Analysis of antagonistic activity of Fusarium lateritium strain Pa2 against strawberry black spot

Abstract: 【Objective】Strawberry is a perennial herb crop with short growth cycle and high economic value, and is cultivated all over the world. However, this fruit is prone to pests and diseases at each stage of production, resulting in significant losses in yield and quality. Strawberry black spot caused by Alternaria alternata is one of the common diseases. It mainly affects fruit, leaves, stems and other parts, and seriously harms the nutritional and economic values of strawberry. At present, the disease has been reported in many countries. Chemical agents are commonly used to control the disease, which generates pesticide residues, causing environmental pollution and endangering human health. Therefore, it is urgent to explore green and safe methods to control this disease. Compared with chemical control, biological control has the advantages of no pollution, no residue, less toxicity and reducing drug resistance. There has been no report on biological control of strawberry black spot with endophyte. Many plant species are infected by Fusarium lateritium, which causes plant diseases. However, it is reported that endophytic F. lateritium can also exert beneficial effects on disease resistance of the host plants and their growth. Therefore, the main purpose of this study was to explore the inhibition of F. lateritium strain Pa2 on strawberry black spot, and to provide reference for the development and utilization ofplant endophytes for biocontrol of strawberry black spot.【Methods】In this experiment, the endophyte F. lateritium strain Pa2 was selected as the test material, and the strawberry black spot was used as the control target. The influences of strain Pa2 on A. alternata mycelial growth, spore germination, cell membrane permeability and control effect of strawberry black spot were analyzed. The inhibition effect of the F. lateritium strain Pa2 on A. alternata was determined by plate confrontation experiment culture method. Spore suspension of strain Pa2 was cultured in a shaker at 28 ℃ and 180 r·min^-1 for 7 days and centrifuged at 4000 r·min-1 for 20 min. The supernatant was filtered through a 0.45 μm filter, and then through a 0.22 μm filter. Finally, sterile supernatant was obtained and PDA medium was prepared according to the volume ratio. The final concentration of the supernatant medium was set to 5% and 10%, and the colony growth status was determined. Meanwhile, 1 mL of spore suspension with a concentration of 1×106 CUF·mL−1 was placed in PDB, cultured at 28 ℃ and 180 r·min-1 for 3 days, centrifuged at 3000 r · min- 1 for 5 min, and the supernatant was removed. After washing twice with sterile water, the spores were placed in 20 mL sterile water containing 5% and 10% Pa2 supernatant, respectively. The control did not contain the supernatant. The values of OD260 and OD280 were measured at 0, 12, 24 and 36 h after treatment, respectively. After culturing on PDA plate for 20 days, 5 mL of sterile water was added to the pathogen plate with a pipette, and the plate colonies were scraped and placed in a sterile centrifuge tube. The cap of centrifuge tube was closed and shook to mix. Then, the spore suspension was filtered through 4 layers of sterile lens paper and spores were counted with a haematocrit plate, and a spore suspension with a concentration of 1×106 CUF · mL− 1 was prepared. The spore suspension and Pa2 supernatant were mixed at volume ratios to prepare mixtures with the final concentration of 5% and 10% . 20 μL of conidial suspension was dropped on hydrophobic glass slide and placed in petri dish (200 mm in diameter). Photographs of the spore germination were taken at 4, 8, 16, and 32 h after treatment. Through the combination of extraction and rotary evaporation, the supernatant of strain Pa2 was extracted with ethyl acetate, concentrated by rotary evaporation to extract, dissolved and diluted with methanol to 20 mg · mL- 1 to determine the inhibition of the extract on strawberry black spot. SYTOX green nucleic acid stain was used to detect the effect of strain Pa2 supernatant on the permeability of mycelium and spore cell membrane of strawberry black spot pathogen. The antagonistic activity of strain Pa2 against strawberry black spot was determined on detached fruit and leaves of strawberry. The fruit and leaves treated with the spore suspension of strain Pa2 were inoculated with the spore suspension of A. alternata on the fruit, and the mycelial plugs on the leaves. Water treatment was used as negative control and prochloraz was used as the positive control. On the fifth day after fruit inoculation, the lesion diameter and incidence were measured, and on the seventh day after leaf inoculation, the measurement was carried out on the leaves.【Results】The plate confrontation experiment showed that the inhibition rate of strain Pa2 on the growth of A. alternata was 80.96%. After the treatment of strain Pa2, it was found that the hyphae of A. alternata showed nodules, terminal enlargement and other malformations, and even developed rupture of the hyphal membrane in the inhibition zone and the leakage of protoplasts under the microscope. The supernatant of strain Pa2 inhibited the mycelial growth and spore germination of A. alternata in a dose-dependent fashion. The colony diameter of strawberry black spot cultured with 10% Pa2 supernatant was 2.30 cm, and the inhibition rate reached 74.15%. The spore germination rate was about 15% and the inhibition rate reached 81.11% at 32 h. Furthermore, the supernatant of strain Pa2 could also damage the cell membrane of hyphae and spores, and the GFP fluorescence was clearly displayed after SYTOX green staining. In addition, control test results suggested that the strain Pa2 could strongly inhibit strawberry black spot caused by A. alternata. Statistical results indicat-ed that the incidence and lesion diameters on the fruit and the leaves of strawberry treated with strain Pa2 were reduced significantly, comparing to control. The inhibition of strawberry black spot by strain Pa2 was comparable to the positive control with prochloraz.【Conclusion】F. lateritium strain Pa2 showed strong inhibitory effect on strawberry black spot, and has potential application value in the biocontrol of strawberry black spot.