Lucas Magalhães de Abreu

The effective control of post‐harvest diseases caused by phytopathogenic fungi is essential to guarantee the quality and productivity of cultivated fruits, motivating the search for alternative and sustainable methods, such as using essential oils (EOs) from unconventional food plants. The objective of this work was to isolate, identify and evaluate the pathogenicity of the causal agents of anthracnose associated with post‐harvest diseases of avocado fruits and to evaluate the potential of EOs from Pelargonium graveolens (geranium) and Cordia verbenacea (black sage, erva baleeira) in controlling the disease. Colletotrichum nymphaeae (UCBV 362) was used from a previous work of ours, and C. siamense (UCBV 136) was isolated from fruits with lesions characteristic of anthracnose. The antifungal activity of geranium and black sage EOs was evaluated at various concentrations. The EOs were evaluated by gas chromatography–mass spectrometry, and 58 components were identified in geranium and 57 in black sage. Pathogenicity testing of the fungal isolates and phylogenetic identification were carried out. Both caused typical anthracnose lesions on avocado fruits. The EOs evaluated showed fungicidal activity at a concentration of 0.25% for geranium and 4.0% for black sage to control Colletotrichum spp. Geranium oil exhibited a greater reduction in mycelium for the two fungi, UCBV 362 and UCBV 136, with the latter showing 100% inhibition. This demonstrates the potential application of EOs as an alternative control method with low environmental impact for controlling anthracnose in avocado.

Species of Clonostachys and Sesquicillium are commonly found in soils and associated with plants as saprophytes or endophytes. Clonostachys also contains mycoparasitic species used as biocontrol agents. Thirty-three isolates of both genera, obtained from different substrates and locations in Brazil, were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and phylogenetic analysis based on acl1 gene sequences. Ex-type and reference strains of eight species were also included in the MALDI-TOF MS analyses. Multivariate cluster analysis of MALDI-TOF MS data grouped the reference strains in species-specific clusters and classified 26 isolates into clusters corresponding to seven known species, Clonostachys chloroleuca (n = 1), C. farinosa (n = 13), C. pseudochroleuca (n = 5), C. rhizophaga (n = 1), C. rogersoniana (n = 2), C. rosea (n = 2), and Sesquicillium lasiacidis (n = 2). Two additional clusters were composed of three and four isolates having morphological characteristics resembling C. compactiuscula. Multilocus phylogenetic analyses using five loci (acl1, tub2, tef1, rpb1, rpb2) grouped the seven strains in a single lineage consisting of two subclades and corresponding to a novel phylogenetic species, herein described as Clonostachys itumiriensis, sp. nov. This species belongs to subgenus Bionectria and is phylogenetically close to C. cylindrica, C. divergens, C. hongkongensis, C. rogersoniana, and C. samuelsii. Strains of this species were mostly obtained from leaf litter. Our results show the robustness of MALDI-TOF MS for delimiting known and novel species of Clonostachys and validate the use of this technique as a tool for classifying fungi from diverse substrates.

Aphelenchoides besseyi sensu lato is an emerging nematode that causes green stem and foliar retention syndrome in soybeans and cotton. As sustainable agriculture gains importance, biological control agents (BCAs) have become a key strategy for pest management. However, the influence of A. besseyi s. l. mycophagous behavior on fungal BCAs remains unclear. This study examined its feeding and reproductive abilities of eight fungal isolates commonly used in disease biological control: Trichoderma afroharzianum (T10), Pochonia chlamydosporia (PC10), Pochonia boninensis (PC17), Clonostachys pseudochroleuca (C05), Clonostachys chloroleuca (C17), Clonostachys farinosa (C35), Clonostachys rogersoniana (C101), and Clonostachys rosea (C133). The isolates were cultured on oatmeal agar (OA) and potato dextrose agar (PDA) and inoculated with A. besseyi s. l. The nematode fed and reproduced on T10, PC10, PC17, and C35 across both media. On C101 and C05, reproduction occurred only on PDA and OA, respectively, with mortality observed on C05 in OA. Feeding or reproduction was absent on C17 and C133, highlighting their potential as BCAs against A. besseyi s. l. These findings emphasize A. besseyi s. l.’s capacity to exploit fungal BCAs in vitro, which may undermine fungal disease control in infested areas.

The Hass cultivar is one of the most cultivated and distributed avocado varieties throughout the world, having high productivity, nutritional quality, market acceptance and adaptation to different climatic zones (Dreher and Davenport 2013). Anthracnose affects avocado production in tropical and subtropical regions, causing economic losses, especially post-harvest (Fuentes-Aragón et al. 2020). Correct identification of Colletotrichum species causing anthracnose is essential, as different species vary in important phenotypes such as virulence and sensitivity to fungicides (Chen et al. 2016). Twelve samples from batches of avocados with anthracnose were collected in Minas Gerais State, Brazil, in September 2023. The observed symptoms were brown to black depressed circular spots, ranging from a few millimeters to 3 cm in diameter on the epicarp of the fruits, covered in center by mucilaginous layers of pathogen sporulation. Isolation was performed directly from the spore masses and monoconidial isolates were cultured in PDA at 25°C for 7 days for morphological characterization and preserved in sterile water at 4°C. One of the morphotypes commonly recovered from lesions, represented by isolate UCBV 362 (Culture Collection COAD 3843), formed fast-growing colonies having white aerial mycelium and intense salmon-colored sporulation. The cylindrical conidia were 13 to 17.5 μm long and 4.5 to 7 μm wide (average 14.5 x 5.7 μm, N=100), produced on conidiophores dispersed in the aerial mycelium or aggregated on melanized conidiomata formed on the agar. The partial sequence of the second largest subunit of the RNA polymerase II gene (RPB2) from isolate UCBV 362 (GenBank: PQ034617, 1116 nt) showed 99% of coverage and 99.37% of nucleotide identity with the RPB2 sequence of the ex-epitype strain of Colletotrichum nymphaeae ICMP 17918 (=CBS 515.78) (GenBank: JN985506). In a Maximum Likelihood phylogenetic tree composed with RBP2 sequences from reference strains of the Colletotrichum acutatum species complex, the isolate UCBV 362 formed a highly supported clade with the ex-epitype and other reference strains of Colletotrichum nymphaeae, occupying the Clade 2 of the species complex together with C. scovillae and C. simmondsii (Damm et al. 2012). This result shows the reliability of RPB2 for phylogeny and species delimitation within Colletotrichum. To confirm pathogenicity, 10-mm discs from a 7-day-old colony were inoculated at 3 different points on healthy-looking avocado fruits and incubated at 28°C. Uninoculated fruits served as controls. The first symptoms appeared 5 days after inoculation and were similar to those observed in the original samples, while the fruits in the control group remained asymptomatic. The pathogen was reisolated from the lesions and identified morphologically, fulfilling Koch's postulates. Colletotrichum nymphaeae has been associated with avocado anthracnose in Mexico (Fuentes-Aragón et al. 2020). In Brazil, a study based on molecular phylogeny identified Colletotrichum siamense and C. karsti as etiological agents of this disease (Soares et al. 2021). This report expands the range of species that cause avocado anthracnose in Brazil and provides etiological information for the implementation and monitoring of control methods, especially chemical control.

Despite the numerous benefits plants receive from probiotics, maintaining consistent results across applications is still a challenge. Cultivation-independent methods associated with reduced sequencing costs have considerably improved the overall understanding of microbial ecology in the plant environment. As a result, now, it is possible to engineer a consortium of microbes aiming for improved plant health. Such synthetic microbial communities (SynComs) contain carefully chosen microbial species to produce the desired microbiome function. Microbial biofilm formation, production of secondary metabolites, and ability to induce plant resistance are some of the microbial traits to consider when designing SynComs. Plant-associated microbial communities are not assembled randomly. Ecological theories suggest that these communities have a defined phylogenetic organization structured by general community assembly rules. Using machine learning, we can study these rules and target microbial functions that generate desired plant phenotypes. Well-structured assemblages are more likely to lead to a stable SynCom that thrives under environmental stressors as compared with the classical selection of single microbial activities or taxonomy. However, ensuring microbial colonization and long-term plant phenotype stability is still one of the challenges to overcome with SynComs, as the synthetic community may change over time with microbial horizontal gene transfer and retained mutations. Here, we explored the advances made in SynCom research regarding plant health, focusing on bacteria, as they are the most dominant microbial form compared with other members of the microbiome and the most commonly found in SynCom studies.

In agriculture, the control of fungal infections is essential to improve crop quality and productivity. This study describes the preparation and fungicidal activity evaluation of 12 glycerol derivatives bearing 1,2,3-triazole fragments. The derivatives were prepared from glycerol in four steps. The key step corresponded to the Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction between the azide 4-(azidomethyl)-2,2-dimethyl-1,3-dioxolane (3) and different terminal alkynes (57-91% yield). The compounds were characterized by infrared spectroscopy, nuclear magnetic resonance (1H and 13C), and high-resolution mass spectrometry. The in vitro assessment of the compounds on Asperisporium caricae, that is, the etiological agent of papaya black spot, at 750 mg L-1 showed that the glycerol derivatives significantly inhibited conidial germination with different degrees of efficacy. The most active compound 4-(3-chlorophenyl)-1-((2,2-dimethyl-1,3-dioxolan-4-yl) methyl)-1H-1,2,3-triazole (4c) presented a 91.92% inhibition. In vivo assays revealed that 4c reduced the final severity (70.7%) and area under the disease severity progress curve of black spots on papaya fruits 10 days after inoculation. The glycerol-bearing 1,2,3-triazole derivatives also present agrochemicallikeness properties. Our in silico study using molecular docking calculations show that all triazole derivatives bind favorably to the sterol 14 alpha-demethylase (CYP51) active site at the same region of the substrate lanosterol (LAN) and fungicide propiconazole (PRO). Thus, the mechanism of action of the compounds 4a-4l may be the same as the fungicide PRO, blocking the entrance/ approximation of the LAN into the CYP51 active site by steric effects. The reported results point to the fact that the glycerol derivatives may represent a scaffold to be explored for the development of new chemical agents to control papaya black spot.

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.

Penicillium setosum represents a Penicillium species recently described, with little up-to-date information about its metabolic and biological potential. Due to this scenario, we performed chemical and biological studies of P. setosum CMLD18, a strain isolated from Swinglea glutinosa (Rutaceae). HRMS-MS guided dereplication strategies and anti-leukemia assays conducted the isolation and characterization of six compounds after several chromatographic procedures: 2-chloroemodic acid (2), 2-chloro-1,3,8-trihydroxy-6- (hydroxymethyl)-anthraquinone (7), 7-chloroemodin (8), bisdethiobis(methylthio)acetylaranotine (9), fellutanine C (10), and 4-methyl-5,6-diihydro-2H-pyran-2-one (15). From the assayed metabolites, (10) induced cellular death against Kasumi-1, a human leukemia cell line, as well as good selectivity for it, displaying promising cytotoxic activity. Here, the correct NMR signal assignments for (9) are also described. Therefore, this work highlights more detailed knowledge about the P. setosum chemical profile as well as its biological potential, offering prospects for obtaining natural products with anti-leukemia capabilities.