ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Pulsed light, as a postharvest technology, is an alternative to traditional fungicides, and can be used on a wide variety of fruit and vegetables for sanitization or pathogen control. In addition to these applications, other effects also are detected in vegetal cells, including changes in metabolism and secondary metabolite production, which directly affect disease control response mechanisms. This study aimed to evaluate pulsed ultraviolet light in controlling postharvest rot, caused by Fusarium pallidoroseum in 'Spanish' melon, in natura, and its implications in disease control as a function of metabolomic variation to fungicidal or fungistatic effects. The dose of pulsed light (PL) that inhibited F. pallidoroseum growth in melons (Cucumis melo var. Spanish) was 9 KJ m-2. Ultra-performance liquid chromatography (UPLC) coupled to a quadrupole-time-of-flight (QTOF) mass analyzer identified 12 compounds based on tandem mass spectrometry (MS/MS) fragmentation patterns. Chemometric analysis by Principal Components Analysis (PCA) and Orthogonal Partial Least Squared Discriminant Analysis (OPLS-DA) and corresponding S-Plot were used to evaluate the changes in fruit metabolism. PL technology provided protection against postharvest disease in melons, directly inhibiting the growth of F. pallidoroseum through the upregulation of specific fruit biomarkers such as pipecolic acid (11), saponarin (7), and orientin (3), which acted as major markers for the defense system against pathogens. PL can thus be proposed as a postharvest technology to prevent chemical fungicides and may be applied to reduce the decay of melon quality during its export and storage.
