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For the first time, Stagonosporopsis caricae is reported causing leaf spots on the non-conventional fruit crop Vasconcellea monoica (Caricaceae). The fungus was identified in Brazil based on morphological and molecular features and its pathogenicity was demonstrated. This is the first pathogen to be reported on V. monoica.

Pelargonium × hortorum – geranium - (Geraniaceae) is a highly popular ornamental worldwide, including in Brazil (Lorenzi 2013). Currently only three fungi are reported to occur on geraniums in Brazil (Mendes and Urben 2019): Alternaria sp., Leptosphaeria pelargonii and Puccinia pelargonii-zonalis. In 2018, a leaf spoton geranium plants grown in a disease demonstration garden - campus of the Universidade Federal de Viçosa (UFV), state of Minas Gerais (MG), Brazil (20°45´14´´ S, 42°52´54´´ W). The disease, in combination with attack by the rust fungus P. pelargonii-zonalis caused severe defoliation of plants. The same situation was observed independently in garden areas of the campus of UFV and also in private gardens in MG and also in the state of Rio de Janeiro. Leaf spots were numerous and initially small whitish dots (1 to 2 mm). Spots grew and became circular, surrounded with a dark brown border. Older spots became dark and coalesced, leading to extensive leaf necrosis (3 to 5 cm) (Figs 1a, b, c). Upon observation under a dissecting microscope a dematiaceous fungus was found growing on the majority of the necrotic spots. A sample of diseased leaves was selected, dried in a plant press and deposited in UFV’s herbarium (Acc. No VIC 47336). Pure cultures of the fungus were obtained by transfer of conidia from individual separate leaf spots, onto PDA plates with the help of a sterile fine pointed needle. Colonies had identical morphology and one was selected and deposited in the local culture collection (Acc. No COAD 2916). Fungal structures were scraped from the surface of necrotic tissues with a scalpel and slides were mounted in lactoglycerol and examined with a light microscope (Olympus BX 51). Measurements were taken from at least 30 structures. The fungus had the following morphology: conidiophores fasciculate, cylindrical, geniculate, proliferating sympodially, 85-130 × 4-6 µm, 3 to 6 septate; conidia acicular to filiform, 92 - 210 × 2,5-4 µm, 9 to 23 septate, hyaline, smooth (Figs 1 d, e). This morphology is typical of Cercospora apii sensu lato (Crous and Braun 2003). Genomic DNA was extracted from COAD 2916 grown on PDA using a Wizard Genomic DNA Purification Kit (Promega) per the manufacturer's instructions. Three loci were PCR amplified, namely: calmodulin (CAL), actin (ACT) and histone h3 (HIS) using primers ACT512F/ACT783R, CYLH3FQ/CYLH3R and CAL228F/CAL2Rd, respectively. The resulting sequences were deposited in GenBank (MT199213, MT199214 and MT199215, respectively). A multilocus Bayesian phylogenetic analysis was performed using sequences from other closely related taxa obtained from GenBank. COAD 2916 clustered with Cercospora sp. H (Groenewald et al. 2013). This is a yet unresolved clade of the C. apii sensu lato assemblage. Inoculation of five 3-month-old healthy geranium plants was performed. COAD 2916 was grown on PDA for 12 days and plugs were taken from the margin of growing colonies and placed on young leaves. Five other healthy plants receiving uncultured sterile PDA plugs on leaves served as controls. All plants were then placed in a dew chamber for 24 h, transferred to a bench in a greenhouse afterwards and observed daily for the emergence of symptoms. Symptoms appeared on all inoculated leaves by seven days. Controls remained healthy. COAD 2916 sporulated on the diseased tissues and the fungus was re-isolated in pure culture. Colonies had the same morphology of those originally used for inoculation. Three species of Cercospora have been reported on Pelargonium spp.: C. apii, C. brunkii and C. cf. flagellaris (Bakhshi et al. 2018; Farr and Rossman 2020). COAD 2916 is phylogenetically distant from C. apii sensu stricto, C. brunkii and C. cf. flagellaris and is morphologically different from C. brunkii. This is the first record of Cercospora apii s. lato ‘sp. H’ causing leaf spot on Pelargonium × hortorum worldwide. The other record of C. apii on Pelargonium is on P.zonale from Iran (Pimia et al. 2012) but no molecular information was included in that publication.

Digitodesmium is a genus of saprobic fungi, generally associated with decaying wood in freshwater habitats or in the soil. As morphologic markers they produce cheiroid, euseptate conidia on sporodochia. During an exam of a necrotic robusta coffee stem sent from Nova Venécia, state of Espírito Santo, to the Plant Clinic at the Universidade Federal de Viçosa (Brazil), for disease diagnosis a fungus, recognized as having the typical features of Digitodesmium was observed. The fungus was isolated in pure culture and DNA was extracted. Sequences of the partial 18S ribosomal RNA gene, large subunit of the nrDNA, internal transcribed spacer and translation elongation factor 1-α were generated. The combination of results of the phylogenetic analysis with the exam of the morphology led to the conclusion that the fungus from coffee stem morphological data showed that this fungus represents a monophyletic distinct lineage within Digitodesmium and an undescribed species for the genus. The concatenate tree also revealed that Digitodesmium is divided in two distinct clades. The novel species can be differentiated morphologically from other species of Digitodesmium by the size of the conidia, the number of arms and the presence of appendages. The new species Digitodesmium polybrachiatum is hence proposed herein. A comparative table of conidial morphology for the species in the genus is also included.

During surveys conducted in South America and Africa to identify natural fungal enemies of coffee leaf rust (CLR), Hemileia vastatrix, over 1500 strains were isolated, either as endophytes from healthy tissues of Coffea species or as mycoparasites growing on rust pustules. Based on morphological data, eight isolates—three isolated from wild or semiwild coffee and five from Hemileia species on coffee, all from Africa—were provisionally assigned to the genus Clonostachys. A polyphasic study of their morphological, cultural and molecular characteristics—including the Tef1 (translation elongation factor 1 alpha), RPB1 (largest subunit of RNA polymerase II), TUB (β-tubulin) and ACL1 (ATP citrate lyase) regions—confirmed these isolates as belonging to three species of the genus Clonostachys: namely C. byssicola, C. rhizophaga and C. rosea f. rosea. Preliminary assays were also conducted to test the potential of the Clonostachys isolates to reduce CLR severity on coffee under greenhouse conditions. Foliar and soil applications indicated that seven of the isolates had a significant effect (p < 0.05) in reducing CLR severity. In parallel, in vitro tests that involved conidia suspensions of each of the isolates together with urediniospores of H. vastatrix resulted in high levels of inhibition of urediniospore germination. All eight isolates showed their ability to establish as endophytes in C. arabica during this study, and some proved to be mycoparasites of H. vastatrix. In addition to reporting the first records of Clonostachys associated with healthy coffee tissues and with Hemileia rusts of coffee, this work provides the first evidence that Clonostachys isolates have potential as biological control agents against CLR.

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Aims Elucidating the identity of an isolate of Aspergillus sp. obtained during searches for anti-coffee leaf rust (CLR) biocontrol agents, from healthy coffee berry samples, preliminarily verify whether it is an aflatoxin-producer, confirm its ability to grow as an endophyte in healthy coffee tissues and assess its biocontrol potential against CLR. Methods and Results One, among hundreds of fungal isolates fungus were obtained from healthy coffee tissues belonged to Aspergillus (isolate COAD 3307). A combination of morphology features and molecular analyses; including four regions—internal transcribed spacer (ITS), second-largest subunit of RNA polymerase (RPB2), β-tubulin (BenA) and calmodulin (CAL)—identified COAD 3307 as Aspergillus flavus. Inoculations of healthy Coffea arabica with COAD 3307 confirmed its establishment as an endophyte in leaves, stems and roots. Treatment of Coffea arabica plants by combinated applications of COAD 3307 on aerial parts and in the soil, significantly (p &gt; 0.0001) reduced CLR severity as compared to controls. Thin Layer Chromatography (TLC) indicated that COAD 3307 is not an aflatoxin-producing isolate. In order to confirm this result, the extract was injected into High Performance Liquid Chromatography (HPLC) system equipped with a fluorescence detector and no evidence of aflatoxin was found. Conclusions COAD 3307 is an endophytic isolate of A. flavus—a species which has never been previously recorded as an endophyte of Coffea spp. It is a non-aflatoxin producing strain which has an anti-CLR effect and merits further evaluation as a biocontrol agent.