Survey, Molecular Detection, and Characterization of Geminiviruses Associated with Cassava Mosaic Disease in Zambia.

A survey was conducted from April to May 2014 in 214 farmers' fields located across six major cassava-producing provinces (Western, Northwestern, Northern, Luapula, Lusaka, and Eastern) of Zambia to determine the status of cassava mosaic disease (CMD) and the species diversity of associated cassava mosaic geminiviruses (CMG). Mean CMD incidence varied across all six provinces but was greatest in Lusaka Province (81%) and least in Northern Province (44%). Mean CMD severity varied slightly between provinces, ranging from 2.78 in Eastern Province to 3.00 in Northwestern Province. Polymerase chain reaction discrimination of 226 survey samples, coupled with complete DNA-A genome sequence analysis, revealed the presence of African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), and East African cassava mosaic Malawi virus (EACMMV) as single or mixed infections of different proportions. Single-virus infections were predominant, occurring in 62.8% (ACMV), 5.8% (EACMMV), and 2.2% (EACMV) of samples relative to mixed-virus infections, which occurred in 19.5% (ACMV + EACMMV), 0.4% (ACMV + EACMV), and 0.9% (ACMV + EACMV + EACMMV) of samples. Phylogenetic analysis revealed the segregation of virus isolates from Zambia into clades specific to ACMV, EACMV, and EACMMV, further confirming the presence of all three viruses in Zambia. The results point to a greater diversity of CMG across major cassava-growing provinces of Zambia and implicate contaminated cassava cuttings in disease spread.

Growth and haematological response of indigenous Venda chickens aged 8 to 13 weeks to varying dietary lysine to energy ratios.

The effect of feeding varying dietary lysine to energy levels on growth and haematological values of indigenous Venda chickens aged 8 - 13 weeks was evaluated. Four hundred and twenty Venda chickens (BW 362 ± 10 g) were allocated to four dietary treatments in a completely randomized design. Each treatment was replicated seven times, and each replicate had fifteen chickens. Four maize-soya beans-based diets were formulated. Each diet had similar CP (150 g/kg DM) and lysine (8 g lysine/kg DM) but varying energy levels (11, 12, 13 and 14 MJ ME/kg DM). The birds were reared in a deep litter house; feed and water were provided ad libitum. Data on growth and haematological values were collected and analysed using one-way analysis of variance. Duncan's test for multiple comparisons was used to test the significant difference between treatment means (p < 0.05). A quadratic equation was used to determine dietary lysine to energy ratios for optimum parameters which were significant difference. Results showed that dietary energy level influenced (p < 0.05) feed intake, feed conversion ratio, live weight, haemoglobin and pack cell volume values of chickens. Dry matter digestibility, metabolizable energy and nitrogen retention not influenced by dietary lysine to energy ratio. Also, white blood cell, red blood cell, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration in female Venda chickens aged 91 days were not influenced by dietary lysine to energy ratio. It is concluded that dietary lysine to energy ratios of 0.672, 0.646, 0.639 and 0.649 optimized feed intake, growth rate, FCR and live weight in indigenous female Venda chickens fed diets containing 8 g of lysine/kg DM, 150 g of CP/kg DM and 11 MJ of ME/kg DM. This has implications in diet formulation for indigenous female Venda chickens.

Occurrence of Cucumber mosaic virus Subgroups IA and IB Isolates in Tomatoes in Nigeria.

Tomato (Solanum lycopersicum L.) is a major economic crop consumed globally in fresh or processed forms. During a routine field survey of major tomato-producing areas of southwestern Nigeria in May/June 2013, tomato plants cv. Roma VF showing virus-like symptoms including stunting, chlorosis, and narrowing of leaf blades were observed in 10 farmers' fields with varying levels of incidence averaging ~27%. Moderate to high aphid infestations were also observed in affected fields, and fruit production was significantly impacted based on visual observations. Since symptoms observed on affected plants are similar to those described for Cucumber mosaic virus (CMV) infection in tomato (5), leaf tissue samples collected from a total of 92 tomato plants across 10 commercial farms were subjected to antigen coated plate (ACP)-ELISA essentially as described previously (2). In ACP-ELISA using a CMV polyclonal antibody, 24 of the 92 samples (26.1%) derived from 7 of the 10 survey locations spread across Oyo, Ogun, Ekiti, and Osun states of southwestern Nigeria tested positive for CMV. Based on the ACP-ELISA results, one randomly selected sample from each of the CMV-positive survey locations, seven samples in total, was subjected to total nucleic acid extraction (1) followed by one step-single tube RT-PCR using primers CMV1/CMV2 and conditions described previously (4) with appropriate virus-positive and -negative controls. A ~500 bp DNA band was amplified from these seven ACP-ELISA-positive samples, thus confirming the presence of CMV. To further confirm these results and to enable molecular typing of CMV isolates from southwest Nigeria, the amplified DNA fragments were precipitated with the addition of 70% ethanol and centrifugation and directly sequenced using the ABI 3130xL Genetic Analyzer (Applied Biosystems, California) at the Bioscience Center of the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Following the removal of primer- and 3'UTR-specific sequences, the remaining 366-bp partial CP-specific sequences (GenBank Accession Nos. KM091952 to 58) and corresponding sequences of global CMV isolates obtained from GenBank were subjected to multiple alignments using the MEGA 6.0 software. This analysis showed that tomato-infecting CMV isolates from southwest Nigeria shared 91.6 to 99.4% and 94.9 to 99.1% nucleotide (nt) and amino acid (aa) identities among themselves and 91.6 to 98.0% and 94.1 to 98.3%, 89.4 to 94.1% and 93.2 to 98.3%, and 75.2 to 78.8% and 84.0 to 87.3% with corresponding nt and aa sequences of representatives of CMV isolates belonging to subgroups IA (D10538), IB (AB008777), and II (M21464), respectively. Maximum likelihood phylogenetic analysis revealed the clustering of four and three CMV isolates obtained in this study into subgroups IA and IB, respectively, with >70% bootstrap support. CMV has been detected in tomato seeds (3) and its very wide host range includes cultivated crops and weed species (5). It is therefore plausible that contaminated seed lots and alternative weed and crop host plants serve as sources of CMV inoculum to cultivated tomato in affected farms. Although CMV has been reported from tomato from several countries worldwide, to our knowledge, this is the first empirical evidence for the occurrence of CMV subgroups IA and IB in cultivated tomato in Nigeria. References: (1) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (2) J. d'A. Hughes and S. A. Tarawali. Trop. Sci. 39:70, 1999. (3) K. H. Park and B. J. Cha. Res. Plant Dis. 8:101, 2002. (4) S. Wylie et al. Aus. J. Agric. Res. 44:41, 1993. (5) T. A. Zitter and J. F. Murphy. Plant Health Instructor. DOI: 10.1094/PHI-I-2009-0518-01, 2009.

First Report of Potato virus Y in Potato in West Java, Indonesia.

Potato (Solanum tuberosum) is an important vegetable crop in Indonesia. A small survey was conducted for virus diseases in November 2011 in Lembang, West Java, as part of assessing the sanitary status of potatoes produced in farmers' fields. Among the six potato fields surveyed, one field had nearly 20% of plants displaying stunted growth with leaves showing mild chlorotic spots and reduced size of lamina. Tubers harvested from symptomatic plants showed no necrosis symptoms. Symptomatic leaves from three representative potato plants were positive for Potato virus Y (PVY) when tested with PVY-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from virus-positive plants were imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR using primers (PVY/Y4A and PVY/Y3S) specific to the coat protein (CP) of PVY (3). Nucleic acid extracts from samples infected with PVY ordinary strain (PVYO), tuber necrosis strain (PVYNTN), tobacco veinal necrosis strains (PVYEU-N and PVYNA-N), and a recombinant strain (PVYN:O) were included as standards to validate RT-PCR assays. The approximately 480-bp DNA fragment, representing a portion of the CP, amplified in RT-PCR was cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA). DNA isolated from four independent recombinant clones was sequenced from both orientations. Pairwise comparison of these sequences (GenBank Accession Nos. KF261310 to 13) showed 100% identity among themselves and 93 to 100% identity with corresponding sequences of reference strains of PVY available in GenBank (JQ743609 to 21). To our knowledge, this study represents the first confirmed report of PVY in potato in West Java, Indonesia. Studies are in progress to assess the prevalence of PVY in other potato-growing regions of Indonesia and document the presence of different strains of the virus (2). Since the majority of farmers in Indonesia plant seed selected from their previous potato crop, there is an increased risk of primary and secondary spread of PVY through the informal seed supply system, leading to its increased significance to potato production in Indonesia. Therefore, strengthening foundation seed potato and supply chain programs will promote the production of virus-free potatoes in Indonesia. References: (1) O. J. Alabi et al. Plant Dis. 96:107, 2012. (2) A. Karasev and S. M. Gray. Am. J. Potato Res. 90:7, 2013. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

First Report of Potato virus Y in Potato in Tajikistan.

Potato (Solanum tuberosum L.) is widely grown as a staple food and cash crop in Tajikistan and is an important food security crop in the country. In June 2011, we conducted a survey of potatoes in farmers' fields in the Buston and Dushanbe regions (about 200 miles apart) of Tajikistan. Potato plants with stunted growth and leaves showing chlorotic spots, curling, and necrotic spots and rings were observed with the disease incidence monitored in 10 fields each in Buston and Dushanbe areas varying between 10 and 60%. Representative samples from symptomatic plants tested positive for Potato virus Y (PVY) using virus-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from symptomatic plants were collected from Buston and Dushanbe areas, imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to the lab at Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR with primers (PVY/Y4A and PVY/Y3S) specific to the coat protein of PVY (3). Samples infected with PVY ordinary strain (PVYO), tuber necrosis strain (PVYNTN), tobacco veinal necrosis strains (PVYEU-N and PVYNA-N), and a recombinant strain (PVYN:O) were included as references to validate RT-PCR results. A single DNA product of approximately 480 bp was amplified from potato samples that tested positive with PVY-specific immunostrips. The amplified fragments from two samples from Dushanbe and six from Buston areas were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA) and two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 90 to 100% identity among the cloned amplicons (GenBank Accession Nos. JQ743609 to JQ743616) and 90 to 100% with corresponding nucleotide sequence of reference PVY strains (GenBank Accession Nos. JQ743617 to JQ743621). A global phylogenetic analysis of sequences revealed the presence of PVYO in both samples from Dushanbe and one sample from Buston regions and presence of PVYNTN in the remaining five samples from the Buston region. Because of the possible occurrence of mixed infections of PVY strains (2), further studies are needed to determine the presence of mixed infections of two or more strains of PVY and their specificity to potato cultivars. To our knowledge, this study represents the first confirmed report of two distinct strains of PVY in potato in Tajikistan. The occurrence of PVYNTN, a quarantine pathogen in many countries (2), warrants additional investigations to improve sanitary status of potato fields and to facilitate the availability of virus-free seed in clean plant programs for significant yield increases in Tajikistan. References: (1) O. J. Alabi et al. J. Virol. Methods 154:111, 2008. (2) S. Gray et al. Plant Dis. 94:1384, 2010. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

Occurrence of Grapevine leafroll-associated virus 1 in Two Ornamental Grapevine Cultivars in Washington State.

Roger's Red, an interspecific hybrid between wild grape (Vitis californica, native to northern California) and the V. vinifera cv. Alicante Bouschet (1), and Claret Vine (V. vinifera cv. Purpurea Nana) are grown for their ornamental value in home gardens and other settings. We collected potted grapevines of Roger's Red and Claret Vine showing dull green-to-scarlet red leaves from two different retail nurseries in the Richland-Kennewick area and Prosser, WA, respectively. Since these symptoms 'mimic' grapevine leafroll disease, we tested petiole samples from four grapevines per cultivar for a panel of grapevine-infecting viruses by single-tube one-step reverse transcription (RT)-PCR (4). All samples tested positive only for Grapevine leafroll-associated virus 1 (GLRaV-1; genus Ampelovirus, family Closteroviridae). To further confirm these results, total RNA was subjected to RT-PCR to amplify a portion of the heat shock protein 70 homolog (HSP70h), coat protein duplicate 2 (CPd2), and ORF 9 (p24) of GLRaV-1. RT was performed at 52°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 5 min final extension step at 72°C. Primers specific to HSP70h (HSP70h/416F: 5'-CAGGCGTCGTTTGTACTGTG and HSP70h/955R: 5'-TCGGACAGCGTTTAAGTTCC), CPd2 (CPd2/F: 5'-GTTACGGCCCTTTGTTTATTATGG and CPd2/R: 5'-CGACCCCTTTATTGTTTGAGTATG) and ORF 9 (p24/F: 5'-CGATGGCGTCACTTATACCTAAG and p24/R (5'-CACACCAAATTGCTAGCGATAGC) were designed based on GLRaV-1 sequence (GenBank Accession No. AF195822) to amplify 540, 398, and 633 base pair (bp) DNA fragments, respectively. To verify that the amplified products were specific to the genome of GLRaV-1, the amplicons were cloned into pCR2.1 vector (Invitrogen Corp, Carlsbad, CA) and three independent clones for each amplicon were sequenced in both directions. Pairwise comparison of HSP70h (Accession Nos. HQ833472 and HQ833473), CPd2 (Accession Nos. HQ833474 and HQ833475), and p24 (Accession Nos. HQ833476 and HQ833477) sequences from Roger's Red and Claret Vine showed 100, 96, and 99% identities, respectively, between them, and 86 to 100, 80 to 97, and 86 to 90% nucleotide sequence identities, respectively, with corresponding sequences of GLRaV-1 isolates deposited in GenBank. We further confirmed the presence of GLRaV-1 in these two ornamental grape cultivars by double antibody sandwich-ELISA using commercially available antibodies (Bioreba AG, Reinach, Germany). Previous studies have reported the presence of GLRaV-2 and -3 (1,3) and Grapevine virus A and B (2,3) in Roger's Red. To our knowledge, this study represents the first report of the occurrence of GLRaV-1 in two Vitis species distributed as ornamental grapes. It is important to prevent virus spread via the supply of virus-tested ornamental grapevines by commercial nurseries. References: (1) G. S. Dangl et al. Am. J. Enol. Vitic. 61:266, 2010. (2) D. A. Golino et al. Phytopathology (Abstr.) 99(suppl.):S44, 2009. (3) V. Klaassen et al. Online publication. doi:10.1094/PDIS-09-10-0621. Plant Dis., 2011. (4) T. A. Mekuria et al. Phytopathology (Abstr.) 99(suppl.):S83, 2009.

The Occurrence of Bean common mosaic virus and Cucumber mosaic virus in Yardlong Beans in Indonesia.

Yardlong bean (Vigna unguiculata subsp. sesquipedalis) is extensively cultivated in Indonesia for consumption as a green vegetable. During the 2008 season, a severe outbreak of a virus-like disease occurred in yardlong beans grown in farmers' fields in Bogor, Bekasi, Subang, Indramayu, and Cirebon of West Java, Tanggerang of Banten, and Pekalongan and Muntilan of Central Java. Leaves of infected plants showed severe mosaic to bright yellow mosaic and vein-clearing symptoms, and pods were deformed and also showed mosaic symptoms on the surface. In cv. 777, vein-clearing was observed, resulting in a netting pattern on symptomatic leaves followed by death of the plants as the season advanced. Disease incidence in the Bogor region was approximately 80%, resulting in 100% yield loss. Symptomatic leaf samples from five representative plants tested positive in antigen-coated plate-ELISA with potyvirus group-specific antibodies (AS-573/1; DSMZ, German Resource Center for Biological Material, Braunschweig, Germany) and antibodies to Cucumber mosaic virus (CMV; AS-0929). To confirm these results, viral nucleic acids eluted from FTA classic cards (FTA Classic Card, Whatman International Ltd., Maidstone, UK) were subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (CIFor: 5'-GGIVVIGTIGGIWSIGGIAARTCIAC-3' and CIRev: 5'-ACICCRTTYTCDATDATRTTIGTIGC-3') (3) and degenerate primers (CMV-1F: 5'-ACCGCGGGTCTTATTATGGT-3' and CMV-1R: 5' ACGGATTCAAACTGGGAGCA-3') specific for CMV subgroup I (1). A single DNA product of approximately 683 base pairs (bp) with the potyvirus-specific primers and a 382-bp fragment with the CMV-specific primers were amplified from ELISA-positive samples. These results indicated the presence of a potyvirus and CMV as mixed infections in all five samples. The amplified fragments specific to potyvirus (four samples) and CMV (three samples) were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 93 to 100% identity among the cloned amplicons produced using the potyvirus-specific primers (GenBank Accessions Nos. FJ653916, FJ653917, FJ653918, FJ653919, FJ653920, FJ653921, FJ653922, FJ653923, FJ653924, FJ653925, and FJ653926) and 92 to 97% with a corresponding nucleotide sequence of Bean common mosaic virus (BCMV) from Taiwan (No. AY575773) and 88 to 90% with BCMV sequences from China (No. AJ312438) and the United States (No. AY863025). The sequence analysis indicated that BCMV isolates from yardlong bean are more closely related to an isolate from Taiwan than with isolates from China and the United States. The CMV isolates (GenBank No. FJ687054) each were 100% identical and 96% identical with corresponding sequences of CMV subgroup I isolates from Thailand (No. AJ810264) and Malaysia (No. DQ195082). Both BCMV and CMV have been documented in soybean, mungbean, and peanut in East Java of Indonesia (2). Previously, BCMV, but not CMV, was documented on yardlong beans in Guam (4). To our knowledge, this study represents the first confirmed report of CMV in yardlong bean in Indonesia and is further evidence that BCMV is becoming established in Indonesia. References: (1) J. Aramburu et al. J. Phytopathol. 155:513, 2007. (2) S. K. Green et al. Plant Dis. 72:994, 1988. (3) C. Ha et al. Arch. Virol. 153:25, 2008. (4) G. C. Wall et al. Micronesica 29:101, 1996.

Occurrence of Banana Bunchy Top Disease Caused by the Banana bunchy top virus on Banana and Plantain (Musa sp.) in Cameroon.

Banana bunchy top virus (BBTV; genus Babuvirus, family Nanoviridae) is a serious pathogen of banana (AAA genome) and plantain (AAB genome) (Musa sp.). It is transmitted by the banana aphid (Pentalonia nigronervosa) in a persistent manner (1). In recent years, BBTV has emerged as a major constraint to banana and plantain production in several countries of Africa and had been previously confirmed in viz., Burundi, Central African Republic, Republic of Congo, Democratic Republic of Congo, Egypt, Equatorial Guinea, Gabon, Malawi, and Rwanda (1) and more recently in Mozambique and Zambia (2) and Angola (3). To assess the potential threat of BBTV in West-Central Africa, we conducted surveys in August and September 2008 in 36 major banana- and plantain-producing regions of Littoral, South, Southwest, and Western Provinces of Cameroon. DNA was extracted from 520 plants and tested by PCR with primers specific for a conserved domain of BBTV DNA-R segment (4). A 240-bp DNA fragment specific to the virus was amplified in 31 samples from 18 plantain and 13 banana plants from Southwest, Western, and Southern Cameroon. Among virus-positive samples, symptoms (upright leaf growth, small leaves with pale chlorotic margins that choked the throat of the plant creating the bunchy appearance at the top) typical of bunchy top disease were observed only in banana (cv. Cavendish Williams) from Muea in the Southwest Province. PCR products obtained from the symptomatic and asymptomatic banana (Cavendish Williams) from Muea and Abang, respectively, were cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and two independent clones from each isolate were sequenced in both directions. Pairwise comparison of these sequences showed 100% sequence homology. A comparison of these sequences (Accession No. F580970) with corresponding sequences in GenBank showed 99% nt sequence identity with a BBTV isolate from Angola (Accession No. EU851977) and 96 to 98% identity with BBTV isolates belonging to the South Pacific group (Australia, Africa, South Asia, and South Pacific). However, the BBTV isolate from Cameroon showed 85 to 90% sequence identity with isolates belonging to the Asian group (China, Indonesia, Japan, Taiwan, Philippines, and Vietnam). To further confirm the virus identity, complete nucleotide sequence of the DNA-SCP segment that encodes for the virus coat protein was determined using PCR amplification of viral DNA (1), cloning of products into pCR2.1 vector, and sequencing. The derived sequence (1,075 nt; Accession No. GQ249344) in BLAST search at NCBI database revealed 98% nt sequence identity with coat protein gene of BBTV isolate from Burundi (Accession No. AF148943). These results, together with phylogenetic analysis, indicate that BBTV isolates from Cameroon have greater affinity to the South Pacific group. To our knowledge, this is the first report of BBTV in West-Central Africa. The occurrence of BBTV in the Western and Southern provinces of Cameroon, neighboring north of Gabon, suggests a possible spread of the virus from Gabon. This report also underscores the need to monitor other countries of West Africa for BBTV and enforce quarantine measures to prevent further spread through infected suckers from endemic areas of West and Central Africa. References: (1) I. Amin et al. Virus Genes 36:191, 2008. (2) W. T. Gondwe et al. InfoMusa 16:38, 2007. (3) P. L. Kumar et al. Plant Pathol. 58:402, 2009. (4) S. Mansoor et al. Mol. Biotechnol. 30:167, 2005.

First Report of the Occurrence of African cassava mosaic virus in a Mosaic Disease of Soybean in Nigeria.

African cassava mosaic virus (ACMV; genus Begomovirus, family Geminiviridae) is one of six viruses documented in cassava (Manihot esculenta Crantz.) plants showing cassava mosaic disease in sub-Saharan Africa (SSA). In addition to cassava, the natural host range of ACMV includes a few wild Manihot species, Jatropha multifida, and Ricinus communis L. in Euphorbiaceae, and Hewittia sublobata in Convolvulaceae. The experimental host range of ACMV includes Nicotiana sp. and Datura sp. in the Solanaceae (2). Recently, natural occurrence of ACMV was reported in Combretum confertum (Benth.), Leucana leucocephala (Lam.) De Witt, and Senna occidentalis (L.) Link belonging to Leguminasae from Nigeria (1,3). During reconnaissance studies conducted on soybean (Glycine max L. Merr.) in September and October of 2007 in the Ibadan (N = 19) and Benue (N = 23) regions and in February of 2008 in Ibadan (N = 16), we observed soybean showing yellow mosaic and mottling symptoms. Samples from these plants (N = 58) were tested by indirect ELISA and symptomatic leaves tested negative to Cucumber mosaic virus, Cowpea mottle virus, Southern bean mosaic virus, Tobacco ringspot virus, Soybean dwarf virus, Cowpea aphid-borne mosaic virus, Blackeye cowpea mosaic virus, Peanut mottle virus, and Broad bean mosaic virus, which have been documented in soybean in SSA. However, 8.6% of these samples (5 of 58) (one each from Ibadan and Benue in the 2007 survey and three from Ibadan in the 2008 survey) tested positive in triple-antibody sandwich-ELISA with a monoclonal antibody (SCR33) to ACMV. ELISA results were further confirmed by PCR with ACMV specific primers AL1/F and AR0/R that amplified a 987-bp DNA fragment corresponding to the intergenic region, AC-4 and AC-1 genes of DNA-A segment (4). The PCR product was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and three independent clones were sequenced in both orientations. Pairwise comparison of the derived consensus sequence (GenBank Accession No. EU367500) with corresponding ACMV sequence of ACMV isolate from Nigeria (GenBank Accession No. X17095) showed 98% identity at the nucleotide level. To further confirm the virus identity, complete nucleotide sequence of the DNA-A segment was determined by PCR amplification of viral DNA with four primers, cloning of overlapping products into pCR2.1 vector and sequencing. The derived sequence (2,781 nucleotides; GenBank Accession No. EU685385) was compared with the DNA sequences available at NCBI database using BLAST. This revealed 97% nucleotide sequence identity with ACMV-[NG:Ogo:90] (Accession No. AJ427910) and ACMV-[NG] (Accession No. X17095) from Nigeria. These results confirm the presence of ACMV in symptomatic soybean leaves. To our knowledge, this is the first report of soybean as a natural host of ACMV in SSA. On the basis of previous reports (1) and the results currently presented it seems that ACMV has a wide host range. References: (1) O. J. Alabi et al. Phytopathology (Abstr.) 97(suppl.):S3, 2007. (2) A. A. Brunt et al., eds. Plant viruses online: Descriptions and lists from the VIDE database. Version 20. Online publication, 1996. (3) F. O. Ogbe et al. Plant Dis. 90:548, 2006; (4) X. Zhou et al. J. Gen. Virol. 78:2101, 1997.

First Report of Cucumber mosaic virus in Yams (Dioscorea spp.) in Ghana, Togo, and Republic of Benin in West Africa.

Yam (Dioscorea spp., family Dioscoreaceae) is one of the most important food crops cultivated in the West African yam zone comprising the forest and savannah areas of Nigeria, Ghana, Côte d'Ivoire, Republic of Benin, and Togo, which account for more than 90% of the 4.59 million ha of yam cultivation worldwide (1). A survey was conducted in 2005 to document viruses in yams in Ghana, Togo, and the Republic of Benin. Samples (1,405) from five species of yam showing mosaic, chlorosis, and stunting as well as asymptomatic plants were tested for Dioscorea bacilliform virus (DBV, genus Badnavirus), Yam mosaic virus (YMV, genus Potyvirus), and Yam mild mosaic virus (YMMV, genus Potyvirus), the three most common viruses infecting yams. In addition, samples were tested for Cucumber mosaic virus (CMV), since CMV was previously reported to infect yams in Côte d'Ivoire (2) and Nigeria (3). In protein-A sandwich-ELISA with polyclonal antibodies to a cowpea isolate of CMV, 23 of the 1,405 samples (6 of 218 samples from Togo, 13 of 628 samples from Ghana, and 4 of 559 samples from Republic of Benin) tested positive for CMV. The CMV-positive samples were from D. alata (N = 16) and D. rotundata (N = 7), whereas all samples from D. cayenensis, D. dumetorum, and D. bulbifera tested negative. CMV was detected as mixed infections with DBV, YMV, or YMMV in 21 of 23 samples. Some of these samples showed puckering, chlorosis, mottling, and crinkling, whereas some plants infected by two or more viruses were asymptomatic. Only two samples from D. rotundata had a single infection of CMV and they showed mild chlorotic symptoms in young leaves that were inconspicuous in mature leaves. In sap inoculations, the virus induced systemic mosaic in Nicotiana glutinosa. The presence of CMV in ELISA-positive yam samples was further confirmed by immunocapture-reverse transcription (IC-RT)-PCR using CMV antibodies as trapping antibody and oligonucleotide primers specific for a 485 nt corresponding to 3' end of the coat protein gene and C-terminal noncoding region of RNA-3 (4). To confirm the specificity of IC-RT-PCR, the 485-bp amplicons from an isolate from the Republic of Benin was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and three independent clones were sequenced from both orientations. Pairwise comparison of a consensus sequence (Accession No. EU274471) with corresponding sequences of other CMV isolates deposited in GenBank showed 99% identity at the nucleotide sequence level (Accession No. U22821) and revealed that the CMV isolate from yam belongs to sub-Group IA. To our knowledge, this is the first report of CMV infection in yams (D. alata and D. rotundata) in Ghana, Togo, and the Republic of Benin. Together with a previous documentation of CMV in D. alata and D. trifida in Côte d'Ivoire and Nigeria (2,3), this report adds to existing knowledge on distribution of CMV in yams with implications for yam production and germplasm distribution in the West Africa Region. References: (1) FAO. Online publication. FAOSTAT, 2007. (2) C. Fauquet and J. C. Thouvenel. Plant Viral Diseases in the Ivory Coast. ORSTROM: Documentation Techniques. Paris, 1987. (3) Jd'A. Hughes et al. Phytopathology 87:S45, 1997. (4) S. Wylie et al. Aus. J. Agric. Res. 44:41, 1993.

Status of Cassava Begomoviruses and Their New Natural Hosts in Nigeria.

A diagnostic survey was conducted in 2002-03 to determine the status of cassava mosaic begomoviruses in Nigeria and to ascertain if the virulent Ugandan variant of East African cassava mosaic virus (EACMV-Ug2) was present. Of the 418 farms visited, 48% had cassava with moderately severe or severe symptoms, whereas 52% had cassava with mild symptoms. These distributions were at random. Of the 1,397 cassava leaf samples examined, 1,106 had symptoms. In polymerase chain reaction tests, 74.1% of the symptom-bearing samples tested positive for African cassava mosaic virus (ACMV) alone, 0.3% for EACMV alone, 24.4% for mixed infections by the two viruses, and 1.2% did not react with any of the primers used. The two viruses also were detected in 32% of the 291 symptomless plants and in the whitefly vector samples. EACMV-Ug2, Indian cassava mosaic virus, and South African cassava mosaic virus were not detected in any of the whitefly or leaf samples. Most farms had ACMV in single infection as well as in mixed infections with EACMV. Most doubly infected plants showed severe symptoms. Two biological variants of ACMV were identified based on symptom expression on cassava in the field. ACMV and EACMV were detected in the leguminous plant Senna occidentalis (L.) Link and the weed Combretum confertum Lams.; these are new natural hosts of the viruses. Although the virulent EACMV-Ug2 was not detected, the occurrence of variants of ACMV and a high proportion of mixed infections by ACMV and EACMV, which could result in recombination events such as the one that produced EACMV-Ug2, demands appropriate measures to safeguard cassava production in Nigeria.

Pseudococcus maritimus (Hemiptera: Pseudococcidae) and Parthenolecanium corni (Hemiptera: Coccidae) are capable of transmitting grapevine leafroll-associated virus 3 between Vitis x labruscana and Vitis vinifera.

The grape mealybug, Pseudococcus maritimus (Ehrhorn), and European fruit lecanium scale, Parthenolecanium corni (Bouché), are the predominant species of Coccoidea in Washington State vineyards. The grape mealybug has been established as a vector of Grapevine leafroll-associated virus 3 (GLRaV-3) between wine grape (Vitis vinifera L.) cultivars, elevating its pest status. The objective of this study was to determine if GLRaV-3 could be transmitted between Vitis x labruscana L. and V. vinifera by the grape mealybug and scale insects. Three transmission experiments were conducted with regard to direction; from V. vinifera to V. x labruscana L., from V. x labruscana L. to V. x labruscana L., and from V. x labruscana L. to V. vinifera. Each experiment was replicated 15 times for each vector species. Crawlers (first-instars) of each vector species were allowed 1-wk acquisition and inoculation access periods. The identities of viral and vector species were confirmed by reverse transcription-polymerase chain reaction, cloning, and sequencing of species-specific DNA fragments. GLRaV-3 was successfully transmitted by both species in all experiments, although Ps. maritimus was a more efficient vector under our experimental conditions. To the best of our knowledge, this study represents the first documented evidence of interspecific transmission of GLRaV-3 between two disparate Vitis species. It also highlights the potential role of V. x labruscana L. in the epidemiology of grapevine leafroll disease as a symptomless source of GLRaV-3 inoculum.

Effect of Aqueous Moringa Oleifera (Lam) Leaf Extracts on Growth Performance and Carcass Characteristics of Hubbard Broiler Chicken

Two hundred and forty day old broiler chicks were used to investigate the effect of aqueous Moringa oleifera leaf extracts (AMOLE) on growth performance and carcass characteristics of broiler chicken. The birds were randomly allocated into six treatments with four replicates, and each replicate containing 10 broiler chicks; the CRD was used. The treatments contained AMOLE0+ (positive control with antibiotic treatment), AMOLE0- (negative control with ordinary water), AMOLE60 (60 ml/l), AMOLE90 (90 ml/l), AMOLE120 (120 ml/l) and AMOLE150 (150 ml/l) inclusion levels of AMOLE, respectively. Birds on positive control had the highest final body weight and growth rate (2392.00 g and 53.61 g respectively) and the ones on 150 ml/l of AMOLE had the least (2042.00 g and 45.37 g respectively). Results of feed intake showed that birds on positive control had the highest (84.70 g) and the ones on 90 ml/litre of AMOLE had the lowest (73.19 g); while the results of feed conversion ratio indicated that birds on AMOLE90 and AMOLE120 performed better than the positive control treatment. Birds on the AMOLE had similar dressing percentages though that of positive control was highest (94.93 %); while those on AMOLE60 and AMOLE150 had the highest large intestine and lung weights respectively. Aqueous Moringa oleifera leaf extract can be included up to 90 ml/litre in the drinking water of broiler chicken for reduced feed intake (12.83 %) and improved feed conversion efficiency (9.11) thus, AMOLE can be used to replace synthetic antibiotics as growth promoter.(AU)

Effect of Aqueous Moringa Oleifera (Lam) Leaf Extracts on Growth Performance and Carcass Characteristics of Hubbard Broiler Chicken

Two hundred and forty day old broiler chicks were used to investigate the effect of aqueous Moringa oleifera leaf extracts (AMOLE) on growth performance and carcass characteristics of broiler chicken. The birds were randomly allocated into six treatments with four replicates, and each replicate containing 10 broiler chicks; the CRD was used. The treatments contained AMOLE0+ (positive control with antibiotic treatment), AMOLE0- (negative control with ordinary water), AMOLE60 (60 ml/l), AMOLE90 (90 ml/l), AMOLE120 (120 ml/l) and AMOLE150 (150 ml/l) inclusion levels of AMOLE, respectively. Birds on positive control had the highest final body weight and growth rate (2392.00 g and 53.61 g respectively) and the ones on 150 ml/l of AMOLE had the least (2042.00 g and 45.37 g respectively). Results of feed intake showed that birds on positive control had the highest (84.70 g) and the ones on 90 ml/litre of AMOLE had the lowest (73.19 g); while the results of feed conversion ratio indicated that birds on AMOLE90 and AMOLE120 performed better than the positive control treatment. Birds on the AMOLE had similar dressing percentages though that of positive control was highest (94.93 %); while those on AMOLE60 and AMOLE150 had the highest large intestine and lung weights respectively. Aqueous Moringa oleifera leaf extract can be included up to 90 ml/litre in the drinking water of broiler chicken for reduced feed intake (12.83 %) and improved feed conversion efficiency (9.11) thus, AMOLE can be used to replace synthetic antibiotics as growth promoter.

Banana bunchy top virus in sub-Saharan Africa: investigations on virus distribution and diversity.

Banana bunchy top virus (BBTV) was first reported from sub-Saharan Africa (SSA) from Democratic Republic of Congo (DRC) in the 1950s, has become invasive and spread into 11 countries in the region. To determine the potential threat of BBTV to the production of bananas and plantains (Musa spp.) in the sub-region, field surveys were conducted for the presence of banana bunchy top disease (BBTD) in the DRC, Angola, Cameroon, Gabon and Malawi. Using the DNA-S and DNA-R segments of the virus genome, the genetic diversity of BBTV isolates was also determined from these countries relative to virus isolates across the banana-growing regions around the world. The results established that BBTD is widely prevalent in all parts of DRC, Malawi, Angola and Gabon, in south and western part of Cameroon. Analysis of the nucleotide sequences of DNA-S and DNA-R indicate that BBTV isolates from these countries are genetically identical forming a unique clade within the 'South Pacific' phylogroup that includes isolates from Australia, Egypt, South Asia and South Pacific. These results imply that farmers' traditional practice of transferring vegetative propagules within and between countries, together with virus spread by the widely prevalent banana aphid vector, Pentalonia nigronervosa, could have contributed to the geographic expansion of BBTV in SSA. The results provided a baseline to explore sanitary measures and other 'clean' plant programs for sustainable management of BBTV and its vector in regions where the disease has already been established and prevent the spread of the virus to as yet unaffected regions in SSA.