Espécies de Bacillus no controle de Meloidogyne incógnita e Meloidogyne javanica in vitro e na cana-de-açúcar

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Reação de genótipos de berinjela ao nematóide-das-galhas(Meloidogyne spp).

Este trabalho teve por objetivo identificar possíveis fontes de resistência ao mematoide-das-galhas em genótipos de berinjela.

Agronomic performance of selected sweet potato cultivars under greenhouse and field infested with meloigogyne incognita and soilborne insect pests.

The root knot nematode (RKN), Meloidogyne incognita, is widespread worldwide and a major pathogen of several cultivated crops. The use of resistant genotypes is the most effective and environmentally sound way to manage RKN. In this study, we screened 16 selected sweet potato cultivars including Amanda, Bárbara, Beatriz, Beauregard, Brazlândia Branca, Brazlândia Rosada, Brazlândia Roxa, BRS Amélia, BRS Cuia, BRS Rubissol, Carolina Vitória, Duda, Júlia, Marcela, PA-26/2009, and Princesa obtained from Embrapa and Universidade Federal do Tocantins? germplasm bank. Studies were conducted under greenhouse and field conditions and the agronomic performance of the cultivars was evaluated in a nematode and soilborne insect-infested field. All 16 sweet potato cultivars tested were rated as resistant to this nematode both under greenhouse and field conditions with reproduction factors < 1. In the field infested with M. incognita, sweet potato cultivars Duda, BRS Amélia, Beauregard, Brazlândia Rosada, and Brazlândia Roxa stood out as superior cultivars, with average yield ranging from 26 to 47 tons per ha. Overall, most cultivars exhibited a fusiform to near fusiform root shape, a good characteristic for the market, and were moderately affected by insects (attack incidence 1 to 30%). As global demand for energy continues to rise, selecting new cultivars of sweet potatoes with increased resistance to nematode diseases and with high yield will be important for food security and biofuel production.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Efeito da solarizacao sobre a producao de tomate de mesa e de industria em uma estufa modelo capela.

A tecnologia de controle de nematóides nos solos de estufas, em uso, envolvem o uso de processos físicos(vapor), físicos/químicos e químicos que são onerosos, insalubres, de difíceis manuseio e poluidores do meio ambiente. Esta pesquisa tem por objetivo adaptar o processo de solarização de solos, para as condições de plantio protegido de tomate do DF. Os testes com 6 cultivares desenvolvidas em áreas infestadas como Meloidogyne incognita raça 1 e Meloidogyne javanica e com solarização evidenciaram a eficácia da tecnologia de solarização no controle de nematóides de galhas.

Manual de identificação de doenças de soja.

Doenças causadas por fungos: Antracnose (Colletotrichum truncatum), Cancro da haste (Diaporthe phaseolorum var. meridionalis e D. phaseolorum var. caulivora), Crestamento foliar de cercóspora e mancha púrpura (Cercospora kikuchii), Ferrugem (Phakopsora pachyrhizi e P. meibomiae), Mancha alvo e podridão radicular de corinéspora (Corynespora cassiicola), Mancha foliar de ascoquita (Ascochyta sojae), Mancha foliar de mirotécio (Myrothecium roridum), Mancha olho-de-rã (Cercospora sojina), Mancha parda (Septoria glycines), Mela ou requeima (Rhizoctonia solani AG1), Míldio (Peronospora manshurica), Tombamento e morte em reboleira de rizoctonia (Rhizoctonia solani), Tombamento e murcha de esclerócio (Sclerotium rolfsii), Oídio (Erysiphe diffusa), Podridão branca da haste (Sclerotinia sclerotiorum), Podridão de carvão da raiz (Macrophomina phaseolina), Podridão parda da haste (Cadophora gregata), Podridão radicular de roselínia (Rosellinia necatrix), Seca da haste e da vagem (Phomopsis spp.), Podridão radicular de fitóftora (Phytophthora sojae), Podridão vermelha da raiz (Fusarium spp.). Doenças causadas por bactérias: Crestamento bacteriano (Pseudomonas savastanoi pv. glycinea), Fogo Selvagem (Pseudomonas syringae pv. tabaci), Pústula bacteriana (Xanthomonas axonopodis pv. glycines). Doenças causadas por vírus: Mosaico cálico (Alfalfa Mosaic Virus - AMV), Mosqueado do feijão (Bean Pod Mottle Virus - BPMV), Mosaico comum da soja (Soybean Mosaic Virus - SMV), Necrose da haste (Cowpea Mild Mottle Virus - CPMMV), Queima do broto (Tobacco Streak Virus - TSV). Doenças causadas por nematóides: Nematóide de cisto (Heterodera glycines), Nematóides de galhas (Meloidogyne incognita e M. javanica), Nematóide das lesões (Pratylenchus spp.), Nematóide reniforme (Rotylenchulus reniformis). Estádios de desenvolvimento da soja.

Doenças de soja: diagnose, epidemiologia e controle.

Página modelo; Simbologia empregada; Doenças causadas por fungos; Míldio da soja (Peronospora manshurica); Oídio da soja (Microsphaera diffusa); Ferrugem asiática (Phakopsora pachyrhizi); Mancha parda da folha (Septoria glycines); Mancha alvo (Corynespora cassiicola); Mancha olho-de-rã (Cercospora sojina); Mancha púrpura (Cercospora kikuchi); Seca da haste e da vagem (Phomopsis spp.); Antracnose (Colletotrichum truncatum); Cancro da haste (Phomopsis phaseoli f. sp. meridionalis); Podridão parda da haste (Phialophora gregata); Podridão vermelha da raiz (Fusarium solani); Mofo branco da haste (Sclerotinia sclerotiorum); Murcha de esclerotium (Sclerotium rolfsii); Podridão da raiz e da haste (Phytophthora megasperma f. sp. glycinea); Mela da folha (Rhizoctonia solani); Tombamento (Rhizoctonia solani); Morte em reboleira (Rhizoctonia solani); Roseliniose (Dematophora necatrix); Podridão negra da raiz (Macrophomina phaseolina); Doenças causadas por nematóides; Nematóide de cisto (Heterodera glycines); Nematóide de galha (Meloidogyne incognita); Doenças causadas por vírus; Mosaico comum da soja; Queima do broto; Doenças causadas por bactérias; Pústula bacteriana (Xanthomonas axonopodis pv. glycines); Fogo selvagem (Pseudomonas syringae pv. tabaci); Crestamento bacteriano (Pseudomonas savastonoi pv. glycinea); Microorganismos que frequentemente causam a morte das sementes a campo; Aspergillus spp.; Penicillium spp.; Bacillus subtilis; Créditos fotográficos; Estádios vegetativos da planta de soja; Estádios reprodutivos da planta de soja.

Doenças de soja: diagnose, epidemiologia e controle.

Mildio da soja (Peronospera manshurica); Oidio da soja (Microsphaera diffusa); Mancha parda da folha (Septoria glycines); Mancha alvo (Corynespora cassiicola); Mancha de alternaria (Alternaria spp.); Mancha olho-de-rã (Cercospora sojina); Mancha purpura (Cercospora kikuchii); Seca da haste e da vagem (Phomopsis spp.); Antracnose (Colletotrichum truncatum); Cancro da haste (Phomopsis phaseoli f. sp. meridionalis); Podridão parda da haste (Phialophora gregata); Podridão vermelha da raiz (Fusarium solani); Mofo branco da haste (Sclerotinia sclerotiorum); Murcha de esclerotium (Sclerotium rolfsii); Podridão da raiz e da haste (Phytophthora megasperma f. sp. glycinea); Mela da folha (Rhizoctonia solani); Tombamento (Rhizoctonia solani); Morte em reboleira (Rhizoctonia solani); Roseliniose (Dematophora necatrix); Podridão negra da raiz (Macrophomina phaseolina); Nematoide de cisto (Heterodera glycines); Nematoide de galha (Meloidogyne incognita); Mosaico comum da soja; Queima do broto; Pustula bacteriana (Xanthomonas campestris pv. glycines); Fogo selvagem (Pseudomonas syringae pv. tabaci); Crestamento bacteriano (Pseudomonas syringae pv. glycinea); Aspergillus spp.; Penicillium spp.; Bacillus subtilis; Créditos fotográficos; Estádios vegetativos da planta de soja; Estádios produtivos da planta de soja.

RNAi Effector Diversity in Nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.

Novel bioassays to examine the host-finding ability of plant-parasitic nematodes

We present two novel bioassays to be used in the examination of plant-parasitic nematode host-finding ability. The host-finding 'pipette-bulb assay' was constructed from modelled Pasteur pipette bulbs and connecting barrels using parafilm fastenings. This assay examines the direction of second-stage juvenile (J2) migration in response to a host seedling, through a moistened sand substrate, which underlies terminal upward-facing 'seedling bulbs', one containing a host seedling in potting compost, the other with only potting compost. An equal watering regime through both upward-facing seedling bulbs creates a directional concentration gradient of host diffusate chemotactic factors. Positive chemotactic stimuli cause the J2 to orientate and migrate towards the host plant. We present validation data collected from assays of the root-knot nematode, Meloidogyne incognita, and the potato cyst nematode, Globodera pallida, which indicate a highly significant positive attraction of J2 of both species to respective host plants. This represents a simple, quick and inexpensive method of assessing host-finding behaviour in the laboratory. We consider that the pipette-bulb assay improves on previous host-finding/chemo-attraction assays through creating a more biologically relevant environment for experimental J2; analysis is quick and easy, allowing the straightforward interpretation of results. In addition, we have developed an 'agar trough' sensory assay variant which we believe can be used rapidly to ratify nematode responses to chemical gustatory or olfactory cues. This was constructed from a water agar substrate such that two counting wells were connected by a raised central trough, all flooded with water. Two small water agar plugs were dehydrated briefly in an oven and then hydrated in either an attractant, repellent or water control; these plugs were then placed in the terminal counting wells and subsequently leached the attractant or repellent to form a concentration gradient along the central trough, which contained the initial J2 innoculum. Our data show that both M. incognita and G. pallida J2 are positively attracted to host diffusates. In addition, they displayed a strong repulsion in response to 1 M NaCl2. J2 of M. incognita displayed a mild aversion to a non-host oak root diffusate, whereas G. pallida J2 displayed a strong aversion to the same non-host diffusate; neither species responded to a compost leachate. We believe that the agar trough assay improves on previous methods by facilitating rapid diffusion of attractant or repellents. Both of the aforementioned assays were designed as tools to assess the impact of RNAi-based reverse genetics screens for gene targets involved in chemosensory orientation.

FMRFamide-like peptides in root knot nematodes and their potential role in nematode physiology

FMRFamide-like peptides (FLPs) are a diverse group of neuropeptides that are expressed abundantly in nematodes. They exert potent physiological effects on locomotory, feeding and reproductive musculature and also act as neuromodulators. However, little is known about the specific expression patterns and functions of individual peptides. The current study employed rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to characterize flp genes from infective juveniles of the root knot nematodes, Meloidogyne incognita and Meloidogyne minor. The peptides identified from these transcripts are sequelogs of FLPs from the free-living nematode, Caenorhabditis elegans; the genes have therefore been designated as Mi-flp-1, Mi-flp-7, Mi-flp-12, Mm-flp-12 and Mi-flp-14. Mi-flp-1 encodes five FLPs with the common C-terminal moiety, NFLRFamide. Mi-flp-7 encodes two copies of APLDRSALVRFamide and APLDRAAMVRFamide and one copy of APFDRSSMVRFamide. Mi-flp-12 and Mm-flp-12 encode the novel peptide KNNKFEFIRFamide (a longer version of RNKFEFIRFamide found in C. elegans). Mi-flp-14 encodes a single copy of KHEYLRFamide (commonly known as AF2 and regarded as the most abundant nematode FLP), and a single copy of the novel peptide KHEFVRFamide. These FLPs share a high degree of conservation between Meloidogyne species and nematodes from other clades, including those of humans and animals, perhaps suggesting a common neurophysiological role which may be exploited by novel drugs. FLP immunoreactivity was observed for the first time in Meloidogyne, in the circumpharyngeal nerve ring, pharyngeal nerves and ventral nerve cord. Additionally, in situ hybridization revealed Mi-flp-12 expression in an RIR-like neuron and Mi-flp-14 expression in SMB-like neurons, respectively. These localizations imply physiological roles for FLP-12 and FLP-14 peptides, including locomotion and sensory perception.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.

Comparative effect of root-knot nematode on severity of Verticillium and Fusarium wilt in cotton

The effect of root-knot nematode (RKN) (Meloidogyne incognita) on Verticillium dahliae and Fusarium oxysporum f.sp. vasinfectum in cotton (Gossypium hirsutum) was investigated. Two different inoculation methods were used, one in which inoculum was added to the soil, so that nematode and fungal inoculum were in close proximity; the other, inoculation into the stem, whereby the two inocula were spatially separated. Invasion of the roots by RKN enhanced disease severity, as measured by the height of vascular browning in the stem, following inoculation with either wilt pathogen. The effect of RKN on Fusarium wilt was more pronounced than that on Verticillium wilt. Nematode-enhanced infection by F. oxysporum is a well known effect but there are few reports of enhanced infection by Verticillium due to RKN. Relative resistance of a number of cotton cultivars to both wilt diseases, as measured by height of vascular browning, was similar to the known field performance of the cultivars. The use of vascular browning as an estimate of disease severity was therefore validated.

Enhancement of germination of spores of Verticillium dahliae and Fusarium oxysporum esp vasinfectum in vascular fluid from cotton plants infected with the root-knot nematode

Root-knot nematode [RKN] (Meloidogyne incognita) can increase the severity of Verticillium (V dahliae) and Fusarium (F oxysporum f.sp. vasinfectum) wilt diseases in cotton (Gossypium hirsutum). This study was conducted to determine some of the physiological responses caused by nematode invasion that might decrease resistance to vascular wilt diseases. The effect of RKN was investigated on spore germination and protein, carbohydrate and peroxidase content in the xylem fluids extracted from nematode-infected plants. Two cotton cultivars were used with different levels of resistance to both of the wilt pathogens. Spore germination was greater in the xylem fluids from nematode-infected plants than from nematode-free plants. The effect on spore germination was greater in the Fusarium-resistant cultivar (51%). Analysis of these fluids showed a decrease in total protein and carbohydrate levels for both wilt-resistant cultivars, and an increase in peroxidase concentration. Fluids from nematode-free plants of the Verticillium-resistant cultivar contained 46% more peroxidase than the Fusarium-resistant cultivar. The results provide further evidence that the effect of RKN on vascular wilt resistance is systemic and not only local. Changes in metabolites in the xylem pass from the root to the stem, accelerating disease development.

A powder X-ray diffraction method for detection of polyprenylated benzophenones in plant extracts associated with HPLC for quantitative analysis

A robust, direct, rapid and non-destructive X-ray diffraction crystallography method to detect the polyprenylated benzophenones 7-epi-clusianone (1) and guttiferone A (2) in extracts from Garcinia brasiliensis is presented. Powder samples of benzophenones 1 and 2, dried hexane extracts from G. brasiliensis seeds and fruit`s pericarp, and the dried ethanolic extract from G. brasiliensis seeds were unambiguously characterized by powder X-ray diffractometry. The calculated X-ray diffraction peaks from crystal structures of analytes 1 and 2, previously determined by single-crystal X-ray diffraction technique, were overlaid to those of the experimental powder diffractograms, providing a practical identification of these compounds in the analyzed material and confirming the pure contents of the powder samples. Using the X-ray diffraction crystallography method, the studied polyprenylated benzophenones were selectively and simultaneously detected in the extracts which were mounted directly on sample holder. In addition, reference materials of the analytes were not required for analyses since the crystal structures of the compounds are known. High performance liquid chromatography analyses also were comparatively carried out to quantify the analytes in the same plant extracts showing to be in agreement with X-ray diffraction crystallography method. (C) 2010 Elsevier B.V. All rights reserved.