Synthesis of bis(ylidene) cyclohexanones and their antifungal activity against selected plant pathogenic fungi.

Botrytis cinerea, Rhizoctonia solani and Hemileia vastatrix are three species of phytopathogenic fungi behind major crop losses worldwide. These have been selected as target models for testing the fungicide potential of a series of bis(ylidene) cyclohexanones. Although some compounds of this chemical class are known to have inhibitory activity against human pathogens, they have never been explored for the control of phytopathogens until now. In the present work, bis(ylidene) cyclohexanones were synthesized through simple, fast and low-cost base- or acid-catalyzed aldol condensation reaction and tested in vitro against B. cinerea, R. solani and H. vastatrix. bis(pyridylmethylene) cyclohexanones showed the highest activity against the target fungi. When tested at 200 nmol per mycelial plug against R. solani., these compounds completely inhibited the mycelial growth, and the most active bis(pyridylmethylene) cyclohexanone compound had an IC50 of 155.5 nmol plug-1. Additionally, bis(pyridylmethylene) cyclohexanones completely inhibited urediniospore germination of H. vastatrix, at 125 µmol L-1. The most active bis(pyridylmethylene) cyclohexanone had an IC50 value of 4.8 µmol L-1, which was estimated as approximately 2.6 times lower than that found for the copper oxychloride-based fungicide, used as control. Additionally, these substances had a low cytotoxicity against the mammalian Vero cell line. Finally, in silico calculations indicated that these compounds present physicochemical parameters regarded as suitable for agrochemicals. Bis(ylidene) cyclohexanones may constitute promising candidates for the development of novel antifungal agents for the control of relevant fungal diseases in agriculture.

First Report of Cercospora apii sensu lato 'sp. Q' Leaf Spot on Hop in Brazil.

Hop - Humulus lupulus (Cannabaceae) - is an important and ancient, herbaceous, temperate, perennial crop. It is a vine which, although having other uses, is cultivated predominantly for the brewing industry (Laws 2013). Cultivation of hops in Brazil is relatively new and has increased in the last twenty years. There is only one published record of a fungal disease affecting hop in Brazil - powdery mildew (Fagherazzi et al 2021). In January 2021, leaf spots appeared on all eight hops plants ithe collection maintained in the Infectarium, a disease demonstration garden on the campus of the Universidade Federal de Viçosa (UFV), Viçosa, Minas Gerais, Brazil (https://www.infectario.ufv.br/). Symptoms were small, sub-circular to irregular spots, up to 5 mm, with a whitish to grayish center, surrounded by a dark brown necrotic margin, followed by a narrow yellowish outer margin. Older lesions became larger, grayish-brown, coalesced leading to extensive necrosis and stem dieback. A sample was collected, dried in a plant press, and deposited in the Herbarium of UFV (Acc. No VIC 47534). A dematiaceous fungus was found sporulating in the center of the lesions, when examined with a dissecting microscope. Fungal structures were scraped with a scalpel, mounted in lactoglycerol and observed with a light microscope (Olympus BX51). A pure culture was obtained after conidia were transferred onto PDA plates with a sterile fine-pointed needle. A representative isolate was deposited in the culture collection of the UFV (COAD 3368). The fungus had the following morphology: conidiophores cylindrical, geniculate, proliferating sympodially, 53 to 380 µm × 3 to 6.5 µm, 3 to 15-septate, smooth, with thickened and dark conidial scars, brown; conidia acicular to filiform, 47 to 210 µm × 2.5 to 5 µm, 3 to 17-septate, thin­walled, smooth, with thickened and dark hila, hyaline. This combination is typical of Cercospora apii sensu lato, as described by Crous and Braun (2003). Genomic DNA was extracted from a 7-day-old culture of COAD 3368 and three loci were PCR amplified, namely: actin (ACT), with 512-F and 783-R primers; calmodulin (CAL), using the primers 228F and 2Rd; and histone H3 gene (HIS3), with the primers CYLH3F and CYLH3R. PCR products were sequenced by Macrogen (Seoul, South Korea) and the resulting sequences were deposited in GenBank (OM677624, OM677625 and OM677626, respectively). A multilocus Bayesian phylogenetic grouped COAD 3368 with Cercospora 'sp. Q' (Groenewald et al. 2013). This is a yet unresolved species complex within C. apii. Six 6­month­old healthy hop plants (cv. Cascade) were sprayed with a COAD 3368 conidial suspension (105 conidia/ mL) whereas another group of six plants was sprayed with sterile distilled water, serving as controls. Plants were placed in a dew chamber for 5 days and then transferred to a greenhouse bench, where they were observed daily. Thirty days after inoculation, symptoms similar to those observed in the field had developed on all inoculated plants, whereas control plants remained healthy. The fungus growing on inoculated hops produced typical Cercospora conidiophores and conidia. Upon reisolation, pure cultures with the same morphology of COAD 3368 were obtained. There are no previous records of Cercospora 'sp. Q' on hops worldwide. Fungi in Cercospora 'sp. Q' are known to have a broad host-range. A previous record of Cercospora 'sp. Q' leaf spots was published from its observation on Dioscorea cayennensis in the Infectarium (Torres et al. 2016). Damage to the hop plants was severe and it is possible that Cercospora leaf spot will become an emerging threat to commercial hop plantations in Brazil.

Synthesis of Tyrosol 1,2,3-Triazole Derivatives and Their Phytotoxic Activity against Euphorbia heterophylla.

The synthesis and phytotoxic activity of a series of tyrosol 1,2,3-triazole derivatives are reported herein. Target compounds were synthesized through the copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), known as click reaction, and these were tested for phytotoxic activity on leaves of wild poinsettia (Euphorbia heterophylla), fleabane (Conyza sumatrensis), and tropical spiderwort (Commelina benghalensis). These are three highly noxious agricultural weeds that challenge available weed control methods, including the use of chemical herbicides. Twenty-five compounds were synthesized and tested. None of the compounds showed phytotoxic activity against C. benghalensis and C. sumatrensis, but almost all of them produced yellowing, bleaching, and necrosis on leaves of E. heterophylla. Two of the tyrosol 1,2,3-triazole derivatives produced more extensive lesions than those produced by the commercial herbicide diquat, used as a positive control (p ≤ 0.05). When applied on leaves of E. heterophylla, these compounds interfered with the stomatal conductance, net photosynthesis, internal carbon concentration, transpiration rate, water-use efficiency, and chlorophyll A and B contents. The interference of such compounds on such photosynthesis-related variables indicates that tyrosol 1,2,3-triazole derivatives may be capable of lowering the competitiveness of E. heterophylla and acting as additional tools for managing this competitive weed in agricultural lands.

Daylight LED promotes photochemical ring contraction of 2-amine-4H-pyran-3-carbonitriles with consequent loss of their antifungal activity.

The initial objective of our work was to synthesize a series of 2-amino-4H-pyran-3-carbonitriles to be tested for their antifungal activities against economically relevant phytopathogenic fungi. Fourteen compounds were prepared in up to 94% yield and shown percentages of Botrytis cinerea inhibition above 70%. Despite the promising biological results, we observed that stock solutions prepared for biological tests showed color changing when kept for a few days on the laboratory bench, under room conditions, illuminated by common LED daylight tubes (4500-6000 k). This prompted us to investigate the possible photo-induced degradation of our compounds. FT-IR ATR experiments evidenced variations in the expected bands for functional of -amino-4H-pyran-3-carbonitriles stored under LED daylight. Following, HPLC-UV analysis showed reductions in the intensity of chromatographic peaks of 2-amino-4H-pyran-3-carbonitriles, and but not for solutions kept in the dark. A solution of (E)-2-amino-8-(4-nitrobenzylidene)-4-(4-nitrophenyl)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile underwent 84.4% of conversion after 72 h of exposure to continuous LED daylight in a BOD chamber, and the reaction product was isolated in 36% yield and characterized as (E)-7-cyano-5-(4-nitrobenzylidene)-8-(4-nitrophenyl)bicyclo[4.2.0]oct-1(6)-ene-7-carboxamide (7*). Despite freshly prepared solutions of 2-amino-4H-pyran-3-carbonitriles produced antifungal activities, these solutions lost biological activity when left on the bench for a week. Besides, compound 7* formed from photo-induced degradation of 7 also showed no antifungal activity. With this, we hope to bring two contributions: (1) production of cyclobutenes through photochemical reactions of 2-amino-4H-pyran-3-carbonitriles can be carried out through exposure to simple white LED daylight; (2) biological applications of such 2-amino-4H-pyran-3-carbonitriles may be impaired by their poor photostability.