A newly emerging alphasatellite affects banana bunchy top virus replication, transcription, siRNA production and transmission by aphids.

Banana bunchy top virus (BBTV) is a six-component ssDNA virus (genus Babuvirus, family Nanoviridae) transmitted by aphids, infecting monocots (mainly species in the family Musaceae) and likely originating from South-East Asia where it is frequently associated with self-replicating alphasatellites. Illumina sequencing analysis of banana aphids and leaf samples from Africa revealed an alphasatellite that should be classified in a new genus, phylogenetically related to alphasatellites of nanoviruses infecting dicots. Alphasatellite DNA was encapsidated by BBTV coat protein and accumulated at high levels in plants and aphids, thereby reducing helper virus loads, altering relative abundance (formula) of viral genome components and interfering with virus transmission by aphids. BBTV and alphasatellite clones infected dicot Nicotiana benthamiana, followed by recovery and symptomless persistence of alphasatellite, and BBTV replication protein (Rep), but not alphasatellite Rep, induced leaf chlorosis. Transcriptome sequencing revealed 21, 22 and 24 nucleotide small interfering (si)RNAs covering both strands of the entire viral genome, monodirectional Pol II transcription units of viral mRNAs and pervasive transcription of each component and alphasatellite in both directions, likely generating double-stranded precursors of viral siRNAs. Consistent with the latter hypothesis, viral DNA formulas with and without alphasatellite resembled viral siRNA formulas but not mRNA formulas. Alphasatellite decreased transcription efficiency of DNA-N encoding a putative aphid transmission factor and increased relative siRNA production rates from Rep- and movement protein-encoding components. Alphasatellite itself spawned the most abundant siRNAs and had the lowest mRNA transcription rate. Collectively, following African invasion, BBTV got associated with an alphasatellite likely originating from a dicot plant and interfering with BBTV replication and transmission. Molecular analysis of virus-infected banana plants revealed new features of viral DNA transcription and siRNA biogenesis, both affected by alphasatellite. Costs and benefits of alphasatellite association with helper viruses are discussed.

New Technologies Provide Innovative Opportunities to Enhance Understanding of Major Virus Diseases Threatening Global Food Security.

Plant viruses pose a continuous and serious threat to crop production worldwide, and globalization and climate change are exacerbating the establishment and rapid spread of new viruses. Simultaneously, developments in genome sequencing technology, nucleic acid amplification methods, and epidemiological modeling are providing plant health specialists with unprecedented opportunities to confront these major threats to the food security and livelihoods of millions of resource-constrained smallholders. In this perspective, we have used recent examples of integrated application of these technologies to enhance understanding of the emergence of plant viral diseases of key food security crops in low- and middle-income countries. We highlight how international funding and collaboration have enabled high-throughput sequencing-based surveillance approaches, targeted field and lab-based diagnostic tools, and modeling approaches that can be effectively used to support surveillance and preparedness against existing and emerging plant viral threats. The importance of national and international collaboration and the future role of CGIAR in further supporting these efforts, including building capabilities to make optimal use of these technologies in low- and middle-income countries, are discussed. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Global Cropland Connectivity: A Risk Factor for Invasion and Saturation by Emerging Pathogens and Pests.

The geographic pattern of cropland is an important risk factor for invasion and saturation by crop-specific pathogens and arthropods. Understanding cropland networks supports smart pest sampling and mitigation strategies. We evaluate global networks of cropland connectivity for key vegetatively propagated crops (banana and plantain, cassava, potato, sweet potato, and yam) important for food security in the tropics. For each crop, potential movement between geographic location pairs was evaluated using a gravity model, with associated uncertainty quantification. The highly linked hub and bridge locations in cropland connectivity risk maps are likely priorities for surveillance and management, and for tracing intraregion movement of pathogens and pests. Important locations are identified beyond those locations that simply have high crop density. Cropland connectivity risk maps provide a new risk component for integration with other factors-such as climatic suitability, genetic resistance, and global trade routes-to inform pest risk assessment and mitigation.

Morphological and molecular characterization of Pratylenchus species from Yam (Dioscorea spp.) in West Africa.

The root-lesion nematodes (RLN), Pratylenchus spp., are among the major plant-parasitic nematodes affecting yam (Dioscorea spp.) production in West Africa. The distribution and diversity of RLN species associated with yam was investigated through a soil and tuber survey of the main producing areas in Nigeria and Ghana. Pratylenchus spp. were detected in the yam rhizosphere in 59% of 81 soil samples from Ghana and 39% of 114 soil samples from Nigeria. Pratylenchus spp. were detected in 24 of 400 tubers examined, in combination with root-knot nematodes (Meloidogyne spp.) and their associated damage of galls and crazy roots (79%), and with yam nematode (Scutellonema bradys) and their associated damage of dry rot (17%), although no specific additional symptoms were observed for Pratylenchus spp. Species of Pratylenchus were identified by their morphological features and by sequences of the D2-D3 region of the 28 S rDNA gene and the mitochondrial cytochrome oxidase I gene (COI). Pratylenchus brachyurus was the most frequent RLN species in both the rhizosphere and tubers of yam. Pratylenchus hexincisus was recovered from one tuber collected in Nigeria. While further investigations are required to establish the host status of yam for this nematode, this appears to be the first record of P. hexincisus on yam. The present taxonomical status of P. scribneri and P. hexincisus is discussed.

Tissue culture and next-generation sequencing: A combined approach for detecting yam (Dioscorea spp.) viruses.

In vitro culture offers many advantages for yam germplasm conservation, propagation and international distribution. However, low virus titres in the generated tissues pose a challenge for reliable virus detection, which makes it difficult to ensure that planting material is virus-free. In this study, we evaluated next-generation sequencing (NGS) for virus detection following yam propagation using a robust tissue culture methodology. We detected and assembled the genomes of novel isolates of already characterised viral species of the genera Badnavirus and Potyvirus, confirming the utility of NGS in diagnosing yam viruses and contributing towards the safe distribution of germplasm.

Prevalence and Genome Characterization of Field Isolates of Sugarcane Mosaic Virus (SCMV) in Nigeria.

Maize and sugarcane are two economically important crops often grown in adjacent fields or co-cultivated in the northern guinea savannah agroecological zone, a major cereal production region of Nigeria. This study was conducted to determine the prevalence of mosaic disease in sugarcane and maize fields in the northern guinea savannah agroecological zone and to molecularly characterize the associated sugarcane mosaic virus (SCMV, genus Potyvirus) isolates. Surveys were conducted from June to July 2015, and sugarcane mosaic disease (SCMD) incidence was assessed across 21 farmer's fields. Mean SCMD incidence varied across states with ∼82% (308/376), ∼66% (143/218), and ∼67% (36/54) recorded in Kaduna, Kano, and Katsina states, respectively. RT-PCR analysis of 415 field-collected samples using genus-specific primers confirmed potyvirus infection in 63.7% (156/245) of sugarcane, 29.7% (42/141) of maize crops, and 45% (13/29) of itch grass samples. Cloning and sequencing of gene-specific DNA amplicons from a subset of 45 samples (sugarcane = 33, maize = 9, itch grass = 3) confirmed their specificities to SCMV. Phylogenetic analysis of the partial gene sequences showed that they all belong to a single monophyletic clade of SCMV. These results were supported by analysis of complete polyprotein sequences of representative maize and sugarcane isolates from Nigeria. Both isolates shared 94.9%/97.3% complete polyprotein nucleotide (nt)/amino acid (aa) identities with each other and 75.2%/97.6% nt/aa identities with corresponding sequences of global SCMV isolates. The detection of identical populations of SCMV isolates in both crop species and a weed host suggests possible vector mediated interspecies spread within cereal landscapes in the study area with implications for the integrated and sustainable management of SCMD in cereal cropping systems in Nigeria.

Rapid detection of potyviruses from crude plant extracts.

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named 'Direct RT-RPA', detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.

Chromogenic detection of yam mosaic virus by closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP).

A closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP) assay was developed for the detection of yam mosaic virus (YMV, genus Potyvirus) infecting yam (Dioscorea spp.). The assay uses a set of six oligonucleotide primers targeting the YMV coat protein region, and the amplification products in YMV-positive samples are visualized by chromogenic detection with SYBR Green I dye. The CT-RT-LAMP assay detected YMV in leaf and tuber tissues of infected plants. The assay is 100 times more sensitive in detecting YMV than standard RT-PCR, while maintaining the same specificity.

Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination.

BACKGROUND: Root and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected "orphan" crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop. RESULTS: To accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage. CONCLUSIONS: Guinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.

Identification and molecular characterization of a novel sugarcane streak mastrevirus and an isolate of the A-strain of maize streak virus from sugarcane in Nigeria.

Sugarcane and maize plants showing symptoms typical of those described for the so-called "African streak viruses" (AfSVs) were encountered during field surveys conducted from February to July 2015 to document viruses infecting both crops across the northern Guinea savannah region of Nigeria. As part of this study, two categories of complete mastrevirus-like genome sequences were obtained from nine samples (maize = 2; sugarcane = 7). In pairwise comparisons, the full-length genomes of the first sequence category (2,687 nt each; maize = 2; sugarcane = 2) shared 96 to 99% identity with global isolates of the A-strain of maize streak virus (MSV-A), indicating that sugarcane may also serve as a reservoir host to MSV-A. Analysis of the complete genomes belonging to the second sequence category (2,757 nt each; sugarcane = 5) showed that they shared 42 to 67% identity with their closest AfSV relatives, thus indicating that they represent sequences of a novel mastrevirus. Both sequence categories shared 61-62% sequence identity with each other. Further analysis revealed that the novel sugarcane-infecting virus, tentatively named as sugarcane chlorotic streak virus (SCSV), arose from a putative interspecific recombination event involving two grass-infecting mastreviruses, eragrostis streak virus and urochloa streak virus, as putative parental sequences. The results of this study add to the repertoire of diverse AfSVs present in cereal and sugarcane mixed cropping landscapes in the northern Guinea savannah region of Nigeria, with implications for disease epidemiology.

Comparative Reliability of Screening Parameters for Anthracnose Resistance in Water Yam (Dioscorea alata).

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the major constraints limiting water yam (Dioscorea alata) production in the tropics. In this region, yam anthracnose control is mostly achieved by the deployment of moderately resistant yam genotypes. Therefore, screening for new sources of anthracnose resistance is an important aspect of yam research in the tropics. The reliability and applicability of different yam anthracnose rating parameters has not been fully examined. Disease severity on detached leaves in the laboratory and leaf severity, lesion size, and spore production on whole plants in the greenhouse were used to screen an F1 yam population and correlate screening results with field evaluations. Anthracnose lesion size had the smallest predicted residual means but whole-plant severity and detached-leaf severity had the best variance homogeneity and relatively small predicted residual means. The concordance correlation coefficient (rc) and κ statistic were used to determine the agreement between anthracnose rating parameters and field evaluations. Detached-leaf (rc = 0.95, κ = 0.81) and whole-plant (rc = 0.96, κ = 0.86) evaluations had high positive agreement with field evaluation but spore production (κ = 0.69) and lesion size (κ = 0.57) had moderate positive agreement. These results suggest that all the evaluated rating parameters can be used to successfully screen yam germplasm for anthracnose resistance but lesion size and spore production data may need to be transformed.

Diversity of Root-knot Nematodes Associated with Tubers of Yam (Dioscorea spp.) Established Using Isozyme Analysis and Mitochondrial DNA-based Identification.

The root-knot nematodes (RKN), Meloidogyne spp., represent an important threat to yam (Dioscorea spp.) production in West Africa. With the aim to establish the diversity of RKN species affecting yam tubers, for control and resistance screening purposes, surveys were conducted in the main yam producing areas of Nigeria. Galled tubers (N = 48) were collected from farmers' stores and markets in nine states in Nigeria and in one district in Ghana. RKN isolated from yam tubers were identified using enzyme phenotyping (esterase and malate dehydrogenase) and mitochondrial DNA (mtDNA) NADH dehydrogenase subunit 5 (Nad5) barcoding. Examination of 48 populations revealed that yam tubers were infested by Meloidogyne incognita (69%), followed by M. javanica (13%), M. enterolobii (2%), and M. arenaria (2%). Most of the tubers sampled (86%) were infected by a single species, and multiple species of RKN were detected in 14% of the samples. Results of both identification methods revealed the same species, confirming their accuracy for the identification of these tropical RKN species. In addition to M. incognita, M. javanica, and M. enterolobii, we report for the first time M. arenaria infecting yam tubers in Nigeria. This finding extends the list of yam pests and calls for caution when developing practices for yam pest management.

Maize Lethal Necrosis (MLN), an Emerging Threat to Maize-Based Food Security in Sub-Saharan Africa.

In sub-Saharan Africa, maize is a staple food and key determinant of food security for smallholder farming communities. Pest and disease outbreaks are key constraints to maize productivity. In September 2011, a serious disease outbreak, later diagnosed as maize lethal necrosis (MLN), was reported on maize in Kenya. The disease has since been confirmed in Rwanda and the Democratic Republic of Congo, and similar symptoms have been reported in Tanzania, Uganda, South Sudan, and Ethiopia. In 2012, yield losses of up to 90% resulted in an estimated grain loss of 126,000 metric tons valued at $52 million in Kenya alone. In eastern Africa, MLN was found to result from coinfection of maize with Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), although MCMV alone appears to cause significant crop losses. We summarize here the results of collaborative research undertaken to understand the biology and epidemiology of MLN in East Africa and to develop disease management strategies, including identification of MLN-tolerant maize germplasm. We discuss recent progress, identify major issues requiring further research, and discuss the possible next steps for effective management of MLN.

First Report of Maize chlorotic mottle virus Infecting Maize in the Democratic Republic of the Congo.

Maize (Zea mays L.) is a major food and fodder crop cultivated on 1.54 million ha in the Democratic Republic of the Congo (DRC). In December 2013, unusually severe chlorotic mottle symptoms and pale green streaks were observed in local varieties (Mudishi 1 and 2, Bambou, Kasayi, H614, H613, and Mugamba) and exotic varieties (H520, H624, H403, HDK8031, and ZM607) in Beni, Lubero, and Rutshuru territories at 1,015 to 1,748 m elevation in North Kivu Province. Symptoms were prominent on newly emerging leaves that later developed marginal necrosis resembling the symptoms of maize lethal necrosis (MLN), caused by a dual infection of Maize chlorotic mottle virus (MCMV, genus Machlomovirus) and Sugarcane mosaic virus (SCMV, genus Potyvirus). Each of these viruses, but particularly MCMV, is also known to cause severe mosaic and mottling symptoms in maize (4). In January 2014, symptomatic and asymptomatic samples (n = 20) from disease-affected fields in Beni and Lubero provinces were collected for virus testing using Whatman FTA Classic Cards (1) and analyzed for MCMV (2681F: 5'-ATGAGAGCAGTTGGGGAATGCG and 3226R: 5'-CGAATCTACACACACACACTCCAGC) and SCMV (8679F: 5'-GCAATGTCGAAGAAAATGCG and 9595R: 5'-GTCTCTCACCAAGAGACTCGCAGC) by reverse transcription (RT)-PCR (4). Samples were also analyzed for Maize streak virus (MSV, genus Mastrevirus), an endemic virus in DRC, by PCR using MSV specific primers (MSV215-234: CCAAAKDTCAGCTCCTCCG and MSV1770-1792: TTGGVCCGMVGATGTASAG) (3). A DNA product of expected size (~520 bp) resulted only for MCMV in all the symptomatic plant samples. None of the samples tested positive for SCMV or MSV. RT-PCR analyses were performed to ascertain the absence of potyviruses using the degenerate potyvirus primers (CIFor: 5'GGIVVIGTIGGIWSIGGIAARTCIAC and CIRev: 5'ACICCRTTYTCDATDATRTTIGTIGC3') (2) were also negative. Occurrence of MCMV in symptomatic samples was further confirmed by antigen-coated plate (ACP)-ELISA using anti-MCMV rabbit polyclonal antibodies produced at the Virology Unit, IITA, Ibadan, Nigeria. The RT-PCR product of MCMV was purified and sequenced in both directions (GenBank Accession No. KJ699379). Pairwise comparison of 518 bp nucleotide sequence corresponding to p32 and p37 open reading frames of MCMV by BLASTn search revealed 99.8% nucleotide sequence identity with an MCMV isolate from Kenya (JX286709), 98 to 99% identity with the isolates from China (JQ982468 and KF010583), and 96% identity with the isolates from the United States (X14736 and EU358605). MCMV is a newly emerging virus in Africa, first detected during a severe MLND outbreak in 2011 in Kenya (4). This disease has since become a serious threat to maize production in East Africa. MCMV has been reported in maize from Kenya, Rwanda, Tanzania, and Uganda. To our knowledge, this is the first report of MCMV occurrence in DRC. This finding confirms the further geographic expansion of MCMV and illustrates the need for further studies to identify vectors and also create awareness about the disease and to strengthen surveillance to prevent its further spread in the continent. References: (1) O. J. Alabi et al. J. Virol. Met. 154:111, 2008. (2) C. Ha et al. Arch. Virol. 153:25, 2008. (3) K. E. Palmer and E. P. Rybicki. Arch. Virol. 146:1089, 2001. (4) A. Wangai et al. Plant Dis. 96:1582, 2012.

An Improved Phenotyping Method for Evaluation of Yam (Dioscorea spp.) Resistance to Nematodes Belonging to the Genera Meloidogyne and Scutellonema.

Phenotyping yam (Dioscorea spp.) germplasm for resistance to parasitic nematodes is hampered by the lack of an efficient screening method. In this study, we developed a new method using rooted yam vine cuttings and yam plantlets generated from semi-autotrophic hydroponics (SAHs) propagation for phenotyping yam genotypes for nematode resistance. The method was evaluated using 26 genotypes of D. rotundata for their reaction to Scutellonema bradys and four root-knot nematode species, Meloidogyne arenaria, M. enterolobii, M. incognita, and M. javanica. Yam plantlets established in nursery bags filled with steam-sterilized soil were used for screening against single nematode species. Plants were inoculated four weeks after planting and assessed for nematode damage eight weeks later. A severity rating scale was used to classify genotypes as resistant, tolerant, or susceptible determine based on the nematode feeding damage on tubers and the rate of nematode multiplication in the roots of inoculated plants. The results demonstrated putative resistance and tolerance against S. bradys in 58% of the genotypes and 88%, 65%, 65%, and 58% against M. arenaria, M. javanica, M. incognita, and M. enterolobii, respectively. The method is rapid, flexible, and seasonally independent, permitting year-round screening under controlled conditions. This method increases the throughput and speed of phenotyping and improves the selection process.

Pathogen-derived resistance using a viral nucleocapsid gene confers only partial non-durable protection in peanut against peanut bud necrosis virus.

Genetic engineering of peanut (Arachis hypogaea L.) using the gene encoding for the nucleocapsid protein (N gene) of peanut bud necrosis virus (PBNV; genus Tospovirus, family Bunyaviridae) was used to impart resistance to bud necrosis disease in peanut (PBND), a disease for which no durable resistance is available in the existing germplasm. Over 200 transgenic lines of peanut var. JL 24 were developed for which integration and expression of the transgenes was confirmed by PCR, Southern hybridization, RT-PCR and western blot analysis. The T(1) and T(2) generation transgenic plants were assayed through virus challenge in the greenhouse by using mechanical sap inoculation at 1:100 and 1:50 dilutions of PBNV, and they showed varying levels of disease incidence and intensity. Greenhouse and field evaluation with T(2) generation plants indicated somewhat superior performance of the three transgenic events that showed considerable reduction in disease incidence. However, only one of these events showed over 75 % reduction in disease incidence when compared to the untransformed control, indicating partial and non-durable resistance to PBND using the viral N-gene.

First Report of Banana bunchy top virus in Banana and Plantain (Musa spp.) in Nigeria.

Plantain and banana (Musa spp.) are among the most important staple crops for food and income generation for the rural and urban populations in the humid forest agroecological zone of West Africa. Until recently, Cucumber mosaic virus (genus Cucumovirus) and Banana streak virus (genus Badnavirus) were the only viruses reported to occur in Musa spp. in West Africa. In 2011, an outbreak of banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBTV; genus Babuvirus, family Nanoviridae) was reported in Ouémé Département (6°30'N and 2°36'E) in the Republic of Benin (2). BBTV is one of the most economically important pathogens of Musa spp. It is well established in Central Africa and also in Angola, Malawi, and Zambia in Southern Africa (2). Plants infected at early growth stages are severely dwarfed and do not bear fruit. BBTV is transmitted by the banana aphid Pentalonia nigronervosa, which is widespread in Africa (1). The regions in the Republic of Benin affected by BBTV border Ogun State (7°00'N and 3°35'E) of Nigeria. Epidemiological investigations were conducted during May 2012 at 31 locations in Ogun State to determine the potential risk of BBTV spreading into Nigeria. Plants with typical symptoms of BBTD (stunting, narrow and shortened leaves, chlorotic streaks on petioles and pseudostem) were observed in four locations: Ilashe, Odan-Itoro, Ido-Ologun, and Igbogila. Total DNA was extracted from 90 leaf samples randomly collected from symptomatic and asymptomatic banana and plantain plants in these areas. Samples were tested for BBTV by polymerase chain reaction (PCR) using primer pairs, mREP-F and mREP-R, which amplifies a 241-bp of BBTV DNA-mRep segment (1), and Scp-F and Scp-R specific for approximately 1075-bp BBTV DNA-S that encodes coat protein gene (1). The amplicons of expected size were obtained from 17 of 90 samples analyzed (18.8%). BBTV in the symptomatic plants was further confirmed by nucleic acid spot hybridization (NASH) assay using DIG-labeled 1,075-bp probe corresponding to coat protein gene and chromogenic detection as per the previously described protocol (3). The DIG-probe specifically reacted with nucleic acid from the symptomatic plants, but not with negative controls, providing conclusive evidence for the BBTV. The PCR products of DNA-mRep segment amplified from three banana plants infected with BBTV collected in Ilashe (Ipokia Local Government Area) were purified and sequenced in both directions. The sequences of these isolates were 100% identical with each other (GenBank Accession Nos. JX290301, JX290302, and JX290303). A BLASTn search revealed 100% nucleotide sequence identity with a BBTV isolate from Benin (JQ437548) and 99 to 100% identity with DNA-mRep sequences of several other BBTV isolates from Africa, Australia, India, and the South Pacific. Further analysis of the 241-bp mRep gene sequences with Neighbor-Joining phylogenetic analysis grouped the BBTV isolate with the South Pacific isolates. To our knowledge, this is the first report of BBTV in Nigeria. This underscores need for surveys to assess the extent of BBTV spread in Nigeria and strict implementation of phytosanitary measures, including restrictions on the movement of planting material from disease-affected regions, to prevent further spread of this important disease. References: (1) P. L. Kumar et al. Virus Res. 159:171, 2011. (2) B. Lokossou et al. New Dis. Rep. 25:13, 2012. (3) W. S. Xie and J. S. Hu. Phytopathol. 85:339, 1995.

Angiotensin II type 1 receptor A1166C gene polymorphism and essential hypertension in Calabar and Uyo cities, Nigeria.

OBJECTIVES: The angiotensin II protein is a vasoconstrictor that exerts most of its influence through the angiotensin II type 1 receptor (AT1R). Inconsistent association between the A1166C polymorphism of the AT1R gene and hypertension has been reported among various populations but not among the peoples of Calabar and Uyo. This study was designed to determine the frequency of the A1166C polymorphism of the AT1R gene and its association with hypertension in a sample population of Calabar and Uyo. MATERIALS AND METHODS: A population-based case control design consisting of total of 1224 participants, 612 each of patients and controls were randomly recruited from hypertension clinics and the general population. Genotyping of the A1166C allele of the AT1R gene to identify variants was performed using polymerase chain reaction and restriction enzyme digestion. Multiple regressions were applied to test whether the A1166 genotypes were predictors of hypertension. RESULTS: 99% of the study population had the wild type AA genotype, and 1% was AC heterozygous carriers of the A1166C polymorphism. CONCLUSION: The A1166C polymorphism was not a predictor of hypertension in the sample population of Calabar and Uyo.

First Report of Mango Malformation Disease Caused by Fusarium tupiense in Senegal.

Mango (Mangifera indica L.) is an economically important export crop for Senegal, producing about 100,000 tons of fruit annually. In April 2009, severe outbreaks of a new disorder occurred in mango orchards in the southeastern part of Casamance. Diseased plants showed abnormal growth of vegetative shoots with short thickened internodes and malformed inflorescence with short leaves interspersed among thickened sterile flowers that aborted early. These symptoms resembled those caused by mango malformation disease (4). To identify the causal agent, floral and vegetative samples from symptomatic mango plants were collected from Kolda district (12°53' N, 14°56' W). Malformed tissues were cut into 4-mm2 pieces, surface sterilized with 75% ethanol for 2 min, dried, and plated on the Fusarium isolation medium Peptone PCNB Agar (PPA) (2). Fungal growth with Fusarium morphology were transferred on PPA and further purified on water agar as single spore isolates. Cultures were identified on the basis of spore characters on carnation leaf agar and colony morphology on PDA (2). Two isolates (I4 and I17) were similar to F. mangiferae/F. sterilihyphosum/F. tupiense complex (3). Macroconidia were slender, slightly falcate, three- to five-septate, 18.5 to 27.7 × 1.1 to 2.3 µm with slightly curved apical cell produced on cream to orange sporodochia. Microconidia were single-celled, oval, 3.7 to 13.6 × 0.75 to 1.1 µm produced on mono- and polyphialides in false heads. Chlamydospores were absent. To confirm the identity, genomic DNA was isolated from pure cultures of I4 and I17, used for amplification of portion of translation elongation factor (TEF-1α). Amplified products (241 bp) were purified and sequenced in both directions (GenBank Accession Nos. JX272929 and JX272930). A BLASTn search revealed 100% sequence identity with F. tupiense (DQ452860), 99% identity with F. mangiferae (HM135531) and F. sterilihyphosum (DQ452858) from Brazil. Phylogenetic analysis inferred from the Clustalw alignment of TEF-1α sequences clustered I4 and I17 isolates with F. tupiense (3). To confirm Koch's postulates, 2-year-old healthy mango seedlings var. Keitt and Kent (12 plants each) were inoculated by placing 20 µl conidial suspension (5 × 107 conidia ml-1) on micro-wounds created in apical and lateral buds. Inoculated buds were covered with filter paper soaked in the same spore suspension (1). Seedlings inoculated similarly with sterile distilled water served as control. Seven months after the inoculation, typical malformation symptoms were observed on vegetative parts on all inoculated plants, but not on control plants. F. tupiense was reisolated from symptomatic shoots of inoculated plants. Based on the morphological characteristics, sequence analysis, and pathogenicity test, the pathogen of mango malformation in Senegal was identified as F. tupiense (3). To our knowledge, this is the first confirmed record in Senegal of mango malformation caused by F. tupiense. This disease is a serious threat to mango production and trade of Senegal. Urgent actions are necessary to stop this emerging epidemic that can spread to other countries in West Africa. References: (1) S. Freeman et al. Phytopathol. 6:456, 1999. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, Blackwell Publishing, Ames, IA, 2006. (4) C. S. Lima et al. Mycologia. 104: in press (doi: 10.3852/12-052). (2) W. F. O. Marasas et al. Phytopathol. 96:667, 2006.