Alternaria tenuissima and use thereof

Abstract

The application provides a kind of Alternaria tenuissima and its application, belong to plant pathogenic microorganism technical field, the application provides the Alternaria tenuissima (Alternaria tenuissima) A-c-qy preservation number is CGMCC No.42437, the application is identified by morphology and molecular biology, confirms that the bacteria is cherry sudden collapse disease pathogenic bacteria;Pathogenicity determination confirms the pathogenicity of the pathogenic bacteria.Biological characteristic analysis shows that the optimal growth temperature of the bacteria is 25 DEG C, and the optimal pH is 6.The application first clearly defines the pathogenic relationship between Alternaria tenuissima and cherry sudden collapse disease, provides an important theoretical basis for efficient use of resources and ecological prevention and control of seedling sudden collapse disease in cherry introduction cultivation in arid areas, and has important significance for ensuring the sustainable use of introduced characteristic forest fruit resources in arid areas.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogenic microorganism technology, and particularly relates to a type of Alternaria spp. and its applications. Background Technology

[0002] cherry( Prunus avium Prunus (L.) is a plant belonging to the genus Prunus in the Rosaceae family and is one of the most economically valuable deciduous fruit trees in northern China. It is a perennial woody plant widely distributed in temperate and subtropical regions of the Northern Hemisphere. The climate and soil conditions in the Hohhot area have somewhat constrained the development of forestry and fruit industry, resulting in a single variety of local fruit trees that cannot meet the diversified demands of the market. To optimize the agricultural industrial structure and increase farmers' income, the region has actively explored cross-regional cherry introduction and cultivation—Dalian, as a well-known cherry-producing area in my country, has become the core source for its seedling introduction. Currently, the scale of cherry planting in Hohhot continues to expand, and a preliminary industrialized planting pattern has been formed. Developing the cherry industry has multi-dimensional value for optimizing the local economic structure: as a high-value-added economic crop, cherries can not only significantly increase the output value per unit of land and broaden the income channels for farmers and herdsmen, but its industrial development can also promote the construction of a distinctive agricultural industrial chain, driving the coordinated development of supporting industries such as processing, logistics, and tourism. Furthermore, although the arid region of Inner Mongolia faces limitations due to insufficient rainfall, ample sunshine and significant diurnal temperature differences are conducive to the accumulation of sugar in cherries, creating a potential advantage for cultivating distinctive and high-quality cherry products. However, introducing cherry varieties native to humid or semi-humid regions into the arid and semi-arid areas of Inner Mongolia presents significant environmental adaptation challenges. For example, during the introduction of Dalian cherry seedlings to Hohhot, damping-off disease occurred on a large scale, becoming a key biological stress factor restricting the healthy development of the industry. This disease causes the death of a large number of seedlings, severely affecting the survival rate and growth quality of cherry seedlings, causing huge economic losses to the introduction work, increasing planting costs, delaying the planting cycle, and hindering the smooth progress of the cherry industry in the local area.

[0003] Damping-off in seedlings is a serious disease threatening forestry seedling cultivation worldwide, with a persistently high incidence rate. The pathogens include both non-infectious and infectious agents, with the infectious agents primarily being fungi such as Fusarium, Rhizoctonia, and Pythium. In recent years, Hohhot, Inner Mongolia, has introduced cherry seedlings from humid regions like Dalian to promote the development of its fruit-growing economy, leading to a large-scale outbreak of damping-off in cherry seedlings. This has become a potentially epidemic disease and a key target for control in the central and western parts of Inner Mongolia. Currently, there are few reports both domestically and internationally on research regarding this pathogen causing damping-off in seedlings. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a *Alternaria spp.* and its application. This invention isolates and screens *Alternaria spp.*, the pathogen causing damping-off disease in cherry seedlings. By analyzing the biological characteristics of *Alternaria spp.*, the key biological characteristics of this pathogen under the environmental conditions of the arid region of Inner Mongolia are elucidated, thereby discovering new control targets. This provides a theoretical basis and technical support for the prevention and control of damping-off disease in cherry seedlings, and lays the foundation for formulating environmentally friendly and resource-saving disease control strategies.

[0005] This invention provides a type of Alternaria spp. ( Alternaria tenuissima Ac-qy, with accession number CGMCC No.42437.

[0006] Preferably, the suitable growth temperature for Alternaria filamentosa Ac-qy is 25~28℃, and the suitable growth pH is 4.5-6.5.

[0007] This invention provides the application of *Alternaria alternata* Ac-qy in inducing damping-off disease in cherry seedlings.

[0008] This invention provides the application of Alternaria alternata Ac-qy in the research and control of damping-off disease in cherry seedlings.

[0009] This invention provides a method for preventing and controlling damping-off disease in cherry seedlings caused by Alternaria alternata, comprising one or more of the following operations (A) to (C): (A) Control the temperature of the seedbed at 18~22℃; (B) Adjust the soil pH to 7.2~7.8; (C) Increase air disinfection.

[0010] Preferably, quicklime is used to adjust the pH value of the soil.

[0011] Preferably, the Alternaria spp. is Alternaria spp. Ac-qy.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention confirms *Alternaria spp.* through a combination of morphological and molecular biological identification. Alternaria tenuissima The pathogen of cherry damping-off (G1) was identified; pathogenicity testing confirmed its pathogenicity. Biological characteristic analysis showed that the optimal growth temperature was 25℃ and the optimal pH was 6. Nutritional affinity group structure analysis screened out the dominant bacterial group G1 and 13 dominant strains including Ac-qy, Ac-qy-2, and Ac-qy-3. A search revealed no reports of *Alternaria spp.* causing cherry damping-off domestically or internationally. This invention clarifies the pathogenic relationship between *Alternaria spp.* and cherry damping-off for the first time, providing an important theoretical basis for the efficient utilization of resources and the ecological control of seedling damping-off in the introduction and cultivation of cherry in arid regions. It is of great significance for ensuring the sustainable utilization of introduced specialty forestry and fruit resources in arid regions. Attached Figure Description

[0013] Figure 1 Macroscopic morphology of the pathogen Ac-qy (cultured for 7 days); Figure 2 The image shows the microscopic morphology (40×) of the pathogen Ac-qy, with the left image showing the conidiophore and the right image showing the conidia. Figure 3 Phylogenetic tree of pathogenic strains constructed based on rDNA-ITS gene sequences (scale bar 0.01 represents sequence difference). Figure 4 To investigate the differences in pathogenicity of different Alternaria spp. strains to cherry seedling stems; Figure 5 The effect of different temperatures on the mycelial growth of Alternaria filamentosa; Figure 6 The effect of different pH values ​​on the mycelial growth of Alternaria filamentosa.

[0014] Biological Preservation Instructions

[0015] Alternaria microphylla ( Alternaria tenuissima Ac-qy is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42437, deposited on January 22, 2026, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0016] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0017] Example 1

[0018] This embodiment employs a repeated experimental design, and the data were analyzed using SPSS 27 software for one-way ANOVA, combined with the Turkey multiple comparison test method. P＜0.05 Conduct data analysis.

[0019] Test materials

[0020] Regional characteristics

[0021] Wusutu (40°49′N, 111°35′E) and Yematu (40°52′N, 111°47′E) are located in Hohhot City and are among the larger orchards and economic forests in the area. The region has a temperate continental climate, characterized by distinct seasons, cold and dry winters, and warm and humid summers; the average annual temperature is 5-8℃, and the average annual precipitation is 350-450 mm, concentrated in summer; prevailing northwesterly winds occur, with occasional sandstorms in spring. The main soil type is chestnut calcareous soil, which is relatively light in texture and has a low organic matter content.

[0022] Test strains

[0023] The experimental materials used were 60 stems of diseased cherry seedlings collected from Wusutu and Yematu.

[0024] The pathogen was isolated using a tissue isolation method (Reference: Fang Zhongda. Plant Disease Research Methods (3rd Edition) [M]. China Agriculture Press, 1998.). Typical symptoms of cherry seedlings were selected. The stems were first rinsed with tap water to remove surface impurities, then dried on clean four-layer gauze. On a clean bench, 0.5 cm × 0.5 cm pieces of diseased tissue were cut at the junction of diseased and healthy tissue using a sterile blade. These pieces were then soaked in 75% alcohol for 30 seconds, disinfected with 1% sodium hypochlorite solution for 5 minutes, rinsed three times with sterile water, and finally dried on sterile filter paper. Three pieces of diseased tissue were inoculated onto PDA plates, sealed, and incubated upside down in a 25℃ constant temperature incubator in the dark (For detailed procedures, see the literature: Liu Qiao. Identification of Pathogens and Indoor Screening of Control Agents for Cherry Leaf Spot Disease in Qinghai Province [D]. Qinghai University, 2020.).

[0025] A total of 114 bacterial strains were isolated from 60 diseased samples. These strains showed a high degree of consistency in colony color, texture, and microstructure. For the convenience of subsequent research, the isolated strains were numbered sequentially according to the isolation order, such as Ac-qy-2, where "A" and "c" represent the Latin scientific name of the genus Alternaria and the first letter of the host cherry, respectively, and "qy-2" represents the initials of the isolater's name and the sequential number of the isolated strain.

[0026] The pathogenic bacterial strain with the fastest growth rate and the most stable colony morphology in subculture was named "Ac-qy" for strain identification.

[0027] Morphological identification

[0028] The isolated and purified strains were inoculated onto PDA solid medium and cultured at 28°C for 12 hours for 7 days. The fungal structure was examined using an Olympus BX53 optical microscope and Olympus Cell-Sens standard software for recording morphological data, and the colony morphology and color were observed. Microscopic slides of the pathogen were prepared on a clean bench using the picking method: a drop of sterile water was placed on a slide, and purified pathogen hyphae were picked up with a sterile inoculation needle and placed on the slide. A coverslip was then placed on top, and excess water was absorbed with absorbent paper. The temporary slide was then prepared and the pathogen structure was observed under an optical microscope.

[0029] Ac-qy culture for 7 days can cover the entire area of ​​a 90 mm diameter petri dish. Colonies are grayish-brown, with a dark brown to black underside of the substrate; the texture is velvety, and the edges are irregular. Figure 1 Conidiophores are solitary, light brown to dark brown, septate, and 110.0 × 3.1 μm in size; conidia are slender, obclavate or fusiform, dark brown, with 3-4 transverse septa and 1-2 longitudinal septa, 42.3 × 8.0 μm in size, with a beak-like cell visible at the apex, without swelling, lighter in color than the spore body, and 5.2-6.0 μm long. Figure 2 Based on the morphological characteristics of the pathogen, it was preliminarily identified as *Alternaria*, a species of fungus belonging to the Deuteromycetes. Alternaria tenuissima ).

[0030] Alternaria alternata commonly causes cherry leaf spot disease, and Dalian is one of the important origins of cherry. Therefore, this study compared *Alternaria spp.*, which can cause damping-off disease and was isolated from the stems of cherry seedlings, with *Alternaria glomerata*, the pathogen causing cherry leaf spot disease in Dalian. Alternaria gaisen (Provided by the College of Plant Protection, Northwest A&F University) for comparison (Table 1). The morphological characteristics of *Alternaria*, the pathogen of cherry leaf spot disease in Dalian, are as follows: Conidiophores are solitary or clustered, straight or geniculate, light brown to brown in color, partially branched or unbranched, septate, often swollen at the base, measuring 24.0–50.0 × 3.5–6.0 μm; Conidia are solitary or in chains, obpyriform, brown, with 4–7 transverse septa and 1–13 longitudinal septa, the septa are distinctly constricted, and the transverse septa are often thickened in older conidia, the spore body is 18.0–61.0 × 11.0–23.5 μm; Pseudobeak is columnar, light brown, often swollen at the apex, measuring 5.0–36.5 × 2.5–6.0 μm.

[0031] Table 1. Comparison of morphological parameters between pathogen Ac-qy and Alternaria collodion

[0032] Alternaria spp. Ac-qy and Alternaria collodion ( A.gaisen Significant differences exist in the size, shape, number of septa, and color of conidia, which can serve as important criteria for morphological classification. This study, through morphological observation and ITS sequence analysis, revealed a clear morphological differentiation between the strain causing damping-off in sweet cherry seedlings introduced from Hohhot and the strain native to Dalian. This fills a gap in research on pathogen adaptability in sweet cherry introduction areas and provides a theoretical basis for cross-regional disease control. Hohhot, as a sweet cherry introduction area, has an arid climate with little rainfall, significantly different from the maritime climate of Dalian. Currently, there are no systematic comparative studies of strains from the two regions, including unclear differences in pathogenic mechanisms and toxin profiles. This study shows that the pathogenic strain causing damping-off in sweet cherry seedlings introduced from Dalian may have undergone adaptive variation. Ac-qy has not yet been reported to cause damping-off in introduced sweet cherry seedlings.

[0033] Molecular biological identification

[0034] Single spores were inoculated onto PDA plates and cultured for 7 days. DNA was extracted using the CTAB method. Using the obtained DNA as a template, PCR amplification was performed using universal fungal primers ITS1 and ITS4. The amplified products were then sequenced. The sequencing results were compared and analyzed on NCBI, and the sequences of relevant standard strains were downloaded from GenBank. Multiple sequence comparisons were performed between the downloaded standard strain sequences and the sequencing results, and a phylogenetic tree was constructed in MEGA 11.0 using the nearest neighbor method.

[0035] Agarose gel electrophoresis results showed that the DNA extraction quality was good, and a specific band of approximately 600 bp was obtained after PCR amplification; phylogenetic tree ( Figure 3 The results showed that the target strain clustered with the Alternaria filamentosa model strain (GenBank accession number: MK798282), with a support rate of 100%. Gel electrophoresis, BLAST alignment, and phylogenetic tree evidence jointly identified the bacterium as Alternaria filamentosa.

[0036] Pathogenicity test

[0037] Koch's postulates were used to conduct a pathogenicity test upon reinoculation. 10 mL of sterile water containing 0.05% Tween was added to an activated pathogen plate. The colony surface was gently scraped with a sterile spreader to collect the spore and hyphae mixture. The mixture was filtered through double-layered sterile gauze to remove hyphae debris, yielding a spore suspension. Spores were counted using a hemocytometer, and the spore concentration was adjusted to 1 × 10⁻⁶ spores with sterile water. 6 ~1×10 7Spores / mL. Sterilized seedling substrate was filled into pots, and cherry seedlings were transplanted and cultured in a greenhouse until they had 2-3 true leaves. A test strain treatment group and a blank control group were set up, with at least three replicates for each group. 1 mL of suspension was pipetted and evenly applied to the base of the stem, then covered with sterile absorbent cotton to maintain moisture. The control group was treated with sterile water containing 0.05% Tween (spore suspension control).

[0038] Cherry seedlings 'Meizao' cherry ( Prunus avium Seedlings of 'Mei Zao' (a specific strain of cherry) exhibited symptoms highly similar to those of cherry seedling damping-off after inoculation with Ac-qy, Ac-qy-2, and Ac-qy-3 strains, respectively: infection began on day 3, with irregular water-soaked lesions appearing at black edges; on day 4, the lesions developed into a completely gray color; on day 5, the lesions were gray with a moldy layer, and the tissue gradually softened. The control treatment showed no lesions. Analysis revealed significant differences in the diameter of lesions induced by Ac-qy, Ac-qy-2, and Ac-qy-3 strains. P＜0.05 For detailed data comparison, see [link / reference]. Figure 4 .

[0039] Biological characteristics study

[0040] Effects of different temperatures on mycelial growth

[0041] In a sterile operating table, the preserved pathogens were made into bacterial discs using a 6 mm diameter punch and inoculated into the center of PDA medium. They were then placed in constant temperature incubators at 15, 20, 25, 30, and 35°C for dark incubation. The treatment was repeated 3 times. After 7 days, the diameter of the bacterial discs was measured using the cross-sectional method.

[0042] Alternaria spp. exhibits a wide growth temperature range (15℃~35℃). At 15℃, mycelial growth is relatively slow; the growth rate is highest at 25℃, and slows down further below 25℃. At 25℃, the mycelial extension lengths of strains Ac-qy, Ac-qy-2, and Ac-qy-3 were 3.8 cm, 3.6 cm, and 3.5 cm, respectively. Above 25℃, the mycelial growth rate gradually decreases with increasing temperature, and at 30~35℃, mycelial growth is extremely weak. Figure 5 ).

[0043] Effects of different pH values ​​on mycelial growth

[0044] The pH of the PDA medium was adjusted to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0 using NaOH and HCl, both at a concentration of 0.1 mol / L. The bacterial pellets were inoculated into the center of the medium and incubated at 25°C. Each treatment was repeated three times. After 7 days, the colony diameter was determined using the cross-hatching method.

[0045] The results showed that *Alternaria spp.* could survive in weakly acidic to weakly alkaline environments, with optimal activity under weakly acidic conditions (4.5–6.5). The highest colony growth rate was observed at pH 6, representing the optimal pH for *Alternaria spp.* growth; growth gradually slowed at pH > 6; and mycelial growth was virtually impossible at pH 10. Figure 6 ).

[0046] Nutritional affinity group structure analysis

[0047] First, the pathogens were activated. Then, a fungal disc was made using a 6 mm diameter sterile punch. The fungal discs of the strains to be tested were simultaneously and symmetrically inoculated onto PDA plates with an inner diameter of 9 cm, with the centers of the two fungal discs 5 cm apart. The inoculated plates were then placed in a 25°C incubator for 7 days.

[0048] Nutritional affinity group structure analysis was performed on 15 *Alternaria spp.* strains with different typical colony morphological characteristics, identifying two hyphal fusion groups in the Hohhot area (Table 2). The criteria for hyphal fusion were as follows: after 7 days of cultivation, observation of the interface between the two mycelial cakes revealed continuous hyphae without obvious antagonistic lines, and the hyphae could interpenetrate and grow, indicating hyphal fusion (+); if a clear inhibition zone, inhibited hyphal growth, or no hyphal connection appeared at the interface, it was considered hyphal non-fusion (-). Confluence group G1 comprises 13 strains: Ac-qy, Ac-qy-2, Ac-qy-3, Ac-qy-4, Ac-qy-5, Ac-qy-6, Ac-qy-7, Ac-qy-8, Ac-qy-9, Ac-qy-10, Ac-qy-11, Ac-qy-13, and Ac-qy-15. Confluence group G2 contains two strains: Ac-qy-12 and Ac-qy-14. Based on the percentage of strains, group G1 accounts for 86.7% (13 / 15) of the total isolated strains, significantly higher than group G2's 13.3% (2 / 15). Combining distribution characteristics, confluence group G1 is identified as the dominant bacterial group, with its 13 strains being the dominant strains. Among them, strain Ac-qy exhibits the fastest growth rate and the strongest pathogenicity.

[0049] Table 2. Identification of hyphal fusion after pairwise culture of 15 strains

[0050] Damping-off disease in cherry seedlings severely restricts the development of the cherry introduction industry in arid and semi-arid regions. This invention is the first to identify *Alternaria alternata* as the pathogen causing damping-off disease in cherry seedlings introduced to the arid region of Inner Mongolia, and clarifies its optimal growth temperature as 25℃ and pH as 6. Furthermore, among 15 isolated strains, the dominant bacterial group G1 contains 13 dominant strains, including Ac-qy-1, Ac-qy-2, and Ac-qy-3. These strains can enhance pathogenicity through synergistic effects. These findings fill a gap in research on related diseases in newly introduced cherry-growing areas such as Inner Mongolia.

[0051] Example 2

[0052] Based on this, targeted prevention and control strategies are proposed: using greenhouse temperature control to keep the seedbed temperature below 25℃; raising the soil pH to above 7 by applying quicklime to reduce pathogen activity; and increasing air disinfection measures within the greenhouse, taking into account the pathogen's airborne transmission characteristics. This research provides theoretical support for the sustainable development of the cherry industry in arid regions, and the relevant technical models can also be extended to the management of diseases in similar introduced seedlings.

[0053] The specific implementation plan is as follows: Set up a control group (CK) and a treatment group; The control group simulated a conventional greenhouse environment with daytime temperatures of 25°C to 28°C and nighttime temperatures of 15°C to 18°C. The average temperature at which pathogens were most likely to be covered was 25°C, and the pH value was 6.2 (in the original soil). No disinfection or sterilization operations were carried out.

[0054] The processing group operations are as follows: ①Use the greenhouse's temperature control system (shading net, water curtain cooling, ventilation adjustment) to strictly set the target temperature range of the seedbed area to 18℃~22℃; ② Before sowing or during substrate preparation, mix quicklime in a certain proportion to raise the soil / substrate pH value from the original slightly acidic 6.0~6.5 to above 7.0, with a target pH of 7.2~7.8; ③ Install air disinfection facilities in the greenhouse and disinfect the air in the greenhouse regularly (kill the suspended conidia and prevent them from settling on the stems and leaves of healthy seedlings and causing secondary infection).

[0055] Results and Analysis: In this experiment, the control group (CK) showed a damping-off incidence rate of 68.5% after 7 days of observation, and the disease index rose to 55.4 after 14 days. Regarding seedling growth, the survival rate of CK seedlings was only 31.5%, and the plant dry weight was measured to be 0.42g. In the treatment group, the treatment conditions were a temperature below 25℃, a pH adjusted to 7.5, and air disinfection measures were implemented. After 7 days, the damping-off incidence rate in this group was 3.5%, and the disease index was only 2.8 after 14 days. The relative control efficacy of the treatment group was excellent, reaching 94.9%. Regarding seedling growth, the survival rate of seedlings in the treatment group was significantly improved, reaching 96.5%, and the plant dry weight increased to 0.88g.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Alternaria microphylla ( Alternaria tenuissima Ac-qy, characterized in that, The accession number is CGMCC No. 42437.

2. The Alternaria filamentosa Ac-qy according to claim 1, characterized in that, The optimal growth temperature for *Alternaria spp.* Ac-qy is 25-28℃, and the optimal growth pH is 4.5-6.

5.

3. The application of Alternaria alternata Ac-qy as described in claim 1 in inducing damping-off disease in cherry seedlings.

4. The application of Alternaria alternata Ac-qy as described in claim 1 in the research and control of damping-off disease in cherry seedlings.

5. A method for preventing and controlling damping-off disease in cherry seedlings caused by *Alternaria spp.*, characterized in that, Includes one or more of the following operations (A) to (C): (A) Control the temperature of the seedbed at 18~22℃; (B) Adjust the soil pH to 7.2~7.8; (C) Increase air disinfection.

6. The method according to claim 5, characterized in that, Quicklime was used to adjust the pH value of the soil.

7. The method according to claim 5, characterized in that, The *Alternaria spp.* is the *Alternaria spp.* Ac-qy* as described in claim 1.

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