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First Report of Pectobacterium brasiliense Causing Banana Soft Rot in Ecuador
Journal
Plant Disease
ISSN
1943-7692
Date Issued
2024-02-01
Author(s)
A. Toaza
R. Caiza
F. J. Garrido
L. A. Ramos
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 ha, mostly concentrated in three coastal provinces, Guayas, Los Ríos, and El Oro.
Between July and September 2022, disease symptomatic banana cultivar 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 threefold 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, U.S.A.) 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 to 99.0%, 98.2%, 99.7 to 99.8%, and 98.4 to 98.9% identity, respectively, with sequences from the Pectobacterium brasiliense type strain LMG_21371 (accession no. JQOE00000000).
The obtained sequences were deposited in GenBank with the following accession numbers: OR392417, OR371545, OR371546, OR727281, OR727282, and OR739074 to OR739080. Using BEAST version 1.10.4 (Suchard et al. 2018), a Bayesian multilocus phylogenetic tree was built with multiple sequence alignments of dnaA, gapA, and gyrB from 22 P. brasiliense isolates and two 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 Mexico and are closely related with the type strain. Pathogenicity tests were conducted through syringe infiltration with 1 ml of 1 × 108 CFU/ml bacterial suspension. Each of the three characterized isolates were inoculated into the pseudostems of five healthy 4-month-old banana plants of the cultivar Williams. 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. The control plants remained symptom-free. Bacteria were reisolated only from symptomatic pseudostems and identified as P. brasiliense with the specific primers Pb1F and Pb1R.
Between July and September 2022, disease symptomatic banana cultivar 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 threefold 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, U.S.A.) 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 to 99.0%, 98.2%, 99.7 to 99.8%, and 98.4 to 98.9% identity, respectively, with sequences from the Pectobacterium brasiliense type strain LMG_21371 (accession no. JQOE00000000).
The obtained sequences were deposited in GenBank with the following accession numbers: OR392417, OR371545, OR371546, OR727281, OR727282, and OR739074 to OR739080. Using BEAST version 1.10.4 (Suchard et al. 2018), a Bayesian multilocus phylogenetic tree was built with multiple sequence alignments of dnaA, gapA, and gyrB from 22 P. brasiliense isolates and two 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 Mexico and are closely related with the type strain. Pathogenicity tests were conducted through syringe infiltration with 1 ml of 1 × 108 CFU/ml bacterial suspension. Each of the three characterized isolates were inoculated into the pseudostems of five healthy 4-month-old banana plants of the cultivar Williams. 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. The control plants remained symptom-free. Bacteria were reisolated only from symptomatic pseudostems and identified as P. brasiliense with the specific primers Pb1F and Pb1R.