV is not only non-pathogenic to host coffee plants, but also has an inhibitory effect on the invasion of spores of the coffee leaf rust pathogen, C. coffeae.

[0038] Example 5: Effect of strain HHV on biomass changes of C. coffeae

[0039] 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 17.688, 16.785, and 17.034, respectively, and the copy number was 1.41×10 8 ,2.81×10 8 ,2.34×10 8However, the coffee rust fungus was not detected in the DNA of the three leaf cakes that were first inoculated with HHV and then inoculated with rust fungus 72 hours later. Figure 6 ).

[0040] Example 6: Growth inhibition of naturally occurring coffee rust by strain HHV

[0041] 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 (Coffee). 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 the bacterial strain HHV. An equal number of lesions were not inoculated with the HHV bacterial strain as a control. Each experiment was repeated 10 times. After inoculation with the HHV bacterial strain, 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 inhibitory effect of the HHV strain on the rust fungus was observed using a scanning electron microscope.

[0042] 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 HHV, 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 hyperparasite fungus growing on the lesions ( Figure 7 B). This indicates that HHV can parasitize the uredia of H. humeroides 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 hunchbacks and short spines on their backs were observed ( Figure 7 C). After the strain HHV 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 HHV has a good effect in controlling coffee rust caused by Puccinia camelus.

[0043] 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 Lecanococcus spenceri, characterized in that: The classification name of the strain is Spencer wax scale fungus Lecanicillium spenceae, and its name is HHV; the strain has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and the deposit number is CGMCC No.41898.

2. Use of the Lecanicillium spenceae HHV according to claim 1 in the preparation of a biocontrol agent, microbial fertilizer or biopesticide for inhibiting coffee rust.

3. Use of the Lecanicillium spenceae HHV according to claim 1 in inhibiting coffee rust.

4. A biocontrol agent for inhibiting coffee rust, wherein the active ingredient is the Lecanicilium spenceae HHV according to claim 1.

5. A microbial fertilizer for inhibiting coffee rust, the active ingredient of which comprises the Lecanicilium spenceae HHV according to claim 1.

6. A biopesticide for inhibiting coffee rust, the active ingredient of which comprises the Lecanicilium spenceae HHV according to claim 1.

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