Morpho-physiological and yield traits for selection of drought tolerant Urochloa grass ecotypes.

Drought has become more recurrent and causes a substantial decline in forage yields leading to strain on feed resources for livestock production. This has intensified the search for drought-tolerant forages to promote sustainable livestock production. The objective of this study was to identify drought-tolerant Urochloa grasses and to discern their morpho-physiological and yield traits to water stress as well as the relationship between these traits and indices of drought resistance. The results showed that the ecotypes, water regimes and their interaction significantly influenced all the studied morpho-physiological and yield traits. There was a significant decrease in plant height, number of leaves and tillers, dry matter yield, relative water content, photosystem II and efficiency of photosystem II with an increase in non-photochemical quenching. The principal component analysis revealed that the performance of Urochloa grass ecotypes was different under water sufficient (WS) and water deficit conditions. Drought tolerance indicators (mean productivity, geometric mean productivity, tolerance index and stress tolerance index) were most effective in identifying Urochloa ecotypes with high biomass production under both water deficient and WS conditions. Ecotypes K17, K7, Kisii, Busia and Kakamega were the most drought tolerant, Basilisk, K6, K10, K19 and Toledo were moderately tolerant whereas, CIAT6385, CIAT16449, K13, K5 and K9 were drought sensitive. The five drought-tolerant Urochloa ecotypes should be tested for sustainable biomass production under field conditions and used in breeding programmes to develop high-yielding drought-tolerant varieties.

Real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of cassava brown streak viruses.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.

Occurrence of passion fruit woodiness disease in the coastal lowlands of Kenya and screening of passion fruit genotypes for resistance to passion fruit woodiness disease.

BACKGROUND: Passion fruit (Passiflora edulis [Sims]) is an important economic fruit crop in Kenya, grown for domestic, regional and international markets. However, passion fruit production is constrained by both biotic and abiotic stresses. Passion fruit woodiness disease (PWD) complex is the most injurious viral disease responsible for yield losses of up to 100%. In East Africa, it is caused by potyviruses. The most effective way to manage PWD is by using resistant cultivars. The objectives of this study were to determine the occurrence of passion fruit woodiness disease in selected counties at the Coastal lowlands of Kenya and screen farmer preferred passion fruit genotypes for resistance to PWD. RESULTS: In the present study, it was established that all surveyed farms in Kwale and Kilifi counties displayed passion fruit woodiness virus disease symptoms. The highest disease incidence of 59.16% and 51.43% was observed at Kilifi and Kwale counties, respectively. A significant difference (p < 0.05) in symptom severity was observed within the tested genotypes with purple and banana passion fruits having the highest and lowest AUDPC values, respectively, both under greenhouse and field conditions. ACP ELISA assays using universal potyvirus antiserum (Agdia Inc., Elkhat, IN) confirmed that the observed characteristic symptoms of woodiness disease were as a result of potyvirus infection. CONCLUSIONS: The findings herein indicate that PWD is widespread in both Kilifi and Kwale counties with low to moderate disease incidence and severity. The observed prevalence, incidence and severity levels of PWD in Kwale and Kilifi counties could be aggravated by poor management practices such as non-sterilization of pruning tools, intercropping with target crops and crop rotation with the same target crops. Response of passion fruit genotypes to woodiness viruses was genotype dependent. There is need to sensitize farmers on the cause and spread of PWD and management strategies in order to increase production and enhance the quality of fruits.

Antimicrobial resistance profiles of salmonella spp. and escherichia coli isolated from fresh nile tilapia (oreochromis niloticus) fish marketed for human consumption.

BACKGROUND: Salmonella spp. and pathogenic strains of Escherichia coli are among the major foodborne zoonotic pathogens. These bacterial pathogens cause human illnesses characterized by hemorrhagic colitis, vomiting, nausea, and other agent-related symptoms. The increasing occurrence of antimicrobial resistance in these pathogens is also a serious public health concern globally. Regular surveillance of phenotypes and genotypes of Salmonella spp. and Escherichia coli from animal-derived foods is necessary for effective reduction and control of these foodborne pathogens. This study was conducted to assess the occurrence, antimicrobial resistance, virulence genes and genetic diversity of Salmonella spp. and E. coli isolates from fresh Nile tilapia obtained from retail markets in Nairobi, Kenya. METHODS: A total of 68 fresh Nile tilapia fish samples were collected from retail markets and used for isolation of Salmonella spp. and E. coli. Antimicrobial susceptibilities of the isolates weretested by Kirby-Bauer agar disc diffusion method. According to the antimicrobial resistance profiles, the multi-drug resistant isolates were identified by 16 S rRNA sequencing and phylogenetic analysis using the Bayesian inference method. The MDR Salmonella spp. and E. coli isolates were subjected to PCR-based screening for the detection virulence and antibiotic resistance genes. RESULTS: The prevalence of contamination of the fish samples with Salmonella spp. and E.coli was 26.47% and 35.29% respectively. Overall phenotypic resistance among the Salmonella spp. ranged from 5.5% for ceftazidime, chloramphenicol, meropenem, nitrofurantoin and streptomycin and 22.2% for penicillin-G. For E. coli phenotypic resistance ranged from 4.2% for ceftazidime and chloramphenicol and 25% for rifampicin. Multi-drug resistance was observed in three Salmonella spp. and two E. coli isolates. Results of 16 S rRNA sequences, sequence alignment and phylogenic trees confirmed the identified MDR isolates as S. typhymurium WES-09, S. typhymurium MAK-22, S. typhimurium EMB-32 and E. coli MAK-26 and E. coli LAN-35. The presence of antibiotic-resistance genes belonging to ß-lactamases, tetracycline, sulfonamide, trimethoprim and aminoglycosides-resistant genes were detected in all the identified MDR isolates. CONCLUSIONS: The findings from this study indicate that Nile tilapia (Oreochromis niloticus) sold in retail markets can acts as reservoirs of Salmonella spp. and E. coli pathogens linked to human disease, some of which were multidrug resistance to critically important antimicrobials. Both microorganisms are of zoonotic significance and represent a significant public health risk to the society.

Identification and Characterization of Colletotrichum Species Causing Sorghum Anthracnose in Kenya and Screening of Sorghum Germplasm for Resistance to Anthracnose.

Anthracnose caused by Colletotrichum species is one of the most destructive fungal diseases of sorghum with annual yield losses of up to 100%. Although the resistance to anthracnose has been identified elsewhere, the usefulness of the resistance loci differs depending on the pathogen species and pathotypes. Accurate species identification of the disease-causing fungal pathogens is essential for developing and implementing suitable management strategies. The use of host resistance is the most effective strategy of anthracnose management and therefore identification of sources for resistance against unique pathogen pathotypes is fundamental. The aims of this study were to identify and characterize Colletotrichum species associated with sorghum anthracnose and screen sorghum germplasm for resistance to anthracnose. Symptomatic sorghum leaf samples were collected from smallholder farmers in lower eastern Kenya and used for the isolation, identification and characterization of Colletotrichum species using morpho-cultural and phylogenetic analyses with the sequences of the rDNA internal transcribed spacer (ITS) region. Pathogenicity tests of the seven fungal isolates showed that there were no significant differences in the pathogenicity on host plants. The fungal isolates were variable in cultural and morphological characters such as colony type and color, colony diameter, mycelia growth and hyaline. The phenotypic characters observed were useful in the identification of the genus Colletotrichum and not the species. Based on the sequence and phylogenetic analysis of ITS, Colletotrichum sublineola was revealed to be associated with anthracnose on sorghum. Germplasm screening for resistance to anthracnose showed differential reactions of sorghum genotypes to anthracnose under greenhouse and field conditions. The results revealed four resistant genotypes and ten susceptible genotypes against Colletotrichum sublineola. Significant (p ≤ 0.05) differences were observed in grain weight, grain yield, weight of 100 seeds and harvest index among the tested sorghum genotypes. The present study indicated that the Kenyan accessions could be an important source of resistance to anthracnose. The findings from this study provide a platform towards devising efficient disease control strategies and resistance breeding.

Distribution and Molecular Diversity of Whitefly Species Colonizing Cassava in Kenya.

The whitefly, Bemisia tabaci (Gennadium, Hemiptera) has been reported to transmit viruses that cause cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) in many parts of sub-Saharan Africa (SSA). Currently, there is limited information on the distribution, species and haplotype composition of the whitefly populations colonizing cassava in Kenya. A study was conducted in the major cassava growing regions of Kenya to address this gap. Analyses of mitochondrial DNA cytochrome oxidase 1 (mtCO1) sequences revealed the presence of four distinct whitefly species: Bemisia tabaci, Bemisia afer, Aleurodicus dispersus and Paraleyrodes bondari in Kenya. The B. tabaci haplotypes were further resolved into SSA1, SSA2 and Indian Ocean (IO) putative species. The SSA1 population had three haplogroups of SSA1-SG1, SSA-SG2 and SSA1-SG3. Application of KASP genotyping grouped the Bemisia tabaci into two haplogroups namely sub-Saharan Africa East and Southern Africa (SSA-ESA) and sub-Saharan Africa East and Central Africa (SSA-ECA). The study presents the first report of P. bondari (Bondar's nesting whitefly) on cassava in Kenya. Bemisia tabaci was widely distributed in all the major cassava growing regions in Kenya. The increased detection of different whitefly species on cassava and genetically diverse B. tabaci mitotypes indicates a significant influence on the dynamics of cassava virus epidemics in the field. The study highlights the need for continuous monitoring of invasive whitefly species population on cassava for timely application of management practices to reduce the impact of cassava viral diseases and prevent potential yield losses.

A simple and fast Agrobacterium-mediated transformation system for passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa).

BACKGROUND: Passion fruit (Passiflora edulis Sims) is an important horticultural crop in the tropics and subtropics, where it has great commercial potential due to high demand for fresh edible fruits and processed juice as well as source of raw materials in cosmetic industries. Genetic engineering shows great potential in passion fruit improvement and can compensate for the limitations of conventional breeding. Despite the success achieved in genetic modification of few passion fruit varieties, transgenic passion fruit production is still difficult for farmer-preferred cultivars. Therefore, it is important to establish a simple and fast Agrobacterium-mediated cell transformation of commercial hybrid passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa). RESULTS: In the present study, we have developed a simple and fast Agrobacterium-mediated transformation system for hybrid passion fruit KPF4 using leaf disc explants. Factors affecting the rate of transient beta (ß)-glucuronidase (gusA) expression and consequently transformation efficiency were optimized as follows: Agrobacterium cell density with an OD600 of 0.5, 30 min infection time, 3 days of co-cultivation duration and the incorporation of 200 µM acetosyringone into Agrobacterium infection suspension medium. Using the optimized conditions, transgenic plants of KPF4 were produced within 2 months with an average transformation efficiency of 0.67%. The ß-glucuronidase (GUS) histochemical staining confirmed the expression and integration of an intron-containing gusA gene into transformed leaf discs and transgenic plant lines of KPF4. The presence of gusA gene in the transgenic plants was confirmed by polymerase chain reaction (PCR). The results confirmed that the gusA gene was efficiently integrated into the passion fruit genome. CONCLUSIONS: The developed transformation protocol is simple and rapid and could be useful for functional genomic studies and transferring agronomically important traits into passion fruit hybrid KPF4. This study developed a method that can be used to transfer traits such as resistance to viral diseases, low fruit quality and short storage life. To the best of our knowledge, this is the first report on genetic transformation system for commercial passion fruit hybrid KPF4.

Overexpression of rice thaumatin-like protein (Ostlp) gene in transgenic cassava results in enhanced tolerance to Colletotrichum gloeosporioides f. sp. manihotis.

Cassava (Manihot esculenta Crantz) is the most important staple food for more than 300 million people in Africa, and anthracnose disease caused by Colletotrichum gloeosporioides f. sp. manihotis is the most destructive fungal disease affecting cassava production in sub-Saharan Africa. The main objective of this study was to improve anthracnose resistance in cassava through genetic engineering. Transgenic cassava plants harbouring rice thaumatin-like protein (Ostlp) gene, driven by the constitutive CaMV35S promoter, were generated using Agrobacterium-mediated transformation of friable embryogenic calli (FEC) of cultivar TMS 60444. Molecular analysis confirmed the presence, integration, copy number of the transgene all the independent transgenic events. Semi-quantitative RT-PCR confirmed high expression levels of Ostlp in six transgenic lines tested. The antifungal activity of the transgene against Colletotrichum gloeosporioides pathogen was evaluated using the leaves and stem cuttings bioassay. The results demonstrated significantly delayed disease development and reduced size of necrotic lesions in leaves and stem cuttings of all transgenic lines compared to the leaves and stem cuttingss of non-transgenic control plants. Therefore, constitutive overexpression of rice thaumatin-like protein in transgenic cassava confers enhanced tolerance to the fungal pathogen C. gloeosporioides f. sp. manihotis. These results can therefore serve as an initial step towards genetic engineering of farmer-preffered cassava cultivars for resistance to anthracnose disease.

Morphological and Genetic Diversity of Rhizobia Nodulating Cowpea (Vigna unguiculata L.) from Agricultural Soils of Lower Eastern Kenya.

Limited nitrogen (N) content in the soil is a major challenge to sustainable and high crop production in many developing countries. The nitrogen fixing symbiosis of legumes with rhizobia plays an important role in supplying sufficient N for legumes and subsequent nonleguminous crops. To identify rhizobia strains which are suitable for bioinoculant production, characterization of rhizobia is a prerequisite. The objective of this study was to assess the morphological and genetic diversity of rhizobia that nodulates cowpea in agricultural soils of lower eastern Kenya. Twenty-eight rhizobia isolates were recovered from soil samples collected from farmers' fields in Machakos, Makueni, and Kitui counties in lower eastern Kenya and characterized based on morphological characteristics. Thirteen representative isolates were selected and characterized using BOX repetitive element PCR fingerprinting. Based on the dendrogram generated from morphological characteristics, the test isolates were distributed into two major clusters at a similarity of 75%. Phylogenetic tree, based on BOX repetitive element PCR, grouped the isolates into two clusters at 90% similarity level. The clustering of the isolates did not show a relationship to the origin of soil samples, although the isolates were genetically diverse. This study is a prerequisite to the selection of suitable cowpea rhizobia to develop bioinoculants for sustainable crop production in Kenya.

Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14.

Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1-2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava.