Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, 'Candidatus Liberibacter asiaticus', 'Candidatus Liberibacter africanus' and 'Candidatus Liberibacter americanus', is essential for the preventive control of the disease. Real-time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of 'Ca. Liberibacter' species was achieved by direct tissue-printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real-time PCR. This simplified method was validated and could detect HLB-liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB-infected trees. The use of direct assays as template showed good agreement with use of purified DNA (j = 0Á76 AE 0Á052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB-infected trees with or without symptoms. A commercial ready-made kit based on this technology showed 96% accuracy in intra-laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.

Sauer, A.V., Zanutto, C.A., Nocchi, P.T.R., Machado, M.A., Bock, C.H, and Nunes, W.M.C. 201X. Seasonal variation in populations of 'Candidatus Liberibacter asiaticus' in citrus trees in Paraná state, Brazil. Plant Disease XX: xx-xx.Huanglongbing (HLB) is considered one of the most destructive diseases of citrus as the plants rapidly become unproductive, enter a decline and eventually die. HLB is caused by the phloem-limited bacterium 'Candidatus Liberibacter' spp. The objective of this study was to evaluate seasonal variation of the in planta population of 'Ca. Liberibacter asiaticus' (Las) in in the foliage of citrus trees in Brazil using real-time PCR (qPCR). Eleven plants (naturally infected, then screened) in the field with very mild and localized symptoms of HLB were confirmed to be Las-infected by conventional PCR, and the canopies were divided into four quadrants. The bacterial population in the trees was tested on a monthly basis for up to 20 months by quantifying Las DNA using qPCR Las-specific primers (As84F/As180R). The average C t (cycle threshold) values, which relate to Las titer, were analyzed using a mixed model. Significant differences were observed in C t values between seasons (F = 8.77, P = 0.0004), and abrupt changes were observed in C t values in different quadrants of the trees.Autumn had the lowest C t values, indicating the highest Las titer and is thus considered the best period to detect Las infection in citrus trees in southern Brazil. In addition to the seasonal changes in C t values, there was an initial decline in the C t value in the months following initial detection, the rate of decline slowing with time. Concomitant with the increase of the bacterial population in the host, there was an increase in severity of HLB symptoms in the trees over time (Spearman's rank correlation, r = -0.4083, P<0.0001). The

Maize white spot, caused by the bacterium Pantoea ananatis, is one of the most detrimental diseases of maize in Brazil and has contributed to significant yield reduction. In this study, P. ananatis isolates were recovered from maize white spot lesions, healthy leaves, corn crop residues and Digitaria horizontalis. All the bacterial isolates were identified and characterized by morphological traits analysis, acid production from sorbitol and glycerol, ice nucleation activity, metabolic fingerprint similarities and molecular analysis by Polymerase Chain Reaction with species-specific primers for P. ananatis (ANAF/ANAR). Results showed that isolates recovered from maize white spot lesions were similar to those isolates collected from other sources. It suggests that corn crop residues, D. horizontalis and healthy leaf surface of the corn plant itself, could be a source of survival for P. ananatis.

Maize white spot lesions caused by Pantoea ananatis has contributed substantially to yield reduction of maize crops in many countries, including Brazil. The initial symptoms of the disease include watersoaked lesions on the leaves, which later become necrotic and straw-colored. Basic knowledge regarding the biology and the infection mechanisms of this pathogen is lacking. In this study, 15 P. ananatis isolates obtained from maize white spot lesions were examined for their ice nucleation activity (INA). The INAs of individual bacterial isolates was determined by tube nucleation tests. Bacterial isolates were grown on tryptic soy broth medium and an aliquot of 0.1 mL of culture was added to test tubes containing 1 mL of sterile distilled water. The tubes were packed in an ice bath, which had a temperature below -10°C, for approximately 2 min. Instantaneous formation of ice in the tube revealed a positive INA phenotype of the isolate. Only 9 of the 15 studied isolates showed the INA + phenotype. Pathogenicity tests were performed using whole plants and detached leaves. Symptoms were reproduced in both tests, but only for the inoculations using INA + isolates. Electron microscopy allowed visualization of protein vesicles under outer cell wall of isolates characterized as INA + . Key words: INA, vesicle protein, transmission electron microscopy ResumoLesões de mancha branca do milho causadas por Pantoea ananatis contribuiram substancialmente para a redução da produtividade nos cultivos de milho, em muitos países, incluindo o Brasil. Os sintomas iniciais da doença incluem lesões anasarcas nas folhas, que mais tarde tornam-se necróticas e de cor palha. O conhecimento básico sobre a biologia e os mecanismos de infecção deste patógeno são escassos. Neste estudo, 15 isolados de P. ananatis obtidos a partir de lesões da mancha branca do milho foram examinados quanto à sua atividade de nucleação de gelo (INA). A INA de isolados bacterianos individuais foi determinada por testes de nucleação em tubos. Isolados bacterianos foram cultivados em meio de caldo de soja tríptico e uma alíquota de 0,1 mL de cultura foi adicionada à tubos de ensaio contendo 1 mL de água destilada esterelizada. Os tubos foram colados em banho de gelo, o qual continha temperatura inferior a -10 ºC, por aproximadamente 2 min. A formação de gelo instantânea no tubo revelou um fenótipo INA positivo do isolado. Apenas 9 dos 15 isolados estudados apresentaram o fenótipo INA+. Testes de patogenicidade foram realizados com plantas inteiras e folhas destacadas.

O objetivo do trabalho foi realizar uma revisão bibliográfica sobre confinamento com dieta a base de grãos de milho inteiro para terminação de bovinos. A revisão foi realizada em trabalhos publicados por autores sobre o tema. O confinamento no Brasil chegou como uma técnica para modernizar a pecuária de corte, elevando os índices de produção e melhorando a qualidade da carne e seus derivados. A utilização de dietas a base de milho vem sendo utilizada para melhorar o desempenho dos animais e diminuir os custos de produção. Conclui-se que o produtor deve ficar atento ao preço do milho e dos animais, para que não tenha prejuízo durante o respectivo processo e maior rendimento por hectare.