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.

Relationship between serum aflatoxin concentrations and the nutritional status of children aged 6-24 months from Zambia.

In Zambia, mothers/caregivers feed their children cereal-based complementary foods that are prone to aflatoxin contamination. This study evaluated the relationship between exposure to aflatoxins and the nutritional status of young children. It covered 400 mothers with children aged 6-24 months. Their nutritional status assessed by measuring weight and height using standard procedures. Serum samples analysed for aflatoxin B1-lysine (AFB1-lys), a reliable biomarker of aflatoxin exposure. Child sickness and age, exposure to aflatoxin in foods, and albumin-normalised AFB1-lys level were found to be significantly (p < .05) associated with child stunting except for child age that was not significant at p = .05. Children with an increase in the blood serum aflatoxin B1 lysine adduct are more likely to be stunted. These results have shown that dietary exposure to aflatoxin could lead to an increase in serum aflatoxin concentrations, both of which are associated with stunting.

Prevalence of Aflatoxin- and Fumonisin-Producing Fungi Associated with Cereal Crops Grown in Zimbabwe and Their Associated Risks in a Climate Change Scenario.

In most sub-Saharan African countries, staple cereal grains harbor many fungi and some produce mycotoxins that negatively impact health and trade. Maize and three small grain cereals (sorghum, pearl millet, and finger millet) produced by smallholder farmers in Zimbabwe during 2016 and 2017 were examined for fungal community structure, and total aflatoxin (AF) and fumonisin (FM) content. A total of 800 maize and 180 small grain samples were collected at harvest and during storage from four agroecological zones. Fusarium spp. dominated the fungi associated with maize. Across crops, Aspergillusflavus constituted the main Aspergillus spp. Small grain cereals were less susceptible to both AF and FM. AF (52%) and FM (89%) prevalence was higher in maize than in small grains (13-25% for AF and 0-32% for FM). Less than 2% of small grain samples exceeded the EU regulatory limit for AF (4 µg/kg), while <10% exceeded the EU regulatory limit for FM (1000 µg/kg). For maize, 28% and 54% of samples exceeded AF and FM Codex guidance limits, respectively. Higher AF contamination occurred in the drier and hotter areas while more FM occurred in the wetter year. AF exposure risk assessment revealed that small grain consumption posed low health risks (≤0.02 liver cancer cases/100,000 persons/year) while maize consumption potentially caused higher liver cancer rates of up to 9.2 cases/100,000 persons/year depending on the locality. Additionally, FM hazard quotients from maize consumption among children and adults were high in both years, but more so in a wet year than a dry year. Adoption of AF and FM management practices throughout the maize value chain coupled with policies supporting dietary diversification are needed to protect maize consumers in Zimbabwe from AF- and FM-associated health effects. The higher risk of health burden from diseases associated with elevated concentration of mycotoxins in preferred maize during climate change events can be relieved by increased consumption of small grains.

Aflatoxin Contamination of Dried Insects and Fish in Zambia.

Dried insects and fish are important sources of income and dietary protein in Zambia. Some aflatoxin-producing fungi are entomopathogenic and also colonize insects and fish after harvest and processing. Aflatoxins are carcinogenic, immune-suppressing mycotoxins that are frequent food contaminants worldwide. Several species within Aspergillus section Flavi have been implicated as causal agents of aflatoxin contamination of crops in Africa. However, aflatoxin producers associated with dried fish and edible insects in Zambia remain unknown, and aflatoxin concentrations in these foods have been inadequately evaluated. The current study sought to address these data gaps to assess potential human vulnerability through the dried fish and edible insect routes of aflatoxin exposure. Caterpillars ( n = 97), termites ( n = 4), and dried fish ( n = 66) sampled in 2016 and 2017 were assayed for aflatoxin by using lateral flow immunochromatography. Average aflatoxin concentrations exceeded regulatory limits for Zambia (10 µg/kg) in the moth Gynanisa maja (11 µg/kg), the moth Gonimbrasia zambesina (Walker) (12 µg/kg), and the termite Macrotermes falciger (Gerstacker) (24 µg/kg). When samples were subjected to simulated poor storage, aflatoxins increased ( P < 0.001) to unsafe levels in caterpillars (mean, 4,800 µg/kg) and fish ( Oreochromis) (mean, 23 µg/kg). The L strain morphotype of A. flavus was the most common aflatoxin producer on dried fish (88% of Aspergillus section Flavi), termites (68%), and caterpillars (61%), with the exception of Gynanisa maja, for which A. parasiticus was the most common (44%). Dried fish and insects supported growth (mean, 1.3 × 109 CFU/g) and aflatoxin production (mean, 63,620 µg/kg) by previously characterized toxigenic Aspergillus section Flavi species, although the extent of growth and aflatoxigenicity depended on specific fungus-host combinations. The current study shows the need for proper storage and testing of dried insects and fish before consumption as measures to mitigate human exposure to aflatoxins through consumption in Zambia.

Nutrient and aflatoxin contents of traditional complementary foods consumed by children of 6-24 months.

The nutrient composition and safety of complementary foods have recently become areas of concern, especially with regard to aflatoxin contamination which has been found to adversely affect health outcomes. This study presents the nutrient and aflatoxin contents of complementary foods consumed by children (6-24 months) and infants and young child feeding practices of mothers from two districts in eastern and southern Zambia. A total of 400 mother-child pairs were recruited from Monze and Chipata districts, and data on breastfeeding and complementary feeding practices were collected twice at 3-month interval using a structured questionnaire. Samples of two traditional complementary foods (Maize Nshima and Maize porridge) were collected from the mothers and analyzed for nutrient contents and aflatoxin contamination. The results showed that there is a high level of awareness on exclusive breastfeeding among mothers. Fat, protein, carbohydrate, and ash contents of Maize nshima from Chipata were significantly lower (p < .05) compared to those from Monze district except for starch and sugar. Monze mothers preferred to prepare a thicker Maize nshima and Maize porridge compared to their Chipata counterparts. The aflatoxin contamination showed that the Maize porridge samples from Chipata were the most contaminated with mean aflatoxin content of 5.8 ± 15.93 mg/100 g, while Maize nshima was the most contaminated of the two complementary foods from Monze districts with mean aflatoxin level of 3.8 ± 6.41 mg/100 g. There were significant (p < .05) positive correlations between fat and aflatoxin contents for Chipata samples (r = .12409) and for Monze samples (r = .13666). The traditional complementary foods studied were found to be low in fat and protein and high in aflatoxin contamination. Thus, it is imperative that best practices and interventions are designed and introduced to reduce the possible lethal health implications of consumption of such complementary foods by children under 5 years.

Aspergillus section Flavi community structure in Zambia influences aflatoxin contamination of maize and groundnut.

Aflatoxins are cancer-causing, immuno-suppressive mycotoxins that frequently contaminate important staples in Zambia including maize and groundnut. Several species within Aspergillus section Flavi have been implicated as causal agents of aflatoxin contamination in Africa. However, Aspergillus populations associated with aflatoxin contamination in Zambia have not been adequately detailed. Most of Zambia's arable land is non-cultivated and Aspergillus communities in crops may originate in non-cultivated soil. However, relationships between Aspergillus populations on crops and those resident in non-cultivated soils have not been explored. Because characterization of similar fungal populations outside of Zambia have resulted in strategies to prevent aflatoxins, the current study sought to improve understanding of fungal communities in cultivated and non-cultivated soils and in crops. Crops (n=412) and soils from cultivated (n=160) and non-cultivated land (n=60) were assayed for Aspergillus section Flavi from 2012 to 2016. The L-strain morphotype of Aspergillus flavus and A. parasiticus were dominant on maize and groundnut (60% and 42% of Aspergillus section Flavi, respectively). Incidences of A. flavus L-morphotype were negatively correlated with aflatoxin in groundnut (log y=2.4990935-0.09966x, R2=0.79, P=0.001) but not in maize. Incidences of A. parasiticus partially explained groundnut aflatoxin concentrations in all agroecologies and maize aflatoxin in agroecology III (log y=0.1956034+0.510379x, R2=0.57, P<0.001) supporting A. parasiticus as the dominant etiologic agent of aflatoxin contamination in Zambia. Communities in both non-cultivated and cultivated soils were dominated by A. parasiticus (69% and 58%, respectively). Aspergillus parasiticus from cultivated and non-cultivated land produced statistically similar concentrations of aflatoxins. Aflatoxin-producers causing contamination of crops in Zambia may be native and, originate from non-cultivated areas, and not be introduced with non-native crops such as maize and groundnut. Non-cultivated land may be an important reservoir from which aflatoxin-producers are repeatedly introduced to cultivated areas. The potential of atoxigenic members of the A. flavus-L morphotype for management of aflatoxin in Zambia is also suggested. Characterization of the causal agents of aflatoxin contamination in agroecologies across Zambia gives support for modifying fungal community structure to reduce the aflatoxin-producing potential.

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.).

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.