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Phyllosticta citricarpa is a fungal pathogen causing citrus black spot (CBS). As a regulated pest in some countries, the presence of the pathogen limits the export of fruit and is therefore of agricultural and economic importance. In this study, we used high throughput sequencing data to infer the global phylogeographic distribution of this pathogen, including 71 isolates from eight countries, Argentina, Australia, Brazil, China, Cuba, Eswatini, South Africa and the United States of America. We assembled draft genomes and used a pairwise read mapping approach for the detection and enumeration of variants between isolates. We performed SSR marker discovery based on the assembled genome with the best assembly statistics, and generated genotype profiles for all isolates with 1987 SSR markers in silico. Furthermore, we identified 32,560 SNPs relative to a reference sequence followed by population genetic analyses based on the three datasets; pairwise variant counts, SSR genotypes and SNP genotypes. All three analysis approaches gave similar overall results. Possible pathways of dissemination among the populations from China, Australia, southern Africa and the Americas are postulated. The Chinese population is the most diverse, and is genetically the furthest removed from all other populations, and is therefore considered the closest to the origin of the pathogen. Isolates from Australia, Eswatini and the South African province Mpumalanga are closely associated and clustered together with those from Argentina and Brazil. The Eastern Cape, North West, and KwaZulu-Natal populations in South Africa grouped in another cluster, while isolates from Limpopo are distributed between the two aforementioned clusters. Southern African populations showed a close relationship to populations in North America, and could be a possible source of P. citricarpa populations that are now found in North America. This study represents the largest whole genome sequencing survey of P. citricarpa to date and provides a more comprehensive assessment of the population genetic diversity and connectivity of P. citricarpa from different geographic origins. This information could further assist in a better understanding of the epidemiology of the CBS pathogen, its long-distance dispersal and dissemination pathways, and can be used to refine phytosanitary regulations and management programmes for the disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Citrus black spot (CBS) and post-bloom fruit drop (PFD), caused by Phyllosticta citricarpa and Colletotrichum abscissum, respectively, are two important citrus diseases worldwide. CBS depreciates the market value and prevents exportation of citrus fruits to Europe. PFD under favorable climatic conditions can cause the abscission of flowers, thereby reducing citrus production by 80%. An ecofriendly alternative to control plant diseases is the use of endophytic microorganisms, or secondary metabolites produced by them. Strain LGMF1631, close related to Diaporthe cf. heveae 1, was isolated from the medicinal plant Stryphnodendron adstringens and showed significant antimicrobial activity, in a previous study. In view of the potential presented by strain LGMF1631, and the absence of chemical data for secondary metabolites produced by D. cf. heveae, we decided to characterize the compounds produced by strain LGMF1631. Based on ITS, TEF1, and TUB phylogenetic analysis, strain LGMF1631 was confirmed to belong to D. cf. heveae 1. Chemical assessment of the fungal strain LGMF1631 revealed one new seco-dihydroisocoumarin [cladosporin B (1)] along with six other related, already known dihydroisocoumarin derivatives and one monoterpene [(-)-(1S,2R,3S,4R)-p-menthane-1,2,3-triol (8)]. Among the isolated metabolites, compound 5 drastically reduced the growth of both phytopathogens in vitro and completely inhibited the development of CBS and PFD in citrus fruits and flowers. In addition, compound 5 did not show toxicity against human cancer cell lines or citrus leaves, at concentrations higher than used for the inhibition of the phytopathogens, suggesting the potential use of (-)-(3R,4R)-cis-4-hydroxy-5-methylmellein (5) to control citrus diseases.

Endophytic fungi have been considered a rich source for bioactive secondary metabolites with novel chemical structures. A high diverse group of endophytes, isolated from different medicinal plants, belongs to the genus Diaporthe. In a previously study performed by our group the crude extract of strain LGMF1583 showed considerable antibacterial activity mainly against Gram-negative bacteria. Based on ITS phylogeny analysis, strain LGMF1583 was identified as belonging to Diaporthe genus and may represent a new species. In the present study, we described the new species Diporthe vochysiae based on multilocus phylogeny analysis and morphological characteristics. The species name refers to the host, from which strain LGMF1583 was isolated, the medicinal plant Vochysia divergens. In view of the biotechnological potential of strain LGMF1583, we have also characterized the secondary metabolites produced by D. vochysiae. Chemical assessment of the D. vochysiae LGMF1583 revealed two new carboxamides, vochysiamides A (1) and B (2), in addition to the known metabolite, 2,5-dihydroxybenzyl alcohol (3). In the biological activity analysis, vochysiamide B (2) displayed considerable antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae (KPC), a producer of carbapenemases, MIC of 80 μg/mL. Carbapenemases are considered a major antimicrobial resistance threat, and infections caused by KPC have been considered a public health problem worldwide, and new compounds with activity against this bacterium are nowadays even more required., (Copyright © 2019. Published by Elsevier B.V.)

The isolation and structure elucidation of one new fungal metabolite, phenguignardic acid butyl ester (1a), and four previously reported metabolites (1b, 2a, 3-4) from the citrus phytopathogen Phyllosticta citricarpa LGMF06 are described. The new dioxolanone phenguignardic acid butyl ester (1a) had low phytotoxic activity in citrus leaves and fruits (at dose of 100 µg), and its importance as virulence factor in citrus black spot disease needs to be further addressed. Beside the phytotoxic analysis, we also evaluated the antibacterial (against methicillin sensitive and resistant Staphylococcus aureus) and cytotoxic (A549 non-small cell lung cancer, PC3 prostate cancer and HEL 299 normal epithelial lung) activities of the isolated compounds, which revealed that compounds 1a, 1b and 2a were responsible for the antibacterial activity of this strain.

The citrus black spot (CBS), caused by Phyllosticta citricarpa, is one of the most important citrus diseases in subtropical regions of Africa, Asia, Oceania, and the Americas, and fruits with CBS lesions are still subject to quarantine regulations in the European Union. Despite the high application of fungicides, the disease remains present in the citrus crops of Central and South America. In order to find alternatives to help control CBS and reduce the use of fungicides, we explored the antifungal potential of endophytic actinomycetes isolated from the Brazilian medicinal plant Vochysia divergens found in the Pantanal biome. Two different culture media and temperatures were selected to identify the most efficient conditions for the production of active secondary metabolites. The metabolites produced by strain Microbacterium sp. LGMB471 cultured in SG medium at 36 °C considerably inhibited the development of P. citricarpa. Three isoflavones and five diketopiperazines were identified, and the compounds 7-O-β-D-glucosyl-genistein and 7-O-β-D-glucosyl-daidzein showed high activity against P. citricarpa, with the MIC of 33 μg/mL and inhibited the production of asexual spores of P. citricarpa on leaves and citrus fruits. Compounds that inhibit conidia formation may be a promising alternative to reduce the use of fungicides in the control of CBS lesions, especially in regions where sexual reproduction does not occur, as in the USA. Our data suggest the use of Microbacterium sp. LGMB471 or its metabolites as an ecological alternative to be used in association with the fungicides for the control of CBS disease.

Citrus black spot disease, caused by the phytopathogen Phyllosticta citricarpa, depreciates the market value of citrus fruits and prevents their exportation to disease-free regions. It may also reduce the productivity of citrus fruit orchards. To identify an alternative to conventional disease control measures, isolates of Diaporthe terebinthifolii, active against P. citricarpa, were selected from an endophytic fungal population of Schinus terebinthifolia leaves. Different culture media were screened to identify the culture medium that afforded the most efficient production of biologically active extracts. A particular fraction (fraction VI) of the extract completely protected orange leaves by inhibiting the germination of P. citricarpa conidia with a minimum inhibitory concentration of 0.003 μg.mL-1. The active constituents in D. terebinthifolii extract fractions were identified by gas chromatography coupled to mass spectrometry as verbanol, phenylethyl alcohol, verbenyl acetate and methyl hexadecanoate. The results obtained strongly suggest the existence of a synergistic effect among the metabolites produced. Thus, these fungal metabolites could be used to control the CBS disease. As the asexual spores of P. citricarpa play an important role in fruit lesion development and disease dispersion, fungal extracts that inhibit the spore germination can be used as an effective alternative for directional disease control., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phyllosticta citricarpa is the epidemiological agent of Citrus Black Spot (CBS) disease, which is responsible for large economic losses worldwide. CBS is characterized by the presence of spores (pycnidiospores) in dark lesions of fruit, which are also responsible for short distance dispersal of the disease. The identification of genes involved in asexual reproduction of P. citricarpa can be an alternative for directional disease control. We analyzed a library of mutants obtained through Agrobacterium tumefaciens transformation system, looking for alterations in growth and reproductive structure formation. Two mutant strains were found to have lost the ability to form pycnidia. The flanking T-DNA insertion regions were identified on P. citricarpa genome by using blast analysis and further gene prediction. The predicted genes containing the T-DNA insertions were identified as Spindle Poison Sensitivity Scp3, Ion Transport protein, and Cullin Binding proteins. The Ion Transport and Cullin Binding proteins are known to be correlated with sexual and asexual reproduction in fungi; however, the exact mechanism by which these proteins act on spore formation in P. citricarpa needs to be better characterized. The Scp3 proteins are suggested here for the first time as being associated with asexual reproduction in fungus. This protein is associated with microtubule formation, and as microtubules play an essential role as spindle machinery for chromosome segregation and cytokinesis, insertions in this gene can lead to abnormal formations, such as that observed here in P. citricarpa. We suggest these genes as new targets for fungicide development and CBS disease control, by iRNA., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The citrus industry is severely affected by citrus black spot (CBS), a disease caused by the pathogen Phyllosticta citricarpa. This disease causes loss of production, decrease in the market price of the fruit, and reduction in its export to the European Union. Currently, CBS disease is being treated in orchards with various pesticides and fungicides every year. One alternative to CBS disease control without harming the environment is the use of microorganisms for biological control. Diaporthe endophytica and D. terebinthifolii, isolated from the medicinal plants Maytenus ilicifolia and Schinus terebinthifolius have an inhibitory effect against P. citricarpa in vitro and in detached fruits. Moreover, D. endophytica and D. terebinthifolii were transformed by Agrobacterium tumefaciens for in vivo studies. The transformants retained the ability to control of phytopathogenic fungus P. citricarpa after transformation process. Furthermore, D. endophytica and D. terebinthifolii were able to infect and colonize citrus plants, which is confirmed by reisolation of transformants from inoculated and uninoculated leaves. Light microscopic analysis showed fungus mycelium colonizing intercellular region and oil glands of citrus, suggesting that these two new species are capable of colonizing citrus plants, in addition to controlling the pathogen P. citricarpa., (Copyright © 2016 Elsevier GmbH. All rights reserved.)