Association of Olpidium bornovanus and Melon necrotic spot virus with Vine Decline of Melon in Guatemala.

Thirty-one soil samples from 14 different fields of Guatemala melon with vine decline symptoms were analyzed for the presence of organisms associated with the disease. With a soil-dilution plating method, only Macrophomina phaseolina was detected in five samples. With a melon bait plant technique, Olpidium bornovanus, often together with Melon necrotic spot virus (MNSV), was found in nearly all the samples, corresponding with all the fields studied. Other pathogens that were detected less frequently included Pythium aphanidermatum, Monosporascus cannonballus, and Rhizoctonia solani. Consequently, O. bornovanus and MNSV were uniquely associated with disease occurrence and thus are the most probable cause of melon vine decline in the fields studied.

New Natural Hosts of Pepino mosaic virus in Spain.

Pepino mosaic virus (PepMV) was first detected in Spain in 2000 (1). The virus infects tomato (Lycopersicon esculentum Mill.) crops and causes a variety of symptoms including leaf distortion, chlorosis, mosaic, blistering of the leaf surface, green striations on the stem and sepals, and fruit discoloration. PepMV is present along the southern and eastern regions of Spain (provinces of Granada, Almeria, Murcia, Alicante, Valencia, and Barcelona), Balearic, and the Canary Islands. In the summer and autumn of 2001 and 2002, virus-like symptoms were observed in native plants growing in or around tomato fields in Murcia and Almeria provinces. To study the alternate hosts that may serve as virus reservoirs, 62 samples of 42 common weed species, including asymptomatic plants, were collected and analyzed for PepMV using double-antibody sandwich enzyme-linked immunosorbent assay with a commercial antiserum (DSMZ As-0554; Biologische Bundesantstal, Braunschweig, Germany). The following weed hosts tested positive for PepMV: Bassia scoparia (L.) Voss., Calystegia sepium (L.) R.Br., Chenopodium murale L., Convolvulus althaeoides L., Convolvulus arvensis L., Conyza albida Willd. ex Spreng., Coronopus sp., Diplotaxis erucoides (L.) DC, Echium creticum L., E. humile Desf., Heliotropium europaeum L., Moricandia arvensis (L.) DC., Onopordum sp., Piptatherum multiflorum (Cav.) Beauv., Plantago afra L., Rumex sp., Sisymbrium irio L., Sonchus tenerrimus L., and Taraxacum vulgare (Lam.) Schrank. The presence of PepMV in these weed species was confirmed using reverse transcription-polymerase chain reaction with primers specific for PepMV (2). Although the number of samples examined may be insufficient to assess precisely the role of weed reservoirs in outbreaks of PepMV, these findings reveal potential virus sources and contribute to further understanding of PepMV epidemiology in Spain. References: (1) C. Jordá et al. Plant Dis. 85:1292, 2001. (2) P. Martínez-Culebras et al. Eur. J. Plant Pathol. 108:887, 2002.

First Report of Alfalfa mosaic virus in Lavandula officinalis.

For several years, in ornamental nurseries in the Mediterranean area of Spain, stunting and yellow leaf spotting have been observed in young plants of Lavandula officinalis. Symptoms eventually disappeared as the plants matured. During the summer of 2003, the number of plantlets affected and the intensity of symptoms increased significantly. Symptomatic plants tested positive using enzyme-linked immunosorbent assay (ELISA) (Phyto-Diagnostics, INRA, France) for the presence of Alfalfa mosaic virus (AMV). ELISA results were verified using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA extracts from symptomatic plants were analyzed using primers designed specifically for the coat protein region of AMV utilizing sequence data from GenBank Accession No. AF215664: AMVcoat-F: GT GGT GGG AAA GCT GGT AAA and AMVcoat-R: CAC CCA GTG GAG GTC AGC ATT. The thermocycling schedule was as follows: reverse transcriptase step at 50°C for 30 min, first PCR cycle at 94°C for 2 min, 35 cycles each of 30 s at 94°C, 30 s at 54°C, 30 s at 72°C, followed by a final extension at 72°C for 10 min. A 700-pb PCR product of the expected size was obtained from plants that were positive for AMV using ELISA. The two systems provide for rapid detection of AMV in L. officinalis. A regular screening program will assist in providing virus-free plants to ornamental nurseries. These results demonstrate the presence of AMV in L. officinalis. Alfalfa (Medicago sativa L.) is a typical source of AMV. However, because the nurseries where L. officinalis is grown are not in the vicinity of alfalfa fields, we suggest the source of the infection originated in the propagation material. AMV has currently been reported in L. officinalis only in Italy and France (1). To our knowledge, this is the first report of AMV in L. officinalis in Spain. Reference: (1): A. Garibaldi et al. Ed. Edagricole-Edisioni Agricole della Calderini s.r.l., Bologna, 2000.

Initiation of the breeding season in a grazing-based dairy by synchronization of ovulation.

Lactating dairy cows (n = 228) in a semiseasonal, grazing-based dairy were subjected to artificial insemination (AI) to start the 23-d breeding season (d 0 to 22) followed by natural service (d 23 to 120). Cows were randomly assigned to: 1) Ovsynch (GnRH, d -10; PGF2,, d -3; GnRH, d -1; timed AI, d 0) followed by AI at estrus (tail paint removal) on d 1 to 22 (Ovsynch; n = 114); or 2) AI at estrus (tail paint removal) throughout 23 d of AI breeding (tail paint; n = 114). Days to first AI service were greater and the 23-d AI service rate was less for tail paint vs. Ovsynch cows (12.0 +/- 0.6 d vs. 0 d; and 84.2 vs. 100%, respectively). However, conception to first AI was greater for tail paint vs. Ovsynch cows (47.3 vs. 27.3%, respectively). Cows in the tail paint group received only one AI, during 23 d of AI, but 46.4% of Ovsynch cows received a second AI, with similar conception (43.1%) to that of tail paint cows at first AI (47.3%). Based on serum progesterone, incomplete luteal regression after PGF2alpha, and poor ovulatory responses to GnRH contributed to lower conception to timed AI in the Ovsynch group. Cumulative pregnancy rates for tail paint and Ovsynch cows did not differ after 23 d of AI breeding (47.3 vs. 46.3%, respectively) nor after 120 d of AI/ natural service breeding (80.5 vs. 83.3%, respectively). Lactating cows in this grazing-based dairy synchronized poorly to Ovsynch resulting in reduced conception to timed AI compared with AI after tail paint removal.

Assessment of a commercially available early conception factor (ECF) test for determining pregnancy status of dairy cattle.

The Early Conception Factor (ECF) test is a commercially available qualitative assay that reportedly detects a pregnancy-associated glycoprotein present in bovine serum within 48 h after conception. One concern with previous assessments of this test is that animals with viable embryos early during pregnancy that subsequently undergo embryonic loss before pregnancy diagnosis increase the rate of false-positive results and bias the assessment. To preclude this possibility, noninseminated Holstein cows (n = 9) and heifers (n = 8) were evaluated as an unequivocal source of nonpregnant animals, and Holstein cows (n = 17) and heifers (n = 1) inseminated at estrus and in which at least one embryo of transferable quality was recovered at a nonsurgical flush 6 d after artificial insemination were evaluated as an unequivocal source of pregnant animals. Blood samples were collected from all animals 6 d after estrus, which was immediately before embryo collection in pregnant animals. Each serum sample was evaluated using two ECF test cassettes (tests 1 and 2), and the result of each test cassette was interpreted by two independent readers (readers 1 and 2). Test sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 86, 4, 49, 23, and 46%, respectively. Although the observed agreement between readers (91% for test 1; 89% for test 2) and between tests for the same serum sample (94% for reader 1; 91% for reader 2) was high, the overall rates of false-positive and false-negative ECF test results were 96 and 14%, respectively. We conclude that the ECF test is an unreliable method for determining pregnancy status of dairy cattle on day 6 after estrus.

Evaluation of two hormonal protocols for synchronization of ovulation and timed artificial insemination in dairy cows managed in grazing-based dairies.

To evaluate the efficacy of two hormonal protocols for synchronization of ovulation and timed artificial insemination (TAI) in dairy cows managed in grazing-based dairies, lactating dairy cows (n = 142) from two grazing-based dairies were randomly assigned to one of three treatment groups. Cows in the first group (Ovsynch) received 50 microg of GnRH (d -10); 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) followed by timed AI on d 0. Cows in the second group (PGF + Ovsynch) received a modified Ovsynch and timed AI similar to Ovsynch but with the addition of 25 mg of PGF2alpha 12 d (d -22) before initiation of Ovsynch. Cows in the third group (control) received standard reproductive management in place on each farm. Luteolysis occurred in 90.5% of cows exhibiting luteal function on d -22 in the PGF + Ovsynch treatment group, whereas none of the cows in the Ovsynch group underwent luteolysis on d -22. Synchronization rate (i.e., ovulatory response at 48 h after the second GnRH injection), conception rates at TAI and pregnancy rates after 35 d of breeding were similar for cows in the Ovsynch and PGF + Ovsynch groups. The proportion of anovular cows at the first GnRH injection of the synchronization protocols (d -10) was similar for cows receiving Ovsynch (28.0%) and PGF + Ovsynch (30.7%), and conception rate at TAI was similar for cycling (45.8%) and anovular (30.0%) cows. The cumulative pregnancy rate was greater for cows receiving TAI compared with control cows after 7 d of breeding (41.2 vs. 20.0%) but did not differ at 35 d of breeding (54.9 vs. 60.0%). Administration of PGF2alpha 12 d before initiation of Ovsynch did not improve synchronization, conception, or pregnancy rate compared with the standard Ovsynch protocol. Synchronization of ovulation to initiate timed AI at the onset of the breeding season resulted in earlier establishment of pregnancy compared with standard reproductive management.

Topography of the 27- and 31-kDa electron transport proteins in the onion root plasma membrane.

Plasma membranes purified from onion roots contain two distinct NAD(P)H-dehydrogenases of 27 and 31 kDa that differ in their physicochemical properties, substrate specificities and inhibitors sensitivities. The 27-kDa enzyme used both NADH and NADPH as electron donors. The 31-kDa enzyme was fully specific for NADH and accounted for the bulk of NADH-ferricyanide oxidoreductase. We have used NADPH- and NADH-ferricyanide oxidoreductase activities as markers for investigating the orientation of the 27- and 31-kDa enzymes at the plasma membrane, respectively. These activities were assayed in right-side-out vesicles isolated by two-phase partition, inside-out vesicles obtained by treatment with the detergent Brij 58 and membranes permeabilized with Triton X-100. Upon addition of Brij 58 to right-side-out plasma membrane vesicles, both NADPH- and NADH-ferricyanide oxidoreductases were activated to the same degree as the plasma membrane H(+)-ATPase. Redox activities were similar when measured in the presence of either Brij 58 or Triton X-100. Our results demonstrate that both enzymes expose their catalytic sites toward the cytoplasmic side of the plasma membrane.