First report of Colletotrichum plurivorum causing anthracnose on Cucumis sativus in Brazil.

Cucumbers have great economic and social importance. Annual worldwide production is approximately 80 million tons (FAOSTAT, 2019), 184 thousand tons of which are produced in Brazil (IBGE, 2020). Leaves with symptoms of anthracnose (necrotic brown or angular spots) were observed on cucumber plants grown in organic systems in September 2021, Pernambuco, Brazil (8°7'45''S, 35°16'167''W). About 40% of the plants fields were infected. Samples were collected and fragments were cut from the margins of the symptomatic tissue. The fragments were superficially disinfected with 70% ethanol (30 s) and 2% sodium hypochlorite (2 min), then washed three times with sterile distilled H2O and dried on sterile filter paper. The fragments were placed on potato dextrose agar (PDA) containing chloramphenicol (50 mg/L) and incubated at 28 ± 2 °C for 3 days. From the fungal isolates obtained, a representative specimen of Colletotrichum spp. was isolated, purified by subculturing from emergent hyphae tips and used for morphological characterization, phylogenetic analysis, and pathogenicity testing. The fungus isolated on PDA formed gray to grayish-black colonies with white aerial mycelia after 7 days. Ascomata were globose to subglobose, 120-200 × 100-150 µm in size (n = 10). Setae formed directly on the hyphae. Asci were 50-70 × 10-12 µm in size, 8-spored, unitunicate, thin-walled, and clavate. Ascospores were 14-22 × 4-5 µm in size (n = 30), hyaline, slightly curved to curved with obtuse to slightly rounded ends. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, the apex and base rounded, and 12-15 × 5 µm in size, (n = 30). For molecular identification, the nuclear ribosomal internal transcribed spacers (nrITS), actin (ACT), beta-tubulin (TUB), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were sequenced (Damm et al. 2019). The sequences obtained were deposited in GenBank (nrITS: OP720945, ACT: OP723523, TUB: OP723525, and GAPDH: OP723524). The sequences from the nrITS region, ACT, TUB2, and GAPDH were highly similar to those from C. plurivorum: nrITS - CBS 125474 (539/539 - 100%; NR_160828); ACT - CBS 125474 (270/271 - 99%; MG600925), TUB2 - CBS 125474 (517/518 - 99%; MG600985); and GAPDH - CBS 125474 (197/197 - 100%; MG600781), respectively. Multilocus phylogenetic analysis was performed using Bayesian inference, which showed that the isolate C. plurivorum FPO04 clustered in the same clade as the ex-type of C. plurivorum (CBS 125474). In the pathogenicity test, leaves of five healthy cucumber plants, previously injured in the middle region with sterile needles, were inoculated with 50 µl of a conidial suspension (1 × 106 spores mL -1) prepared from 7-day-old of colonies of C. plurivorum. Sterile distilled water was used as negative controls. The inoculated plants were maintained in a humid greenhouse chamber for 24 hours. After 7 days, the same anthracnose symptoms seen in the field were observed on the inoculated plants. Control plants remained healthy. Colletotrichum plurivorum was reisolated from symptomatic leaves, fulfilling Koch's postulates. This species has been reported from several crops, including Abelmoschus esculentus (okra) (Damm et al. 2019) and Glycine max (soybeans) (Zaw et al. 2019). To our knowledge, this is the first report of C. plurivorum causing anthracnose on cucumber leaves in Brazil. This report lays the groundwork for future studies to determine management practices for control of this disease in C. sativus.

Cassava (Manihot esculenta) defensins: Prospection, structural analysis and tissue-specific expression under biotic/abiotic stresses.

Defensins are a prominent family of antimicrobial peptides. They play sophisticated roles in the defense against pathogens in all living organisms, but few works address their expression under different conditions and plant tissues. The present work prospected defensins of Manihot esculenta Crantz, popularly known as cassava. Five defensin candidates (MeDefs) were retrieved from the genome sequences and characterized. Considering chromosome distribution, only MeDef1 and 2 occupy adjacent positions in the same chromosome arm. All 3D structures had antiparallel ß-sheets, an α-helix, and amphipathic residues distributed throughout the peptides with a predominance of cationic surface charge. MeDefs expression was validated by RT-qPCR, including two stress types (biotic: fungus Macrophomina pseudophaseolina, and abiotic: mechanical injury) and a combination of both stresses (fungus+injury) in three different tissues (root, stem, and leaf). For this purpose, ten reference genes (RGs) were tested, and three were chosen to characterize MeDef expression. MeDef3 was up-regulated at roots in all stress situations tested. MeDef1 and MeDef5 were induced in leaves under biotic and abiotic stresses, but not in both stress types simultaneously. Only MeDef2 was down-regulated in the stem tissue also with biotic/abiotic combined stresses. These results indicate that although defensins are known to be responsive to pathogen infection, they may act as preformed defense or, still, have tissue or stress specificities. Aspects of their structure, stability and evolution are also discussed.

Phylogeny, Identification, and Pathogenicity of Lasiodiplodia Associated with Postharvest Stem-End Rot of Coconut in Brazil.

Coconut palm (Cocos nucifera L.) is one of the most important perennial tropical crops. Stem-end rot is the major postharvest disease of coconut in Brazil. The fungus Lasiodiplodia theobromae is the only species that has been reported to be associated with this disease. However, a comprehensive study elucidating the true identity of this pathogen with molecular tools has never been conducted. In recent years, new species of Lasiodiplodia have been proposed after molecular studies were performed, indicating the existence of a species complex. The aims of this research were to study the etiology of the postharvest stem-end rot of immature coconut based on a combination of morphological and phylogenetic analyses, to establish the phylogenetic position of such taxa, and to assess the pathogenicity of each taxon. Four species were identified: L. brasiliense, L. egyptiacae, L. pseudotheobromae, and L. theobromae. All of the species were distinguished morphologically and phylogenetically and were proven to be pathogenic to coconut following artificial inoculation. L. theobromae was the most common and the most aggressive species. This study represents the first report of three additional species of Lasiodiplodia as causal agents of postharvest stem-end rot of immature coconut in Brazil.