RESUMO
Bacterial spot caused by Xanthomonas phaseoli pv. manihotis (Xpm) is considered the main bacterial disease that affects cassava, causing significant losses when not properly managed. In the present study, a fast, sensitive, and easy-to-apply method to detect Xpm via colorimetric loop-mediated isothermal amplification (LAMP) was developed. In order to ensure the use of a unique to the target pathovar core region for primer design, 74 complete genomic sequences of Xpm together with different bacterial species and pathovars were used for comparative genomics. A total of 42 unique genes were used to design 27 LAMP primer sets, from which nine primers were synthesized and only one (Xpm_Lp1 primer set) showed sufficient efficiency in preliminary tests. The sensitivity, assessed by a serial dilution of the type strain (IBSBF 278) DNA, yielded high sensitivity, detecting up to 100 fg. The LAMP primers showed high specificity, not cross-reacting with other bacterial species or other pathovars tested, and amplifying only the Xpm isolates. Tests confirmed the high efficiency of the protocol using infected or inoculated macerated cassava leaves, without the need for additional sample treatment. The LAMP test developed in this study was able to detect Xpm in a fast, simple, and sensitive way, and it can be used to monitor the disease under laboratory and field conditions.
RESUMO
Banana (Musa spp.) is the second most-consumed fruit in Brazil, the fourth-largest producer globally, with 7 million tons in 2021 (IBGE 2021). Studies about the morphological and pathogenic characteristics revealed that the etiology of Fusarium wilt in banana cultivars in Brazil had been related to the Fusarium oxysporum f. sp. cubense (Foc) (E.F. Smith) Snyder and Hansen species (Costa et al. 2015; Cordeiro et al. 2016; Araújo et al. 2017). Phylogenetic studies have shown the existence of distinct genetic lineages for Foc, which has come to be called the Fusarium oxysporum Species Complex (FOSC) (O'Donnell et al. 1998; Maryani et al. 2019). Symptoms of Fusarium wilt were observed in banana trees at the headquarters of Embrapa Roraima (02°45'26.89"N and 60°43'52.78"W), Roraima-Brazil, in 2016. Samples were collected and sterilized with 70% ethanol for 30 s, followed by 3% NaClO for 1 min, rinsed three times in sterile distilled water, seeded on potato dextrose agar (PDA), and incubated at 25 °C for three days. Two isolates obtained from a pure culture (LPPC130) were submitted to the morphological characterization by Leslie and Summerell (2006) protocol. The fungal colony showed vinaceous color, progressing to livid red (Rayner 1970), with a mean diameter of 41 mm (± 0.1) at three days of incubation in a PDA culture medium. The fungus produced abundant macroconidia in spezieller nährstoffarmer agar (SNA) culture medium containing clove leaf (CLA) after 14 days of incubation at 25 °C. The sporodochium conidia presented a falcate shape, moderately curved, with 3 to 5 septa and dimensions ranging from 38.8 (48.0) 56.2 x 3.5 (4.4) 6.0 µm (n=50). The conidia of the aerial mycelium presented ovoid to ellipsoid shape, slightly curved, aseptic, measuring 6.0 (12.0) 18.0 x 2.8 (3.3) 5.0 µm (n=50). The genomic DNA of the isolate was extracted (Murray and Thompson 1980), and fragments of the elongation factor 1-α (TEF1) and RNA Polymerase II (RPB2) gene regions were amplified and sequenced in both directions (O'Donnell et al. 1998; O'Donnell et al. 2010) (GenBank accession numbers: Seq1 OL802918 and Seq2 OL802919). Multiple alignments of the combined dataset of the isolates and representative sequences obtained from GenBank were submitted to phylogenetic analysis with 1,000 bootstrap replicates. The micromorphological characteristics together to phylogenetic inference on the TEF1 and RPB2 genes, allowed a robust analysis, generating 42 more parsimonious trees and making it possible to identify the LPPC130 isolate as Fusarium kalimantanense, a species belonging to the F. oxysporum species complex (FOSC), with 100% bootstrap support (Maryani et al. 2019). The pathogenicity of the isolate was evaluated in five micropropagated seedlings of banana cv. Silk 75 days old, grown in pots with 5 kg of sterile formulation of sand and soil, in 1:1. Seedlings were inoculated by wounding the roots and depositing a suspension of conidia and chlamydospores at 105 spores mL-1. The inoculating of the isolate in 35 micropropagated seedlings of banana was based on Koch's postulates. The seedlings were transplanted into plastic bags (2 kg of sterile formulation: sandy soil and substrate, in 2:1) and inoculated with 10 mL of the chlamydospore suspension (107 CFU mL-1) at transplanting, and after 30 days of transplanting. Seedlings treated only with water were used as control. Evaluation of the symptoms of the disease was carried out 90 days after inoculation, and revealed that the isolate (LPPC130) was pathogenic by inducing the same symptoms of Fusarium wilt. F. kalimantanense was first reported associated with the pseudostems of Musa acuminata var. Pisang Ambon, and proved to be non-pathogenic to cv. Gros Michel and the bananas of the Cavendish group (Maryani et al. 2019). In Brazil, this fungus was recently associated with the Fusarium rot on melon fruits (Araújo et al. 2021); however, this is the first report of its pathogenicity in banana trees cv. Silk.
