Caracterização molecular de dois isolados brasileiros de Lettuce mosaic virus apresentando propriedades biológicas distintas

Efetuou-se a clonagem e seqüenciamento do gene que codifica a proteína capsidial de dois isolados do vírus do mosaico da alface (Lettuce mosaic virus, LMV) provenientes do estado de São Paulo, previamente caracterizados como pertencentes aos patótipos II (AF198, incapaz de infetar cultivares com os genes de resistência mo1¹ ou mo1²) e IV (AF199, capaz de quebrar a resistência propiciada pelos genes mo1¹ e mo1²), com base na virulência em cultivares diferenciadoras. Análise comparativa das seqüências de nucleotídeos de isolados provenientes da Europa, América do Norte, Oriente Médio e os dois isolados brasileiros não permitiu sua separação em estirpes, pois as porcentagens de homologia foram sempre superiores a 95%. Entretanto, análise filogenética dos isolados sugere uma origem comum entre o isolado AF-198 e os isolados LMV-R e LMV-0 (patótipo II, provenientes dos Estados Unidos e da França, respectivamente). O isolado AF199 apresentou uma alta homologia de seqüência com os isolados LMV-Aud e LMV-13, ambos provenientes da França. Esses isolados também são relacionados a isolados provenientes do Chile, embora uma origem comum não seja proposta. Eventos independentes de mutação podem estar ocorrendo em diferentes partes do mundo, propiciando o surgimento de novas estirpes de LMV capazes de quebrar a resistência conferida pelos genes mo1¹ e mo1².

Quantitative control of Lettuce mosaic virus fitness and host defence inhibition by P1-HCPro

Two Lettuce mosaic virus isolates capable of overcoming the resistance afforded by the resistance gene mo1² in lettuce, LMV-AF199 from Brazil, and LMV-E, an European isolate, were evaluated for the rapidity and severity of symptoms induced on the lettuce variety Salinas 88 (mo1²). The mosaic symptoms on Salinas 88 plants inoculated with LMV-AF199 appeared 7 days post-inoculation (dpi) and 15 dpi for LMV-E. The symptoms induced by LMV-AF199 in this cultivar were also more severe than those induced by LMV-E. In order to identify the region of the viral genome responsible for this phenotype, recombinant viruses were constructed between these isolates and the phenotype of each recombinant was analysed. The region encoding proteins P1 and HcPro from LMV-AF199 was associated with the increased virulence in Salinas 88.

Frequência de Lettuce big-vein associated virus e Mirafiori lettuce big-vein virus em Plantas de alface no Estado de São Paulo e transmissão através de extrato vegetal e solo

A presença de sintomas de 'big vein' ou engrossamento das nervuras em alface e a associação do Lettuce big-vein associated virus (LBVaV) e Mirafiori lettuce big-vein virus (MLBVV) foram verificadas por RT-PCR utilizando-se oligonucleotídeos específicos para ambos os vírus. Foram coletadas 366 plantas sintomáticas nas regiões de Bauru, Campinas e Mogi das Cruzes no estado de São Paulo nos meses de junho e setembro de 2004 e abril e julho de 2005, e 18 plantas assintomáticas na região de Mogi das Cruzes no mês de dezembro de 2004. Infecção mista do LBVaV e MLBVV foi detectada em 76,2% das plantas sintomáticas, em 11,5% somente o MLBVV foi detectado e em 6,6% somente o LBVaV. Nas plantas assintomáticas coletadas em dezembro na região de Mogi das Cruzes, em áreas de alta incidência da doença durante o inverno, foi detectada a presença de MLBVV em 9 amostras e LBVaV em 7 amostras. Quatro dessas amostras apresentaram infecção mista, indicando que o desenvolvimento de sintomas depende de fatores abióticos, como temperatura. A inoculação via extrato vegetal a partir de plantas de alface com infecção mista pelo MLBVV e LBVaV foi realizada a 16°C/ 10°C (dia/noite) e fotoperíodo de 11 horas, verificando-se que o MLBVV foi transmitido para Nicotiana tabacum TNN, N. rustica, N. occidentalis, Chenopodium quinoa e para as cultivares de alface Trocadero e White Boston, enquanto o LBVaV foi transmitido apenas para a alface White Boston. Entretanto, nenhuma destas plantas apresentou sintomas da doença, com exceção de C. quinoa que apresentou sintomas de lesões locais. Plantio em solo proveniente de áreas com histórico da doença de Mogi das Cruzes permitiu a transmissão do LBVaV para alface cv. White Boston e do MLBVV para alface cv. Trocadero e White Boston, embora apenas a cv. White Boston tenha apresentado sintomas da doença. Em conjunto, estes resultados demonstram a dificuldade de transmissão de ambos os vírus, mesmo em condições de laboratório.

Prevalence of Lettuce mosaic virus - common strain on three lettuce producing areas from São Paulo State

LMV is one of the most important pathogens of lettuce worldwide. Based on their ability to overcome the resistance genes mo1¹ and mo1² in lettuce, isolates can be divided in two types: LMV-Most, which can infect and are seed-borne in cultivars containing the mo1 gene and LMV-Common, which do not cause symptoms on these cultivars and are seed transmitted only in susceptible cultivars. To evaluate the occurrence of these two types of LMV isolates, a survey was carried out during 2002-2005 in three lettuce production areas from São Paulo State. Total RNA was used for the diagnosis of LMV isolates by RT-PCR using universal primers for the variable N-terminus of the capsid protein, in the 3' end of the genome. Positives samples were analyzed by a second RT-PCR using specifics primers for LMV-Most isolates designed to amplify a fragment from the central region (CI-VPg) of the genome. A total of 1362 samples showing mosaic symptoms were collected and 504 (37.29 %) were positives for LMV. On susceptible lettuce cultivars, LMV-Common was prevalent (77.3%). LMV-Most was found frequently associated with tolerant (mo1¹) lettuce cultivars. Susceptible cultivars correspond today for most of the area of lettuce production. So, despite the ability of LMV-Most isolates to overcome the resistance provided by the recessive mo1¹ gene, they are not prevalent in the conditions of São Paulo State.

Sawfly larval poisoning in cattle: Report on new outbreaks and brief review of the literature

Sawfly larval poisoning (SLP) is an acute hepatotoxicosis documented in livestock in Australia, Denmark and in countries of South America. It is caused by the ingestion of the larval stage of insects of the suborder Symphyta, order Hymenoptera, commonly known as "sawfly". Three species of sawfly are reportedly involved in the toxicosis. The insect involved in Australian SLP is Lophyrotoma interrupta (Pergidae), in Denmark the cause of SLP is the ingestion of the larvae Arge pullata (Argidae), and in South American countries documented outbreaks of SLP were caused by the ingestion of yet another sawfly, Perreyia flavipes (Pergidae). In all geographical areas where it occurred, SLP causes important livestock losses. In cattle, as well as in other affected species, the disease has a short clinical course and in many outbreaks affected cattle can be found dead. When observed, clinical signs include apathy, recumbence, tremors, paddling movements and death in 24-48 hours. Neurological signs such aggressiveness attributable to hepatic encephalopathy are also observed. In cases with a more protracted course icterus and photodermatitis may develop. Gross findings included ascites, petechiae and ecchymosis over serosal surfaces of thoracic and abdominal cavities, and an enlarged liver that displays accentuation of the lobular pattern and edema of the gall bladder wall. Sawfly larval body fragments and heads are consistently found in the fore stomachs and occasionally abomasum of affected cattle. Main microscopic lesions are restricted to the liver and consist of centrolobular (periacinar) to massive hepatocellular necrosis. In most lobules necrotic areas extended up to the portal triads where only a few viable hepatocytes remain. Mild to moderate lymphocyte necrosis is seen in lymphatic tissues. Cases occur in the winter months when the larval stages of the sawfly are developing. D-amino acid-containing peptides have been found to be the toxic principle in each sawfly involved in SLP. The octapeptide lophyrotomin is the major toxin in the in the larvae of Australian and Danish sawflies and is present in small amounts in the larvae of South American sawfly. The heptadecapeptide pergidin is the main toxin in the South American sawfly while small amounts of pergidin have been found in the other two species of toxic sawfly. During the winter of 2011 (July-August) four outbreaks of SLP were diagnosed in the State of Rio Grande do Sul, Brazil. The findings in those outbreaks are reported here and a brief review of the literature regarding SLP around the world is provided.

Seneciosis in cattle associated with photosensitization

Senecio spp. poisoning is the main cause of cattle mortality in the central region of Rio Grande do Sul. This paper reports an outbreak of seneciosis in cattle with high prevalence of photosensitization, where 83 out of 162 cows (51.3%) presented this clinical sign. The outbreak occurred in September 2013, affecting adult cows that were held in a 205 hectare-pasture from April to October 2013 with abundant Senecio brasiliensis infestation. Main clinical signs were weight loss, excessive lacrimation or mucopurulent ocular discharge, nasal serous discharge, ventral diphteric glossitis, crusts in the nose, teats, dorsum of ears, and vulva. Liver biopsy was performed in all the cows under risk; the histopathological findings in the liver biopsies consisted of fibrosis, megalocytosis, and biliary ductal proliferation and were present in 73.4% of the biopsied animals. Six cows had increased serum activity of gamma glutamyl transferase. Three affected cows were necropsied. The main necropsy findings were a hard liver, distended gall bladder, edema of the mesentery and abomasum. Liver histological changes in the necropsied cows were similar to those of the biopsied livers. Spongiosis was detected in the brain of necropsied cows and is characteristic of hepatic encephalopathy.

Anatomy and ontogenesis of hymenopteran leaf galls of Struthanthus vulgaris Mart. (Loranthaceae)

Leaves of Struthanthus vulgaris Mart. (Loranthaceae) exhibit galls induced by a Hymenoptera. These galls pass through five developmental stages. In the first stage, a small brown swelling is observed on the surface of the leaf. Internally, the chlorenchyma cells around the eggs of the gall-makers are divided. In the second stage, the gall enlarges and its surface assumes a wavy appearance with a depressed region in its center. Within this depression, an incompletely divided gall chamber with embryos is observed. Neoformed parenchyma is present around the chamber and the secondary walls of fibers and sclereids are no longer observed. The vascular parenchyma shows hyperplasia. In the third stage, the gall grows larger and adopts an ellipsoidal shape. Fissures appear on the gall epidermis and the neoformed parenchyma is conspicuous, with a cortical and a medullar region. In the medullar region, each gall chamber, with one inducer in larval phase, is lined with 1-2 layers of nutritive tissue. The gall is larger still at the fourth stage of development and a periderm coats most of the gall. New vascular bundles, sclereids, and fibers are formed. The gall-makers are in advanced larval phase and no nutritive tissue cells are observed. In the fifth stage, the gall reaches its definitive size and the inducers are in the pupa phase. At this stage, the cortical region undergoes slight hypertrophy. The senescent gall shows the orifices of the exit channel made by the adult gallmakers. The anatomical studies of the hymenopteran gall enabled to compare this gall with a dipteran one, previously discribed in the same plant host. It is suggested that during the maturation of the gall, specific key processes are triggered, which bring about a specific cecidogenesis.

Anatomical and developmental aspects of leaf galls induced by Schizomyia macrocapillata Maia (Diptera: Cecidomyiidae) on Bauhinia brevipes Vogel (Fabaceae)

Samples of healthy leaves and galls induced by Schizomyia macrocapillata Maia on Bauhinia brevipes Vogel were submitted to routine techniques to investigate gall anatomy and development. Pouch galls are induced on the abaxial surface of unfolded immature leaves, and become spheroid with long reddish hairs covering their external surface. Galls occur isolated or coalesce when in larger numbers. Gall development was divided into six phases: 1) initiation; 2) tissue re-arrangement; 3) tissue differentiation; 4) maturation; 5) growth phase; and 6) dehiscence. This last phase corresponds to gall senescence, which takes place just after the larva exits the chamber to pupate. An important developmental phase of tissue reorientation was recorded after the initiation phase. The presence of hyphae close to the covering layer characterizes this gall as an ambrosia gall and the feeding mode of the gall migde is discussed. Few hyphae were found during the first developmental phases and fungi may play an important role during gall morphogenesis. Neoformed trichomes may provide not only photoprotection but also protection against natural enemies and water loss. The neoformation of phloematic bundles suggests host manipulation and indicates the establishment of a deviating sink.

Morphological, anatomical and biochemical studies on the foliar galls of Alstonia scholaris (Apocynaceae)

Morphological, anatomical and biochemical alterations in foliar galls of Alstonia scholaris R. Br. induced by the insect Pauropsylla tuberculata (Psyllidae) are described and quantified. Galls occur isolated or agglomerated on the abaxial surface of the leaf. The insect along with the egg deposits some physiologic fluid which act as a stimulant for the induction of the gall. This stimulus brings about hypertrophy followed by hyperplasia of cells next to the location of the deposited eggs. The psyllid presents three nymphal instars, from eclosion of the egg to the adult. Hyperplasia in the palisade cells is very distinctly noticed. Hypertrophy followed by hyperplasia takes place and brings about elevation of hypodermal and palisade parenchyma which undergoes repeated anticlinal divisions. Neoformation of phloematic bundles were distinctly noticed close to the site of infection. With an increase in the growth of the gall, chlorophyll content in the gall tissue decreases. A steady increase of sugar content is noticed. The immature galled tissue showed almost two fold increases in the protein content. The mature galled tissue showed a very high increase in the proline content compared to the immature galled tissue indicating a stressed condition of the galled tissue.

The radioprotective effect of a new aminothiol (20-PRA)

We examined the radioprotective effect of aminothiol 2-N-propylamine-cyclo-hexanethiol (20-PRA) on a human leukemic cell line (K562) following various radiation doses (5, 7.5 and 20 Gy) using a source of 60Co g-rays. At 5 Gy and 1 nM 20-PRA, a substantial protective effect (58%) was seen 24 h after irradiation, followed by a decrease at 48 h (11%). At the high radiation dose (20 Gy) a low protective effect was also seen (35%). In addition, the antitumorigenic potential of 10 nM 20-PRA was shown by the inhibition of crown gall formation induced by Agrobacterium tumefaciens. The radioprotective potency of 20-PRA is 105-106 times higher than that of the aminothiol WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) whose protective effect is in the 0.1 to 1.0 mM range.

Huitlacoche yield in some maize varieties in the Mediterranean region of Turkey

Huitlacoche is the Aztecs name given to the smut galls on ears of maize caused by the pathogenic plant fungus Ustilago maydis [(DC) Corda.)]. It is known as maize mushroom, and it has been considered a delicacy and in Mesoamerica. The aim of the present study was to determine the responses of some maize varieties to the growth of the fungus in order to evaluate the prospect production of these smutty ears as a maize mushroom. A 2-year study was conducted in the Mediterranean region of Turkey in 2010 and 2011. Inoculations were performed by injecting inoculum into the ear through the silk channel of plants in plots. Each treatment had control plots. Average ear-gall (huitlacoche) severity and incidence of all the varieties were at the rates of 4.0 and 41.6%, respectively. However, the highest severity of ear-gall (6.5) and incidence (60.6%) were found in Karadeniz Yıldızı flint maize variety; colossal smutty ears were formed in the maize cultivars. This study showed that certain maize cultivars (flint corn and dent corn) can be used efficiently in the production of huitlacoche.

Food webs in the era of molecular revolution – Like resolving the Gordian knot with a tricorder

Living nature consists of countless organisms, which are classified into millions of species. These species interact in many ways; for example predators when foraging on their prey, insect larvae consuming plants, and pathogenic bacteria drifting into humans. In addition, abiotic nature has a great initiative impact on life through many factors (including sunlight, ambient temperature, and water. In my thesis, I have studied interactions among different life forms in multifaceted ways. The webs of these interactions are commonly referred to as food webs, describing feeding relationships between species or energy transfer from one trophic level to another. These ecological interactions – whether they occur between species, between individuals, or between microorganisms within an individual – are among the greatest forces affecting natural communities. Relationships are tightly related to biological diversity, that is, species richness and abundances. A species is called a node in food web vocabulary, and its interactions to other species are called links. Generally, Artic food webs are considered to be loosely linked, simple structures. This conception roots into early modern food webs, where insects and other arthropods, for example, were clumped under one node. However, it has been shown that arthropods form the greatest part of diversity and biomass both in the tropics and in Arctic areas. Earlier challenges of revealing the role of insects and microorganisms in interactions webs have become possible with the help of recent advances in molecular techniques. In the first chapter, I studied the prey diversity of a common bat, Myotis daubentonii, in southwestern Finland. My results proved M. daubentonii being a versatile predator whose diet mainly consists of aquatic insects, such as chironomid midges. In the second chapter, I expanded the view to changes in seasonal and individual-based variation in the diet of M. daubentonii including the relationship between available and observed prey. I found out that chironomids remain the major prey group even though their abundance decreases in proportion to other insect groups. Diet varied a lot between individuals, although the differences were not statistically significant. The third chapter took the study to a large network in Greenland. I showed that Artic food webs are very complex when arthropods are taken into account. In the fourth chapter, I examined the bacterial flora of M. daubentonii and surveyed the zoonotic potential of these bacteria. I found Bartonella bacteria, of which one was described as a new species named after the locality of discovery. I have shown in my thesis that Myotis daubentonii as a predator links many insect species as well as terrestrial and aquatic environments. Moreover, I have exposed that Arctic food webs are complex structures comprising of many densely linked species. Finally, I demonstrated that the bacterial flora of bats includes several previously unknown species, some of which could possibly turn in to zoonosis. To summarize, molecular methods have untied several knots in biological research. I hope that this kind of increasing knowledge of the surrounding nature makes us further value all the life forms on earth.

Meloidogne incognita (nematode) parasitism of Lycopersicon esculentum (tomato) plants : Ethylene action in susceptible and resistant host responses

Involvement of ethylene in the etiology of tomato plants (Lycopersicon esculentum) infected with the root-knot nematode (Meloidogyne incognita) was investigated. Endogenous root concentrations of ethylene were not significantly different in uninfected resistant var. Anahu and susceptible var. Vendor plants. Exposure of resistant plants to high doses of infectious nematode larvae did not affect root ethylene concentrations during the subsequent 30 day period. The possibility that ethylene may be involved in the mechanism of resistance is therefore not supported by these experiments. In no experiments did ethylene concentrations in roots of susceptible plants increase significantly subsequent to ~ incognita infestation. This result is not consistent with the hypothesis in the literature which suggests that increased ethylene production accompanies gall formation. Growth of susceptible tomato plants was affected by ~ incognita infestation such that root weights increased (due to galling), stem heights decreased and top weights increased. The possibility that alterations in stem growth resulted from increased production of 'stress' ethylene is discussed. Growth of resistant plants was unaffected by exposure to high doses of ~ incognita and galls were never detected on the roots of these plants. Root ethane concentrations generally varied in parallel with root ethylene concentrations although ethane concentrations were without exception greater. In 4 of 6 experiments conducted ethane/ethylene ratios increased significantly with time. These results are discussed in the light of published data on the relationship between ethane and ethylene synthesis. The term infested is used throughout this thesis in reference to plants whose root systems had been exposed to nematodes and does not distinguish between the susceptible and resistant response.