First report of Lasiodiplodia iraniensis causing crown rot on banana fruits in Brazil.

Banana is a fruit of great importance in Brazil and crown rot cause considerable damage and losses (Ploetz et al. 2003). The disease is associated with fungal complexes, especially the Lasiodiplodia theobromae sensu lato (Kamel et al. 2016; Renganathan et al. 2020; Waliullah et al. 2022). Three asymptomatic bunches of banana cv. 'Prata Catarina' were collected in Russas, Brazil (04°58'11.6"S, 38°01'44.5"W), in 2017. The samples were disinfected (NaClO, 200 ppm), and incubated in a moist chamber at 28 °C, with 12 h light/12 h dark for 3 days. With the appearance of the symptoms (32% of severity), the isolation was conducted in potato dextrose agar (PDA). A monosporic culture (BAN14) was obtained from a typical crown rot lesion, which was subjected to morphological characterization, showing abundant aerial mycelium of olivaceous grey color on the surface and greenish grey on the back (Rayner 1970) in PDA after 15 days at 28 °C. The growth rate was 28.2 mm. day-1. The fungus produced pycnidia and conidia on water agar medium containing pine needles, with 3-4 weeks at 28 °C, presenting conidia initially aseptate, subglobose to subcylindrical, becoming pigmented with 1-central transverse septum and longitudinal striations 23.5 (18.7) 26.0 x 12.7 (9.7) 14.8 µm (n=50). Paraphyses, hyaline, cylindrical, thin-walled, apparently coenocytic with rounded apex, with length and width dimensions of 34 (43.8) 53.2 x 2.1 (2.5) 3.2 µm (n=30). Conidiophore absent, conidiogenous cells hyaline, smooth and with thin walls. The genomic DNA was extracted and amplified by PCR with primers TEF1-688F/TEF1-1251R, ITS1/ITS4, and Bt2a/Bt2b, and sequenced in both directions (O'Donnell et al. 1998; O'Donnell et al. 2010) (GenBank accession ON975017 [TEF1], ON986403 [TUB2], and ON921398 [ITS]). BLASTn analysis of TEF1, TUB2 and ITS sequences in NCBI database showed 99 to 100% nucleotide identity to a representative isolate of Lasiodiplodia iraniensis (IRAN921). Phylogenetic analysis using maximum parsimony based on the combined TEF1, TUB2 and ITS sequences indicated that the BAN14 formed a supported clade (82% bootstrap value) to L. iraniensis. The pathogenicity was evaluated in 20 banana fruit cv. 'Prata Catarina', at the point of harvest. For inoculation, the bananas were washed with water and soap, and disinfected with NaClO (200 ppm). Posteriorly, two wounds were made on the extremities of the fruits, in which were deposited mycelial discs of 5 mm in diameter, with 7 days of the growth on PDA. After inoculation, the fruits were incubated in plastic boxes in a wet chamber at 25 °C, with 12 h light/12 h dark for 5 days. The control fruits were not inoculated with the pathogen, only with PDA discs. The experiments were repeat twice. The BAN14 isolate was pathogenic to the banana cv. 'Prata Catarina'. The BAN14 was grouped with the species L. iraniensis described by Abdollahzadeh et al. (2010) in Iran. This species is distributed in Asia, South and North America, Australia, and Africa. In Brazil it was reported in association to Anacardium occidentale, Annona muricata, A. squamosa, Annona ×cherimola-squamosa, Citrus sp., Eucalyptus sp., Jatropha curcas, Mangifera indica, Manihot esculenta, Nopalea cochenillifera, Vitis sp. and V. vinifera. Until the moment, there is not description of the relation between banana crown rot and L. iraniensis (Farr and Rossman 2022). Our work is the first report on the pathogenicity of this species on banana fruit cv. 'Prata Catarina' worldwide.

First report of Lasiodiplodia brasiliensis causing crown rot on banana in Brazil.

Postharvest diseases compromise banana quality and cause high economic losses in Brazil. Among them, the crown rot prevails and its causal agents belong to distinct fungal species such as Colletotrichum musae (Berk. & Curt.) von Arx, Fusarium spp., and Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (Griffee and Burden 1976; Ploetz et al. 2003). Symptoms of crown rot were observed on banana fruits of cv. Williams in a commercial area in Assu, Rio Grande do Norte, Brazil (04°54'0.06"S, 37°22'6.02"W) in 2017. The samples were collected, superficially disinfected with NaClO (2%), and incubated in a wet chamber at 25 °C, with a 12 h photoperiod, for approximately 3 days. After the appearance of disease symptoms and pathogen signs, mycelia were transferred from the lesions to obtain pure cultures on a potato dextrose agar (PDA) medium. Thus, a monosporic culture was obtained (isolate BAN82). The fungus produced pycnidia with conidia on potato carrot agar (PCA) culture medium containing pine needles, after four weeks of incubation at 28 °C. The conidia were hyaline when immature and brown with central transverse septum when mature. The presence of conidiogenous cells, paraphyses, and conidiophores also were observed. The conidia present ovoid format measuring 20-28 x 11-14 µm (n=50). The fungal colony produced abundant aerial mycelia of mouse grey coloration, progressing to dark mouse grey (Rayner 1970), on PDA for 15 days to 28 °C. The growth rate was 29.3 mm/day on PDA. The genomic DNA was extracted and amplified PCR with primers TEF1-688F/TEF1-1251R, ITS1/ITS4, and Bt2a/Bt2b and sequenced in both directions. The TEF1 and TUB2 sequences showed 100%, and the ITS showed 93.06% identity with the sequences of Lasiodiplodia brasiliensis (GenBank accession numbers: ON623895, TEF1, ON623896, TUB2, and ON599012, ITS. Multiple alignments of the combined dataset of the isolate and representative sequences obtained from GenBank were submitted phylogenetic analyses to bayesian inference (IB) with posterior probabilities of 10,000,000 generations. The morphological characteristics together with multigenic analysis of the three genomic regions made it possible to identify the BAN82 isolate as Lasiodiplodia brasiliensis, showing bootstrap support of posterior probabilities of 0,98 in the IB analysis. The pathogenicity was evaluated on 16 banana fruits from cv. Prata Catarina, at the point of harvest. For inoculation, the bananas were disinfected with water, soap, and, NaClO (2%). Posteriorly, the fruits were wounded on both ends, followed by the deposition of 5mm diameter mycelial plugs from the fungal culture, within 7 days of the growth. After the inoculation, the fruits were incubated in plastic boxes in a wet chamber at 25 °C, with 12 h photoperiod, for 3 days. To complete Koch's postulates, the isolate was inoculated again into 16 other banana fruits from cv. Prata Catarina. The negative control fruits were not inoculated with the pathogen, only with PDA discs. The BAN82 isolate was pathogenic to the banana cv. Prata Catarina. In the Brazilian Northeast, L. brasiliensis was described in 2014 as being associated with papaya stem rot. Up to the moment, there are no reports of L. brasiliensis as the causal agent of crown rot on bananas from Brazil (Netto et al. 2014; Farr and Rossman 2022). Thus, our work is the first to report L. brasiliensis causing crown rot on banana fruits cv. Prata Catarina in Brazil.

First report of Fusarium kalimantanense causing Fusarium wilt on banana tree in Brazil.

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.