Abstract: 【Objective】To reduce the use of chemical agents and achieve safe and effective
control of walnut tree rot disease, we selected indigenous bacterial strains that can control walnut rot and other fungal diseases while promoting the growth of walnut seeds and seedlings. 【Methods】Bacteria were isolated from the rhizosphere soil of walnut trees using confronting plate methods and streaking on agar to screen for antagonistic strains. The broad-spectrum antibacterial activity of the selected antagonistic strains against walnut rot disease was evaluated. Morphological characteristics, physicochemical properties, and molecular biology methods were employed to identify the screened strains. The thermal stability of the antagonistic strains was determined using mycelial growth assays under different temperature gradients. Inoculation experiments were conducted to ascertain the effects of the antagonistic strains on walnut rot symptoms. Pot experiments in a controlled environment were carried out to analyze the impact of varying concentrations of fermented filtrate on the germination of walnut seeds, germination rates, and the rate of poor-quality seeds. Mature, unspoiled walnuts weighing approximately 12 grams each were selected as experimental materials and air-dried naturally. The fermented filtrate of the antagonistic strain WS-04 was added to sterilized distilled water, setting five concentrations (50 mg/mL, 150 mg/mL, 250 mg/mL, 350 mg/mL, and 450 mg/mL) for treatment. Sterilized distilled water was used as a control, and each concentration was applied to 20 seeds with three repetitions for each experiment. Prior to soaking, the walnut shells were opened to ensure contact between the fermented filtrate and walnut kernels. After soaking for one day, the seeds were rinsed with clean water and placed in a humid germination box within an incubator at a constant temperature of 30℃ to promote germination. Daily observations were made regarding seed germination, and any rotten or moldy seeds were promptly removed. Germinated seeds were transplanted into pots with a diameter of 20 cm, with two seeds planted per hole, covered with 1 cm of substrate (vermiculite: perlite: peat soil: soil in a volume ratio of 1:1:1:1). The number of germinated seeds was recorded, and the rate of blanching was calculated. Following the emergence of two leaves, the germination rate and the rate of poor-quality seeds were calculated. Simultaneously, both indoor pot experiments and field pot experiments were conducted to study the optimal concentration of the fermented filtrate's effect on the growth of walnut seedlings. Hulled walnut seeds underwent pretreatment: walnuts were gently split open to avoid contact between the kernel and the fermented filtrate of the antagonistic bacteria. A concentration of 150 mg/mL of the antagonistic culture was assigned to the experimental group, while the control group consisted of a medium without antagonistic culture; each group comprised 25 walnut seeds, with five repetitions. After germination, the treatments involved single well plates (32 wells, 6 cm × 4.5 cm), with one seed placed per well. Seedling treatment in plates involved sowing in the wells and initiating irrigation with the antagonistic filtrate (experimental group received 150 mg/mL of fermented filtrate), administering 50 mL every five days. After 20 days of growth, the seedlings from the well plates were transplanted outdoors. For pot experiments, the walnut seedlings were transplanted into experimental plots (using pots with a diameter of 40 cm), with each pot receiving 500 mL of fermented filtrate (once every ten days). After 90 days of transplanting, five plants were randomly selected to measure biological indicators, including dry weight, seedling height, main root length, and root count. 【Results】 A total of 157 bacterial strains were isolated from walnut rhizosphere soil, of which five strains were found to inhibit walnut rot disease, with strain WS-04 demonstrating an inhibition rate of 87.00%. The inhibition rates for Cytospora nivea, Valsa mali, Valsa ambiens, Cytospora chrysosperma, Cytospora leucostoma, Alternaria alternata, Verticillium dahliae, and Fusarium oxysporum were all above 70.00%. Through morphological characteristics, physicochemical properties, and molecular biological confirmation, strain WS-04 was ultimately identified as Pseudomonas chlororaphis. The antibacterial activity of C. chrysosperma was assessed using the mycelial growth rate method, revealing that WS-04 exhibited significant inhibitory effects on C. chrysosperma, with inhibition rates exceeding 80% as the concentration of fermented filtrate increased. At a concentration of 15%, the inhibition rate reached 93.65%. Thermal stability assays indicated that as the temperature increased, the inhibition rate of WS-04 gradually declined; however, it remained above 85% against C. chrysosperma, indicating favorable thermal stability. Treatment at 95℃ for 30 minutes demonstrated that increased temperature progressively led to the loss of the fermented filtrate's activity. Isolation protection tests suggested that WS-04 has good preventive effects against walnut rot, with an average disease lesion area of 0.85 cm² for treated samples compared to 6.32 cm² for the control group, resulting in a control efficacy of 84.96%. Pot experiments revealed that the germination rate of walnut seeds improved with increasing concentration after treatment. At concentrations of 150 mg/mL and 250 mg/mL, significant promotion of walnut seed germination was observed. The germination rate was notably higher in the treatment groups with concentrations of 250 mg/mL > 150 mg/mL > 350 mg/mL > 50 mg/mL > 450 mg/mL compared to the control group (P ＜ 0.05). As the concentration increased, the germination rate initially rose and then declined; within the range of 50 to 250 mg/mL, the germination rate of walnut seeds steadily increased, but when the concentration of the fermented filtrate exceeded 250 mg/mL, the germination rate began to decrease. At a concentration of 150 mg/mL, the percentage of poor-quality seeds was at its lowest, reaching 6.68%. When the concentration of the antagonistic bacteria exceeded 150 mg/mL, the rate of poor-quality seeds exhibited an upward trend; however, the rates across all concentrations remained significantly lower than those in the control group (P ＜ 0.05). In field experiments, we found that WS-04 could promote the growth of walnut seedlings, increasing seedling height, root length, leaf area, and dry weight by 25.40%, 83.33%, 48.02%, 24.76%, and 98.40%, respectively. 【Conclusion】A biocontrol bacterium with broad antibacterial spectrum and growth promotion effect on walnut seeds and seedlings was isolated and screened from the soil of walnut orchard. This provides a novel strain source for the biological control of walnut rot disease.
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