J. Albersio A. Lima

Identificação sorológica de espécies de vírus que infetam cucurbitáceas em áreas produtoras do Maranhão

Viruses represent serious obstacles for the success of producing vegetables, consisting the correct identification of those that infect cucurbits in a region, a fundamental point for establishing strategies for their control. Surveys were carried out at cucurbit fields in producing areas of the State of Maranhao. Leaf samples were collected from 118 plants showing virus like symptoms, 46 from squash (Cucurbita moschata); 30 from watermelon (Citrullus lanatus); 23 from Cucumis anguria; 13 from cucumber (C. sativus) and six from melon (C. melo). All samples were tested against antisera specific to the main virus species from the families Bromoviridae, Comoviridae and Potyviridae that infect cucurbits in Northeastern Brazil by enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion technique. The results revealed that Papaya ringspot virus was serologically detected in 64.4% of samples analyzed, followed by Watermelon mosaic virus-2 in 15.2%, Cucumber mosaic virus in 6.8%, Squash mosaic virus in 3.4% and Zucchini yellow mosaic virus in 3.4%. This survey confirmed the prevalence of PRSV in cucurbit species grown in the State of Maranhao.

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.

Identificação sorológica de espécies de potyvirus em melancia, no estado de Roraima

Leaf samples were collected from watermelon plants (Citrullus lanatus) from 21 different fields in the state of Roraima from May/2003 to March/2004. The samples were sent to the Plant Virus Laboratory of the Federal University of Ceara, to be tested by indirect enzyme linked immunosorbent assay (Elisa) against antisera specific to Cucumber mosaic virus (CMV), Papaya ringspot virus strain watermelon (PRSV-W), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). The Elisa tests were processed using the universal conjugate, anti-imunoglobulim (IgG) from rabbit produced in goat, conjugated to alkaline phosphatase. All samples were also tested by double immunodifusion against antiserum specific to Squash mosaic virus (SqMV). The presence of PRSV-W was detected in 84.2% of the samples collected in May/2003, in 7.1% of the samples collected in December/2003 and 55,6% in March/2004. The presence of ZYMV was detected in 10.5% of the samples collected in May, in 21.4% in December/2003 and in 25.9% in March/2004. The WMV was detected only in eight of the samples collected in March/2004 (29.6%). These results confirm the world widespread dispersion of PRSV-W in cultivated cucurbit fields in Brazil and rise a concern about the ZYMV expansion, which has caused serious yield lost in other parts of the world.

Genes diferentes podem conferir resistência ao Cowpea severe mosaic virus em caupi

Cowpea (Vigna unguiculata) is an important vegetable crop in Northeast Brazil and has been traditionally cultivated by small farmers. Virus diseases are considered to be the main factor in yield limiting cowpea yield in the region. The severe mosaic disease caused by Cowpea severe mosaic virus (CpSMV), family Comoviridae, genus Comovirus, seems to be one of the most prevalent diseases responsible for high losses. Resistant cultivars may be considered the best alternative for disease control, and several promising sources of resistance such as Macaibo, CNC 0434 cultivars, and the line L 254.008 have been reported. More recent investigations into the genetic basis of these resistant plant genotypes have pointed to similar results, and the inheritance of this resistance has been recognized as monogenic recessive. On the other hand, any research aimed to investigate if the resistance genes are alleles or not. In the present investigation a test was conducted to elucidate this question. Plants of the genotypes Macaibo, CNC 0434 and L 254.008 were bred in reciprocal and direct ways producing six populations. Evaluation of those cowpea genotypes using one isolate of CpSMV indicated that the resistance gene of Macaibo is the same as that for CNC 0434 and distinct from the gene L 254.008 found in that line.

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.

PRODUCTION OF POLICLONAL ANTISERA SPECIFIC TO PLANT VIRUSES BY RABBIT ORAL IMMUNIZATION

Serological techniques are of great importance for plant virus identification and characterization. The major limiting factor for using these techniques for plant virus identification is the requirement of a good virus purified preparation to be used in immunizing animals for antiserum production. In the present study, two New Zealand rabbits were orally immunized with extracts from cowpea (Vigna unguiculata) plants systemically infected with Cowpea severe mosaic virus (CPSMV) and with extracts from papaya (Carica papaya) infected with Papaya lethal yellowing virus (PLYV). The leaf extracts were prepared in saline solution 0.15 M in the rate of 1:1 (w/v) and clarified by a centrifugation of 10,000 g for 10 min. The clarified extracts containing the viruses were orally administered to the New Zealand rabbits in two series of five daily doses of 1.0 ml each. The obtained policlonal antisera were shown to be very specific to their respective viruses in double immunodiffusion and indirect ELISA. These seem to be the first antisera specific for plant virus obtained by rabbit oral immunization. The results open up some possibilities for producing antisera to plant viruses of difficult purification. It is a simple, fast and inexpensive method for production of antisera for plant viruses when compared to the traditional techniques that involve rabbit injections with purified virus preparations.

Serological identification of Dasheen mosaic virus in Anthurium sp. in the State of Ceará.

RESUMO Identificacao sorologica de Dashim mosaic virus em Anturio no estado do Ceara Os anturios (Anthurium spp.) sao cultivados nas serras umidas do estado do Ceara, especialmente, no Macico de Baturite e vem despontando como promissora ornamental no mercado internacional. Plantas com sintomas de virus foram constatadas em visitas de inspecao a plantios comerciais de anturios no Macico de Baturite. Testes sorologicos de Elisa indireto com amostras de flores e de folhas de plantas com sintomas apresentaram resultados positivos para Dasheen mosaic virus, familia Potyviridae, genero Potyvirus. A constatacao de inclusoes citoplasmatica em celulas da epiderme de plantas infetadas confirmou a infeccao por virus do genero Potyvirus. Este e o primeiro registro da incidencia do DMV no Ceara.

Sintomas atípicos em frutos de meloeiro e de melancia ocasionados por Watermelon mosaic virus-2

The Northeast of Brazil presents great potencial for melon (Cucumis melo) and watermelon (Citrullus lanatus) production. Melon fruit with white spots and watermelon fruits showing chlorotic spots were demonstrated to be caused by Watermelon mosaic virus-2, through indirect enzyme linked-immuno sorbent assay.

Methods of diagnosis, stability, transmission, and host interaction of Papaya lethal yellowing virus in papaya

Papaya ( Carica papaya ) is a tropical fruit consumed worldwide for its quality and acceptance. Unfortunately, virus infections seriously affect the quality and quantity of papaya production. Virus diseases have been responsible for the greatest losses in papaya production throughout the world. The most important viruses that infect papaya in Northeastern Brazil are Papaya ringspot virus (PRSV) in the family Potyviridae, genus Potyvirus ; Papaya lethal yellowing virus (PLYV) in the genus Sobemovirus ; and Papaya meleira virus (PMeV). Lethal yellowing is a disease caused by PLYV that occurs only in Northeastern Brazil; it is characterized by progressive leaf yellowing and greenish circular spots on the fruits. The virion of PLYV is isometric, ca. 30 nm, with a genome of single-stranded RNA (ssRNA) of ca. 1.6 × 10 6 Da and a single component coat protein of ca. 34.7 kDa. Although no biologic vector has been confirmed, the virus spreads every year, probably by infected plantlets and contaminated tools. The virus infects only C. papaya, Jacaratia heterophyla, J. spinosa , Vasconcellea cauliflora, V. quercifolia , and V. monoica , all members of the family Caricaceae. None of the other 82 species from 16 families become infected when inoculated with PLYV. The virus is very stable and can be detected in dried roots and leaves maintained for up to 120 days. The virus has the following physical properties: thermal inactivation point 80 o C, longevity in vitro 60 days, and dilution end point 10 −6 . High concentration of virus particles can be purified from infected papaya, and good polyclonal antisera can be obtained.