Aline Kelly Queiroz do Nascimento

Serology Applied to Plant Virology

Virus infections affect seriously the quality and quantity of agricultural products around the world, especially in less developed countries. For this reason, the development and the adaptation of efficient and rapid techniques for diagnosis and control of plant viruses constitute an imperative and relevant necessity. Attempting to control plant diseases including those caused by viruses without sufficient information about their causal agents, their dissemination and surviving properties, usually results in inadequate control and many times, in a total failure. So, any attempt to establish a control program for a plant disease must be, always, preceded by a correct and precise laboratory diagnosis.

Serological survey of virus in cucurbit species in the Lower Middle São Francisco River Basin, Brazil

Aiming to get information about the virus that most frequently infects cucurbit plants of the species Benincasa hispida,Citrullus spp., Cucurbita spp., Cucumis melo, Lagenaria sp., Momordica charantia and also some F1 hybrids of C. lanatus with C. lanatus var. citroides in irrigated areas of Petrolina-PE and Juazeiro-BA, experimental and farming areas were surveyed in the first and second half of each year (2005 to 2007). The samples were tested by indirect enzyme-linked immunosorbent assay (ELISA) against antisera specific for Papaya ringspot virus, type watermelon(PRSV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV), and by Agar Double Diffusion against antiserum specific for Squash mosaic virus (SqMV) at the Plant Virus Laboratory of the Federal University of Ceara. Leaf samples from 967 plants were analyzed and 608 of them were infected. Among 125 samples from symptomless plants, 42 allow replication. PRSV-W was predominant, followed by WMV and ZYMV, and mixed infections occurred in all infected species. CMV was detected only in one sample and SqMV was not found. This is an indication that a breeding program for virus resistance in cucurbit species in the region must consider the three potyviruses.

Biological, serological and molecular comparison between isolates of Cowpea severe mosaic virus.

ABSTRACT Cowpea, Vigna unguiculata, can be affected by several diseases, and those caused by viruses are considered of great importance. Cowpea severe mosaic virus (CPSMV), which belongs to the family Comoviridae, genus Comovirus, stands out for its severity and degree of incidence. Its genetic variability can give rise to different strains around the world, including in Brazil. The objective of the present research was to establish a biological, serological and molecular comparison between five CPSMV isolates obtained from naturally infected plants in different States of the Northeast of Brazil. Isolates were obtained from cowpea: CPSMV-AL (State of Alagoas), CPSMV- CE (Ceara); CPSMV-MC (Piaui), CPSMV-PE (Pernambuco) and an isolate obtained from Crotalaria paulinea, in the State of Maranhao - CPSMV-CROT. A host range study evidenced biological differences among the virus isolates, especially in cowpea genotypes. The isolates were showed to be serologically and molecularly related by polymerase chain reaction �� PCR, using degenerated primers which amplified two conserved regions in the coat protein and in the replicase genes. Cloning and sequence of CPSMV-PE made possible its comparison with other CPSMV isolates and other virus species from the genus Comovirus.

Seleção de acessos e progênies de Citrullus spp. para resistência a três potyvirus

Six watermelon (Citrullus spp.) accessions (87-019, 87-029, 91-080, PI-244018, 91-043 and PI-195927) and two samples (PI-244019A and PI-244019B) from the acession PI-244019B from Embrapa Cucurbit Germplasm Bank in Petrolina-PE, Northeast of Brazil were evaluated in an attempt to select accessions and progenies of watermelon as sources of resistance to Papaya ringspot virus type watermelon (PRSV-W), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). Endogamic and open pollinated progenies from these accessions were also evaluated for resistance to PRSV-W, WMV and ZYMV. The evaluations were carried out in a greenhouse at the Plant Virus Laboratory of the Federal University of Ceara where the plants were mechanically inoculated and serological screened by Elisa. The selected plants were grown in the Experimental Station of Bebedouro in Petrolina-PE, where the virus inoculations were made, naturally, by vectors. According to the results, plants were not infected by PRSV-W in the following accessions: 87-019, PI-244019A, 91-080, PI-244018, PI-244019B and PI-195927; plants were not infected by WMV in the accessions: 87-019 and 87-029; and plants were not infected by ZYMV in the accessions: PI-244019A, 87-029, 91-080,91-043, PI-244019B and PI-195927. The progenies showed differentiated behavior with the percentage of selected plants ranging from 20 to 100% for PRSV-W and 60 to 100% for WMV. The progenies evaluated against ZYMV did not show resistant plants. Therefore, it can be postulated that resistance for PRSV-W and WMV in the progenies differs from that for ZYMV, since all progenies were submitted to the same number of self-pollinations.

Crotalaria paulinea, a new natural host of Cowpea severe mosaic virus.

Leaf samples from Crotalaria paulinea showing mosaic were collected in the city of Sao Luiz, MA and sent to the Plant Virus Laboratory at the UFC. The leaf samples were tested by indirect enzyme-linked immunosorbent assay (Elisa) against antisera specific to Cowpea aphid-borne mosaic virus (CABMV) and Cucumber mosaic virus (CMV) and by gel double-diffusion against antiserum to Cowpea severe mosaic virus (CPSMV). The samples reacted only with the antiserum to CPSMV indicating that C. paulinea is one more natural host of the virus. Leaf extracts from infected C. paulinea were mechanically inoculated in cowpea (Vigna unguiculata subsp. unguiculata) plants maintained in greenhouse. Ten days after inoculation, the plants started to exhibit mosaic and the presence of CPSMV was confirmed by serology. In a host range study involving eight plant species, the CPSMV isolate obtained from C. paulinea (CPSMV-Cp) infected only cowpea cultivars. The results of RT-PCR revealed a band in the agarose gel of 594 pb for CPSMV-Cp similar to those of other CPSMV isolates. The CPSMV-Cp was increased in cowpea cv. Pitiuba and purified by clarification using n-butanol, virus particle precipitation with polyethylene glycol (PEG) and ultra centrifugation. The purified virus preparation presented an ultraviolet light absorption spectrum typical of nucleoprotein, with a ratio A260/A280 equal to 1.7. A White New Zealand rabbit immunized with the purified virus preparation produced polyclonal antiserum reactive to CPSMV in agar double-diffusion. This is the first report about natural infection of C. paulinea by CPSMV.

Sequence variability in the coat protein gene of Cowpea severe mosaic virus isolates from northeastern Brazil

The comovirus Cowpea severe mosaic virus (CPSMV) causes a disease in cowpea (Vigna unguiculata) but, despite its importance, there are no studies on the genetic diversity of viral populations. We have determined the nucleotide sequences of part of the coat protein (CP) gene of six Brazilian isolates of CPSMV. Genomic fragments (521 nucleotides) were RT-PCR amplified, cloned, and their sequences were compared with each other and with other comoviruses. Sequence comparisons indicated a high degree of conservation for the CP gene, with 92-100% nucleotide and 97-100% amino acid sequence identity among the isolates. There was no correlation between geographical origin and sequence identity or phylogeny among the isolates.

The presence of Papaya ringspot virus and Papaya lethal yellowing virus in commercialized papaya fruits

Considering the high incidence of virus in papaya (Carica papaya) orchards and their typical symptoms in the fruits, a survey was carried out in the Central Market in the State of Ceara (CEASA) for the presence of papaya fruits infected with Papaya ringspot virus (PRSV) and Papaya lethal yellowing virus (PLYV). CEASAs are the most important fruit commercial centers, and a great number of plant products sold in supermarkets are obtained in CEASA. Fruits were randomly collected from CEASA stalls and brought to the Plant Virus Laboratory at the Federal University of Ceara (UFC) to be analyzed for typical virus symptoms and tested by indirect enzyme linked immunosorbent assay (ELISA) against antisera specific to PRSV and PLYV. According to the results obtained by symptom analysis, which were confirmed by serology, among the 8,400 fruits analyzed 5.5% were infected with PRSV and 1.2% with PLYV, showing the need to implement a control program involving eradication of virus sources in the field, represented by infected plants in papaya orchards, abandoned orchards and infected plants identified in backyards.

Biological and serological of an isolate of squash mosaic virus and effects of mixed infection with a virus of the genus Potyvirus

O Nordeste brasileiro possui condicoes edafoclimaticas favoraveis ao cultivo do meloeiro (Cucumis melo). No entanto, diferentes problemas fitossanitarios afetam sua produtividade, destacando-se as doencas ocasionadas por virus. A presente pesquisa objetivou efetuar a caracterizacao biologica e sorologica de um isolado de Squash mosaic virus (SqMV) obtido no Ceara. Em estudos de gama de hospedeiros o virus infetou sistemicamente somente cinco especies da familia Cucurbitaceae e ocasionou lesoes locais em Chenopodium amaranticolor e C. quinoa. Estudos da infeccao mista do SqMV com virus do genero Potyvirus demonstraram efeito sinergico entre os mesmos. A purificacao quimica do SqMV foi possivel a partir de plantas de meloeiro e a imunizacao em coelho com a preparacao purificada possibilitou a producao de antissoro policlonal especifico. O antissoro produzido apresentou titulos de 1:10.000 quando avaliado por plate trapped antigen enzyme linked immune absorbent assay (PTA-ELISA), de 1:160.000 em immune-precipitation ELISA (IP-PTA-ELISA) e de 1:1.024 em Dupla Difusao em Agar. Estudos do relacionamento sorologico entre SqMV e Cowpea severe mosaic virus (CPSMV) do genero Comovirus contra seus antissoros homologos e heterologos em Dupla Difusao em Agar demonstraram relacionamento unilateral entre os mesmos.

Biological stability of a strain of Cowpea severe mosaic virus over 20 years.

Cowpea (Vigna unguiculata) is an important crop of the traditional agriculture system in the Northeast of Brazil. It can be infected by more than 20 virus species and Cowpea severe mosaic virus (CPSMV) is one of the most important pathogens that naturally infect cowpea in Brazil. Several CPSMV isolates were obtained and characterized in the Plant Virus Laboratory at the Federal University of Ceara: CPSMV-CE - the first characterized isolate of the virus obtained from cowpea in the State of Ceara; CPSMV-AL - isolated from cowpea in Alagoas; CPSMV-PE - isolated from cowpea in Pernambuco; CPSMV-PR - obtained from soybean (Glycine max) in Parana and CPSMV-CROT - isolated from Crotalaria paulinea, in Maranhao. An isolate of CPSMV with the property to infect the cv. Macaibo, a cowpea cultivar immune to most of CPSMV isolates was also biologically and serologically characterized as a new strain of the virus (CPSMV-MC). The CPSMV-MC was isolated in January 1990 and has been evaluated over 20 years by host range studies and maintenance in vivo by periodical mechanical inoculations in cowpea. The results of this periodical evaluation revealed that the biological integrity and the serological properties of CPSMV-MC were preserved over 20 years, indicating that the genetic preservation of a virus strain could occur over the years. Molecular studies involving part of the coat protein (CP) gene of CPSMV-MC and five other Brazilian CPSMV isolates indicated a high degree of conservation, with 92-100% nucleotide sequence identity among the isolates.

Sorologia e sobrevivência do vírus do amarelo letal do mamoeiro

The Papaya lethal yellowing virus (PLYV) is responsible for one of the most important disease of papaya (Carica papaya) in the Northeast of Brazil. The PLYV can be transmitted through the soil, irrigation water, agriculture tools and contaminated hands. The present research had the objective to characterize biologically, serologically and physically a PLYV isolate and evaluates how long it survives in infected dried tissues. The PLYV was identified by indirect enzyme linked immunoabsorbent assay (ELISA), isolated in young papaya plants by mechanical inoculations and maintained at green house conditions for virus purification. Approximately 309.5 mg of virus was purified per kg of infected papaya leaves and a polyclonal antiserum was obtained from an immunized rabbit. The antiserum obtained was shown to be highly specific to PLYV with a titer of 1:128 in double immune-diffusion and a titer of 1:1.024.000 in indirect ELISA. The virus was detected in dried roots and leaves maintained at room temperature up to 120 days, confirming its high stability within nonliving plant tissues, which could explain its dissemination by contaminated hands, tools, water and soil. The physical properties determined for the virus revealed a thermal inactivation point of 80 o C, longevity in vitro over 50 days and dilution end point ac. of 10 -6 .