First report of Colletotrichum tropical causing leaf spot on Cariniana legalis in Brazil.

Cariniana legalis (Mart.) Kuntze (Lecythidaceae), commonly known as "Jequitibá Rosa," an endemic tree from Brazil, is one of the largest trees that grows in the Atlantic Rain Forest. Jequitibá Rosa has been extensively exploited due to the excellent quality of its wood, which has resulted in the species being placed as "vulnerable" on the IUCN red list of threatened species (IUCN, 2021). Thus, conservation of the species is paramount. In October 2018, in the municipality of Ilhéus, Bahia (14º 79' 67" S, 39º 17' 32" W), trees of C. legalis were observed with approximately 80% of leaves presenting light to dark brown necrotic lesions with yellow edges at the tips and margins of the leaves; the lesions gradually expanded through the leaves and coalesced to form wider lesions, which resulted in death of the leaves and weakening of the trees. The symptoms indicate infection caused by a Colletotrichum spp., important pathogens known to cause diseases in several economically important plants. Symptomatic leaves were collected and lesions examined under a stereomicroscope at 20x magnification (Supplementary Figure S1. A). Conidia were transferred directly and aseptically from the leaf spots to potato-dextrose-agar (PDA) and incubated in the dark at 25 ± 1 ºC for 72 h. Subsequently, pure cultures were obtained by monosporic isolation. All fungal structures were measured using a Leica DM500 microscope at 1000x magnification. A pathogenicity test was performed both on hosts in the field and using detached leaves by inoculation with a conidial suspension 106 conidia/mL prepared from a culture grown on PDA incubated in the dark at 25 ± 1 ºC for 10 days. Six healthy and fully developed detached leaves were inoculated with two 10 µL drops of conidial suspension on the adaxial surface. Six leaves were inoculated with two drops of sterile water were used as controls. The inoculated leaves were kept in a humid chamber at 25º C with a 12 h photoperiod. Four trees of C. legalis growing in the field were selected and 120 healthy leaves (30 per tree) sprayed with a conidial suspension and covered with transparent polyethylene plastic bags, with a piece of wet cotton inside, to maintain moisture for 72 h. A further 30 leaves per tree were sprayed with sterile water as controls. DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method following the protocol described by Oliveira et al. (2016). Internal transcribed spacer (ITS), partial histone3 (HIS3), and partial glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene sequences were amplified by polymerase chain reaction (PCR) using ITS1/ITS4 (White et al. 1990), CYLH3F/CYLH3R (Crous et al. 2004), and GDF1/GDR1 (Guerber et al. 2003) primers. The PCR for the ITS amplification were performed according to Oliveira et al. (2014), and for the HIS3 and GAPDH according Damm et al. (2012). Maximum likelihood analysis (with support estimated by a bootstrap analysis with 1,000 replicates) was performed using PhyML 3.0 (Guindon and Gascuel 2003) and launched from Topali 2.5. The colony produced abundant white to light gray aerial mycelium, and an orange viscous mass of conidia (Supplementary Figure S1. B). Conidia were single-celled, smooth, hyaline and sub-cylindrical with rounded ends, 13 (11-15) × 3.5 (3-4) µm (Supplementary Figure S1. C); appressoria were aseptate, brown, subglobose to clavate, 11 (10-12) × 7 (6-8) µm (Supplementary Figure S1. D). BLASTn analysis revealed that the partial gene sequences of ITS (URM 8381 ‒ MZ158701), GAPDH (URM 8381 ‒ MZ189259) and HIS3 (URM 8381 ‒ MZ189260) were 100% identical to Colletotrichum tropicale (CPO 27.830 ‒MN744296/CBS 129983 ‒ MH865615), (CMM 4071 ‒ KC517181/CPO 27.719 ‒MN737355) and (CBS 124949 ‒ KY856395). A maximum likelihood phylogenetic tree was generated by combining all sequenced loci. The phylogenetic tree revealed that sequences of the isolate URM8381 formed a clade with the sequences from the type species of C. tropicale E.I. Rojas, S.A. Rehner & Samuels (CBS 124949) with a high support value (95), which is distinct from other related species (Supplementary Figure S1. E). The sequences from the isolate were deposited in GenBank under the following accession numbers: ITS: MZ158701; GAPDH: MZ189259 and HIS3: MZ189260. C. tropicale was reisolated from the inoculated leaves, and had the same cultural and morphological characteristics as the original isolate. Both detached leaves and leaves on trees were inoculated in the field and presented leaf spot symptoms (Supplementary Figure S1.F), at 6 and 8 days after inoculation, respectively, which further confirms C.tropicale as the causal agent of the symptoms observed on the leaves of C. legalis. The controls did not develop any symptoms (Supplementary Figure S1. G). C. tropicale belongs to the C. gloeosporioides species complex. In Brazil, C. tropicale causes anthracnose on various hosts, including Annona muricata L. (soursop) and Myrciaria dubia (Kunth) McVaugh ("camu-camu") (Costa et al. 2019; Matos et al. 2020). To our knowledge, this is the first report of C. tropicale causing leaf spot on C. legalis. References: Costa, J. F. O., et al. 2019. Eur. J. Plant Pathol. 153:1119. Crous, P. W., et al. 2004. Stud. Mycol. 50:415. Damm, U., et al. 2012. Stud. Mycol. 73:1. Guerber, J. C., et al. 2003. Mycol. 95:87. Guindon, S.; Gascuel, O. 2003. Syst Biol 52(5): 696-704. https://doi.org/10.1080/10635150390235520. IUCN. 2021. http://www.iucnredlist.org. Matos, K. S., et al. 2020. Plant Dis. 104. https://doi.org/10.1094/PDIS-04-19- 0882-PDN Oliveira R. J. V., et al. 2016. N. Hedw. 103:185. White, T. J., et al. 1990. In: Innis, M.A., et al. (Eds.) Academic Press, San Diego, pp. 315-322. http://dx.doi.org/10.1016/b978-0-12-372180-8.50042-1.

Endophytic fungi of Brunfelsia uniflora: isolation, cryopreservation, and determination of enzymatic and antioxidant activity.

Brunfelsia uniflora (Pohl.) D. Don (Solanaceae), commonly known as manacá-de-cheiro, is widely distributed in Brazil and used by local indigenous peoples as an antirheumatic, antisyphilitic, depurative, emetic, vermifuge, and purgative agent. Several studies have examined the biological activities and phytochemical profile of Brunfelsia; however, few have focused on the diversity of endophytic microorganisms that colonize members of the genus. This study aimed to isolate and cryopreserve endophytic fungi from B. uniflora and determine their cellulase, laccase, and antioxidant activities. Endophytic fungi were isolated from B. uniflora stems, cultured on wheat grains, immersed in a 150 g L-1 aqueous sucrose solution, and cryopreserved at - 80 °C for 1 and 6 months. Cellulase activity was determined by a qualitative test using carboxymethylcellulose medium and laccase activity by a quantitative test based on the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). Prior to antioxidant activity assays, fungi were grown in malt extract broth for production of mycelial biomass. A methanolic extract was prepared for evaluation of DPPH· scavenging activity, FRAP activity, and total phenolic content. A total of 46 endophytic fungal isolates were obtained from B. uniflora stems and classified into 24 groups according to morphological similarities. B. uniflora was shown to harbor different genera of ascomycete fungi as endophytic organisms. Mycelial viability was observed after 1 and 6 months of cryopreservation at - 80 °C. Fungi exhibited cellulase and laccase activities. Isolate CE23 had the highest laccase activity after 7 days of cultivation. Twelve isolates were found to have low total phenolic contents and DPPH· and FRAP activities.

High-throughput screening for distinguishing nitrilases from nitrile hydratases in Aspergillus and application of a Box-Behnken design for the optimization of nitrilase.

Nitrilases and nitrile hydratases/amidases hydrolyze nitriles into carboxylic acids and/or amides, which are used in industrial chemical processes. In the present study, 26 microorganisms, including yeasts and filamentous fungi, in a minimum solid mineral medium supplemented with glucose and phenylacetonitrile were screened to evaluate their biocatalytic potential. Of these microorganisms, five fungi of the genus Aspergillus were selected and subjected to colorimetry studies to evaluate the production and distinction of nitrilase and nitrile hydratase/amidase enzymes. Aspergillus parasiticus Speare 7967 and A. niger Tiegh. 8285 produced nitrilases and nitrile hydratase, respectively. Nitrilase optimization was performed using a Box-Behnken design (BBD) and fungus A. parasiticus Speare 7967 with phenylacetonitrile volume (µl), pH, and carbohydrate source (starch:glucose; g/g) as independent variables and nitrilase activity (U ml-1 ) as dependent variable. Maximum activity (2.97 × 10-3  U ml-1 ) was obtained at pH 5.5, 80 µl of phenylacetonitrile, and 15 g of glucose. A. parasiticus Speare 7967 showed promise in the biotransformation of nitriles to carboxylic acids.

Endophytic fungal diversity in Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) trees and their potential for growth promotion and biocontrol of black-pod disease.

The endophytic niches of plants are a rich source of microbes that can directly and indirectly promote plant protection, growth and development. The diversity of culturable endophytic fungi from stems and branches of Theobroma cacao (cacao) and Theobroma grandiflorum (cupuaçu) trees growing in the Amazon region of Brazil was assessed. The collection of fungal endophytic isolates obtained was applied in field experiments to evaluate their potential as biocontrol agents against Phytophthora palmivora, the causal agent of the black-pod rot disease of cacao, one of the most important pathogens in cocoa-producing regions worldwide. The isolated endophytic fungi from 60 traditional, farmer-planted, healthy cacao and 10 cupuaçu plants were cultured in PDA under conditions inducing sporulation. Isolates were classified based upon the morphological characteristics of their cultures and reproductive structures. Spore suspensions from a total of 103 isolates that could be classified at least up to genus level were tested against P. palmivora in pods attached to cacao trees in the field. Results indicated that ∼70% of isolates showed biocontrol effects to a certain extent, suggesting that culturable endophytic fungal biodiversity in this system is of a mostly mutualistic type of interaction with the host. Eight isolates from genera Trichoderma (reference isolate), Pestalotiopsis, Curvularia, Tolypocladium and Fusarium showed the highest level of activity against the pathogen, and were further characterized. All demonstrated their endophytic nature by colonizing axenic cacao plantlets, and confirmed their biocontrol activity on attached pods trials by showing significant decrease in disease severity in relation to the positive control. None, however, showed detectable growth-promotion effects. Aspects related to endophytic biodiversity and host-pathogen-endophyte interactions in the environment of this study were discussed on the context of developing sustainable strategies for biological control of black-pod rot of cacao.

A simplified DNA extraction method for PCR analysis of Camarotella spp

This work aimed to optimize an efficient and simple protocol for DNA extraction of Camarotella species, an obligate plant pathogen that cause verrucosis or "lixa" on coconut tree and other palms, facilitating the molecular studies of these biotrophic microorganisms. The method proposed enabled a fast, reproducible and reliable DNA extraction from Camarotella species.

A extração e amplificação de DNA são etapas fundamentais para a aplicação de métodos moleculares e para tal, a origem do material é relevante. As espécies do gênero Camarotella que causam as lixas do coqueiro e outras palmeiras são biotróficas e o crescimento em meio artificial é controverso, embora Oliveira et. al, (2004) tenham registrado seu cultivo in vitro, utilizando meio líquido completo. No entanto, não ficou provado que o micélio formado correspondia ao micélio de Camarotella. Em conseqüência das dificuldades com o cultivo dos agentes etiológicos das lixas, foi otimizado um protocolo para extração de DNA genômico a partir do himênio ascógeno in natura e um protocolo de PCR para sua amplificação. Nossos resultados são importantes para o estudo da família Phyllachoraceae, pois possibilitará a análise molecular que, nessa família, é limitada pela dificuldade de obtenção de DNA.

New Annulohypoxylon species from Brazil.

Four new Annulohypoxylon species are described from northeastern Brazil: A. austrobahiense, A. macrodiscum, A. hemicarpum and A. purpureopigmentum. A key is provided.

Hemibeltrania urbanodendrii sp. nov. and Pseudobeltrania angamosensis: new fungal records from the brazilian tropical seasonal semi-deciduous montane forest

The new species Hemibeltrania urbanodendrii, associated to leaf-spots on Urbanodendron verrucosum (Lauracea) and Pseudobeltrania angamosensis, associated with leaf-spots on Virola gardneri (Myristicaceae), are recorded for the first time in Brazil. They represent additions to the mycobiota of the Tropical Seasonal Semi-Deciduous Montane Forest in Viçosa (Minas Gerais, Brazil), a highly threatened ecosystem.

Novas ocorrências de fungos relacionados a manchas foliares são apresentadas: Hemibeltrania urbanodendrii sp. nov., associado a Urbanodendron verrucosum (Lauracea) e Pseudobeltrania angamosensis, associado a Virola gardneri (Myristicaceae). Eles representam adições à micobiota da Floresta Tropical Estacional Semidecidual Montana de Viçosa (Minas Gerais), um ecossistema fortemente ameaçado.