RESUMEN

Over the last ten years, global raspberry production has increased by 47.89%, based mainly on the red raspberry species (Rubus idaeus). However, the black raspberry (Rubus occidentalis), although less consumed, is resistant to one of the most important diseases for the crop, the late leaf rust caused by Acculeastrum americanum fungus. In this context, genetic resistance is the most sustainable way to control the disease, mainly because there are no registered fungicides for late leaf rust in Brazil. Therefore, the aim was to understand the genetic architecture that controls resistance to late leaf rust in raspberries. For that, we used an interspecific multi-parental population using the species mentioned above as parents, two different statistical approaches to associate the phenotypes with markers (GWAS and copula graphical models), and two phenotyping methodologies from the first to the seventeenth Day After Inoculation (D.A.I.) (high-throughput phenotyping with a multispectral camera and traditional phenotyping by disease severity scores). Our findings indicate that a locus of higher effect, at position 13.3 Mb on chromosome five, possibly controls late leaf rust resistance, as both GWAS and the network suggested the same marker. Of the twelve genes flanking its region, four were possible receptors, three were likely defense executors, one gene was likely part of signaling cascades, and four were classified as non-defense related. Although the network and GWAS indicated the same higher effect genomic region, the network identified other different candidate regions, potentially complementing the genetic control comprehension.

RESUMEN

Late leaf rust is a fungal disease in raspberries caused by Aculeastrum americanum (Farl.) M. Scholler U. Braun (syn. Thekopsora americana (Farl.) Aime McTaggart) leading to early defoliation and yield losses. Red raspberries (Rubus idaeus L.) are susceptible to this pathogen, although this susceptibility varies among cultivars. In contrast, black raspberries were previously reported to be more resistant (Rubus occidentalis L.) and immune (Rubus niveus Thunb.) to this pathogen, raising their importance in plant breeding programs. However, what features make them respond differently to the same pathogen? In this study, we characterize for the first time the pre- and post-formed structural and biochemical defense mechanisms of R. idaeus cv. Autumn Bliss, R. occidentalis and R. niveus. Ultrastructural and histopathological analyses were used to uncover the interactions between these raspberries and A. americanum. The ultrastructural results indicate that the pathogen germinates on both leaf surfaces but can only form appressoria on the stomata. Although the three raspberry species were infected and colonized by A. americanum, a clear difference in susceptibility was observed between them. A compact mesophyll, pre- and post-formed phenolic compounds, and post-formed pectic compounds were the main plant defense mechanisms against fungal colonization. These findings provide new information about raspberries' defense mechanisms in response to A. americanum and elucidate the interactions occurring in these pathosystems.

RESUMEN

The constitutive characters of plants can be structural or biochemical and play an important role in their defence against pathogens. Citrus postbloom fruit drop (PFD) caused by Colletotrichum spp. is one of the most important fungal diseases of citrus. The pathogen infects the flowers, leading to premature fruit drop and reducing citrus production. However, flower buds smaller than 8 mm long are usually not infected by Colletotrichum spp. Thus, this study investigated whether there are constitutive mechanisms in flower buds related to Colletotrichum spp. infection. We studied flower buds that were 2, 3, 4, 8, 12 and 15 mm long and petals, after anthesis, of sweet orange 'Valência' using light and scanning electron microscopy and histochemistry. We evaluated the effect of volatile organic compounds (VOCs) in flowers (R-limonene and linalool) on the in vitro growth of Colletotrichum acutatum. We found that the arrangement of the epidermal papillae in the petal primordia, the occurrence of prismatic crystals and the distribution of oil glands are the main differences between buds smaller than 8 mm and buds 8-15 mm long. Osmophores at the tips of petals produced and accumulated phenols, terpenes and lipophilic compounds. Flower buds smaller than 8 mm long have constitutive structural and biochemical barriers to Colletotrichum spp. infection. In addition, this is the first time that osmophores have been reported in citrus. Our study shows that natural terpenes of Citrus flowers inhibit the fungal growth in vitro, highlighting the potential use of terpenes for the chemical control of PFD in citrus.

RESUMEN

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 µM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

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RESUMEN

The Alternaria brown spot (ABS) is a disease caused in tangerine plants and its hybrids by the fungus Alternaria alternata f. sp. citri which has been found in Brazil since 2001. Due to the recent occurrence in Brazilian orchards, the epidemiology and genetic variability of this pathogen is still an issue to be addressed. Here it is presented a survey about the genetic variability of this fungus by the characterization of twenty four pathogenic isolates of A. alternata f. sp. citri from citrus plants and four endophytic isolates from mango (one Alternaria tenuissima and three Alternaria arborescens). The application of two molecular markers Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) had revealed the isolates clustering in distinct groups when fingerprintings were analyzed by Principal Components Analysis (PCA). Despite the better assessment of the genetic variability through the AFLP, significant modifications in clusters components were not observed, and only slight shifts in the positioning of isolates LRS 39/3 and 25M were observed in PCA plots. Furthermore, in both analyses, only the isolates from lemon plants revealed to be clustered, differently from the absence of clustering for other hosts or plant tissues. Summarizing, both RAPD and AFLP analyses were both efficient to detect the genetic variability within the population of the pathogenic fungus Alternaria spp., supplying information on the genetic variability of this species as a basis for further studies aiming the disease control.

A mancha marrom ou mancha de Alternaria é uma doença causada pelo fungo Alternaria alternata f. sp. citri, encontrada no Brasil desde 2001 em plantas de tangerina e seus híbridos. Por se tratar de uma doença recente no Brasil, a epidemiologia e variabilidade genética deste patógeno compõem importantes pontos a serem estudados. Este trabalho teve como objetivo avaliar a variabilidade genética deste patógeno por meio da caracterização de vinte e quatro isolados de A. alternata f. sp. citri de plantas de citros juntamente com quatro isolados endofíticos de manga, sendo um deles identificado como Alternaria tenuissima e outros três como Alternaria arborescens. A análise de componentes principais (PCA) do perfil de bandas obtidos pela aplicação de duas técnicas de marcadores moleculares, Amplificação Aleatória de Polimorfismos de DNA (RAPD) e Polimorfismo de Comprimento de Fragmentos Amplificados (AFLP), mostrou a formação de quatro grupos distintos. Apesar do mais amplo perfil de análise por meio da técnica de AFLP, não foi observado nenhuma modificação significativa dentro dos grandes grupos obtidos quando comparado ao RAPD, exceto no posicionamento dos isolados LRS 39/3 e 25M. Além disso, em ambas as análises, somente os isolados de plantas de limão agruparam entre si. Considerando outros hospedeiros ou tecidos de planta não foi possível encontrar grupos específicos. Concluindo, ambas as análises (RAPD e AFLP) são eficientes no estudo de variabilidade genética de Alternaria spp., fornecendo informações sobre a diversidade genética desta espécie, servindo como base para futuramente correlacionar este estudo com estudos adicionais objetivando o controle da doença.

RESUMEN

The Alternaria brown spot (ABS) is a disease caused in tangerine plants and its hybrids by the fungus Alternaria alternata f. sp. citri which has been found in Brazil since 2001. Due to the recent occurrence in Brazilian orchards, the epidemiology and genetic variability of this pathogen is still an issue to be addressed. Here it is presented a survey about the genetic variability of this fungus by the characterization of twenty four pathogenic isolates of A. alternata f. sp. citri from citrus plants and four endophytic isolates from mango (one Alternaria tenuissima and three Alternaria arborescens). The application of two molecular markers Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) had revealed the isolates clustering in distinct groups when fingerprintings were analyzed by Principal Components Analysis (PCA). Despite the better assessment of the genetic variability through the AFLP, significant modifications in clusters components were not observed, and only slight shifts in the positioning of isolates LRS 39/3 and 25M were observed in PCA plots. Furthermore, in both analyses, only the isolates from lemon plants revealed to be clustered, differently from the absence of clustering for other hosts or plant tissues. Summarizing, both RAPD and AFLP analyses were both efficient to detect the genetic variability within the population of the pathogenic fungus Alternaria spp., supplying information on the genetic variability of this species as a basis for further studies aiming the disease control.