Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

Evaluación del comportamiento agronómico de once cultivares de tomate (Lycopersicon esculentum Mill), bajo el manejo del productor en el valle de Sébaco, Matagalpa

Se evaluaron once cultivares de tomate de los cuales cuatro son originarios de Estados Unidos, uno de la India, tres del AVRDC, y tres de la Universidad Hebrea de Israel. El ensayo se estableció en el Valle de Sébaco con el objetivo de identificar los mejores materiales con las características deseables de rendimiento y tolerancia a enfermedades virales para posteriores estudios . Entre los cultivares evaluados se incluyeron tres testigos de uso local, uno altamente susceptible a geminivirus Río Grande y dos tolerante, TY-13 y TY-4; además, se evaluó un testigo introducido susceptible a virosis, CLN2026D. El diseño experimental que se utilizó fue un bloque comp letamente al azar donde se evaluaron las variables de crecimiento y desarrollo, periodo en anaquel del fruto, resistencia a geminivirus e incidencia de mosca blanca ( Bemisia tabaci Genn) y rendimiento comercial. Los resultados evidencian que el cultivar que presentó la mayor altura fue FLA456-4 con 124.6 cm, destacándose por presentar resistencia a virosis. El mayor diámetro lo obtuvo el cultivar FLA478-6-3-0 con 17.5 mm. Con respecto a la incidencia del vector mosca blanca, en el cultivar que más se encontró fue en CLN2026D con promedio de 9.9 mosca por planta. El mayor rendimiento comercial lo presento el cultivar TLCV7(híbrido) con 27.5 ton ha -1 seguido por los testigos TY-13 y CLN2026D con 25.5y 24.8 tonha -1 respectivamente.

Comportamiento agronómico de siete genotipos de tomate (Lycopersicum esculentum Mill) provenientes de AVRDC resistentes a virosis transmitida por mosca blanca, Tisma, Masaya, 2012

Se evaluaron siete genotipos de tomate de los cuales cinco son provenientes del Centro de Investigación y Desarrollo Vegetal de Asia (AVRDC) y dos testigos , uno de origen israelí y uno de origen chino. El ensayo se estableció en Tisma - Masaya en el año 2012 con el objetivo de generar información de cinco genotipos de tomate ( Lycopersicum esculentum Mill ) resistentes a virus transmitidos por mosca blanca ( Bemisia tabaci ) a fin de que sean alternativas para los productores. Entre los genotipos evaluados se incluyeron dos variedades comerciales ampliamente utilizadas por los productores : Butero , una variedad altamente susceptible a geminivirus y Shanty , comercializada como tolerante. El diseño experimental fue en Bloques Completamente al azar (BCA) con siete tratamientos y cuatro réplicas. Los resultados obtenidos demuestran que los genotipos en estudio presentaron diferencias altamente significativas para todas las variables. En cuanto a la severidad de la virosis se presentó un comportamiento diferencial, cuatro genotipos fueron ligeramente susceptibles al igual que Shanty. Entre los genotipos en estudio destaca AVTO1173 que muestra el mayor rendimiento, AVTO1082 , AVTO1883, AVTO1203 son estadísticame nte similares a Shanty, AVTO1031 y Butero obtuvieron el menor rendimiento, este ú ltimo debido a que es muy susceptible a virus . AVTO1173 resultó ser un genotipo promisorio por mostrar parámetros aceptables con respecto a Shanty y cuyo rendimiento fue superior a los demás genotipos en las condiciones ambientales en que se desarrolló el experimento.

Manejo integrado da mosca-branca Bemisia argentifolii.

Historico; Classificacao sistematica, descricao morfologica, biologia e danos; Ecologia; Hospedeiras e febologia; Metodos de controle de pragas; Manejo integrado.

Adult attractiveness and oviposition preference of Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) B-biotype in cotton genotypes

Considerada importante praga do algodoeiro, a mosca-branca Bemisia tabaci biótipo B pode através de sua alimentação, diminuir o vigor das plantas, trasmitir vírus e prejudicar a qualidade da fibra. Visando avaliar a resistência de genótipos de algodoeiro, Gossypium hirsutum (L.), à mosca-branca Bemisia tabaci biótipo B, realizaram-se testes de atratividade e não-preferência para oviposição, com e sem chance de escolha, em telado, a temperatura ambiente. Verificou-se baixa atratividade das plantas dos genótipos Fabrika, CNPA Ita 90, Makina, Coodetec 407 e IAC 01-639 CPA 02-24 a adultos dessa mosca-branca, o que pode representar um componente de resistência destes materiais genéticos ao inseto. Os genótipos BRS Aroeira, Coodetec 406, Fabrika e Coodetec 401 apresentaram resistência do tipo não-preferência para oviposição, nos testes com e sem chance de escolha. Os números de tricomas e de glândulas de gossipol por cm² não foram adequados para se avaliar a não-preferência para oviposição de adultos da mosca-branca em genótipos de algodoeiro.

Evaluation of resistance to Tomato severe rugose virus (ToSRV) in Capsicum spp. genotypes

Tomato severe rugose virus (ToSRV) is the predominant species of begomovirus in São Paulo State, Brazil, and infects primarily tomato and pepper plants. There is no information about genetic resistance of pepper to this virus, so in this work the reaction of 29 genotypes of Capsicum spp. was evaluated by inoculation of two ToSRV isolates: ToSRV-Sk (isolated from a tomato plant) and ToSRV-PJU (isolated from a pepper plant). For both isolates, two C. annuun genotypes (Catarino Cascabel - México and Silver) showed no symptoms 30 days after inoculation (d.a.i). In a second experiment, these two genotypes were evaluated for 150 d.a.i and, again, no symptoms could be observed. However, the virus was detected by RCA-PCR, indicating that both genotypes are susceptible, but less affected by ToSRV infection. Catarino Cascabel - México and Silver can be indicated for use in breeding programs for resistance of pepper to ToSRV.

Avaliação de genótipos de tomateiro tipo Santa Cruz no período de verão, em Araguari, MG

O trabalho foi desenvolvido na fazenda Jordão (município de Araguari, MG), na época do verão (período das águas), com o objetivo de verificar o desempenho agronômico de genótipos de tomateiro tipo Santa Cruz. Utilizou-se o delineamento experimental blocos casualizados, com 16 tratamentos (genótipos) e quatro repetições. A parcela experimental foi constituída por duas fileiras com 12 plantas cada, no espaçamento de 1,00 m entre linhas e 0,55 m entre plantas (1 planta/cova). Efetuaram-se 17 colheitas, sendo a primeira aos 69 dias após o transplante. Vários genótipos apresentaram um bom desempenho agronômico, principalmente Saladinha, Débora Plus, SM-16 e Atlas, podendo ser cultivados no período de verão. Apenas Saladinha e Atlas ultrapassaram 140 g de peso médio, destacando-se também em frutos do tipo extra AA. Observou-se uma correlação significativa e negativa com r = -0,52 e -0,54 na primeira avaliação, e r = -0,55 e -0,45 na segunda avaliação para a produção total e produção comercial, respectivamente, em relação à incidência de geminivírus nos diferentes genótipos. Os híbridos Saladinha e SM-16 apresentaram o menor número de plantas viróticas, enquanto Santa Clara Importada, Santa Clara, Jumbo AG-592 e IAC Santa Clara, apresentaram o maior número.

Diversidade genética de begomovírus em cultivos de tomateiro no Centro-Oeste Paulista

A diversidade genética de vírus pertencentes ao gênero Begomovirus em tomateiro (Lycopersicon esculentum Mill) foi analisada em regiões produtoras do Centro-Oeste paulista. No período de janeiro de 2003 a fevereiro de 2004, cento e sessenta e seis amostras de tomate foram coletadas e a presença de begomovírus observada em 60% das amostras, por PCR, utilizando-se oligonucleotídeos universais para o gênero Begomovirus. O sequenciamento direto do produto de PCR de 16 dessas amostras indicou a possível presença do Tomato severe rugose virus (ToSRV), Sida mottle virus (SiMoV-[BR]) e da espécie tentativa Tomato yellow vein streak virus (ToYVSV-[BR]). em duas amostras foi detectada uma possível nova espécie de begomovírus. A presença do ToSRV e do SiMoV ainda não havia sido verificada em tomateiro no estado de São Paulo. Estes resultados indicam a existência de diversidade de espécies de begomovírus infectando o tomateiro nesta região, servindo como um alerta para melhoristas que trabalham na busca de fontes de resistência a esse importante grupo de patógenos.

Interação de Tomato severe rugose virus com Bemisia tabaci biótipo B, a acessos de Capsicum spp. e ocorrência de espécies de mosca-branca no Estado de São Paulo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tolerance to high temperature in F-5 inbred lines of tomato

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

Evaluación de insecticidas químicos alternados con botánicos como opciones de manejo de mosca blanca (Bemisia tabaci, Gennadius) y otros insectos plagas en tomate (Solanum lycopersicum L.), en Tisma, Masaya 2016

La mosca blanca (Bemisia tabaci, Genn.) y los Geminivirus son los principales agentes de daño causantes de problemas fitosanitarios severos para los productores de tomate (Solanum lycopersicum, L.), este insecto provoca importantes pérdidas económicas, disminuyendo los rendimientos al afectar la calidad de frutos. En base a esta esta situación se realizó un estudio en el municipio de Tisma, Masaya en el período comprendido entre Julio y Septiembre del 2015 para evaluar la efectividad que tiene para controlar plagas los insecticidas químicos alternados con botánicos, los tratamientos evaluados fueron: Engeo alternado con Chile+Ajo+Jabón, Imidacloprid alternado con Madero Negro, Abamectina alternado con Neem, Monarca alternado con Chile+ajo y testigo (agua). Las variables evaluadas fueron, número de mosca blanca por planta, incidencia y severidad del daño de virosis por planta, número de Halticus sp por planta, número de Aphis sp por planta, número de Liriomyza sp por planta, numero de Arañas por planta, rendimiento en kg/ha. De los tratamientos evaluados, el menor número de moscas blancas, Halticus sp y menor porcentaje de incidencia y severidad lo presentó el tratamiento Abamectina alternado con Neem. El análisis económico determinó que el tratamiento que obtuvo el mayor rendimiento comercial fue Abamectina alternado con Neem. Se concluye que Abamectina alternado con Neem es el tratamiento que resultó más efectivo para el manejo de mosca blanca y otros insectos plagas.

'Abnormal vertical growth' : A disorder threatening the viability of the Australian macadamia industry

'Abnormal vertical growth' (AVG) was recognised in Australia as a dysfunction of macadamia (Macadamia spp.) in the mid-1990s. Affected trees displayed unusually erect branching, and poor flowering and yield. Since 2002, the commercial significance of AVG, its cause, and strategies to alleviate its affects, has been studied. The cause is still unknown, and AVG remains a serious threat to orchard viability. AVG affects both commercial and urban macadamia. It occurs predominantly in the warmer-drier production regions of Queensland and New South Wales. An estimated 100,000 orchard trees are affected, equating to an annual loss of $ 10.5 M. In orchards, AVG occurs as aggregations of affected trees, affected tree number can increase by 4.5% per year, and yield reduction can exceed 30%. The more upright cultivars 'HAES 344' and '741' are highly susceptible, while the more spreading cultivars 'A4', 'A16' and 'A268' show tolerance. Incidence is higher (p<0.05) in soils of high permeability and good drainage. No soil chemical anomaly has been found. Fine root dry weight of AVG trees (0-15 cm depth) was found lower (p<0.05) than non-AVG. Next generation sequencing has led to the discovery of a new Bacillus sp. and a bipartite Geminivirus, which may have a role in the disease. Trunk cinctures will increase (p<0.05) yield of moderately affected trees. Further research is needed to clarify whether a pathogen is the cause, the role of soil moisture in AVG, and develop a varietal solution.