First Report of Pseudomonas oryzihabitans Causing Walnut Leaf Spot Disease in China.

China ranks first in the production and harvest area of walnut ( Juglans regia L.) worldwide. Currently, the poor health and low yield of walnut caused by pathogen infection is of concern. In 2022, severe walnut leaf spot disease was observed on the seedlings of four walnut nurseries (0.08 to 0.23 ha) in Liaocheng, Shandong, China, with an average incidence of 48.6% (from 34.6% to 65.3% on the cultivar Xiangling). From August to October, leaf spots mainly appeared on the edges of the leaflets, and occasionally between veins. The lesions were initially soft and rotten, and then light brown, round to semi-circular. Subsequently, the adjacent lesions fused, and the edges of the leaflets and entire leaflets showed symptoms of browning and wilting. For pathogen isolation, five leaflets with representative symptoms from one of the nurseries were collected and wiped three times with sterile absorbent cotton dipped in 75% alcohol and washed with distilled water. Leaflet pieces at the junction of the lesion and healthy tissues were removed, crushed in a sterile mortar, and soaked in a small amount of distilled water for 10 min. The diseased tissue suspension was streaked on a nutrient agar medium (NA) with a sterile inoculation ring and incubated at 28°C for 24 to 72 h. The bacterial colonies obtained were further cultured on NA. The purified colonies were uniform in shape, round, and yellow, with a raised, shiny surface and smooth margin. The isolates were Gram-negative, and the electron microscope analysis showed that the pathogens were short rods (0.35 to 0.52 × 0.90 to 1.24 μm, average = 0.44 ± 0.05 × 1.08 ± 0.11 μm, n = 25). For bacterial species identification, a single-colony culture was subjected to genomic DNA extraction and gene amplification and sequencing of 16S rRNA, rpo D, and gyr B. The universal primers 27F/1492R (Lane 1991) were used to amplify the 16S rRNA gene and the specific primers 70F/70R and UP-1E/APrU (Yamamoto et al. 2000) were used to amplify the rpo D and gyrB genes, respectively. In the BLAST analysis, the 16S rRNA sequence (GenBank OR195734) of the isolate shared 99% similarity (1409/1410 bp) with Pseudomonas oryzihabitans strain IAM 1568T (AM262973.1), and the rpo D (OR709708) and gyr B (OR709707) sequences showed >98% identity to rpo D (707/717 bp; FN554494.1) and gyr B (787/801 bp; FN554210.1) of P. oryzihabitans strain LMG 7040T. Based on the above results, the isolated bacterium was identified as P. oryzihabitans . For the pathogenicity test, healthy leaflets from 10 two-year-old potted walnut seedlings (cv. Xiangling) were used as inoculation materials. The leaflets were punctured with a sterile inoculation needle of 0.4 mm, and three small holes on each leaflet at an interval of about 5 mm were covered with a piece of sterile cotton. A bacterial suspension (1 ml) at 10 ⁷ CFU/ml was spread onto the cotton, and wrapped with plastic film for 24 h. Water was used as a negative control. The inoculations were performed five times. Plants were grown outdoors at a daily average temperature of 22°C with relative humidity over 45%. Two days after inoculation, the disease began to develop in the leaflets with similar symptoms to those observed in the field. In contrast, control plants remained healthy and symptomless. Bacteria were reisolated from the inoculated walnut plants, and the morphology and 16S rRNA gene sequences of the isolates were the same as those of the original strains. Since it was discovered as an opportunistic human pathogenic bacterium in the 1970s (Keikha et al. 2019), P. oryzihabitans has also been shown to cause certain plant diseases, such as panicle blight and grain discoloration on rice (Hou et al. 2020), fruit black rot on prickly ash (Liu et al. 2021), and stem and leaf rot on muskmelon (Li et al. 2021). As far as we know, this is the first report of P. oryzihabitans causing walnut leaf spot disease in China. Leaf spot caused by P. oryzihabitans may be a threat to walnut cultivation, and this report of its occurrence is the first step in determining potential spread and effective control measures.