First report of Pectobacterium brasiliense causing banana soft rot in Ecuador

Abstract

Banana (Musa spp.) is the most economically important crop in Ecuador, with exports representing 35% of the agricultural GDP of the country. It covers 230,000 hectares, mostly concentrated in three coastal provinces, Guayas, Los Ríos, and El Oro. Between July and September 2022, disease symptomatic banana cv. Williams plants were observed in commercial plantations located in two parishes in the province of Guayas (Naranjito and Lorenzo de Garaicoa) and one parish in the province of Santo Domingo de los Tsáchilas (La Concordia), with an incidence that ranged from 5% to 15%. Symptoms included soft rot of the pseudostem and rhizome decay, characterized by a fetid odor. Three symptomatic pseudostems from each location were collected, washed with running water to remove any debris, and dried with absorbent paper. From the lesion of each pseudostem, seven pieces of 2 cm² were taken, surface-sterilized, and macerated in 9 ml of sterile peptone water (0.1% w/v). The macerate was diluted three fold in sterile water, plated on nutrient agar, and incubated at 30°C for 24 h. Eight randomly picked colonies, with convex elevation and creamy white color, were isolated on nutrient agar. Each of the bacterial isolates was biochemically profiled by the Biolog system (Biolog Inc., USA) and identified as Pectobacterium. Three isolates, one from each parish (FP220416, FP220694, and FP220904), were selected for testing Koch's postulates and further identification. Sequences from fragments of the 16S, dnaA, gapA and gyrB genes were obtained from these isolates, following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), showing 98.1-99.0%, 98.2%, 99.7-99.8%, and 98.4-98.9% identitity, respectively, with sequences from the P. brasiliense type strain LMG_21371 (Acc. number JQOE00000000). The obtained sequences were deposited in GenBank with the following accession numbers: OR392417, OR371545,OR371546, OR727281, OR727282, and OR739074-OR739080. Using BEAST v.1.10.4 (Suchard et al.,2018), a bayesian multilocus phylogenetic tree was built with multiple sequence alignments of dnaA, gapA, ang gyrB from 22 P. brasiliense isolates and 2 P. aquaticum isolates used as outgroup. The phylogenetic analysis showed that the Ecuadorian isolates cluster with P. brasiliense BF20, isolated from Opuntia ficus-indica in México and are closely related with the type strain. Pathogenicity tests were conducted through syringe infiltration with 1 ml of 1 × 10^8 CFU ml-1 bacterial suspensions. Each of the three characterized isolates were inoculated into the pseudostems of five healthy 4-month-old banana plants of the Williams cultivar. Negative control plants were infiltrated with sterile distilled water. The plants were incubated at 25°C and 74% relative humidity. Black lesions started to appear 11 days after inoculation and 5 weeks after inoculation plants showed clear symptoms of soft rot of the pseudostem, including fetid odor associated with plant tissue decomposition. Control plants remained symptom-free. Bacteria were re-isolated only from symptomatic pseudostems and identified as P. brasiliense with specific primers Pb1F and Pb1R. Soft rot of banana caused by different enterobacteria including Dickeya zeae, Erwinia carotovora, and Erwinia chrysanthemi hasve been previously reported (Jingxin et al. 2022, Arun et al. 2012, Loganathan, et al. 2019). This is the first report of P. brasiliense causing soft rot of banana in Ecuador, the biggest exporter of the fruit in the world.