Members of Botryosphaeriaceae family are associated with serious diseases in different plants across the world. In cashew nut plants (Anacardium occidentale), the fungus Lasiodiplodia theobromae causes a severe group of symptoms related to gummosis that results in decreased nut production. The aim of this work was to develop an indirect enzyme-linked immunosorbent assay (ELISA) with sufficient sensitivity and specificity to detect the fungus both in vitro and in planta (artificially and naturally infected) and to increase the detection specificity within the fungi group using primers specific for the internal transcribed spacer (ITS) sequences. A collection of L. theobromae isolates was obtained, and antisera against the fungus were raised in rabbits. Cross-reactivity against Neofusicoccum sp., Colletotrichum gloeosporioides, Phomopsis anacardii and Pestalotiopsis guepinii was examined. Naturally and artificially infected vegetal material were employed in the ELISAs. The fungi ITS sequences were determined, and single nucleotide polymorphisms were identified and used for primer design. For the naturally infected plants, there was an approximately fourfold variation in the absorbance values. Some positive readings for asymptomatic samples were detected. For the artificially infected samples, an ELISA-based weekly timecourse analysis was conducted, and the values for samples from 0 and 7 days were lower than the threshold value. Beginning on day 14, the infection could be detected, with rates varying from 40% on day 14 to 80% on day 21 and 100% by the end of the experiment. The ITS sequencing revealed few polymorphisms among the L. theobromae isolates, but for C. gloeosporioides, P. anacardii, P. guepinii and Neofusicoccum sp., the sequences were sufficient to permit reliable discrimination. The feasibility of ELISA as an early detection technique to assist in gummosis management was demonstrated. PCR amplification based on ITS regions increases and complements serological specificity.
Tambaqui, Colossoma macropomum, is the main native freshwater fish in Brazilian aquaculture. Therefore, intensive research pressure has been applied to the species to support new technologies for tambaqui farming. Molecular biology represents a tool that can be used to investigate every field of applied biology, from fish physiology to the effects of climate change. Based on the importance of reference genes for the relative or absolute quantification of gene transcripts, we cloned and sequenced three candidate reference genes in tambaqui (18S ribossomal RNA - 18s, glyceraldehyde-3-phosphate dehydrogenase - gapdh, and actin beta - β-actin), and validated a set of primers for each gene for use in real-time quantitative PCR. The results were evaluated by RefFinder, which indicated that β-actin is the most suitable reference gene for tambaqui among those studied, followed by 18s.
ABSTRACT. Manioc, Manihot esculenta, is economically important in many tropical and subtropical countries. The genetic variability of the species has not been fully explored, and new information may help expand its use. Molecular markers based on retrotransposons have good potential for analysis of genetic diversity given their abundance in the genome. Eight long terminal repeat retrotransposons were selected for the development of interretrotransposon-amplified polymorphism markers. To test these primers, we analyzed 32 varieties from Anori, 30 from Manicoré and 10 Mandiocabas from the Manioc Germplasm Bank at Embrapa Western Amazonia. The six informative primer pairs yielded 20-60 polymorphic bands, averaging 92% polymorphism (51.7-98.4) and 0.37 heterozygosity (0.17 to 0.40), with a Shannon information index of 0.54 (0.26-0.59). These markers can be used to explore the genetic diversity of manioc.
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