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 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.

Diversity of Colletotrichum species associated with torch ginger anthracnose.

Anthracnose caused by Colletotrichum species is one of the most important diseases of torch ginger. The disease leads to loss of aesthetic and commercial value of torch ginger stems. This study aimed to characterize Colletotrichum species associated with torch ginger anthracnose in the production areas of Pernambuco and Ceará. A total of 48 Colletotrichum isolates were identified using molecular techniques. Pathogenicity tests were performed on torch ginger with representative isolates. Phylogenetic analyses based on seven loci-DNA lyase (APN2), intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), calmodulin (CAL), intergenic spacer between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a hypothetical protein (GAP2-IGS), glutamine synthetase (GS), and ß-tubulin (TUB2)-revealed that they belong to five known Colletotrichum species, namely, C. chrysophilum, C. fructicola, C. siamense, C. theobromicola, and C. tropicale, and three newly discovered species, described here as C. atlanticum, C. floscerae, and C. zingibericola. Of these, C. atlanticum was the most dominant. Pathogenicity assays showed that all isolates were pathogenic to torch ginger bracts. All species are reported for the first time associated with torch ginger in Brazil. The present study contributes to the current understanding of the diversity of Colletotrichum species associated with anthracnose on torch ginger and demonstrates the importance of accurate species identification for effective disease management strategies.

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.

Trichoderma longibrachiatum as a biostimulant of micropropagated banana seedlings under acclimatization.

The use of growth-promoting microorganisms with biostimulant characteristics is an important biological asset for the acclimatization of micropropagated seedlings. The present study aimed to evaluate the efficacy of the application of Trichoderma spp. on the promotion of the growth of micropropagated banana seedlings during acclimatization. The experiment was performed in an 8 × 6 completely randomized design using the following treatments: water, seedlings fertilized with controlled-release fertilizer, commercial biological inputs (A: T. asperellum, B/C: T. harzianum), and LPPC299 and LPPC300 strains. Plant height, pseudostem diameter, number of leaves, total leaf area, root length, fresh and dry mass of the plant, and accumulation of sodium, macronutrients, and micronutrients were evaluated 60 days after inoculation. Strains LPPC299 and LPPC300 were subjected to molecular identification by DNA sequencing of the ITS/5.8S locus. In vitro detection of growth promotion-related mechanisms and mycelial growth of biostimulants were performed using scanning electron microscopy. LPPC299 and LPPC300 had a greater similarity to T. longibrachiatum. LPPC299 was able to promote greater pseudostem diameter, number of leaves, and total leaf area in banana seedlings. T. asperellum (A) favored seedling performance in terms of fresh and dry mass of the plants. The strains were able to produce siderophores, indoleacetic acid, and catalase in vitro. Seedlings inoculated with the strains accumulated Mn, S (LPPC300), and Mg (LPPC299). LPPC299 from the banana rhizosphere was efficient in promoting performance in banana seedlings, showing its potential as a biostimulant for this crop.