Screening and identification of antagonistic bacterium Pseudomonas chlororaphis for walnut rot disease and its effect on promoting germination of seeds and growth of seedlings in walnut

【Objective】The study aimed to select indigenous bacterial strain that could control walnut rot and other fungal diseases and promote the germination of seeds and growth of seedlings of walnut. 【Methods】Bacteria were isolated from the rhizosphere soil of walnut (Juglans regia L.) trees using confronting plate methods and streaked on agar to screen for antagonistic strains. The broad-spectrum antibacterial activity of the selected antagonistic strains against walnut rot disease was evaluated. The morphological characteristics, physicochemical properties, and molecular biological characters were employed to identify the screened strains. The thermal stability of the antagonistic strains was determined using mycelial growth assays under different temperature gradients. The inoculation experimentswere conducted to ascertain the effects of the antagonistic strains on the walnut rot symptoms. The 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 and the rate of poor-quality seeds. The mature, unspoiled walnuts weighing approximately 12 grams each were selected as experimental materials and dried in the air naturally. The fermented filtrate of the antagonistic strain WS-04 was diluted by sterilized distilled water, and five concentrations (50, 150, 250, 350, and 450 mg ·mL- 1 ) were set for treatment. The sterilized distilled water was used as a control, and each concentration was applied to 20 seeds with three repeats for each experiment. Prior to soaking, the walnut shells were opened to ensure that the kernels would contact the fermented filtrate. 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 during the course of seed germination, and any rotten or moldy seeds were promptly removed. The 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 effect of different concentrations of the fermented filtrate on the growth of walnut seedlings. The pretreatment of hulled walnut seeds for the experiment of promotion of growth on seedlings was as follows: the walnuts were gently split open to ensure the contact between the kernel and the fermented filtrate of the antagonistic bacteria. A concentration of 150 mg · mL- 1 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 repeats. After germination, the single well plates (32 wells, 6 cm × 4.5 cm) were used for the treatments, one seed per well. The seedling treatment in plates involved sowing in the wells and initiating irrigation with the antagonistic filtrate (experimental group received 150 mg ·mL-1 of fermented filtrate), administering 50 mL every five days. After 20 days of growth, the seedlings from the well plates were transplanted outdoors. For the pot experiments, the walnut seedlings were transplanted into experimental plots (with a diameter of 40 cm), with each pot irrigated with 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 the morphological characteristics, physicochemical properties, and molecular biological confirmation, the 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%. The thermal stability assays indicated that as the temperature increased, the inhibition rate of the WS- 04 gradually declined; however, it remained above 85% against C. chrysosperma, indicating favorable thermal stability. The treatment at 95 ℃ for 30 minutes demonstrated that increased temperature progressively led to the loss of the activity of the fermented filtrate. The isolation protection tests suggested that theWS-04 would have good preventive effects on anti- walnut rot, with an average disease lesion area of 0.85 cm² for treated samples compared with 6.32 cm² for the control group, resulting in a control efficacy of 84.96% . The pot experiments revealed that the germination rate of the walnut seeds was improved with increase of the concentrations after treatments. At concentrations of 150 mg·mL-1 and 250 mg·mL-1 , significant promotion of walnut seed germination was observed. The germination rate was notably higher in the treatment groups with concentrations of 250 mg·mL-1 ＞ 150 mg·mL-1 ＞ 350 mg·mL-1 ＞ 50 mg · mL- 1 ＞ 450 mg · mL- 1 compared with 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- 1 , the germination rate of walnut seeds steadily increased, but when the concentration of the fermented filtrate exceeded 250 mg·mL-1 , the germination rate began to decrease. At a concentration of 150 mg·mL-1 , the percentage of the poor-quality seeds was the lowest, reaching 6.68%. When the concentration of the antagonistic bacteria exceeded 150 mg ·mL-1 , the rate of poor-quality seeds exhibited an upward trend; however, the rates of poor quality seeds of all concentrations remained significantly lower than those in the control group (p ＜ 0.05). In the field experiments, we found that the 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 would provide a novel strain source for the biological control of the walnut rot disease.