First Report of Maize chlorotic mottle virus and Maize Lethal Necrosis in Kenya.

In September 2011, a high incidence of a new maize (Zea mays L.) disease was reported at lower elevations (1,900 m asl) in the Longisa division of Bomet County, Southern Rift Valley, Kenya. The disease later spread to the Narok South and North and Naivasha Districts. By March 2012, the disease was reported at up to 2,100 m asl. Diseased plants had symptoms characteristic of virus diseases: a chlorotic mottle on leaves, developing from the base of young whorl leaves upward to the leaf tips; mild to severe leaf mottling; and necrosis developing from leaf margins to the mid-rib. Necrosis of young leaves led to a "dead heart" symptom, and plant death. Severely affected plants had small cobs with little or no grain set. Plants frequently died before tasseling. All maize varieties grown in the affected areas had similar symptoms. In these regions, maize is grown continuously throughout the year, with the main planting season starting in November. Maize streak virus was present, but incidence was low (data not shown). Infected plants were distributed throughout affected fields, with heavier infection along field edges. High thrips (Frankliniella williamsi Hood) populations were present in sampled fields, but populations of other potential disease vectors, such as aphids and leafhoppers, were low. Because of the high thrips populations and foliar symptoms, symptomatic plants were tested for the presence of Maize chlorotic mottle virus (MCMV) (3) using tissue blot immunoassay (TBIA) (1). Of 17 symptomatic leaf samples from each Bomet and Naivasha, nine from Bomet and all 17 from Naivasha were positive for MCMV. However, the observed symptoms were more severe than commonly associated with MCMV, suggesting the presence of maize lethal necrosis (MLN), a disease that results from maize infection with both MCMV and a potyvirus (4). Therefore, samples were tested for the presence of Sugarcane mosaic virus (SCMV), which is present in Kenya (2). Twenty-seven samples were positive for SCMV by TBIA, and 23 of 34 samples were infected with both viruses. Virus identities were verified with reverse-transcription (RT)-PCR (Access RT-PCR, Promega) and MCMV or SCMV-specific primers. MCMV primers (2681F: 5'-ATGAGAGCAGTTGGGGAATGCG and 3226R: 5'-CGAATCTACACACACACACTCCAGC) amplified the expected 550-bp product from three leaf samples. Amplicon sequences were identical, and had 95 to 98% identity with MCMV sequences in GenBank. SCMV primers (8679F: 5'-GCAATGTCGAAGAAAATGCG) and 9595R: 5'-GTCTCTCACCAAGAGACTCGCAGC) amplified the expected 900-bp product from four leaf samples. Amplicon sequences had 96 to 98% identity, and were 88 to 96% identical with SCMV sequences in GenBank. To our knowledge, this is the first report of MCMV and of maize coinfection with MCMV and SCMV associated with MLN in Kenya and Africa. MLN is a serious threat to farmers in the affected areas, who are experiencing extensive to complete crop loss. References: (1) P. G. S. Chang et al. J. Virol. Meth. 171:345, 2011. (2) Delgadillo Sanchez et al. Rev. Mex. Fitopat. 5:21, 1987. (3) Jiang et al., Crop Prot. 11:248, 1992. (4) R. Louie, Plant Dis. 64:944, 1980.

The complete genome sequence of the Tanzanian strain of Cassava brown streak virus and comparison with the Ugandan strain sequence.

The complete genome sequence for an isolate of the Ugandan and Tanzanian strain types of Cassava brown streak virus have been determined using the novel approach of non-directed next generation sequencing. Comparison of the genome sequences revealed that CBSV is highly heterogeneous at the isolate level as well as the strain level. The isolate of the Ugandan strain was found to have a genome 9,070 nucleotides long coding for a polypeptide with 2,902 amino acid residues. The isolate of the Tanzanian strain was 9,008 nucleotides long and coded for a polypeptide with 2,916 amino acid residues. Nucleotide identity between the isolates across the genome was 76%, with protein encoding regions 57-77% and individual proteins had 65-91% amino acid similarity. In addition between the two strains four protein products (PIPO, CI, NIa-Vpg and coat protein) varied in size and an unusual HAM1-like protein, whilst of identical nucleotide length, was found to have the lowest homology. The implication of diversity of CBSV is discussed in the context of speciation, evolution, development of diagnostics, and breeding for resistance.

First Report of a Begomovirus Infecting Sweetpotato in Kenya.

Previous surveys for viruses in sweetpotatoes (Ipomoea batatas) in Africa did not assay for the presence of begomoviruses such as Sweet potato leaf curl virus (SPLCV), which have been found recently in the Americas and Asia. Symptomatic sweetpotato plants, including some with leaf curling symptoms similar to those observed in SPLCV-infected sweet-potato plants (2), were collected from a germplasm collection plot at Kakamega Research Station in Western Kenya during February 2005. Whiteflies, the vectors for begomoviruses, were observed in the same plots. Ipomoea setosa plants graft-inoculated with scions from the symptomatic sweetpotato developed leaf curl, leaf roll, interveinal chlorosis, and stunting symptoms similar to those caused by infection with SPLCV alone or in combination with Sweet potato feathery mottle virus. Total DNA was isolated from 10 I. setosa plants using the GenElute Plant Genomic DNA Kit (Sigma-Aldrich Inc., St. Louis, MO). Sweetpotato cuttings from 39 clones, selected from the Kenyan germplasm collection for their resistance or susceptibility to sweetpotato virus disease (SPVD), were sent to the Plant Germplasm Quarantine Office of USDA-ARS. The cuttings were planted in a greenhouse. Total DNA was extracted from sweetpotato leaves 1 month later using a cetyltrimethylammoniumbromide (CTAB) extraction method (1). Degenerate primers SPG1/SPG2, developed for PCR detection of begomoviruses (1), amplified a 912-bp DNA fragment from 3 of 10 DNA extracts from I. setosa and 5 of 39 sweetpotato plants held in quarantine. The primers anneal to regions of open reading frame (ORF) AC2 and ORF AC1 that are highly conserved in begomoviruses infecting sweetpotato. SPLCV-specific primers PW285-1/PW285-2 (2) amplified a 512-bp DNA fragment of ORF AC1 from seven samples (two from I. setosa and five from I. batatas). Amplicons from three independent PCR assays of two samples and single PCR assays of four additional samples were cloned into the pGEM-T Easy vector. Clone inserts were sequenced, and compared with sequences deposited in GenBank using the basic local alignment search tool (BLAST). Sequences were closely related to SPLCV (GenBank Accession No. AF104036) with nucleotide sequence identities varying from 93% (GenBank Accession No. DQ361004) to 97% (GenBank Accession No. DQ361005). The presence of the virus poses a challenge to the dissemination of planting materials in the region because begomovirus-infected plants often do not show symptoms. To our knowledge, this is the first report of a begomovirus infecting sweetpotato in Kenya or the East African Region. References: (1) R. Li et al. Plant Dis. 88:1347, 2004. (2) P. Lotrakul et al. Plant Dis. 82:1253, 1998.

Loop-mediated isothermal amplification for rapid detection of the causal agents of cassava brown streak disease.

The causal agents of cassava brown streak disease have recently been identified as Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Primers have been developed for rapid detection of these viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP). Performance of the RT-LAMP assays compared favourably with published RT-PCR and real-time RT-PCR methods. Furthermore, amplification by RT-LAMP is completed in 40 min and does not require thermal cycling equipment. Modification of the RT-LAMP reactions to use labelled primers allowed rapid detection of amplification products using lateral flow devices containing antibodies specific to the incorporated labels, avoiding the need for fluorescence detection or gel electrophoresis.