Unraveling the antifungal composition of bitter orange decoction against the melon pathogen Fusarium jinanense.

Bitter orange (Citrus aurantium) is an important source of essential oils with high antimicrobial activities, however the composition and antifungal potential of the decoction peels is little explored. This study assessed the peel decoction's chemical profile at the secondary metabolism level and its antifungal activity against the melon phytopathogen Fusarium jinanense. The decoction's antifungal potential was investigated using a bioassay-guided fractionation approach based on Solid-Phase Extraction (SPE) and LC-HRMS/MS analysis. Coumarins and flavones were the most abundant classes of compounds in the high-value fractions responsible for up to 61% of the mycelial inhibition of F. jinanense. Overall, this study has presented for the first time the chemical composition, the antifungal potential of the decoction of C. aurantium peels and the compounds associated with these results. This strategy can guide the exploration of under-explored food sources and add value to compounds or fractions enriched with bioactive compounds.

First Report of Colletotrichum tropicale Causing Anthracnose on Pitaya (Hylocereus costaricensis) in Brazil.

Pitaya, Hylocereus costaricensis, is a species of the Cactaceae family and originated in the Americas (Ortiz & Livera, 1995). It has been cultivated in Brazil and has shown a great potential for fruit production and is currently present in several markets (Faleiro et al. 2021). In July 2018, infected plants of pitaya with symptoms of anthracnose were obtained from an orchard in Fortaleza, Ceará Brazil, (3°44'24.5"S 38°34'30.8"W), with 50% disease incidence. The symptoms observed consisted of well-defined and depressed stains, that initially appeared as reddish-orange spots and were surrounded by a border of dark-brown color. As the lesion progressed, the center became light brown or whitish in color, with black dots appearing later. Four cladodes were collected with anthracnose symptoms. The pathogen was isolated from symptomatic cladodes, which were surface disinfected with 1% v/v NaClO and 70% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA) medium and incubated under a light/dark (12h/12h) photoperiod. Two isolates were recovered from the lesions on cladodes. Pure cultures were obtained from single conidia produced on colonies grown on PDA medium, using an inoculation needle under a microscope. Colonies on PDA exhibited white aerial mycelia with an orange conidial mass. The colonies were light grey in the front and light orange in the reverse of the plate. Morphological features suggested that the isolates had the same characteristics as previously described for Colletotrichum spp. (Weir et al., 2012). In order to identify the species of the isolates, the genomic DNA of UFCM 0684 and UFCM 0685 isolates was extracted using the CTAB method and the ITS region, TUB2, ACT, GS, GAPDH gene fragments were amplified. PCR products were sequenced and the resulting sequences were submitted to phylogenetic analyses based on maximum likelihood for the combination of the genes. The isolates grouped within Colletotrichum tropicale with 99% bootstrap support. The sequences obtained in this study were deposited in GenBank as ACT (accession no. OL799311, OL799312), TUB2 (OL799313; OL799314), GAPDH (OL799315, OL799316), GS (OL799317; OL799318) and ITS (OL799319; OL799320). After that, the UFCM 0685 isolate was selected to study for further characterization. Conidia (n = 50) were 13.7 (length) × 4.7 µm (width) in average, hyaline, aseptate and cylindrical. To complete Koch's postulates, pathogenicity tests were performed in moist chamber for one week at 25°C with 80% relative humidity on a 12 h fluorescent light/dark photoperiod. The cladodes were wounded using a sterilized needle and inoculated with 10 µl of a conidial suspension (1 × 106 conidia/ml) on three cladodes with five wounds each. The same number of uninoculated cladode was used as control. The experiment was performed twice. Two weeks later, all inoculated cladodes showed necrotic symptoms, which were similar to the symptoms previously observed in the field. The uninoculated cladode remained symptomless. The fungus was reisolated from the inoculated cladode and its morphological characteristics were similar to the original isolate. Colletotrichum tropicale has been reported to cause anthracnose on H. costaricensis in Mexico (Nunez-Garcia et al. 2023), H. undatus, H. monocanthus and H. megalanthus (Evallo et al. 2022). For the best of our knowledge, this is the first report of anthracnose caused by C. tropicale in H. costaricensis in Brazil.

First report of stem gray blight on Hylocereus megalanthus and Hylocereus polyrhizus caused by Diaporthe arecae in Brazil.

In November 2021, stem gray blight symptoms were seen on two dragon fruit (pitaya) species (Hylocereus megalanthus and H. polyrhizus) in an orchard with 100% disease incidence in Fortaleza, Ceará, Brazil (3°44'24.5"S 38°34'30.8"W). The symptoms were initially yellowish to dark brown lesions, and as the symptoms progressed, the lesions turned grayish with small black pycnidia in the center. Isolation was carried out by disinfecting small pieces of the symptomatic stems in 70% ethanol for 1 min, followed by 1% NaOCl for 1 min, and then rinsed three times with sterile distilled water. Excess water was removed using sterile filter paper. Then the stem fragments were placed on PDA media. Colonies produced small black pycnidia with conidia and some were sterile after 68 days of incubation. Two monosporic isolates were obtained from the colonies: UFCM 0708 from H. megalanthus and the UFCM 0710 from H. polyrhizus, which were used for pathogenicity test, morphological and molecular identification. The colony on PDA was smoke gray with aerial mycelium and the reverse was smoke grey to dark grey. The α-conidia from UFCM 0708 and UFCM 0710 were hyaline, aseptate and fusiform and measured 6.4 to 9.7 (8.0) x 1.2 to 2.4 (1.7) µm and 6 to 13.1 (8.2) x 1.7 to 2.4 (2.0) µm, respectively. The ß-conidia from UFCM 0708 and UFCM 0710 were hyaline, aseptate and filiform and measured 15 to 22.5 (18.8) x 0.6 to 1.7 (1.0) µm, and 17.2 to 27.5 (22.3) x 0.5 to 1.0 (0.8) µm (n=30), respectively. This morphology placed the isolates as Diaporthe sp. (Udayanga et al. 2012). For further confirmation, genomic DNA was extracted from the isolates (UFCM 0708 and UFCM 0710), and beta-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1) gene fragments were amplified. BLASTn search results with isolates TEF1 and TUB2 sequences varied from 98.58% to 99.52% identity to the ex-type sequence of Diaporthe arecae (CBS 161.64). Phylogenetic analysis of concatenated sequences alignment carried out using the Maxinum-likelihood and Bayesian Inference analysis placed the isolates within D. arecae clade with 86% bootstrap and 0.99 posterior probabilities support. The sequences obtained in this study were deposited in GenBank (TEF1: OP534720 and OP534722; TUB2: OP534717 and OP534719). The isolates were confirmed as D. arecae based on molecular analysis and morphological characteristics (Gomes et al. 2013). Koch's postulates were completed as described by Karim et al. (2019) through the inoculation of six stems of each dragon fruit (pitaya) species. The stems were wounded by removing a 5 mm diameter disc and after that they were inoculated with a 5 mm diameter mycelial plug from 5 days old PDA plates. PDA plugs were used as control. Each stem was covered with a plastic bag and sterilized water was added into the sterilized filter paper to maintain humidity. The bags were kept in a room at day and night temperature of 25 ± 2 °C. The same symptoms seen in the field appeared on the stems 21 days after inoculation. The control stems remained symptomless. Diaporthe arecae have been reported on H. polyrhizus in Malaysia (Huda-Shakirah et al. 2021). To our knowledge, this is the first report of D. arecae on H. megalanthus and H. polyrhizus in Brazil.

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.