First Report of Garlic Bulb Rot Caused by Bacillus pumilus in China

Garlic (Allium sativum L.) is cultivated throughout the world and is an important horticultural crop used both for culinary and medicinal purposes. In 2023, China cultivated garlics across 840,200 hectares, producing 20.7 million tons—the highest output and largest cultivation area globally (FAO-STAT, 2023). On October 20th, 2023, an unusual bulb rot was observed on garlic plants in Handan, Hebei Province (36.583°N, 114.483°E). In sporadic field occurrences, approximately 10–20 plants per mu (0.067 hectares) exhibited the following symptoms: leaf chlorosis, stunted growth, and partial wilting. The affected garlic bulbs had light-brown lesions and roots were corroded and discolored. Symptomatic tissue blocks (~0.5 × 0.5 cm) of 10 samples from 5 fields were excised from the junction of healthy and diseased tissue. After surface sterilization with 1% NaClO (2 min) and 75% ethanol (3 min), the blocks were rinsed 3–5 times with sterile water (1 min each). Dried tissues were ground in a mortar, and the homogenate was serially diluted. Aliquots (100 µL) of each dilution were plated on nutrient agar (NA) and incubated at 30°C for 24 h. Bacterial colonies of the predominant morphology were purified, transferred to NA slants, and stored at 4°C. Two colonies with the same morphology were transferred to NA plates by repetitive streaking until pure cultures were obtained. The colonies were white, round, wavy edges and wrinkled. Bacterial isolates were Gram-positive, motile and rod-shaped with endospores. DNA was extracted from two isolate, XJG-84X and XJG-8-4. Both isolates were initially identified as Bacillus pumilus based on PCR amplification of the 16S rDNA, gyrB, gyrA, and pta genes using primers described by Frank et al. (2008), Liu et al. (2013), Liu et al. (2022), and Presecan-Siedel et al. (1999). The information of four housekeeping genes and BLASTn analysis results shown in Table S1. Whole genome sequencing of strain XJG-84X was performed using PacBio Sequel IIe sequencer & Illumina NovaSeq6000 system (Majorbio BioPharm Technology Co., Ltd., Shanghai, China) (accession ID: CRA029592, https://ngdc.cncb.ac.cn/gsa) (CNCB-NGDC 2025). The strain XJG-84X was identified as Bacillus pumilus via MLSA analysis of 31 house-keeping genes. Pathogenicity tests were conducted using five 10-days-old healthy garlic seedlings per treatment by clipping the leaves with scissors dipped in the different bacterial suspensions (1×105 - 1×109 CFU/mL) of sterile distilled water. Negative control garlic seedlings were clipped and inoculated with sterile distilled water. All experiments were repeated three times. Both inoculated and control garlic plants were incubated at 25℃ for 20 d. After 20 d, the control plants remained healthy and asymptomatic. Garlic plants inoculated with B. pumilus XJG-84X developed leaf chlorosis, bulb lesions, and root discoloration, consistent with the symptoms initially observed in the field. The colonies re-isolated from all the inoculated plants were morphologically identical and matched the 16S rRNA sequencing results with 100% identity, fulfilling Koch's postulates. B. pumilus has been reported to cause fruit rot on muskmelon in China (Song et al. 2018) and trunk bulges on rubber tree in Malaysia (Mazlan et al. 2019). This is the first report in China of B. pumilus causing bulb rot in garlic seedlings. Identifying B. pumilus as the causative agent of the observed garlic bulb rot is significant for future prevention and control of this disease.