Metagenomic Analysis of Plant Virus Occurrence in Common Bean (Phaseolus vulgaris) in Central Kenya.

Two closely related potyviruses, bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV), are regarded as major constraints on production of common bean (Phaseolus vulgaris L.) in Eastern and Central Africa, where this crop provides a high proportion of dietary protein as well as other nutritional, agronomic, and economic benefits. Previous studies using antibody-based assays and indicator plants indicated that BCMV and BCMNV are both prevalent in bean fields in the region but these approaches cannot distinguish between these potyviruses or detect other viruses that may threaten the crop. In this study, we utilized next generation shotgun sequencing for a metagenomic examination of viruses present in bean plants growing at two locations in Kenya: the University of Nairobi Research Farm in Nairobi's Kabete district and at sites in Kirinyaga County. RNA was extracted from leaves of bean plants exhibiting apparent viral symptoms and sequenced on the Illumina MiSeq platform. We detected BCMNV, cucumber mosaic virus (CMV), and Phaseolus vulgaris alphaendornaviruses 1 and 2 (PvEV1 and 2), with CMV present in the Kirinyaga samples. The CMV strain detected in this study was most closely related to Asian strains, which suggests that it may be a recent introduction to the region. Surprisingly, and in contrast to previous surveys, BCMV was not detected in plants at either location. Some plants were infected with PvEV1 and 2. The detection of PvEV1 and 2 suggests these seed transmitted viruses may be more prevalent in Eastern African bean germplasm than previously thought.

Viral metagenomics of aphids present in bean and maize plots on mixed-use farms in Kenya reveals the presence of three dicistroviruses including a novel Big Sioux River virus-like dicistrovirus.

BACKGROUND: Aphids are major vectors of plant viruses. Common bean (Phaseolus vulgaris L.) and maize (Zea mays L.) are important crops that are vulnerable to aphid herbivory and aphid-transmitted viruses. In East and Central Africa, common bean is frequently intercropped by smallholder farmers to provide fixed nitrogen for cultivation of starch crops such as maize. We used a PCR-based technique to identify aphids prevalent in smallholder bean farms and next generation sequencing shotgun metagenomics to examine the diversity of viruses present in aphids and in maize leaf samples. Samples were collected from farms in Kenya in a range of agro-ecological zones. RESULTS: Cytochrome oxidase 1 (CO1) gene sequencing showed that Aphis fabae was the sole aphid species present in bean plots in the farms visited. Sequencing of total RNA from aphids using the Illumina platform detected three dicistroviruses. Maize leaf RNA was also analysed. Identification of Aphid lethal paralysis virus (ALPV), Rhopalosiphum padi virus (RhPV), and a novel Big Sioux River virus (BSRV)-like dicistrovirus in aphid and maize samples was confirmed using reverse transcription-polymerase chain reactions and sequencing of amplified DNA products. Phylogenetic, nucleotide and protein sequence analyses of eight ALPV genomes revealed evidence of intra-species recombination, with the data suggesting there may be two ALPV lineages. Analysis of BSRV-like virus genomic RNA sequences revealed features that are consistent with other dicistroviruses and that it is phylogenetically closely related to dicistroviruses of the genus Cripavirus. CONCLUSIONS: The discovery of ALPV and RhPV in aphids and maize further demonstrates the broad occurrence of these dicistroviruses. Dicistroviruses are remarkable in that they use plants as reservoirs that facilitate infection of their insect replicative hosts, such as aphids. This is the first report of these viruses being isolated from either organism. The BSRV-like sequences represent a potentially novel dicistrovirus infecting A. fabae.

Tools for Defusing a Major Global Food and Feed Safety Risk: Nonbiological Postharvest Procedures To Decontaminate Mycotoxins in Foods and Feeds.

Mycotoxin contamination of foods and animal feeds is a worldwide problem for human and animal health. Controlling mycotoxin contamination has drawn the attention of scientists and other food and feed stakeholders all over the world. Despite best efforts targeting field and storage preventive measures, environmental conditions can still lead to mycotoxin contamination. This raises a need for developing decontamination methods to inactivate or remove the toxins from contaminated products. At present, decontamination methods applied include an array of both biological and nonbiological methods. The targeted use of nonbiological methods spans from the latter half of last century, when ammoniation and ozonation were first used to inactivate mycotoxins in animal feeds, to the novel techniques being developed today such as photosensitization. Effectiveness and drawbacks of different nonbiological methods have been reported in the literature, and this review examines the utility of these methods in addressing food safety. Particular consideration is given to the application of such methods in the developing world, where mycotoxin contamination is a serious food safety issue in staple crops such as maize and rice.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains.

In vitro experimental environments are used to study interactions between microorganisms, and to predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to match closely the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation were studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25 and 30°C). Competition experiments showed interaction between the main effects of aflatoxin accumulation and the environment at 25°C, but not so at 30°C. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-h incubation in different experimental environments. Whereas all fungi incubated within the soil environment survived, in the cotton wool environment none of the competitors of A. flavus survived at 30°C. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post-harvest.

Bean Common Mosaic Virus and Bean Common Mosaic Necrosis Virus: Relationships, Biology, and Prospects for Control.

The closely related potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) are major constraints on common bean (Phaseolus vulgaris) production. Crop losses caused by BCMV and BCMNV impact severely not only on commercial scale cultivation of this high-value crop but also on production by smallholder farmers in the developing world, where bean serves as a key source of dietary protein and mineral nutrition. In many parts of the world, progress has been made in combating BCMV through breeding bean varieties possessing the I gene, a dominant gene conferring resistance to most BCMV strains. However, in Africa, and in particular in Central and East Africa, BCMNV is endemic and this presents a serious problem for deployment of the I gene because this virus triggers systemic necrosis (black root disease) in plants possessing this resistance gene. Information on these two important viruses is scattered throughout the literature from 1917 onward, and although reviews on resistance to BCMV and BCMNV exist, there is currently no comprehensive review on the biology and taxonomy of BCMV and BCMNV. In this chapter, we discuss the current state of our knowledge of these two potyviruses including fundamental aspects of classification and phylogeny, molecular biology, host interactions, transmission through seed and by aphid vectors, geographic distribution, as well as current and future prospects for the control of these important viruses.

Genetic diversity of Kenyan native oyster mushroom (Pleurotus).

Members of the genus Pleurotus, also commonly known as oyster mushroom, are well known for their socioeconomic and biotechnological potentials. Despite being one of the most important edible fungi, the scarce information about the genetic diversity of the species in natural populations has limited their sustainable utilization. A total of 71 isolates of Pleurotus species were collected from three natural populations: 25 isolates were obtained from Kakamega forest, 34 isolates from Arabuko Sokoke forest and 12 isolates from Mount Kenya forest. Amplified fragment length polymorphism (AFLP) was applied to thirteen isolates of locally grown Pleurotus species obtained from laboratory samples using five primer pair combinations. AFLP markers and internal transcribed spacer (ITS) sequences of the ribosomal DNA were used to estimate the genetic diversity and evaluate phylogenetic relationships, respectively, among and within populations. The five primer pair combinations generated 293 polymorphic loci across the 84 isolates. The mean genetic diversity among the populations was 0.25 with the population from Arabuko Sokoke having higher (0.27) diversity estimates compared to Mount Kenya population (0.24). Diversity between the isolates from the natural population (0.25) and commercial cultivars (0.24) did not differ significantly. However, diversity was greater within (89%; P > 0.001) populations than among populations. Homology search analysis against the GenBank database using 16 rDNA ITS sequences randomly selected from the two clades of AFLP dendrogram revealed three mushroom species: P. djamor, P. floridanus and P. sapidus; the three mushrooms form part of the diversity of Pleurotus species in Kenya. The broad diversity within the Kenyan Pleurotus species suggests the possibility of obtaining native strains suitable for commercial cultivation.

Physiological and molecular characterization of drought responses and identification of candidate tolerance genes in cassava.

Cassava is an important root crop to resource-poor farmers in marginal areas, where its production faces drought stress constraints. Given the difficulties associated with cassava breeding, a molecular understanding of drought tolerance in cassava will help in the identification of markers for use in marker-assisted selection and genes for transgenic improvement of drought tolerance. This study was carried out to identify candidate drought-tolerance genes and expression-based markers of drought stress in cassava. One drought-tolerant (improved variety) and one drought-susceptible (farmer-preferred) cassava landrace were grown in the glasshouse under well-watered and water-stressed conditions. Their morphological, physiological and molecular responses to drought were characterized. Morphological and physiological measurements indicate that the tolerance of the improved variety is based on drought avoidance, through reduction of water loss via partial stomatal closure. Ten genes that have previously been biologically validated as conferring or being associated with drought tolerance in other plant species were confirmed as being drought responsive in cassava. Four genes (MeALDH, MeZFP, MeMSD and MeRD28) were identified as candidate cassava drought-tolerance genes, as they were exclusively up-regulated in the drought-tolerant genotype to comparable levels known to confer drought tolerance in other species. Based on these genes, we hypothesize that the basis of the tolerance at the cellular level is probably through mitigation of the oxidative burst and osmotic adjustment. This study provides an initial characterization of the molecular response of cassava to drought stress resembling field conditions. The drought-responsive genes can now be used as expression-based markers of drought stress tolerance in cassava, and the candidate tolerance genes tested in the context of breeding (as possible quantitative trait loci) and engineering drought tolerance in transgenics.

An antiviral defense role of AGO2 in plants.

BACKGROUND: Argonaute (AGO) proteins bind to small-interfering (si)RNAs and micro (mi)RNAs to target RNA silencing against viruses, transgenes and in regulation of mRNAs. Plants encode multiple AGO proteins but, in Arabidopsis, only AGO1 is known to have an antiviral role. METHODOLOGY/PRINCIPAL FINDINGS: To uncover the roles of specific AGOs in limiting virus accumulation we inoculated turnip crinkle virus (TCV) to Arabidopsis plants that were mutant for each of the ten AGO genes. The viral symptoms on most of the plants were the same as on wild type plants although the ago2 mutants were markedly hyper-susceptible to this virus. ago2 plants were also hyper-susceptible to cucumber mosaic virus (CMV), confirming that the antiviral role of AGO2 is not specific to a single virus. For both viruses, this phenotype was associated with transient increase in virus accumulation. In wild type plants the AGO2 protein was induced by TCV and CMV infection. CONCLUSIONS/SIGNIFICANCE: Based on these results we propose that there are multiple layers to RNA-mediated defense and counter-defense in the interactions between plants and their viruses. AGO1 represents a first layer. With some viruses, including TCV and CMV, this layer is overcome by viral suppressors of silencing that can target AGO1 and a second layer involving AGO2 limits virus accumulation. The second layer is activated when the first layer is suppressed because AGO2 is repressed by AGO1 via miR403. The activation of the second layer is therefore a direct consequence of the loss of the first layer of defense.

Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an Agrobacterium rhizogenes-based functional screen.

The genetic regulation of programmed cell death (PCD) is well characterized in animal systems, but largely unresolved in plants. This research was designed to identify plant genes that can suppress PCD triggered in plants by Fumonisin B1 (FB1). Agrobacterium rhizogenes was used to transform individual members of a cDNA library into tomato roots, which were then screened for resistance to FB1. Cellular changes elicited during FB1-induced PCD include chromatin condensation, fragmentation into pycnotic DNA bodies, TUNEL positive reactions, ROS accumulation, and eventual loss of membrane integrity. Several cDNA library members collectively overexpressed in a transformed root population revealed PCD suppressive action and were recovered by PCR. One of the FB1 suppressive genes was homologous to metallothionein, and shared sequence homology to the animal ortholog reported to suppress PCD through interference with formation or activity of reactive oxygen species (ROS). The metallothionein recovered in this screen suppressed ROS accumulation in FB1-treated roots and prevented symptoms of PCD. Anti-PCD genes recovered by this screen represent potential sources of resistance to PCD-dependent plant diseases, while the screen should be useful to identify genes capable of suppressing PCD triggered by other effectors, including those expressed by root pathogens during infection.