Genetically engineered virus-resistant plants in developing countries: current status and future prospects.

Plant viruses cause severe crop losses worldwide. Conventional control strategies, such as cultural methods and biocide applications against arthropod, nematode, and plasmodiophorid vectors, have limited success at mitigating the impact of plant viruses. Planting resistant cultivars is the most effective and economical way to control plant virus diseases. Natural sources of resistance have been exploited extensively to develop virus-resistant plants by conventional breeding. Non-conventional methods have also been used successfully to confer virus resistance by transferring primarily virus-derived genes, including viral coat protein, replicase, movement protein, defective interfering RNA, non-coding RNA sequences, and protease, into susceptible plants. Non-viral genes (R genes, microRNAs, ribosome-inactivating proteins, protease inhibitors, dsRNAse, RNA modifying enzymes, and scFvs) have also been used successfully to engineer resistance to viruses in plants. Very few genetically engineered (GE) virus resistant (VR) crops have been released for cultivation and none is available yet in developing countries. However, a number of economically important GEVR crops, transformed with viral genes are of great interest in developing countries. The major issues confronting the production and deregulation of GEVR crops in developing countries are primarily socio-economic and related to intellectual property rights, biosafety regulatory frameworks, expenditure to generate GE crops and opposition by non-governmental activists. Suggestions for satisfactory resolution of these factors, presumably leading to field tests and deregulation of GEVR crops in developing countries, are given.

Molecular characterization of whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) populations infesting cassava.

Bemisia tabaci (Gennadius) populations, collected from cassava and other plants in major cassava-cultivation areas of Sub-saharan Africa and from elsewhere around the world, were studied to determine their biotype status and genetic variation. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers were used to examine the genetic structure of the populations. The dendogram obtained using the neighbour joining method (NJ) split the cassava-associated populations from the non-cassava types with a 100% bootstrap probability. Analysis of molecular variance (AMOVA) of the RAPD fragments revealed that 63.2% of the total variation was attributable to differences among populations, while the differences among groups (host) and within populations accounted for 27.1 and 9.8% respectively. Analysis of the internally transcribed spacer region I (ITS 1) of the ribosomal DNA confirmed that the cassava populations of B. tabaci populations were distinct from non-cassava populations. Experiments to establish whitefly populations on various host plants revealed that cassava-associated populations were restricted to cassava only, whereas B. tabaci from other hosts were polyphagous but did not colonize cassava. Hence, populations of B. tabaci from cassava in Africa represent a distinct group.

Variants of East African cassava mosaic virus and Its Distribution in Double Infections with African cassava mosaic virus in Nigeria.

In a survey for cassava mosaic begomoviruses conducted in 1997 and 1998 in Nigeria, East African cassava mosaic virus (EACMV) was detected by the polymerase chain reaction together with African cassava mosaic virus (ACMV) in 27 out of 290 cassava leaf samples of infected plants from 254 farmers' fields in five agroecological zones. One plant was infected with EACMV only. Five variant isolates of EACMV were observed based on their reactions to primers that could detect Cameroonian and East African strains of EACMV. Isolates of variants 1 and 3 occurred mostly in the derived or coastal and southern Guinea savannahs, while variants 4 and 5 predominated in the humid forest region. Isolates of variant 2 were widely distributed across the three agroecologies. EACMV was not detected in the northern Guinea savannah and arid and semiarid zones. Most doubly infected plants showed more severe symptoms than plants with single infection. Occurrence of EACMV variants together with ACMV detection and information about their distribution in Nigeria could be used for the selection of cassava clones in cassava mosaic disease resistance programs.

Purification and characterization of a Bacillus cereus exochitinase.

Five extracellular chitinases of Bacillus cereus 6E1 were detected by a novel in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant violet 5R (CM-chitin-RBV) as a substrate. The major chitinase activity was associated with a 36-kDa (Chi36) gel band. Chi36 was purified by a one-step, native gel purification procedure derived from the new in-gel chitinase assay. The purified Chi36 has optimal activity at pH 5.8 and retains some enzymatic activity between pH 2.5-8. The temperature optimum for Chi36 was 35 degrees C, but the enzyme was active between 4-70 degrees C. Based on its ability to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta-D-glucosaminide)(2), Chi36 is characterized as a chitobiosidase, a type of exochitinase. The N-terminal amino acid sequence of mature Chi36 was determined (25 amino acids). Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36. The N-terminal sequence of Chi36 demonstrated highest similarity with Bacillus circulans WL-12 chitinase D and significant similarity with several other bacterial chitinases.

Molecular cloning and structural analysis of the gene encoding Bacillus cereus exochitinase Chi36.

The chi36 gene encoding exochitinase Chi36 was cloned from a Bacillus cereus 6E1 subgenomic library. The chi36 open reading frame is 1080 bp long encoding a Chi36 precursor protein of 360 amino acids, consisting of a 27 amino acid N-terminal signal peptide and a 333 amino acid sequence found in the mature Chi36 protein of 36.346 kDa. Chi36 shows significant amino acid sequence similarity to many bacterial chitinases, but has highest similarity to B. circulans WL-12 chitinase D. Chi36 belongs to subfamily B of bacterial chitinases in family 18 of glycosyl hydrolases. Chi36 shows a simple and compact structural organization composed of an N-terminal signal peptide and a C-terminal (beta/alpha)8-barrel catalytic domain (CaD). The Chi36 signal peptide is recognized by Escherichia coli, allowing Chi36 secretion. Chi36 is the first one-domain (CaD) bacterial chitinase cloned from B. cereus.

Detection of episomal banana streak badnavirus by IC-PCR.

A polymerase chain reaction (PCR) based strategy to detect episomal banana streak badnavirus (BSV) in banana and plantain plants that carry integrated BSV sequences was developed. Antisera used in immuno-capture polymerase chain reaction (IC-PCR) are capable of binding a large number of BSV serotypes. The primers used for PCR are capable of annealing to and amplifying across the aspartic protease-reverse transcriptase domain boundaries of both episomal and integrated BSV sequences and result in similar or identical sequence size fragments from either template. However, we show that under the conditions selected for IC-PCR, nuclear, mitochondrial or chloroplast genomic sequences are not amplified and thus only captured episomal BSV is amplified. IC-PCR is suitable for the large-scale screening of Musa for episomal BSV which is necessary for germplasm movement.

First Report of East African Cassava Mosaic Begomovirus in Ghana.

Virus species causing cassava mosaic disease have been categorized into three classes based on their reaction with monoclonal antibodies (MAbs) and their distribution (2). These viruses have different, scarcely overlapping distribution: African cassava mosaic begomovirus (ACMV) occurs in Africa west of the Rift Valley and in South Africa; East African cassava mosaic (EACMV) occurs in Africa east of the Rift Valley and in Madagascar; and Indian cassava mosaic virus (ICMV) occurs in India and Sri Lanka (2). During 1998, surveys were conducted in farmers' fields in Ghana to assess the incidence and reaction of local cassava cultivars to cassava mosaic disease. Leaf samples from symptomatic plants were indexed by triple antibody sandwich-enzyme-linked immunosorbent assay with crude extracts and monoclonal antibodies obtained from the International Institute of Tropical Agriculture (IITA). Each sample was assayed with monoclonal antibody SCR 23, which detects ACMV and EACMV, SCR 33, which detects ACMV, and SCR 58, which detects ICMV. None of the samples reacted with SCR 58. Two of the samples collected from the western region of Ghana produced strong reactions with MAb SCR23 but did not react with ACMV-specific MAb SCR 33. This result was consistent in three separate experiments conducted on the samples, confirming that the virus was EACMV and not ACMV. The results extend the work by Ogbe et al. (1) and provide further evidence of the occurrence of EACMV in west Africa. References: (1) F. O. Ogbe et al. Plant Dis 83:398, 1999. (2) M. M. Swanson and B. D. Harrison. Trop. Sci. 34:15, 1994.

A New Virus on Maize in Nigeria: Maize Mild Mottle Virus.

Maize (Zea mays) and itch grass (Rottboellia cochinchinensis) plants exhibiting a mild mosaic or mottle were collected from a farmer's field near Mokwa, Nigeria, in 1993. Icosahedral virions (approximately 28 to 30 nm) were purified from symptomatic tissue by differential centrifugation in 0.1 M phosphate buffer, pH 7.0. The virions are composed of one single-stranded positive-sense RNA of approximately 4,000 nucleotides and, as estimated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a capsid protein of approximately 28 kDa. The virus was readily detected in infected plants by enzyme-linked immunosorbent assay and immunoblot assays with a polyclonal rabbit antibody derived from purified virions. A partial cDNA library was generated with random primers. Sequence analyses of a cDNA clone representing a portion of the putative replicase gene aligned most closely with related sequences of viruses within the Tombusviridae. In particular, a region of 78 predicted amino acids surrounding the "GDD" replicase motif shares 73% identity with panicum mosaic virus and 61% identity with maize chlorotic mottle virus. The virus is readily transmitted by mechanical inoculation to sweet and dent corn, millet, and wheat. Currently it is not considered of economic importance in Nigeria. The data suggest that "maize mild mottle virus" is a newly identified virus infecting maize.

First Report of East African Cassava Mosaic Begomovirus in Nigeria.

Cassava (Manihot esculenta Crantz) is an important food crop in sub-Saharan Africa. One of the major production constraints is cassava mosaic disease caused by African cassava mosaic (ACMV) and East African cassava mosaic (EACMV) begomoviruses. ACMV is widespread in its distribution, occurring throughout West and Central Africa and in some eastern and southern African countries. In contrast, EACMV has been reported to occur mainly in more easterly areas, particularly in coastal Kenya and Tanzania, Malawi, and Madagascar. In 1997, a survey was conducted in Nigeria to determine the distribution of ACMV and its strains. Samples from 225 cassava plants showing mosaic symptoms were tested with ACMV monoclonal antibodies (MAbs) in triple antibody sandwich enzyme-linked immunosorbent assay (1). Three samples reacted strongly with MAbs that could detect both ACMV and EACMV. One of them did not react with ACMV-specific MAbs while the other two reacted weakly with such MAbs. With polymerase chain reaction (2), the presence of EACMV and a mixture of EACMV and ACMV in the respective samples was confirmed. These samples were collected from two villages: Ogbena in Kwara State and Akamkpa in Cross River State. Co-infection of some cassava varieties with ACMV and EACMV leads to severe symptoms. More importantly, a strain of mosaic geminivirus known as Uganda variant arose from recombination between the two viruses (2). This report provides evidence for the presence of EACMV in West Africa. References: (1) J. E. Thomas et al. J. Gen. Virol. 67:2739, 1986. (2) X. Zhou et al. J. Gen. Virol. 78:2101, 1997.

Properties of a virus causing mosaic and leaf curl disease of Celosia argentea L. in Nigeria.

A sap transmissible virus, causing mosaic and leaf curl disease of Celosia argentea, was isolated at vegetable farms in Amuwo Odofin, Tejuoso, and Abule Ado, Lagos, Nigeria. The virus had a restricted host range confined to a few species of the Amaranthaceae, Chenopodiaceae and Solanaceae families. It failed to infect several other species of the Aizoaceae, Brassicaceae, Cucurbitaceae, Fabaceae, Lamiaceae, Malvaceae, Poaceae and Tiliaceae families. The virus was transmitted in a non-persistent manner by Aphis spiraecola and Toxoptera citricidus but not by eight other aphid species tested. There was no evidence of transmission by seeds of C. argentae varieties. The viral coat protein had a relative molecular mass (M(r)) of about 30.2 K. Electron microscopy of purified virus preparations revealed flexuous rod shaped particles of about 750 nm in length. Serological studies were performed using the enzyme-linked immunosorbent assay (ELISA), immunosorbent electron microscopy (ISEM) and Western blot analysis. The virus reacted positively with an universal potyvirus group monoclonal antibody (MoAb) and MoAb P-3-3H8 raised against peanut stripe potyvirus. It also reacted with polyclonal antibodies raised against several potyviruses including asparagus virus-1 (AV-1), turnip mosaic virus (TuMV), maize dwarf mosaic virus (MDMV), watermelon mosaic virus (WMV-2), plum pox virus (PPV), soybean mosaic virus (SoyMV), lettuce mosaic virus (LMV), bean common mosaic virus (BCMV) and beet mosaic virus (BMV) in at least one of the serological assays used. On the basis of host range, mode of transmission, and available literature data, the celosia virus seems to be different from potyviruses previously reported to infect vegetables in Nigeria. The name celosia mosaic virus (CIMV) has been proposed for this virus.

Effect of Temperature on Symptom Expression and Reliability of Banana Streak Badnavirus Detection in Naturally Infected Plantain and Banana (Musa spp.).

The effect of temperature on symptom expression and detection of banana streak badnavirus (BSV) by immunosorbent electronmicroscopy (ISEM) and enzyme-linked immunosorbent assay of 12 in vitro-propagated plantain hybrids (genome AAB × AA), 3 ABB cooking banana, and 3 AAB plantain landraces was studied. Experiments were done for 2 years under two temperature regimes, 28 to 35°C in a screenhouse and 22°C in a temperature-controlled room. Most BSV-infected plants of plantain hybrids expressed symptoms under both conditions. Symptom expression was enhanced when plants were continuously grown at 22°C, but later became indiscernible when plants were continuously grown at 28 to 35°C. Plants grown at 22°C and showing severe symptoms contained significantly higher virus titer than plants grown at 28 to 35°C. When asymptomatic plants with very low virus titer at 28 to 35°C were transferred back to 22°C, there was a significant increase in both symptom severity and concentration of virus (greater than 3 to 5 times) in leaf tissues after 9 months. In contrast, the concentration of virus and symptom severity decreased in plants after transfer from 22°C to 28 to 35°C. Micropropagated plants of AAB plantain landrace cv. Mimi Abue and ABB cooking bananas (cvs. Bluggoe, Cardaba, and Pelipita) did not express visible symptoms under either temperature regime, but BSV was detected by ISEM in 23% of the plants. After 2 years at 22°C, virus was detected in 64% of the plants, but the concentration of virus remained low. Implications of these results on quarantine screening of in vitro plants and virus diagnosis are discussed.

Cowpea embryo rescue. 1. Influence of culture media composition on plant recovery from isolated immature embryos.

Factors responsible for successful rescue of immature embryos of cowpea [Vigna unguiculata (L.) Walp.] and V. vexillata (L.) and for in vitro embryo development were studied. A new basal medium for embryo development in vitro was formulated on the basis of the mineral composition of embryos. Sucrose, fructose and glucose were compared as carbohydrate sources. The highest frequency of embryos developing into plants was obtained with sucrose. Adding casein hydrolysate to the medium increased plant recovery by 30%. Among the plant growth factors used, cytokinins, zeatin, 6-benzylaminopurine and kinetin were the most effective in promoting embryo maturation and development. A method that can routinely ensure high plant recovery from cultured immature cowpea embryos is proposed.

Identification of okra mosaic virus from Indigofera spicata in Nigeria.

Okra mosaic virus (OMV, tymovirus group) was isolated from Indigofera spicata plants growing at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria. Its identity was established on the basis of particle morphology, analysis of viral coat protein and nucleic acid and serology. In reciprocal agar gel diffusion tests, the virus isolate from I. spicata and an OMV isolate from okra in Ibadan (OMV-Ibadan isolate) were found to be serologically identical. However, because the isolates differ in symptom induction in various host plants, the name OMV-Indigofera isolate is suggested. This is the first report on the occurence of OMV in I. spicata.

Evidence that cowpea aphid-borne mosaic and blackeye cowpea mosaic viruses are two different potyviruses.

The immunoreactivity of a panel of polyclonal antibodies and monoclonal antibodies (MAbs) raised against African isolates of potyviruses from cowpea and African yam bean was examined in ELISAs. A serological study including reference isolates followed by further characterization in differential hosts resulted in separation of the potyviruses into two distinct serogroups, one containing blackeye cowpea mosaic virus (BlCMV) and the other containing cowpea aphid-borne mosaic virus (CAMV). Using biotin-labelled MAbs, the BlCMV isolates were further subdivided into two serotypes and the CAMV isolates into five serotypes. Because both BlCMV and CAMV induce a very similar mosaic disease in cowpea, different ELISA procedures using mixed MAbs were evaluated and a single protocol was developed which allowed reliable diagnosis of both viruses.

Origin and phylogeny of Guinea yams as revealed by RFLP analysis of chloroplast DNA and nuclear ribosomal DNA.

The origin and phylogeny of the Guinea yams, consisting of the white yam (Dioscorea rotundata Poir.) and the yellow yam (D. cayenensis Lamk.), has been investigated. Fourteen cultivars of Guinea yams were sampled with 12 accessions from seven wild yam species. A total of 26 accessions were surveyed for restriction fragment length polymorphisms (RFLP) in chloroplast DNA (cpDNA) and nuclear ribosomal DNA (rDNA) using seven restriction endonucleases and various heterologous probes. Chloroplast DNA probes covering 80% of the total chloroplast genome revealed nine restriction site changes and one length mutation among the cpDNAs of Guinea yams and their wild relatives. The estimated numbers of nucleotide substitutions per site (d) among these cpDNAs were very low (0.0005-0.0027), indicating a rather recent divergence of this group. On the basis of these ten mutations, five chloroplast genome types (A-E) were recognized. It was revealed that two cultivated species (D. rotundata and D. cayenensis) display the same chloroplast genome type, type A, as the three wild species D. praehensilis, D. liebrechtsiana and D. abyssinica. Chloroplast genome types B, C, D and E were found in D. minutiflora, D. burkilliana, D. smilacifolia and D. togoensis, respectively. Maximum parsimony analysis produced a hypothetical phylogeny of three primary lineages among cpDNAs of Guinea yams and their relatives: the genome type A lineage, the genome type B lineage and one lineage including genome types C, D and E.Using rDNA clones of rice and taro as probes, we detected ribosomal DNA variation, presumably at the intergenic spacer region, in Guinea yams and their wild relatives. The survey of rDNA together with that of cpDNA indicates that D. rotundata (white yam) was domesticated from either D. abyssinica, D. liebrechtsiana or D. praehensilis or their hybrid, and that D. cayenensis (yellow yam) is derived from hybridization between a male plant of either D. burkilliana, D. minutiflora or D. smilacifolia and a female plant of either D. rotundata, D. abyssinica, D. liebrechtsiana or D. praehensilis. We propose that the previous nomenclature of white yam should be retained, D. rotundata Poir. nomen nudum, and that yellow yam should be treated as a variety of D. rotundata, denoted as D. rotundata var. x 'cayenensis'.