First report of Lasiodiplodia brasiliensis causing crown rot on banana in Brazil.

Postharvest diseases compromise banana quality and cause high economic losses in Brazil. Among them, the crown rot prevails and its causal agents belong to distinct fungal species such as Colletotrichum musae (Berk. & Curt.) von Arx, Fusarium spp., and Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (Griffee and Burden 1976; Ploetz et al. 2003). Symptoms of crown rot were observed on banana fruits of cv. Williams in a commercial area in Assu, Rio Grande do Norte, Brazil (04°54'0.06"S, 37°22'6.02"W) in 2017. The samples were collected, superficially disinfected with NaClO (2%), and incubated in a wet chamber at 25 °C, with a 12 h photoperiod, for approximately 3 days. After the appearance of disease symptoms and pathogen signs, mycelia were transferred from the lesions to obtain pure cultures on a potato dextrose agar (PDA) medium. Thus, a monosporic culture was obtained (isolate BAN82). The fungus produced pycnidia with conidia on potato carrot agar (PCA) culture medium containing pine needles, after four weeks of incubation at 28 °C. The conidia were hyaline when immature and brown with central transverse septum when mature. The presence of conidiogenous cells, paraphyses, and conidiophores also were observed. The conidia present ovoid format measuring 20-28 x 11-14 µm (n=50). The fungal colony produced abundant aerial mycelia of mouse grey coloration, progressing to dark mouse grey (Rayner 1970), on PDA for 15 days to 28 °C. The growth rate was 29.3 mm/day on PDA. The genomic DNA was extracted and amplified PCR with primers TEF1-688F/TEF1-1251R, ITS1/ITS4, and Bt2a/Bt2b and sequenced in both directions. The TEF1 and TUB2 sequences showed 100%, and the ITS showed 93.06% identity with the sequences of Lasiodiplodia brasiliensis (GenBank accession numbers: ON623895, TEF1, ON623896, TUB2, and ON599012, ITS. Multiple alignments of the combined dataset of the isolate and representative sequences obtained from GenBank were submitted phylogenetic analyses to bayesian inference (IB) with posterior probabilities of 10,000,000 generations. The morphological characteristics together with multigenic analysis of the three genomic regions made it possible to identify the BAN82 isolate as Lasiodiplodia brasiliensis, showing bootstrap support of posterior probabilities of 0,98 in the IB analysis. The pathogenicity was evaluated on 16 banana fruits from cv. Prata Catarina, at the point of harvest. For inoculation, the bananas were disinfected with water, soap, and, NaClO (2%). Posteriorly, the fruits were wounded on both ends, followed by the deposition of 5mm diameter mycelial plugs from the fungal culture, within 7 days of the growth. After the inoculation, the fruits were incubated in plastic boxes in a wet chamber at 25 °C, with 12 h photoperiod, for 3 days. To complete Koch's postulates, the isolate was inoculated again into 16 other banana fruits from cv. Prata Catarina. The negative control fruits were not inoculated with the pathogen, only with PDA discs. The BAN82 isolate was pathogenic to the banana cv. Prata Catarina. In the Brazilian Northeast, L. brasiliensis was described in 2014 as being associated with papaya stem rot. Up to the moment, there are no reports of L. brasiliensis as the causal agent of crown rot on bananas from Brazil (Netto et al. 2014; Farr and Rossman 2022). Thus, our work is the first to report L. brasiliensis causing crown rot on banana fruits cv. Prata Catarina in Brazil.

Metabolomic Profile of Volatile Organic Compounds from Leaves of Cashew Clones by HS-SPME/GC-MS for the Identification of Candidates for Anthracnose Resistance Markers.

Anthracnose caused by Colletotrichum gloeosporioides affects the leaves, inflorescences, nuts, and peduncles of cashew trees (Anacardium occidentale). The use of genetically improved plants and the insertion of dwarf cashew clones that are more resistant to phytopathogens are strategies to minimize the impact of anthracnose on cashew production. However, resistance mechanisms related to the biosynthesis of secondary metabolites remain unknown. Thus, this study promoted the investigation of the profile of volatile organic compounds of resistant cashew clone leaves ('CCP 76', 'BRS 226' and 'BRS 189') and susceptible ('BRS 265') to C. gloeosporioides, in the periods of non-infection and infection of the pathogen in the field (July-December 2019 - Brazil). Seventy-eight compounds were provisionally identified. Chemometric analyses, such as Principal Component Analysis (PCA), Discriminating Partial Least Squares Analysis (PLS-DA), Discriminating Analysis of Orthogonal Partial Least Squares (OPLS-DA), and Hierarchical Cluster Analysis (HCA), separated the samples into different groups, highlighting hexanal, (E)-hex-2-enal, (Z)-hex-2-en-1-ol, (E)-hex-3-en-1-ol, in addition to α-pinene, α-terpinene, γ-terpinene, ß-pinene, and δ-3-carene, in the samples of the resistant clones in comparison to the susceptible clone. According to the literature, these metabolites have antimicrobial activity and are therefore chemical marker candidates for resistance to C. gloeosporioides in cashew trees.

Effects of centipedic acid on acute lung injury: A dose-response study in a murine model.

Centipedic Acid (CPA), a natural diterpene from Egletes viscosa, an endemic species of the Caatinga biome, has shown antioxidant and anti-inflammatory properties. However, no report on the CPA on respiratory system mechanics has been so far advanced. We aimed to investigate the dose-response behavior of CPA on E. coli lipopolysaccharide (LPS)-triggered acute lung injury (ALI). Forty-eight C57BL/6 mice were randomly divided into six groups: control (SS), induced to ALI (LPS), 4 groups induced to ALI pre-treated with 12.5, 25, 50 and 100 mg/kg of CPA (CPA12.5, CPA25, CPA50 and CPA100 groups). CPA 100 mg/kg could prevent inflammatory cell infiltration, alveolar collapse, changes in tissue micromechanics and lung function (airway resistance, tissue elastance, tissue resistance and Static compliance). These results indicate preventive potential of this compound in the installation of ALI.

Antagonism of volatile organic compounds of the Bacillus sp. against Fusarium kalimantanense.

Fusarium kalimantanense is a genetic lineage of Fusarium oxysporum f. sp. cubense (Foc) and belongs to the Fusarium oxysporum species complex (FOSC). This pathogen is a causative agent of Panama disease, an infection that has caused damage to the banana crop worldwide. Bacillus sp. (LPPC170) showed preliminary antagonist activity against F. kalimantanense (LPPC130) in vitro tests from the cultivation of axenic culture and co-culture with inhibition of mycelial growth of phytopathogen of 41.23%. According to these findings, volatile organic compounds (VOCs) emitted from Bacillus sp. were obtained by solid-phase microextraction and identified by gas chromatography coupled with a mass spectrometer (GC-MS). The multivariate data analysis tool (PLS-DA and Heatmap) identified short-chain organic acids as the main antagonistic VOCs responsible for inhibiting the mycelial growth of LPPC130. Acetic acid, propanoic acid, butanoic acid, valeric acid, and isovaleric acid exhibited a strong inhibitory effect on the mycelial growth of LPPC130, with inhibition of 20.68%, 33.30%, 26.87%, 43.71%, and 53.10%, respectively. Scanning electron microscopy revealed that VOCs caused damage to the vegetative and reproductive structures of the fungus. These results suggest Bacillus LPPC170 as an excellent biocontrol tool against the phytopathogen causative agents of Panama disease.

First report of Fusarium kalimantanense causing Fusarium wilt on banana tree in Brazil.

Banana (Musa spp.) is the second most-consumed fruit in Brazil, the fourth-largest producer globally, with 7 million tons in 2021 (IBGE 2021). Studies about the morphological and pathogenic characteristics revealed that the etiology of Fusarium wilt in banana cultivars in Brazil had been related to the Fusarium oxysporum f. sp. cubense (Foc) (E.F. Smith) Snyder and Hansen species (Costa et al. 2015; Cordeiro et al. 2016; Araújo et al. 2017). Phylogenetic studies have shown the existence of distinct genetic lineages for Foc, which has come to be called the Fusarium oxysporum Species Complex (FOSC) (O'Donnell et al. 1998; Maryani et al. 2019). Symptoms of Fusarium wilt were observed in banana trees at the headquarters of Embrapa Roraima (02°45'26.89"N and 60°43'52.78"W), Roraima-Brazil, in 2016. Samples were collected and sterilized with 70% ethanol for 30 s, followed by 3% NaClO for 1 min, rinsed three times in sterile distilled water, seeded on potato dextrose agar (PDA), and incubated at 25 °C for three days. Two isolates obtained from a pure culture (LPPC130) were submitted to the morphological characterization by Leslie and Summerell (2006) protocol. The fungal colony showed vinaceous color, progressing to livid red (Rayner 1970), with a mean diameter of 41 mm (± 0.1) at three days of incubation in a PDA culture medium. The fungus produced abundant macroconidia in spezieller nährstoffarmer agar (SNA) culture medium containing clove leaf (CLA) after 14 days of incubation at 25 °C. The sporodochium conidia presented a falcate shape, moderately curved, with 3 to 5 septa and dimensions ranging from 38.8 (48.0) 56.2 x 3.5 (4.4) 6.0 µm (n=50). The conidia of the aerial mycelium presented ovoid to ellipsoid shape, slightly curved, aseptic, measuring 6.0 (12.0) 18.0 x 2.8 (3.3) 5.0 µm (n=50). The genomic DNA of the isolate was extracted (Murray and Thompson 1980), and fragments of the elongation factor 1-α (TEF1) and RNA Polymerase II (RPB2) gene regions were amplified and sequenced in both directions (O'Donnell et al. 1998; O'Donnell et al. 2010) (GenBank accession numbers: Seq1 OL802918 and Seq2 OL802919). Multiple alignments of the combined dataset of the isolates and representative sequences obtained from GenBank were submitted to phylogenetic analysis with 1,000 bootstrap replicates. The micromorphological characteristics together to phylogenetic inference on the TEF1 and RPB2 genes, allowed a robust analysis, generating 42 more parsimonious trees and making it possible to identify the LPPC130 isolate as Fusarium kalimantanense, a species belonging to the F. oxysporum species complex (FOSC), with 100% bootstrap support (Maryani et al. 2019). The pathogenicity of the isolate was evaluated in five micropropagated seedlings of banana cv. Silk 75 days old, grown in pots with 5 kg of sterile formulation of sand and soil, in 1:1. Seedlings were inoculated by wounding the roots and depositing a suspension of conidia and chlamydospores at 105 spores mL-1. The inoculating of the isolate in 35 micropropagated seedlings of banana was based on Koch's postulates. The seedlings were transplanted into plastic bags (2 kg of sterile formulation: sandy soil and substrate, in 2:1) and inoculated with 10 mL of the chlamydospore suspension (107 CFU mL-1) at transplanting, and after 30 days of transplanting. Seedlings treated only with water were used as control. Evaluation of the symptoms of the disease was carried out 90 days after inoculation, and revealed that the isolate (LPPC130) was pathogenic by inducing the same symptoms of Fusarium wilt. F. kalimantanense was first reported associated with the pseudostems of Musa acuminata var. Pisang Ambon, and proved to be non-pathogenic to cv. Gros Michel and the bananas of the Cavendish group (Maryani et al. 2019). In Brazil, this fungus was recently associated with the Fusarium rot on melon fruits (Araújo et al. 2021); however, this is the first report of its pathogenicity in banana trees cv. Silk.

Spirostanol glucosides from the leaves of Cestrum laevigatum L.

Two new steroidal saponins, (25R)-spirost-5-ene-3ß,26ß-diol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[(1 → 2)-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (1) and (25R)-spirost-6-ene-3ß,5ß-diol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[(1 → 2)-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (2), along with the known diosgenin 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranoside (3), chonglouoside SL-5 (4) and Paris saponin Pb (5) were isolated from the leaves of Cestrum laevigatum. The structures of the compounds were determined using spectroscopic analyses including HRESI-MS, 1D and 2D NMR data, followed by comparison with data from the literature. Among them, two are particularly unique, compound 1 is the first (6)Δ-spirostanol saponin and compound 2 has an unusual C-26 hydroxyl in the (5)Δ-spirostanol skeleton. Antifungal testing showed a potent activity to formosanin C against Candida albicans and Candida parapsilosis. Evaluation of the cytotoxic activity indicated that compound 1 has a moderate activity against HL-60 and SF-295 cell lines, while compound 2 were active only against HL-60.

New unusual alkaloids from the ascidian Eudistoma vannamei.

The MeOH extract of the ascidian Eudistoma vannamei was found to contain three novel compounds, the adenine alkaloid derivatives 9-[N-(leucyl)-isoleucyl]- adenine (1) and 8-hydroxy-8-isopentyl-7,8-dihydroadenine (2), and the phenylalanine peptide derivative N-[N-(leucyl)-isoleucyl]phenethylamine (3). Other previously related compounds isolated from this extract include thymidine, 2'-deoxyuridine and phenylalanine. The structures of the new compounds were elucidated through the use of NMR and mass spectrometry.

Structure elucidation of casbane diterpenes from Croton argyrophyllus.

Two novel casbane diterpenes 1-hydroxy-(2E,6Z,12E)-casba-2,6,12-triene-4,5-dione (1) and 6E,12E-casba-1,3,6,12-tetraen-1,4-epoxy-5-one (2) were isolated from the ethanol extract of the stems of Croton argyrophyllus. Structural characterization including the relative stereochemistry of all compounds was established on the basis of spectroscopic methods, mainly 1D and 2D NMR, and HRESIMS.

Structure elucidation and NMR assignments of two unusual monoterpene indole alkaloids from Psychotria stachyoides.

Two unusual monoterpene indole alkaloids, stachyoside (1) and nor-methyl-23-oxo-correantoside (2), have been isolated from the aerial parts of Psychotria stachyoides. The structural elucidation of both compounds was performed by the aid of HRESIMS, FT-IR, and 1D- and 2D-NMR techniques including COSY, HSQC, HMBC, and NOESY.

Complete NMR data assignments for novel pterocarpans from Harpalyce brasiliana.

A NMR study of two new pterocarpans isolated from the roots of Harpalyce brasiliana is described. In addition to 1D NMR, 2D shift-correlated NMR pulse sequences ((1)H-(1)H-COSY, HSQC and HMBC) were used to establish the structures, and unambiguously perform the (1)H and (13)C chemical shift assignments.

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells.

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.

Trypanocidal activity of a new pterocarpan and other secondary metabolites of plants from Northeastern Brazil flora.

Two hundred fifteen compounds isolated from plants of Northeastern Brazil flora have been assayed against epimastigote forms of Trypanosoma cruzi, using the tetrazolium salt MTT as an alternative method. Eight compounds belonging to four different species: Harpalyce brasiliana (Fabaceae), Acnistus arborescens and Physalis angulata (Solanaceae), and Cordia globosa (Boraginaceae) showed significant activity. Among them, a novel and a known pterocarpan, a chalcone, four withasteroids, and a meroterpene benzoquinone were the represented chemical classes.

Piplartine induces inhibition of leukemia cell proliferation triggering both apoptosis and necrosis pathways.

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} is an alkaloid/amide component of Piper species. The purpose of the present study was to examine the antiproliferative effects of piplartine on human leukemia cell lines HL-60, K562, Jukart, and Molt-4 using the trypan blue exclusion method, as well as the effect of piplartine on DNA synthesis. The viability of all human leukemia cell lines were not affected by piplartine after 6 h, 9 h, and 12 h exposure, whereas a steady decline was seen after an exposure time of 24 h. The antiproliferative activity of piplartine seemed to be related to the inhibition of DNA synthesis, as revealed by the reduction of 5-bromo-2'-deoxyuridine (BrdU) incorporation after 24h of incubation. Piplartine-mediated reduction in cell number was associated with an increasing number of dead cells at a concentration of 10 microg/ml. These findings were corroborated by morphologic analysis. However, at the lowest concentration (2.5 microg/ml), piplartine-treated cells exhibited typical apoptotic morphological changes. The increase in caspase-3 activity was also observed in lysates of piplartine-treated cells (2.5 microg/ml). Our findings suggest that piplartine can suppress leukemia growth and reduce cell survival, triggering both apoptosis and/or necrosis, depending on the concentration used.

Antiproliferative effects of two amides, piperine and piplartine, from Piper species.

The present work evaluated the cytotoxicity of piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-trans-2-propenyl]-2(1H)pyridinone} and piperine {1-[5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine}, components obtained from Piper species. The substances were tested for their cytotoxicity on the brine shrimp lethality assay, sea urchin eggs development, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using tumor cell lines and lytic activity on mouse erythrocytes. Piperine showed higher toxicity in brine shrimp (DL50 = 2.8 +/- 0.3 microg/ml) than piplartine (DL50 = 32.3 +/- 3.4 microg/ml). Both piplartine and piperine inhibited the sea urchin eggs development during all phases examined, first and third cleavage and blastulae, but in this assay piplartine was more potent than piperine. In the MTT assay, piplartine was the most active with IC50 values in the range of 0.7 to 1.7 microg/ml. None of the tested substances induced hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of piplartine and piperine was not related to membrane damage.

Total NMR assignments of new [C7-O-C7'']-biflavones from leaves of the limonene-carvone chemotype of Lippia alba (Mill) N. E. Brown.

Phytochemical analysis of leaves of the limonene-carvone chemotype of Lippia alba led to the isolation of two biflavonoids with a new structural pattern with an ether linkage: 5,5''-dihydroxy-6,4',6'',3''',4'''-pentamethoxy-[C(7)--O--C(7'')]-biflavone (1) and 4',4,5,5''-tetrahydroxy-6,6'',3'''-trimethoxy-[C(7)--O--C(7'')]-biflavone (2). Structural elucidation of the new compounds was established on the basis of spectral data, through the use of 1D NMR and several 2D shift correlated NMR pulse sequences (COSY, HMQC, HMBC and NOESY).

Spectral assignments of new diterpenes from Hyptis martiusii Benth.

The structural characterization of two new abietanes and a new spiro-fused tricyclic diterpene isolated from the roots of Hyptis martiusii is described. The first member of a new class of rearranged abietane diterpenoids designated martiusane was characterized by the use of 1D NMR and several 2D shift correlated NMR pulse sequences (1H,1H-COSY, HMQC, HMBC and NOESY). Unambiguous 1H and 13C chemical shift assignments for all compounds are reported.