Bisifusarium lunatum causing cladode soft rot in pear cactus (Nopalea cochenillifera L. Salm-Dyck) in Brazil.

Cactus pear var. miúda (Nopalea cochenillifera L. Salm-Dyck) is an important crop for the Northeast region of Brazil, composing one of the main sources of animal feed. By April 2021, cladode rot caused death of several cactus pear plants in a production area located in Itaporanga, Paraíba state, Brazil (7°21'55.35" S and 38°11'38.68" W). The infected cladodes showed brown circular necrotic spots, and soft rot with perforations that extended throughout the cladode, followed by tipping over and death of the infected plants. The incidence of the disease ranged from 10 to 30% of the plants. Bisifusarium strains were isolated and cultured on potato dextrose agar (PDA) and syntetic-nutrient-poor-agar (SNA). The colonies showed purple color on PDA. On SNA, macroconidia (n = 100) were abundant, hyaline, slightly falcate, three-septate, measuring 11.0-23.1 x 2.3-4.1 µm. Microconidia (n = 100) were oval, generally aseptate, measuring 4.1-8.7 x 2.3-3.0 µm. Conidiogenic cells formed into short monophialides. Chlamydospores were not observed. According to these morphological features, the pathogen was initially identified as Bisifusarium lunatum (Gryzenhoutm et al. 2017). For further confirmation of the identification, the partial sequences of translation elongation factor 1-alpha (TEF1-α) and the second largest subunit of RNA polymerase II (RPB2) genes were sequenced for a representative isolate (CMA 34: GenBank accession no: TEF1-α: OR536502; and RPB2: OR553509) and compared to other Bisifusarium species from GenBank database. Subsequently, it was subjected to a phylogenetic analysis of maximum likelihood including previously published sequences. According to BLAST searches, the TEF1-α and RPB2 sequences were 99% (637/640 nt) and 100% (312/312 nt) similar to B. lunatum (COUFAL0213: TEF1-α (MK640219), and RPB2 (MK301291)), respectively. The isolate was also clustered in a clade containing the ex-type of B. lunatum with 100% support (SH-aLRT and UFboot), being confidently assigned to this species. The pathogenicity test was performed after Medeiros et al. (2015), by using healthy two months old cactus pear seedlings (n = 10) cultivated in a greenhouse. Sterile toothpicks were distributed over colonies of the representative isolate grown on PDA at 25 ± 2 °C for seven days. Seedling cladodes were stuck with the toothpicks, moistened with sterile water and covered with transparent plastic bags for 24h, thus simulating a humid chamber. Following three months, all control plants (stuck with sterile toothpicks) remained healthy, while those inoculated with the representative isolate exhibited rot symptoms. This test was performed twice. B. lunatum was reisolated from symptomatic cladodes and identified as previously described, thus fulfilling the Koch's postulates. To our best knowledge, this is the first report of B. lunatum causing soft rot on N. cochenillifera in Brazil. Besides N. cochenillifera, this species was also reported on Opuntia ficus-indica in India (Gryzenhoutm et al., 2017), which raises concern regarding its ability to infect other forage sources for cattle feed in Brazilian semiarid regions. The present study highlights that the precise identification of B. lunatum is a key factor to adjust control strategies and management of the disease to prevent the spread of this disease to prevent its spread to other crops.

Fusarium pseudocircinatum causing stunting and malformation of sunflower plants in Brazil.

Sunflower (Helianthus annuus L.) is among the main oleaginous crops used in Brazil. During January, 2017, at CCA/UFPB laboratory and greenhouses (Areia/Brazil, 6°58'12″ S; 35°42'15″ W), we observed various sunflower seeds (cultivar Olisun 3, 2017-2018 crop) highly infested with Fusarium. Those seeds were from crops in the municipality of Alagoinha -PB/Brazil (06º57'00'' S; 35º32'42'' W), supplied by Empresa Brasileira de Pesquisa Agropecuária/EMBRAPA. The emerged seedlings from these seeds were also contaminated, with 5% to 26% of them exhibiting stunting and malformation. Fusarium strains were isolated from symptomatic plants, and a single spore was used to grow pure colonies on potato-dextrose-agar (PDA) and synthetic-nutrient-poor-agar (SNA) media. Mycelia of PDA colonies were floccous and dense varying from yellow to orange. Fungal colonies developed aerial mycelium, producing orange pigments. On SNA, hyaline macroconidia, measuring 2.9-4.1 x 32.4-65.0 µm, slightly falcate with three to six septa. Oval microconidia, measuring 2.4-3.6 x 5.1-9.0 µm, were abundant in false heads forming on monophyalides. Chlamydospores were absent. Sterile hyphae were rarely formed. Colectively, the morphological features corresponded to species that belong to the Fusarium fujikuroi species complex (Leslie & Summerell, 2006). To assure the species identity, we sequenced the elongation factor 1α region of two representative isolates (i.e., F2 and F3, GenBank access numbers: MZ666934 and MZ666935, respectively) and compared them to the other Fusarium species found at Fusarium-ID and GenBank databases. Subsequently, we performed a maximum likelihood phylogenetic analysis including previously published sequences (Nicolli et al., 2020). Both isolates exhibited 100% similarity with Fusarium pseudocircinatum (MN386745), and clustered with its ex-type at 100% bootstrap values. The isolates were then grown on PDA amended with manitol to adjust the osmotic pressure to -1.0 Mpa, at 25 ± 2 ° C, for seven days (Sousa et al., 2008). A total of 100 disinfested sunflower seeds (cultivar Olisun 3, 2018-2019 crop) were distributed over the colonies and 48h later they were sown on sterile substrate maintained inside a greenhouse. About 30 days after inoculation, the emerged plants exhibited symptoms of stunting and malformation (60%) compared to controls, which were healthy. F. pseudocircinatum was reisolated from the symptomatic plants, completing Koch's postulates and identified based on above morphological and molecular biological methods. This test was performed twice. Fusarium pseudocircinatum is a broadly distributed and ecologicaly diverse species that infects several wild and cultivated plants. For instance, it was reported on seeds of the wild 'Peroba Rosa' (Aspidosperma polyneuron Muell. Arg.) in Brazil (Mazarotto et al. 2020). Infection of sunflowers may cause plant stand failures, thus resulting in yield and economic losses for Brazilian growers. The correct identification of any pathogen, especialy a generalist one such as F. pseudocircinatum, is crucial to develop eficient management strategies. To our best knowledge, this is the first report of F. pseudocircinatum causing stunting and malformation of sunflower plants in Brazil.

Fungal endophytic communities associated to the phyllosphere of grapevine cultivars under different types of management.

Fungal endophytes present in different asymptomatic grapevine plants (Vitis vinifera L.) located in different vineyards within Alentejo, a highly important viticulture region in Portugal, were identified in this study. Sampled grapevine plants included the three most representative cultivars in the region, Syrah, Cabernet Sauvignon, and Aragonez, growing under two different modes of management, conventional and biological. Sixteen fungal taxa were identified through sequencing of the internal transcribed spacer region. Total number of endophytic fungi isolated showed significant differences both in management mode and in cultivars, with higher numbers in grapevines under conventional mode and from Syrah cultivar. The composition of fungal endophytic communities did not show significant differences among cultivars, but differences were observed between fungal communities isolated from grapevines under biological or conventional modes. The most fungal taxa isolated from grapevines cultivated under biological mode were Alternaria alternata, Cladosporium sp., and Nigrospora oryzae, and under conventional mode Botrytis cinerea, Epicoccum nigrum, and Epicoccum sp. These differences suggest that the different products used in grapevine production have impacts in fungal endophytic composition. Further investigation of the identified fungi with respect to their antagonistic characteristics and potential use in plant protection to ensure food safety is now in course.