Survey for Virus Diversity in Common Bean (Phaseolus vulgaris) Fields and the Detection of a Novel Strain of Cowpea polerovirus 1 in Zambia.

The production of common bean (Phaseolus vulgaris L.) is adversely affected by virus-like diseases globally, but little is known about the occurrence, distribution, and diversity of common bean-infecting viruses in Zambia. Consequently, field surveys were conducted during the 2018 season in 128 fields across six provinces of Zambia and 640 common bean leaf tissue samples were collected with (n = 585) or without (n = 55) symptoms. The prevalence of symptomatic fields was 100%, but incidence of symptomatic plants ranged from 32 to 67.5%. Metagenomic analyses of nine composite samples and a single plant sample of interest revealed the occurrence of isolates of Bean common mosaic necrosis virus, Bean common mosaic virus, Cowpea aphid-borne mosaic virus, Peanut mottle virus, Southern bean mosaic virus (SBMV), Cucumber mosaic virus, Phaseolus vulgaris alphaendornavirus 1 (PvEV-1), PvEV-2, Ethiopian tobacco bushy top virus (ETBTV), and a novel strain of Cowpea polerovirus 1 (CPPV1-Pv) of 5,902 nt in length. While CPPV1-Pv was consistently detected in mixed infection with ETBTV and its satellite RNA molecule, based on results of mechanical transmission assays it does not appear to be involved in disease etiology, suggesting that its role may be limited to being a helper virus for the umbravirus. Screening of the survey samples by real-time PCR for the viruses detected by high-throughput sequencing revealed the prevalence of single (65.2% or 417/640) over mixed (1.9% or 12/640) infections in the samples. SBMV was the most frequently detected virus, occurring in ∼29.4% (188/640) of the samples and at a prevalence rate of 58.6% (75/128) across fields. The results showed that diverse virus species are present in Zambian common bean fields and the information will be useful for the management of common bean viral diseases.

First report of Ethiopian tobacco bushy top virus and its associated satellite RNA infecting common bean (Phaseolus vulgaris L.) in Zambia.

During surveys for common bean viruses in Central Province of Zambia in April 2018, symptoms of bushy top, deep green curled branches and patchy leaf chlorosis were observed on five plants in a 2-ha farmer's field. Total RNA was isolated from symptomatic leaf samples using the CTAB method (Chang et al. 1993). The RNA from one sample (CP414-1) was used to construct a cDNA library with the Illumina TruSeq RNA Library Prep Kit (Illumina, San Diego, CA), followed by high-throughput sequencing (HTS) on the Illumina MiSeq platform that generated ~3.1M single-end raw reads of ~300 nucleotides (nt) each. A total of 355,885 reads showed hits to Ethiopian tobacco bushy top virus (ETBTV; Umbravirus), ETBTV satellite RNA (satRNA) and peanut mottle virus (PeMoV, Potyvirus) based on BLASTn analysis. The full-length genomes of ETBTV (4239-nt; MT225089), its satRNA (521-nt; MT225092) and PeMoV (9,643-nt) were assembled from the HTS reads using Geneious R11.1.2 (Biomatters, Auckland, New Zealand). The obtained complete genome sequences of ETBTV (MT225089) and ETBTV satRNA (MT225092) shared 88% and 95% nt identities, respectively with the corresponding viral (KJ918748) and satRNA (KJ918747) sequences of isolate 18-2 (Abraham et al. 2014). The near complete PeMoV genome was 89% identical to isolate Liaoning (MH270528). The HTS results were validated by two-step RT-PCR analyses of the five field-collected samples using newly designed primer pairs (data not shown). All five samples gave the expected 988-bp ETBTV-specific and 521-bp satRNA-specific DNA bands while three samples produced the expected 2100-bp PeMoV-specific fragment. The virus specificities of the agent specific PCR fragments were ascertained by Sanger sequencing (ETBTV: MT225090-91; ETBTV satRNA: MT225093-94; PeMoV: MT900843-44) and they shared 98-100% identities with their corresponding HTS-derived sequences. To further probe for the presence of an ETBTV helper virus, the samples were screened by RT-PCR with the degenerate primer pair Lu1-mod-F/C2R3 that was modified from Robertson et al. (1991). The expected 245-bp DNA bands was obtained from all five samples, indicating the presence of a possible luteovirus or polerovirus target in these samples. The BLASTn analyses of the two Sanger sequenced gel-eluted products (MT900845-46) showed that they shared 100% identity with each other and 96% nt identity with cowpea polerovirus 1 (CPPV1, KX599163). Leaf tissue extracts from a common bean plant that was confirmed by RT-PCR to be positive for all four agents were rub-inoculated onto Nicotiana occidentalis and common bean (Sutter Pink) plants (n=5 each) at the three fully expanded leaf stage, with a buffer inoculation as control. Systemic foliar symptoms consisting of leaf deformation, stunting and leaf bushy top were observed on all ten plants, 10 days post-inoculation whereas the control plants remained symptomless. All the test plants were screened by RT-PCR as described above. The results showed that all five N. occidentalis plants were positive for ETBTV+ETBTVsatRNA, the five common bean plants tested positive for ETBTV+satRNA+PeMoV, and all 10 plants of both species were negative for CPPV1. To the best of our knowledge, this is the first report of ETBTV, ETBTV satRNA and CPPV1 infecting common bean in Zambia, and the first molecular based confirmation of PeMoV occurrence in the country. Ongoing studies are focused on determining the extent of the disease spread and assessment of its economic impact.

Molecular detection and characterization of genotype 1 bovine leukemia virus from beef cattle in the traditional sector in Zambia.

Whilst bovine leukemia virus (BLV) causes considerable economic losses to the dairy industry worldwide, information on its molecular epidemiology and economic impact in beef cattle is limited. Here, blood from 880 animals from Zambia's major cattle-rearing provinces was screened for BLV by nested PCR. Positive pools were sequenced and phylogenetically analyzed. The estimated pooled prevalence was 2.1%. All strains belonged to genotype 1 and formed a distinct phylogenetic cluster. The study suggests circulation of genotype 1 BLV in beef cattle in these regions. This is the first report on molecular detection and characterization of BLV from beef cattle in Africa.

First genetic detection and characterization of canine parvovirus from diarrheic dogs in Zambia.

Although canine parvovirus (CPV) causes severe gastroenteritis in dogs globally, information on the molecular epidemiology of the virus is lacking in many African countries. Here, 32 fecal samples collected from diarrheic dogs in Zambia were tested for CPV infection using molecular assays. CPV was detected in 23 samples (71.9%). Genetic characterization revealed the predominance of CPV-2c (91.3%). This finding differs from previous reports in Africa, which indicated that CPV-2a and CPV-2b were most prevalent. Phylogenetically, most Zambian CPVs formed a distinct cluster. This is the first report on the molecular characterization of CPV in Zambia.

Direct Biomarkers of Microbial Translocation Correlate with Immune Activation in Adult Zambians with Environmental Enteropathy and Hepatosplenic Schistosomiasis.

Microbial translocation is a poorly understood consequence of several disorders such as environmental enteropathy (EE) and hepatosplenic schistosomiasis (HSS). Herein, we compared biomarkers of microbial origin and immune activation in adults with these disorders and in healthy controls. A cross-sectional study was conducted in participants with EE recruited from Misisi compound, Lusaka, Zambia; HSS patients and healthy controls from the University Teaching Hospital, Lusaka. Plasma lipopolysaccharides (LPSs) was measured by limulus amoebocyte lysate assay, plasma 16S ribosomal RNA (16S rRNA) gene copy number was quantified by quantitative real-time polymerase chain reaction, Toll-like receptor ligand (TLRL) activity by QUANTI-Blue detection medium, and cytokines from cell culture supernatant by Cytometric Bead Array. In univariate analysis LPS, 16S rRNA gene copy number, and TLR activity were all high and correlated with each other and with cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, and IL-4 secreted by the RAW-Blue cells. After controlling for baseline characteristic, biomarkers of microbial translocation in blood were predictors of TNF-α, IL-6, and IL-10 activation in cell culture supernatant from EE participants and HSS patients but not in healthy controls. TLR activity showed the strongest correlation with TNF-α. These data provide correlative evidence that microbial translocation contributes to systemic cytokine activation in two disorders common in the tropics, with total TLR ligand estimation showing the strongest correlation with TNF-α (r = 0.66, P < 0.001).

Identification of groundnut (Arachis hypogaea) SSR markers suitable for multiple resistance traits QTL mapping in African germplasm

Background This study aimed to identify and select informative Simple Sequence Repeat (SSR) markers that may be linked to resistance to important groundnut diseases such as Early Leaf Spot, Groundnut Rosette Disease, rust and aflatoxin contamination. To this end, 799 markers were screened across 16 farmer preferred and other cultivated African groundnut varieties that are routinely used in groundnut improvement, some with known resistance traits. Results The SSR markers amplified 817 loci and were graded on a scale of 1 to 4 according to successful amplification and ease of scoring of amplified alleles. Of these, 376 markers exhibited Polymorphic Information Content (PIC) values ranging from 0.06 to 0.86, with 1476 alleles detected at an average of 3.7 alleles per locus. The remaining 423 markers were either monomorphic or did not work well. The best performing polymorphic markers were subsequently used to construct a dissimilarity matrix that indicated the relatedness of the varieties in order to aid selection of appropriately diverse parents for groundnut improvement. The closest related varieties were MGV5 and ICGV-SM 90704 and most distant were Chalimbana and 47-10. The mean dissimilarity value was 0.51, ranging from 0.34 to 0.66. Discussion Of the 376 informative markers identified in this study, 139 (37%) have previously been mapped to the Arachis genome and can now be employed in Quantitative Trait Loci (QTL) mapping and the additional 237 markers identified can be used to improve the efficiency of introgression of resistance to multiple important biotic constraints into farmer-preferred varieties of Sub-Saharan Africa.

Sub-lethal levels of electric current elicit the biosynthesis of plant secondary metabolites.

Many secondary metabolites that are normally undetectable or in low amounts in healthy plant tissue are synthesized in high amounts in response to microbial infection. Various abiotic and biotic agents have been shown to mimic microorganisms and act as elicitors of the synthesis of these plant compounds. In the present study, sub-lethal levels of electric current are shown to elicit the biosynthesis of secondary metabolites in transgenic and non-transgenic plant tissue. The production of the phytoalexin (+)-pisatin by pea was used as the main model system. Non-transgenic pea hairy roots treated with 30-100 mA of electric current produced 13 times higher amounts of (+)-pisatin than did the non-elicited controls. Electrically elicited transgenic pea hairy root cultures blocked at various enzymatic steps in the (+)-pisatin biosynthetic pathway also accumulated intermediates preceding the blocked enzymatic step. Secondary metabolites not usually produced by pea accumulated in some of the transgenic root cultures after electric elicitation due to the diversion of the intermediates into new pathways. The amount of pisatin in the medium bathing the roots of electro-elicited roots of hydroponically cultivated pea plants was 10 times higher 24 h after elicitation than in the medium surrounding the roots of non-elicited control plants, showing not only that the electric current elicited (+)-pisatin biosynthesis but also that the (+)-pisatin was released from the roots. Seedlings, intact roots or cell suspension cultures of fenugreek (Trigonella foenum-graecum), barrel medic, (Medicago truncatula), Arabidopsis thaliana, red clover (Trifolium pratense) and chickpea (Cicer arietinum) also produced increased levels of secondary metabolites in response to electro-elicitation. On the basis of our results, electric current would appear to be a general elicitor of plant secondary metabolites and to have potential for application in both basic and commercial research.

Inactivation of pea genes by RNAi supports the involvement of two similar O-methyltransferases in the biosynthesis of (+)-pisatin and of chiral intermediates with a configuration opposite that found in (+)-pisatin.

(+)-Pisatin, the major phytoalexin of pea (Pisum sativum L.), is believed to be synthesized via two chiral intermediates, (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] and (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol [(-)-DMDI]; both have an opposite C-3 absolute configuration to that found at C-6a in (+)-pisatin. The expression of isoflavone reductase (IFR), which converts 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone (DMD) to (-)-sophorol, sophorol reductase (SOR), which converts (-)-sophorol to (-)-DMDI, and hydroxymaackiain-3-O-methyltransferase (HMM), believed to be the last step of (+)-pisatin biosynthesis, were inactivated by RNA-mediated genetic interference (RNAi) in pea hairy roots. Some hairy root lines containing RNAi constructs of IFR and SOR accumulated DMD or (-)-sophorol, respectively, and were deficient in (+)-pisatin biosynthesis supporting the involvement of chiral intermediates with a configuration opposite to that found in (+)-pisatin in the biosynthesis of (+)-pisatin. Pea proteins also converted (-)-DMDI to an achiral isoflavene suggesting that an isoflavene might be the intermediate through which the configuration is changed to that found in (+)-pisatin. Hairy roots containing RNAi constructs of HMM also were deficient in (+)-pisatin biosynthesis, but did not accumulate (+)-6a-hydroxymaackiain, the proposed precursor to (+)-pisatin. Instead, 2,7,4'-trihydroxyisoflavanone (TIF), daidzein, isoformononetin, and liquiritigenin accumulated. HMM has a high amino acid similarity to hydroxyisoflavanone-4'-O-methyltransferase (HI4'OMT), an enzyme that methylates TIF, an early intermediate in the isoflavonoid pathway. The accumulation of these four compounds is consistent with the blockage of the synthesis of (+)-pisatin at the HI4'OMT catalyzed step resulting in the accumulation of liquiritigenin and TIF and the diversion of the pathway to produce daidzein and isoformononetin, compounds not normally made by pea. Previous results have identified two highly similar "HMMs" in pea. The current results suggest that both of these O-methyltransferases are involved in (+)-pisatin biosynthesis and that one functions early in the pathway as HI4'OMT and the second acts at the terminal step of the pathway.

Methyl salicylate is a critical mobile signal for plant systemic acquired resistance.

In plants, the mobile signal for systemic acquired resistance (SAR), an organism-wide state of enhanced defense to subsequent infections, has been elusive. By stimulating immune responses in mosaic tobacco plants created by grafting different genetic backgrounds, we showed that the methyl salicylate (MeSA) esterase activity of salicylic acid-binding protein 2 (SABP2), which converts MeSA into salicylic acid (SA), is required for SAR signal perception in systemic tissue, the tissue that does not receive the primary (initial) infection. Moreover, in plants expressing mutant SABP2 with unregulated MeSA esterase activity in SAR signal-generating, primary infected leaves, SAR was compromised and the associated increase in MeSA levels was suppressed in primary infected leaves, their phloem exudates, and systemic leaves. SAR was also blocked when SA methyl transferase (which converts SA to MeSA) was silenced in primary infected leaves, and MeSA treatment of lower leaves induced SAR in upper untreated leaves. Therefore, we conclude that MeSA is a SAR signal in tobacco.