João Paulo Rodrigues Marques

Lesões foliares e de ramos de laranjeira-doce causadas pela leprose-dos-citros

The objective of this work was to present the anatomical description of tissues lesioned by Citrus leprosis virus cytoplasmatic type (CiLV-C), in leaves and twigs of Pera sweet orange (Citrus sinensis (L.) Osbeck) providing new data to understand the interactions between the virus and the plant host, in Brazil. Leprosis lesions were cut on a rotary microtome (5-7 µm thick), stained and mounted in synthetic resin. For scanning electron microscope analysis, fixed tissues were immersed in glycerin 30% and criofractured, dehydrated in a graded ethylic series, critical point drying and coated with gold. Foliar lesions began as necrotic points surrounded by a yellow halo that inhibited the lesion expansion. The twigs presented two different lesions: depressed ones and pustules with ruptures. There was hyperplasia of the cortical and phloematic parenchyma cells causing the obliteration of phloematic vessels. These histological alterations of the phloem tissue could explain why the virus does not become systemic even occasionally infecting phloem parenchyma cells. The development of gummosis traumatic ducts in twig lesions was described here.

Histopathology of postbloom fruit drop caused by Colletotrichum acutatum in citrus flowers

Postbloom fruit drop (PFD) is caused by both Colletotrichum acutatum and C. gloeosporioides and is a potentially serious disease in citrus that occurs when flowering coincides with rainfall. The fungus incites necrotic lesions in petals and stigmas leading to premature fruit drop and reduced yield. The mechanisms of infection and survival of the causal agents remain to be fully elucidated. In the present study, we investigated the histopathology of PFD caused by C. acutatum in the petals and stigmas of sweet oranges using electron and light microscopy. In the petals, pathogen penetration occurred intra and intercellularly and also through the stomata, with intercellular penetration occurring most frequently. The distinct tissues of the petals were colonised, including the vascular system, particularly the xylem. Acervuli were observed on both sides of the petals. Although the fungus did not penetrate through the epidermal cells of the stigma, C. acutatum caused necrosis and an increase of phenolics in this tissue. A protective layer rich in lipophilic and phenolic compounds was formed under the necrotic area and crystals of oxalate were associated with the sites where the pathogen was present.

Comparative morpho-anatomical studies of the lesions caused by citrus leprosis virus on sweet orange.

The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck Pêra. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 microm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.

Structural and biochemical characteristics of citrus flowers associated with defence against a fungal pathogen

The constitutive characters of plants can be structural or biochemical and play an important role in their defense against pathogens. We found that Citrus 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 are reported in Citrus sinensis. Our study shows that natural terpenes of Citrus flowers present a potential use for chemical control of Colletotrichum spp.

Histopathology of black spot symptoms in sweet oranges

In Brazil, citrus black spot (CBS) caused by Guignardia citricarpa is a major disease that has different symptoms on fruit. In this study, fruit of Citrus sinensis infected by G. citricarpa and showing the symptoms false melanosis, freckle spot and hard spot were cross-sectioned and analysed anatomically and histochemically by light microscopy. Immuno-histological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False melanosis lesions did not contain pycnidia and remained restricted to the epicarp or to the first layers of the mesocarp. The stomata in this type of lesion showed phenolic compounds in the guard cells and in the sub-stomatal chamber. In some samples, the guard cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot and hard spot lesions had very similar histological alterations to the epicarp and mesocarp, but in our samples only hard spot lesions contained pycnidia. Both of these symptoms were accompanied by protein inclusions. Epidermal and sub-epidermal cells located in the oil-gland region were obliterated, causing alterations in these structures. All symptoms had regions that stained strongly for lipids and phenols.

Infection of citrus pollen grains by Colletotrichum acutatum

Postbloom fruit drop (PFD), an important disease caused by Colletotrichum spp., affects citrus yields in Brazil. PFD is characterised by the presence of necrotic lesions on the petals and stigmas of citrus flowers and by the subsequent abscission of young fruit. PFD epidemics have high disease progress rates, which is unusual for a pathogen that produces acervuli and is dispersed by rain. It is possible that other dispersal agents, such as insects and pollen, are involved in the spread of this disease. The objective of this work was to test whether citrus pollen grains can be colonised by Colletotrichum acutatum. Studies using light and electron microscopy showed that the pollen of Citrus sinensis can be infected by C. acutatum. This pathogen can penetrate and colonise citrus pollen grains 24xa0h after inoculation with the pathogen. The germ tube of conidia either penetrates the pollen sporodermis directly or passes through pollen germ pores. A single hypha can colonise more than one pollen grain. On the surface of the stigma, conidium formation can be observed. This study shows that Citrus sinensis pollen may, in fact, play a role in the spread of C. acutatum in citrus orchards.

Sugarcane smut: shedding light on the development of the whip-shaped sorus

Background and Aims Sugarcane smut is caused by the fungus Sporisorium scitamineum (Ustilaginales/Ustilaginomycotina/Basidiomycota), which is responsible for losses in sugarcane production worldwide. Infected plants show a profound metabolic modification resulting in the development of a whip‐shaped structure (sorus) composed of a mixture of plant tissues and fungal hyphae. Within this structure, ustilospores develop and disseminate the disease. Despite the importance of this disease, a detailed histopathological analysis of the plant‐pathogen interaction is lacking. Methods The whip‐shaped sorus was investigated using light microscopy, scanning and transmission electron microscopy, histochemical tests and epifluorescence microscopy coupled with deconvolution. Key Results Sorus growth is mediated by intercalary meristem activity at the base of the sorus, where the fungus causes partial host cell wall degradation and formation of intercellular spaces. Sporogenesis in S. scitamineum is thallic, with ustilospore initials in intercalary or terminal positions, and mostly restricted to the base of the sorus. Ustilospore maturation is centrifugal in relation to the ground parenchyma and occurs throughout the sorus median region. At the apex of the sorus, the fungus produces sterile cells and promotes host cell detachment. Hyphae are present throughout the central axis of the sorus (columella). The plant cell produces callose around the intracellular hyphae as well as inside the papillae at the infection site. Conclusions The ontogeny of the whip‐shaped sorus suggests that the fungus can cause the acropetal growth in the intercalary meristem. The sporogenesis of S. scitamineum was described in detail, demonstrating that the spores are formed exclusively at the base of the whip. Light was also shed on the nature of the sterile cells. The presence of the fungus alters the host cell wall composition, promotes its degradation and causes the release of some peripheral cells of the sorus. Finally, callose was observed around fungal hyphae in infected cells, suggesting that deposition of callose by the host may act as a structural response to fungal infection.

Anatomia de lesões foliares causadas pelo vírus da Mancha Clorótica do Clerodendrum, transmitido pelo ácaro Brevipalpus phoenicis em diferentes espécies

O genero botânico Clerodendrum pertence a familia Lamiaceae e compreende varias especies ornamentais, Manchas cloroticas e necroticas em folhas de coracao-sangrento foram observadas pela primeira vez em um jardim de Piracicaba, SP, associadas a infestacao com Brevipalpus phoenicis (Acari: Tenuipalpidae). Exames de seccoes de tecidos das lesoes foliares ao microscopio eletronico revelaram ocorrencia de efeitos citopaticos do tipo nuclear e concluiu-se que os sintomas eram causados por um virus transmitido por Brevipalpus (VTB), o qual foi designado de mancha clorotica de Clerodendrum (Clerodendrum Chlorotic Spot Virus- ClCSV). O ClCSV e transmitido mecanicamente de coracao-sangrento para coracao-sangrento. Em ensaios preliminares foi transmitido por B. phoenicis e mecanicamente para varias outras plantas, alem da ocorrencia de sua disseminacao natural por este acaro para outras especies. Visando complementar a caracterizacao do ClCSV foram feitos estudos sobre alteracoes anatomicas em folhas de plantas infectadas pelo ClCSV. Foram examinadas seccoes histologicas de folhas sadias e infectadas pelo ClCSV de C. x speciosum e de outras hospedeiras como Hibiscus schizopetalus, Salvia leucantha, Malvaviscus arboreus e Annona muricata. Constatou-se que o ClCSV causa alteracoes celulares semelhantes nas diferentes hospedeiras e os sintomas causados por este virus sao similares aos causados por outros virus transmitidos por Brevipalpus como o virus da leprose dos citros citoplasmatico (Citrus Lepros Virus Cytoplasmic- CiLV-C) e nuclear (Citrus Leprosis Virus Nuclear- CiLV-N), mancha anular do cafeeiro (Coffee Ringspot Virus- CoRSV), mancha anular de Solanum violaefolium (Solanum violaefolium Ringspot Virus- SvRSV) e Orchid Fleck Virus (OFV), representadas por hipertrofia e hiperplasia frequentemente acompanhadas de necrose nos tecidos do parenquima palicadico e lacunoso.

Sugarcane cell wall-associated defense responses to infection by Sporisorium scitamineum

The plant cell wall is known to be the first barrier against plant pathogens. Detailed information about sugarcane cell wall-associated defense responses to infection by the causal agent of smut, Sporisorium scitamineum, is scarce. Herein, (immuno)histochemical analysis of two smut resistant and two susceptible sugarcane cultivars was conducted to understand host cell wall structural and compositional modifications in response to fungal infection. Results showed that the fungus grew on the surface and infected the outermost bud scale of both susceptible and resistant cultivars. The present findings also supported the existence of early (24 h after inoculation) and later (72–96 h after inoculation) inducible histopathological responses related to the cell wall modification in resistant cultivars. Lignin and phenolic compounds accumulated during early stages of infection. Later infection response was characterized by the formation of a protective barrier layer with lignin, cellulose and arabinoxylan in the cell walls. Overall, the results suggest possible induction of cell wall-modified responses in smut resistant cultivars to prevent initial entry of the fungus into the meristematic tissues.

Ultrastructural changes in the epidermis of petals of the sweet orange infected by Colletotrichum acutatum

Postbloom fruit drop (PFD) is an important disease caused by the fungus Colletotrichum acutatum. PFD is characterised by the formation of necrotic lesions on the petals and stigmas of flowers as well as premature abscission of the fruit in Citrus spp. We compare the ultrastructure of the epidermis of uninoculated Citrus sinensis petals with that of petals inoculated with the fungus to understand the changes that occur upon C. acutatum infection. Healthy petals have a cuticle with parallel striations covering the uniseriate epidermis. This pattern consists of vacuolated parietal cells whose cytoplasm contains mitochondria, plastids with an undeveloped endomembrane system and a slightly dense stroma, a poorly developed rough endoplasmic reticulum, polysomes, few lipid droplets, and a nucleus positioned near the inner periclinal wall. In damaged regions, the cytoplasm of some cells is densely packed with well-developed endoplasmic reticulum, a large number of hyperactive dictyosomes, numerous mitochondria, and many lipid droplets. The plastids have an electron-dense stroma, starch grains, and a large amount of electron-dense lipid droplets, which can be released into vacuoles or the endoplasmic reticulum. Multivesicular bodies and myelin bodies are frequently observed in the vacuole, cytoplasm, and periplasmic space. Vesicles migrate through the cell wall and are involved in the deposition of cuticular material. In the later stages of infection, there is deposition of new cuticle layers in plaques. The outer periclinal walls can be thick. These observations indicate that epidermal cells respond to the pathogen, resulting in cuticular and parietal changes, which may limit further infection.