Occurrence and Distribution of Physiological Races of Exserohilum turcicum in Maize-Growing Regions of Mexico.

Turcicum leaf blight (TLB) is a common foliar disease of maize in Mexico that is caused by the fungal pathogen Exserohilum turcicum. The most effective management strategy against TLB is monogenic race-specific resistance. Among the 140 E. turcicum isolates from symptomatic leaves collected from maize fields in Mexico, 100 were obtained from tropical (Veracruz) and temperate areas (Estado de México) between 2010 and 2019, and 40 isolates were obtained from tropical (Sinaloa, Tamaulipas, Veracruz, and Chiapas), subtropical (Nayarit, Jalisco, and Guanajuato), and temperate areas (Estado de Mexico, Hidalgo, and Puebla) collected in 2019. All the isolates caused TLB symptoms on the positive control (ht4), showing that they were all pathogenic. Six physiological races of E. turcicum (2, 3, 23, 3N, 23N, and 123N) were identified based on resistant or susceptible responses displayed by five maize differential genotypes (A619Ht1, A619Ht2, A619Ht3, B68HtN, and A619ht4). The most common was race 23, accounting for 68% of the isolates, followed by races 23N, 123N, 3, 2, and 3N at 15, 8, 6, 2, and 1%, respectively. Race 123N was able to infect the greatest number of maize differential genotypes used in the study. Race 123N was detected in Sinaloa and Estado de México. Race 3 was detected in Nayarit and Jalisco. Race 2 was detected in Jalisco, Estado de México, and Veracruz, and race 3N was detected in Tamaulipas. Race 23 was equally dominant in the tropical, subtropical, and temperate regions, while race 123N was more common in the tropical environment, and race 23N was more common in the tropical and temperate environments. There was no evidence for shifts in the races between 2010 and 2019.

Maize Lethal Necrosis disease: review of molecular and genetic resistance mechanisms, socio-economic impacts, and mitigation strategies in sub-Saharan Africa.

BACKGROUND: Maize lethal necrosis (MLN) disease is a significant constraint for maize producers in sub-Saharan Africa (SSA). The disease decimates the maize crop, in some cases, causing total crop failure with far-reaching impacts on regional food security. RESULTS: In this review, we analyze the impacts of MLN in Africa, finding that resource-poor farmers and consumers are the most vulnerable populations. We examine the molecular mechanism of MLN virus transmission, role of vectors and host plant resistance identifying a range of potential opportunities for genetic and phytosanitary interventions to control MLN. We discuss the likely exacerbating effects of climate change on the MLN menace and describe a sobering example of negative genetic association between tolerance to heat/drought and susceptibility to viral infection. We also review role of microRNAs in host plant response to MLN causing viruses as well as heat/drought stress that can be carefully engineered to develop resistant varieties using novel molecular techniques. CONCLUSIONS: With the dual drivers of increased crop loss due to MLN and increased demand of maize for food, the development and deployment of simple and safe technologies, like resistant cultivars developed through accelerated breeding or emerging gene editing technologies, will have substantial positive impact on livelihoods in the region. We have summarized the available genetic resources and identified a few large-effect QTLs that can be further exploited to accelerate conversion of existing farmer-preferred varieties into resistant cultivars.

Comparative study of physicochemical, nutritional, phytochemical, and sensory properties of bread with plantain and soy flours partly replacing wheat flour.

Plantain flour (PLF) and soy flour (SF) were used to substitute wheat flour (10% and 20% w/w) in composite bread. Physicochemical, phytochemical, and sensory properties were investigated. Partial substitution by PLF significantly increased (p < .05) starch, amylose, ascorbic acid, and potassium content in bread samples. In contrast, a significant improvement (p < .05) in protein, fat, amylopectin, and calcium content was observed with SF substitution. Composite bread with PLF and SF together lowered the hydrolysis index (HI) and glycemic index (GI) as compared with whole wheat flour. The molar phytate to minerals (iron, zinc, and calcium) ratio in all composite loaves was lower than reported critical values, except for phytate to iron. Significant differences (p < .05) were found in color, specific volume, and texture characteristics of loaves made from partial substitution with PLF and SF. Sensory evaluation revealed that bread with 10% PLF exhibited better scores for appearance and willingness to pay than the control. In contrast, SF negatively affected (p < .05) the appearance, texture, color, overall acceptance, and willingness to pay. The trade-off analysis indicated that PLF can be utilized to produce bread that meets consumers' demands, while incorporating SF as an alternative high-nutrient density bread will be beneficial to health.

First Report of Lasiodiplodia theobromae Causing Dieback Symptoms on Plantain (Musa AAB subgroup) in Nigeria.

Bananas (banana and plantains) rank sixth among staple food crops (FAO 2018), with production challenged by biotic factors, mainly fungal diseases that may cause a total loss in some orchards (Jones 2018). In April 2017, dieback symptoms (progressive blackening and necrotic aerial plant parts, leaves, fruits and peduncles) were observed on plantain (Musa AAB subgroup), in Onne, Rivers State, Nigeria (4°42'55.4012″N, 7°10'35.92128″E). Diseased plants (n=112) were either wilted with blackened necrotic areas, or dead (Fig. S1). Nearly 10% of the plants had blackened pseudostems and fruits with slate gray to black internal tissues when sliced (Fig. S1) and black, erumpent pycnidia were observed on diseased fruits. A fungal species was consistently isolated when surface disinfected pieces of diseased samples were cultured on PDA plates. Plates were incubated at 25±2°C for 4 to 15 d to observe conidia. Isolates had colonies and conidia consistent with members of the Botryosphaeriaceae family (Phillips et al. 2013). Immature conidia were single-celled, ellipsoidal and hyaline while mature conidia were two-celled, had a thick wall, a central septum, longitudinal striations, and a dark brown, cinnamon-like color. Size of mature conidia (n = 20) ranged 22.9 to 30.0 × 14.2 to 18.4 µm ( = 27.0 × 15.6 µm; Fig. S1). DNA templates of three isolates (23688-2_R16; 19144-18_R15 and PITA_22-1) were amplified using primers ITS1 and ITS4 for the ITS locus, EF1-688F and EF1-1251R for the translation elongation factor 1-α (TEF-1α) locus (Phillips et al. 2013) and sequenced (GenBank accession Nos. MZ413346, MZ413347, and MZ413348 for ITS; and MZ420177, MZ420178, and MZ420179 for TEF-1α). BLASTn query showed 100% identity with reference sequences of various isolates of Lasiodiplodia theobromae. Based on morphological characters and nucleotide homology, the isolates were identified as L. theobromae (Fig. S1 & S2). To fulfil Koch's postulates, 4-month-old plants of plantain hybrid PITA 24, and mature fruits from three genotypes (PITA 24, plantain cultivar Obino L'ewai) were inoculated with mycelial plugs from the margins of 5-d-old cultures of the three L. theobromae isolates. Pseudostems were drilled with a sterile 5 -mm cork borer, a mycelial plug placed down into the wound, covered with sterilized cotton, and sealed with parafilm. Sterile water was injected every third day to maintain moisture at the inoculated area. Toothpicks containing mycelia were used to inoculate fruits, placed in plastic Crisper boxes. Sterile PDA plugs or toothpicks were used for the controls. Inoculated plants and fruits were kept in a screenhouse at room temperature (~26°C) for 14 d. All inoculated materials developed symptoms similar to the diseased plants in the field. Control plants and fruits remained asymptomatic. L. theobromae was re-isolated from the artificially inoculated plant parts and its identity was confirmed. The fungus L. theobromae is distributed in tropical and subtropical regions and has a wide host range (Phillips et al. 2013; Mehl et al. 2017). This fungus was previously reported in grey literature as the causal agent of Musa spp. basal rot at Onne, Nigeria (Mwangi et al. 2005) but its molecular identification was not conducted; it was unknown whether the isolates were indeed L. theobromae or other cryptic species (L. pseudotheobromae or L. parva) (Alves et al. 2008). Over 15 years later, the present study confirms L. theobromae as the causal agent of basal rot of bananas based on nucleotide homology, and to our knowledge, this is the first report of L. theobromae causing dieback disease on plantain in Nigeria and in Africa. There is need to conduct a more comprehensive distribution surveys and develop appropriate control strategies in Nigeria.

Identification and fine mapping of a major QTL (qRtsc8-1) conferring resistance to maize tar spot complex and validation of production markers in breeding lines.

KEY MESSAGE: A major QTL of qRtsc8-1 conferring TSC resistance was identified and fine mapped to a 721 kb region on chromosome 8 at 81 Mb, and production markers were validated in breeding lines. Tar spot complex (TSC) is a major foliar disease of maize in many Central and Latin American countries and leads to severe yield loss. To dissect the genetic architecture of TSC resistance, a genome-wide association study (GWAS) panel and a bi-parental doubled haploid population were used for GWAS and selective genotyping analysis, respectively. A total of 115 SNPs in bin 8.03 were detected by GWAS and three QTL in bins 6.05, 6.07, and 8.03 were detected by selective genotyping. The major QTL qRtsc8-1 located in bin 8.03 was detected by both analyses, and it explained 14.97% of the phenotypic variance. To fine map qRtsc8-1, the recombinant-derived progeny test was implemented. Recombinations in each generation were backcrossed, and the backcross progenies were genotyped with Kompetitive Allele Specific PCR (KASP) markers and phenotyped for TSC resistance individually. The significant tests for comparing the TSC resistance between the two classes of progenies with and without resistant alleles were used for fine mapping. In BC5 generation, qRtsc8-1 was fine mapped in an interval of ~ 721 kb flanked by markers of KASP81160138 and KASP81881276. In this interval, the candidate genes GRMZM2G063511 and GRMZM2G073884 were identified, which encode an integral membrane protein-like and a leucine-rich repeat receptor-like protein kinase, respectively. Both genes are involved in maize disease resistance responses. Two production markers KASP81160138 and KASP81160155 were verified in 471 breeding lines. This study provides valuable information for cloning the resistance gene, and it will also facilitate the routine implementation of marker-assisted selection in the breeding pipeline for improving TSC resistance.

Subterranean Microbiome Affiliations of Plantain (Musa spp.) Under Diverse Agroecologies of Western and Central Africa.

Plantain (Musa spp.) is a staple food crop and an important source of income for millions of smallholder farmers in sub-Saharan Africa (SSA). However, there is a paucity of knowledge on soil microbial diversity in agroecologies where plantains are grown. Microbial diversity that increases plant performance with multi-trophic interactions involving resiliency to environmental constraints is greatly needed. For this purpose, the bacterial and fungal communities of plantain fields in high rainfall forests (HR) and derived savannas (SV) were studied using Illumina MiSeq for 16S rDNA and ITS amplicon deep sequencing. Microbial richness (α- and ß-diversity), operational taxonomic units, and Simpson and Shannon-Wiener indexes (observed species (Sobs), Chao, ACE; P < 0.05) suggested that there were significant differences between HR and SV agroecologies among the most abundant bacterial communities, and some specific dynamic response observed from fungal communities. Proteobacteria formed the predominant bacterial phylum (43.7%) succeeded by Firmicutes (24.7%), and Bacteroidetes (17.6%). Ascomycota, Basidiomycota, and Zygomycota were the three most dominant fungal phyla in both agroecologies. The results also revealed an immense array of beneficial microbes in the roots and rhizosphere of plantain, including Acinetobacter, Bacillus, and Pseudomonas spp. COG and KEGG Orthology database depicted significant variations in the functional attributes of microbes found in the rhizosphere to roots. This result indicates that the different agroecologies and host habitats differentially support the dynamic microbial profile and that helps in altering the structure in the rhizosphere zone for the sake of promoting synergistic host-microbe interactions particularly under resource-poor conditions of SSA.

How Does Cultivar, Maturation, and Pre-Treatment Affect Nutritional, Physicochemical, and Pasting Properties of Plantain Flours?

The effect of cultivar, ripening stage, and pre-treatment method were investigated on the nutritional, physicochemical, and pasting properties of plantain flours from two plantains and two plantain hybrids. There were significant variations (p < 0.05) in chemical composition and physical properties influenced by the interaction of cultivars, ripening stages, and pre-treatment methods. The highest levels of amylose, water-holding capacity (WHC), and oil-holding capacity (OHC) were observed in unripe flours and acid-treated flour recorded the highest content of resistant starch (RS). Flour after pre-blanching contained the highest level of total phenolic (TP), carotenoid contents, and browning index (BI) value. In contrast, acid-treated flours had the lowest BI value. As ripening progressed, peak viscosity and breakdown values increased but final viscosity, setback, and pasting temperature values were reduced. Untreated flour samples showed the highest peak viscosity. Higher breakdown values were found in acid-treated samples and higher setback values in pre-blanched samples.

Consumer Preferences and Socioeconomic Factors Decided on Plantain and Plantain-Based Products in the Central Region of Cameroon and Oyo State, Nigeria.

Plantain is a key staple food in Central and West Africa, but there is limited understanding of its market in Africa. In addition, the cooking methods for enhancing the nutritional value, consumer preference, and willingness to pay for plantain and plantain-based products are not well understood. The knowledge gaps in the market and consumer dimension of the food chain need to be known to increase plantain utilization and guide breeding efforts. This research contributes by examining the cooking methods, consumer preference, and willingness to pay for plantain and plantain-based products in Cameroon and Nigeria. A household survey sample of 454 Cameroonian consumers in four divisions of Central Region and 418 Nigerian consumers in seven government areas of Oyo State in southwest Nigeria was the basis for the analysis. The results showed some levels of similarity and difference in the consumption and cooking of boiled, roasted, and fried plantain in both countries. The trend in consumption of all plantain-based products was constant in Cameroon but increased in Nigeria. The most important factor influencing Cameroonian consumers' choice of plantain and its products was taste, while the nutrition trait influenced Nigerian consumers. Both Cameroonian and Nigerian consumers considered packaging, location of produce, and size and quantity as the least important factors. In addition, socioeconomic characteristics were significant determinants of consumers' choices to consume plantain and its products. Gender significantly influenced (p < 0.05) taste, while nutrition was significantly driven (p < 0.05) by education and annual income. Household size played a significant role (p < 0.05) in consumers' choices when the price was considered. These findings serve as a guideline to improve existing products to match the needs of consumers in each country and develop products for different consumer segments and potentially increase production.

Physiological and ecological warnings that dodders pose an exigent threat to farmlands in Eastern Africa.

Invasive holoparasitic plants of the genus Cuscuta (dodder) threaten African ecosystems due to their rapid spread and attack on various host plant species. Most Cuscuta species cannot photosynthesize and hence rely on host plants for nourishment. After attachment through a peg-like organ called a haustorium, the parasites deprive hosts of water and nutrients, which negatively affects host growth and development. Despite their rapid spread in Africa, dodders have attracted limited research attention, although data on their taxonomy, host range, and epidemiology are critical for their management. Here, we combine taxonomy and phylogenetics to reveal the presence of field dodder (Cuscuta campestris) and C. kilimanjari (both either naturalized or endemic to East Africa), in addition to the introduction of the giant dodder (C. reflexa), a south Asian species, in continental Africa. These parasites have a wide host range, parasitizing species across 13 angiosperm orders. We evaluated the possibility of C. reflexa to expand this host range to tea (Camelia sinensis), coffee (Coffea arabica), and mango (Mangifera indica), crops of economic importance to Africa, for which haustorial formation and vascular-bundle connections in all three crops revealed successful parasitism. However, only mango mounted a successful postattachment resistance response. Furthermore, species distribution models predicted high habitat suitability for Cuscuta spp. across major tea- and coffee-growing regions of Eastern Africa, suggesting an imminent risk to these crops. Our findings provide relevant insights into a poorly understood threat to biodiversity and economic wellbeing in Eastern Africa, and provide critical information to guide development of management strategies to avert Cuscuta spp. spread.

Phytosanitary Interventions for Safe Global Germplasm Exchange and the Prevention of Transboundary Pest Spread: The Role of CGIAR Germplasm Health Units.

The inherent ability of seeds (orthodox, intermediate, and recalcitrant seeds and vegetative propagules) to serve as carriers of pests and pathogens (hereafter referred to as pests) and the risk of transboundary spread along with the seed movement present a high-risk factor for international germplasm distribution activities. Quarantine and phytosanitary procedures have been established by many countries around the world to minimize seed-borne pest spread by screening export and import consignments of germplasm. The effectiveness of these time-consuming and cost-intensive procedures depends on the knowledge of pest distribution, availability of diagnostic tools for seed health testing, qualified operators, procedures for inspection, and seed phytosanitation. This review describes a unique multidisciplinary approach used by the CGIAR Germplasm Health Units (GHUs) in ensuring phytosanitary protection for the safe conservation and global movement of germplasm from the 11 CGIAR genebanks and breeding programs that acquire and distribute germplasm to and from all parts of the world for agricultural research and food security. We also present the challenges, lessons learned, and recommendations stemming from the experience of GHUs, which collaborate with the national quarantine systems to export and distribute about 100,000 germplasm samples annually to partners located in about 90 to 100 countries. Furthermore, we describe how GHUs adjust their procedures to stay in alignment with evolving phytosanitary regulations and pest risk scenarios. In conclusion, we state the benefits of globally coordinated phytosanitary networks for the prevention of the intercontinental spread of pests that are transmissible through plant propagation materials.

Karnal Bunt: A Re-Emerging Old Foe of Wheat.

Wheat (Triticum aestivum L.) crop health assumes unprecedented significance in being the second most important staple crop of the world. It is host to an array of fungal pathogens attacking the plant at different developmental stages and accrues various degrees of yield losses owing to these. Tilletia indica that causes Karnal bunt (KB) disease in wheat is one such fungal pathogen of high quarantine importance restricting the free global trade of wheat besides the loss of grain yield as well as quality. With global climate change, the disease appears to be shifting from its traditional areas of occurrence with reports of increased vulnerabilities of new areas across the continents. This KB vulnerability of new geographies is of serious concern because once established, the disease is extremely difficult to eradicate and no known instance of its complete eradication using any management strategy has been reported yet. The host resistance to KB is the most successful as well as preferred strategy for its mitigation and control. However, breeding of KB resistant wheat cultivars has proven to be not so easy, and the low success rate owes to the scarcity of resistance sources, extremely laborious and regulated field screening protocols delaying identification/validation of putative resistance sources, and complex quantitative nature of resistance with multiple genes conferring only partial resistance. Moreover, given a lack of comprehensive understanding of the KB disease epidemiology, host-pathogen interaction, and pathogen evolution. Here, in this review, we attempt to summarize the progress made and efforts underway toward a holistic understanding of the disease itself with a specific focus on the host-pathogen interaction between T. indica and wheat as key elements in the development of resistant germplasm. In this context, we emphasize the tools and techniques being utilized in development of KB resistant germplasm by illuminating upon the genetics concerning the host responses to the KB pathogen including a future course. As such, this article could act as a one stop information primer on this economically important and re-emerging old foe threatening to cause devastating impacts on food security and well-being of communities that rely on wheat.

Variation in Occurrence and Aflatoxigenicity of Aspergillus flavus from Two Climatically Varied Regions in Kenya.

Aflatoxins are carcinogenic chemical metabolites produced by Aspergillus spp. of the section Flavi. In Kenya, Aspergillus flavus is the most prevalent and has been associated with several acute and chronic aflatoxin outbreaks in the past. In this study, we evaluated the occurrence of A. flavus in soils from two agro-ecological regions with contrasting climatic conditions, aflatoxin contamination histories and cropping systems. Aspergillus spp. were first isolated from soils before the identification and determination of their aflatoxigenicity. Further, we determined the occurrence of Pseudomonas and Bacillus spp. in soils from the two regions. These bacterial species have long been associated with biological control of several plant pathogens including Aspergillus spp. Our results show that A. flavus occurred widely and produced comparatively higher total aflatoxin levels in all (100%) study sites from the eastern to the western regions of Kenya. For the western region, A. flavus was detected in 4 locations (66.7%) that were previously under maize cultivation with the isolates showing low aflatoxigenicity. A. flavus was not isolated from soils under sugarcane cultivation. Distribution of the two bacterial species varied across the regions but we detected a weak relationship between occurrence of bacterial species and A. flavus. We discuss these findings in the context of the influence of climate, microbial profiles, cropping systems and applicability in the deployment of biological control remedies against aflatoxin contamination.

Efficacy of chemotherapy and thermotherapy in elimination of East African cassava mosaic virus from Tanzanian cassava landrace.

Cassava mosaic disease is caused by cassava mosaic begomoviruses (CMBs) and can result in crop losses up to 100% in cassava (Manihot esculenta) in Tanzania. We investigated the efficacy of chemotherapy and thermotherapy for elimination of East African cassava mosaic virus (EACMV) of Tanzanian cassava. In vitro plantlets from EACMV-infected plants obtained from coastal Tanzania were established in the greenhouse. Leaves were sampled from the plants and tested to confirm the presence of EACMV. Plantlets of plants positive for EACMV were initiated in Murashige and Skoog (MS) medium. On the second subculture, they were subjected into chemical treatment in the medium containing salicylic acid (0, 10, 20, 30 and 40 mg/L) and ribavirin (0, 5, 10, 15 and 20 mg/L). In the second experiment, EACMV-infected plantlets were subjected to temperatures between 35 and 40°C with 28°C as the control. After 42 days of growth, DNA was extracted from plant leaves and PCR amplification was performed using EACMV specific primers. It was found that plant survival decreased with increasing levels of both salicylic acid and ribavirin concentrations. In general, plants treated with salicylic acid exhibited a lower plant survival % than those treated with ribavirin. However, the percentage of virus-free plants increased with an increase in the concentration of both ribavirin and salicylic acid. The most effective concentrations were 20 mg/L of ribavirin and 30 mg/L of salicylic acid; these resulted in 85.0% and 88.9% virus-free plantlets, respectively. With regard to thermotherapy, 35°C resulted in 79.5% virus-free plantlets compared to 69.5% at 40°C. Based on virus elimination, ribavirin at 20 mg/L, salicylic acid 30 mg/L and thermotherapy at 35°C are recommended for production of EACMV free cassava plantlets from infected cassava landraces.

Downregulation of transcription factor aflR in Aspergillus flavus confers reduction to aflatoxin accumulation in transgenic maize with alteration of host plant architecture.

KEY MESSAGE: We report success of host-induced gene silencing in downregulation of aflatoxin biosynthesis in Aspergillus flavus infecting maize transformed with a hairpin construct targeting transcription factor aflR. Infestation of crops by aflatoxin-producing fungi results in economic losses as well as negative human and animal health effects. Currently, the control strategies against aflatoxin accumulation are not effective to the small holder farming systems in Africa and this has led to widespread aflatoxin exposure especially in rural populations of sub-Saharan Africa that rely on maize as a staple food crop. A recent strategy called host-induced gene silencing holds great potential for developing aflatoxin-resistant plant germplasm for the African context where farmers are unable to make further investments other than access to the germplasm. We transformed maize with a hairpin construct targeting the aflatoxin biosynthesis transcription factor aflR. The developed transgenic maize were challenged with an aflatoxigenic Aspergillus flavus strain from Eastern Kenya, a region endemic to aflatoxin outbreaks. Our results indicated that aflR was downregulated in A. flavus colonizing transgenic maize. Further, maize kernels from transgenic plants accumulated significantly lower levels of aflatoxins (14-fold) than those from wild type plants. Interestingly, we observed that our silencing cassette caused stunting and reduced kernel placement in the transgenic maize. This could have been due to "off-target" silencing of unintended genes in transformed plants by aflR siRNAs. Overall, this work indicates that host-induced gene silencing has potential in developing aflatoxin-resistant germplasm.

A virus-induced gene silencing (VIGS) system for functional genomics in the parasitic plant Striga hermonthica.

BACKGROUND: Striga hermonthica is a hemiparasitic weed that infects cereals in Sub Sahara Africa (SSA) resulting in up to 100% grain yield loss. This significant loss in grain yields is a major contributor to food insecurity and poverty in the region. Current strategies to control the parasite are costly, unavailable and remain unpracticed by small-scale farmers, underscoring the need for more economical and sustainable control strategies. Development of resistant germplasm is the most sustainable strategy in the control of S. hermonthica, but is constrained by paucity of resistance genes for introduction into crop germplasm. RNA interference (RNAi) has potential for developing host-derived resistance against S. hermonthica by transformation of host crops with RNAi sequences targeted at critical Striga genes. The application of RNAi in management of S. hermonthica is however constrained by lack of efficient high throughput screening protocols for the candidate genes for silencing, as well as sub optimal delivery of siRNAs into the parasite. In comparison to stable transformation, viral induced gene silencing (VIGS) is a rapid and powerful tool for plant functional genomics and provides an easy and effective strategy in screening for putative candidate genes to target through RNAi. In addition, VIGS allows for a secondary amplification of the RNAi signal increasing the siRNA threshold and facilitates siRNA transport through viral movement proteins. We tested the efficiency of the Tobacco rattle virus (TRV1 and TRV2) VIGS vectors in silencing S. hermonthica phytoene desaturase (PDS) gene through agrodrench and agro-infiltration. RESULTS: We report the validation of VIGS in S. hermonthica using a silencing cassette generated from TRV with a PDS gene insert. Agro-infiltrated and agro-drenched S. hermonthica leaves showed photo-bleaching phenotypes typical for PDS silencing within 7 and 14 days post infection respectively. In both cases S. hermonthica plants recovered from photo-bleaching effects within 28 days post inoculation. The transformation efficiency of the VIGS protocol in S. hermonthica was (60 ± 2.9)%. CONCLUSION: These results demonstrate that the TRV-VIGS system work in S. hermonthica and can be used for candidate gene validation for their role in the parasite development and parasitism, with the ultimate goal of developing resistant transgenic maize.

A new double right border binary vector for producing marker-free transgenic plants.

BACKGROUND: Once a transgenic plant is developed, the selectable marker gene (SMG) becomes unnecessary in the plant. In fact, the continued presence of the SMG in the transgenic plant may cause unexpected pleiotropic effects as well as environmental or biosafety issues. Several methods for removal of SMGs that have been reported remain inaccessible due to protection by patents, while development of new ones is expensive and cost prohibitive. Here, we describe the development of a new vector for producing marker-free plants by simply adapting an ordinary binary vector to the double right border (DRB) vector design using conventional cloning procedures. FINDINGS: We developed the DRB vector pMarkfree5.0 by placing the bar gene (representing genes of interest) between two copies of T-DNA right border sequences. The ß-glucuronidase (gus) and nptII genes (representing the selectable marker gene) were cloned next followed by one copy of the left border sequence. When tested in a model species (tobacco), this vector system enabled the generation of 55.6% kanamycin-resistant plants by Agrobacterium-mediated transformation. The frequency of cotransformation of the nptII and bar transgenes using the vector was 66.7%. Using the leaf bleach and Basta assays, we confirmed that the nptII and bar transgenes were coexpressed and segregated independently in the transgenic plants. This enable separation of the transgenes in plants cotransformed using pMarkfree5.0. CONCLUSIONS: The results suggest that the DRB system developed here is a practical and effective approach for separation of gene(s) of interest from a SMG and production of SMG-free plants. Therefore this system could be instrumental in production of "clean" plants containing genes of agronomic importance.

Bioactivity determination of methanol and water extracts for roots and leaves of Kenyan Psidium guajava L landraces against pathogenic bacteria.

Guava (Psidium guajava L) is native to South America and exists as both wild and cultivated. Guava has been used as a source of food and raw materials for pharmaceuticals. The aim of this study was to determine bioactivity of methanol and water extracts from root and leaves of Kenyan guava landraces against selected pathogenic bacteria. Study samples were collected from Western and South Coast of Kenya. One hundred grams of leaf and root ground powders were used for sequential extraction using methanol and water. Extracts were evaporated and 0.2gms dissolved using the extraction solvent and tested against gram positive (Staphylococcus aureus, Bacillus subtilis) and negative bacteria (Escherichia coli). Data on inhibition zone was taken in mm and analyzed at 95% confidence interval. Extracts from Western region had significant inhibition compared to Coastal region. The two regions have different climatic conditions that result in these plants having different compounds even though they are the same species. Roots had higher inhibition compared to the leaves as they contain high levels of tannins compared to leaves. Water as an extracting solvent had higher inhibition than methanol as it is more polar and it absorbs more bioactive compounds. S. aureus was most inhibited followed by E. coli and B. subtilis respectively. There was no significant difference between the gram positive and negative bacteria. Remarkably, some methanol and water root extracts had significant inhibition against bacteria when compared to some commercial antibiotics used. Results of this study indicate that Kenyan guava roots from Western Kenya extracted with methanol and water have a potential to be used as a source of active compounds in treatment of gram positive and gram negative bacteria pathogens.

Prevalence and diversity of cystic echinococcosis in livestock in Maasailand, Kenya.

Cystic echinococcosis (CE) is a zoonotic disease caused by several members of the Echinococcus granulosus species complex. In East Africa, several species/strains are known to occur in livestock and humans, but host preferences, relative frequencies and spatial distribution of these taxa are poorly known. Here, we contribute livestock data for Maasailand of southern Kenya. Total CE prevalence was 25.8 % in cattle (151/587), 16.5 % in sheep (71/430) and 10.8 % in goats (21/194), which is a significant increase compared to surveys done about three decades ago. The majority of cysts occurred in the liver (56 % in cattle, 70 % in sheep and 65 % in goats). Molecular characterization by PCR-RFLP and sequencing of parts of the mitochondrial nad-1 gene was done for a subsample of 285 cysts. E. granulosus G1 was dominant in all host species (200 of 201 cysts from cattle, 68 of 69 from sheep and 11 of 15 from goats); the remaining taxa were Echinococcus canadensis G6 (one cyst from sheep, four from goats) and Echinococcus ortleppi (one cyst from cattle). Considering cyst fertility, sheep appear to be the most important hosts for E. granulosus G1, while goats were found to be suitable hosts for E. canadensis G6 (three of four cysts were fertile). For the first time, E. ortleppi was found in cattle from southern Kenya. Our data show an intense and possibly increasing level of CE transmission in southern Kenya, and the predominance of E. granulosus G1, which appears to be particularly pathogenic to humans, calls for urgent control measures.

Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism.

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

Striga parasitizes transgenic hairy roots of Zea mays and provides a tool for studying plant-plant interactions.

BACKGROUND: Striga species are noxious root hemi-parasitic weeds that debilitate cereal production in sub-Saharan Africa (SSA). Control options for Striga are limited and developing Striga resistant crop germplasm is regarded as the best and most sustainable control measure. Efforts to improve germplasm for Striga resistance by a non-Genetic Modification (GM) approach, for example by exploiting natural resistance, or by a GM approach are constrained by limited information on the biological processes underpinning host-parasite associations. Additionaly, a GM approach is stymied by lack of availability of candidate resistance genes for introduction into hosts and robust transformation methods to validate gene functions. Indeed, a majority of Striga hosts, the world's most cultivated cereals, are recalcitrant to genetic transformation. In maize, the existing protocols for transformation and regeneration are tedious, lengthy, and highly genotype-specific with low efficiency of transformation. RESULTS: We used Agrobacterium rhizogenes strain K599 carrying a reporter gene construct, Green Fluorescent Protein (GFP), to generate transgenic composite maize plants that were challenged with the parasitic plant Striga hermonthica. Eighty five percent of maize plants produced transgenic hairy roots expressing GFP. Consistent with most hairy roots produced in other species, transformed maize roots exhibited a hairy root phenotype, the hallmark of A. rhizogenes mediated transformation. Transgenic hairy roots resulting from A. rhizogenes transformation were readily infected by S. hermonthica. There were no significant differences in the number and size of S. hermonthica individuals recovered from either transgenic or wild type roots. CONCLUSIONS: This rapid, high throughput, transformation technique will advance our understanding of gene function in parasitic plant-host interactions.