Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation

Carnauba wax p-methoxycinnamic diesters: Characterisation, antioxidant activity and simulated gastrointestinal digestion followed by in vitro bioaccessibility.

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Monitoring cashew seedlings during interactions with the fungus Lasiodiplodia theobromae using chlorophyll fluorescence imaging

The chlorophyll (Chl) fluorescence imaging technique was applied to cashew seedlings inoculated with the fungus Lasiodiplodia theobromae to assess any disturbances in the photosynthetic apparatus of the plants before the onset of visual symptoms. Two-month-old cashew plants were inoculated with mycelium of L. theobromae isolate Lt19 or Lt32. Dark-adapted and light-acclimated whole plants or previously labelled, single, mature leaf from each plant were evaluated weekly for Chl fluorescence parameters. From 21 to 28 days, inoculation with both isolates resulted in the significantly lower maximal photochemical quantum yield of PSII (Fv/Fm) than those for control samples, decreasing from values of 0.78 to 0.62. In contrast, the time response of the measured fluorescence transient curve from dark-acclimated plants increased in both whole plants and single mature leaves in inoculated plants compared with controls. The Fv/Fm images clearly exhibited photosynthetic perturbations 14 days after inoculation before any visual symptoms appeared. Additionally, decays in the effective quantum yield of PSII photochemistry and photochemical quenching coefficient were also observed over time. However, nonphotochemical quenching increased during the evaluation period. We conclude that Fv/Fm images are the effective way of detecting early metabolic perturbations in the photosynthetic apparatus of cashew seedlings caused by gummosis in both whole plants and single leaves and could be potentially employed in larger-scale screening systems

Polyclonal Antibody-based ELISA in combination with specific PCR amplification of ITS 1 regions for the detection and quantitation of Lasiodiplodia theobromae, causal agent of 2 gummosis in cashew nut plants

Members of Botryosphaeriaceae family are associated with serious diseases in different plants 18 across the world. In cashew nut plants (Anacardium occidentale L.), the fungus Lasiodiplodia 19 theobromae causes a severe group of symptoms related to gummosis that results in decreased nut 20 production. The aim of this work was to develop an indirect enzyme-linked immunosorbent 21 assay (ELISA) with sufficient sensitivity and specificity to detect the fungus both in vitro and in 22 planta (artificially and naturally infected) and to increase the detection specificity within the 23 fungi group using primers specific for the internal transcribed spacer (ITS) sequences. A 24 collection of L. theobromae isolates was obtained, and antisera against the fungus were raised in 25 rabbits. Cross-reactivity against Neofusicoccum sp., Colletotrichum gloeosporioides, Phomopsis 26 anacardii and Pestalotiopsis guepinii was examined. Naturally and artificially infected vegetal 27 material was employed in the ELISAs. The fungi ITS sequences were determined, and single 28 nucleotide polymorphisms were identified and used for primer design. For the naturally infected 29 2 plants, there was an approximately 4-fold variation in the absorbance values. Some positive 1 readings for asymptomatic samples were detected. For the artificially infected samples, an 2 ELISA-based weekly time-course analysis was conducted, and the values for samples from 0 and 3 7 days were lower than the threshold value. Beginning on day 14, the infection could be 4 detected, with rates varying from 40% on day 14 to 80% on day 21 and 100% by the end of the 5 experiment. The ITS sequencing revealed few polymorphisms among the L. theobromae isolates, 6 but for Colletotrichum gloeosporioides, Phomopsis anacardii, Pestalotiopsis guepinii and 7 Neofusicoccum sp., the sequences were sufficient to permit reliable discrimination. The 8 feasibility of ELISA as an early detection technique to assist in gummosis management was 9 demonstrated. PCR amplification based on ITS regions increases and complements serological 10 specificit

Estudo preliminar toxicológico, antibacteriano e fitoquímico do extrato etanólico das folhas de Jatropha mollissima (Pohl) Baill. (pinhão-bravo, Euphorbiaceae), coletada no Município de Tauá, Ceará, Nordeste Brasileiro

RESUMO A cada dia, cepas bacterianas estão tornando-se resistentes a diversos antibióticos, o que faz necessária a busca de novas substâncias eficazes para o tratamento de doenças. Desta forma, este trabalho reporta o estudo preliminar toxicológico, antibacteriano e fitoquímico do extrato etanólico das folhas de Jatropha mollissima (pinhão-bravo, Euphorbiaceae), coletada no Município de Tauá, Ceará, Nordeste Brasileiro. Inicialmente, realizou-se o teste de toxicidade do extrato contra Artemia salina. Na sequencia, foi realizado o ensaio antibacteriano contra quatro cepas bacterianas Gram-negativas (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Hafnia alvei ATCC 51873, Klebsiella pneumoniae ATCC 13883) e uma cepa Gram-positiva (Enterococcus faecalis ATCC 29212). Finalmente, fez-se a análise fitoquímica preliminar do extrato ativo para detecção das principais classes de metabólitos especiais. Como resultado, o extrato etanólico das folhas de J. mollissima se mostrou tóxico para Artemia salina, pois apresentou CL50 igual a 406,02 μg/mL. Quanto à ação antibacteriana, o extrato se mostrou ativo contra a bactéria Gram-positiva Enterococcus faecalis ATCC 29212, apresentando moderada atividade antibacteriana (halo de inibição igual a 7,03 mm). Evidenciou-se no extrato bioativo a presença de cumarinas, fenóis, taninos, flavonoides (flavonóis e flavanonas), alcaloides e esteroides, ambas as classes reportadas como antimicrobianos. Portanto, esse extrato tem potencial para ser usado na produção de fármacos contra infecções causadas por bactérias Gram-positivas. No entanto, as informações direcionam estudos futuros para o isolamento e identificação dos compostos bioativos, monitorados sob a ação antibacteriana mais expressiva