First Report of <i>Pectobacterium brasiliense</i> Causing Soft Rot on Avocado in Hawai‘i, U.S.A.

Avocado (Persea americana Mill.) is commonly grown throughout Hawai‘i. While predominately used for local consumption, Hawai‘i accounts for ~1% of the production in the United States (USDA-NASS 2023). In July 2022, avocado fruits (accession N18-2) collected at the USDA Agricultural Research Service (ARS) germplasm repository in Hilo and held at ambient room temperature, developed a soft rot within three days post-harvest. Six fruits were brought to the USDA-ARS-PBARC facility for examination. All fruits had darkened areas on the skin, which were soft and slightly sunken. Internally the flesh was water-soaked, discolored, soft, and contained a distinct foul, musky odor. Symptomatic flesh tissue ~0.5 cm 3 in size was excised from two fruits, disinfected in 0.5% sodium hypochlorite for 60 seconds, rinsed with sterile distilled water, placed in a sterile 1.7 ml microcentrifuge tube filled with 200 µl sterile distilled water (SDW), and macerated with a pellet pestle. After 5 minutes, the suspension was streaked onto Luria-Bertani (LB) agar and incubated at 28 o C for 48 h. Nearly pure cultures of cream-white convex bacterial colonies were isolated from diseased tissue and single colonies were selected and purified on fresh LB. Bacterial genomic DNA was extracted using the NucleoSpin Plant II mini kit (Macherey-Nagel, Allentown, PA) using the manufacturer’s specifications. A partial 16S rRNA gene product (1230 bp, accession no. PX861301) was produced using the primer set Y1/Y3 (Cruz et al. 2001) and used to query GenBank, which yielded a 99.84% (1228/1230) identical match to the type strain of Pectobacterium brasiliense (NR_118224). Bacterial identity was further confirmed using the Pectobacterium-specific primers Pec.dnaA-F1 and Pec.dnaA-R1 of DnaA (Dobhal et al. 2020). Accession no. PV593709 (859 bp) was compared in GenBank and was found 99.53% (855/859) identical to P. brasiliense FP220416 (OR739079). To prove pathogenicity, strain P22-98 was grown on LB for 48 h at 28 o C, suspended in sterile water at 1-2 x 10 6 CFU/ml, and injected into six mature avocado fruits using a sterile 31G BD insulin syringe. Fruits injected with sterile water using the same methods served as negative controls. Immediately after injection the sites were covered with autoclave tape and any excess inoculum was removed with 80% ethanol and lab tissues. The inoculated fruits were stored in clear plastic bins and kept at ambient laboratory temperature. The test was conducted twice. Within four days the skin and flesh of the sites injected with P. brasiliense became discolored, water-soaked, soft and sunken with a foul, musky odor, identical to the original symptoms observed. The avocado skin and flesh of the fruits inoculated with SDW remained firm and odor-free. Bacterial colonies were consistently re-isolated from the bacterial-inoculated fruits and morphologically (LB) and molecularly (16s rRNA, Pec.dnaA) identified as P. brasiliense, thus fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of postharvest bacterial soft rot of avocado caused by P. brasiliense in Hawai‘i. The host range of P. brasiliense in Hawai‘i (potato, kale, mizuna, and pak choi) (Arizala et al. 2020; Boluk et al. 2020; Klair et al. 2021a,b) continues to expand since its first discovery in 2019. Additional research is needed to determine if this disease poses a threat to the local avocado industry.