Aspergillus population diversity and its role in aflatoxin contamination of cashew nuts from coastal Kenya.

Cashew nuts are among the main cash crops in coastal Kenya, due in large part to their high nutritional value. Unfortunately, they also make them highly susceptible to mold contamination, resulting in biodeterioration of the nutritional value and potential contamination with toxic secondary metabolites, such as aflatoxins, that cause them to be rejected for sale at the market. We determined the population diversity of the Aspergillus species and their role in aflatoxin contamination in cashew nuts in selected coastal regions of Kenya. Fifty raw cashew nut samples were collected from post-harvest storage facilities across three counties in Kenya's coastal region and examined for moisture content and the presence of Aspergillus fungi. About 63 presumptive isolates were recovered from the cashew nuts. ITS and 28S rDNA regions were sequenced. The aflD, aflM and aflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins' presence on the isolates was screened using UV and the ammonia vapour test on coconut milk agar and validated using ELISA assay. A comparison of cashew moisture content between the three counties sampled revealed a significant difference. Sixty-three isolates were recovered and identified to section based on morphological characters and their respective ITS regions were used to obtain species identifications. Three sections from the genus were represented, Flavi and Nigri, and Terrei with isolates from the section Nigri having slightly greater abundance (n = 35). The aflD, aflM and aflR genes were amplified for all isolates to assess the presence of the aflatoxin biosynthesis pathway, indicating the potential for aflatoxin production. Less than half of the Aspergillus isolates (39.68%) contained the aflatoxin pathway genes, while 22.22% isolates were aflatoxigenic, which included only the section Flavi isolates. Section Flavi isolates identification was confirmed by calmodulin gene. The presence of species from Aspergillus section Flavi and section Nigri indicate the potential for aflatoxin or ochratoxin in the cashew nuts. The study established a foundation for future investigations of the fungi and mycotoxins contaminating cashew nuts in Kenya, which necessitates developing strategies to prevent infection by mycotoxigenic fungi, especially during the storage and processing phases.

Host Specificity Controlled by PWL1 and PWL2 Effector Genes in the Finger Millet Blast Pathogen Magnaporthe oryzae in Eastern Africa.

Magnaporthe oryzae, a devastating pathogen of finger millet (Eleusine coracana), secretes effector molecules during infection to manipulate host immunity. This study determined the presence of avirulence effector genes PWL1 and PWL2 in 221 Eleusine blast isolates from eastern Africa. Most Ethiopian isolates carried both PWL1 and PWL2. Kenyan and Ugandan isolates largely lacked both genes, and Tanzanian isolates carried either PWL1 or lacked both. The roles of PWL1 and PWL2 towards pathogenicity on alternative chloridoid hosts, including weeping lovegrass (Eragrostis curvula), were also investigated. PWL1 and PWL2 were cloned from Ethiopian isolate E22 and were transformed separately into Ugandan isolate U34, which lacked both genes. Resulting transformants harboring either gene gained varying degrees of avirulence on Eragrostis curvula but remained virulent on finger millet. Strains carrying one or both PWL1 and PWL2 infected the chloridoid species Sporobolus phyllotrichus and Eleusine tristachya, indicating the absence of cognate resistance (R) genes for PWL1 and PWL2 in these species. Other chloridoid grasses, however, were fully resistant, regardless of the presence of one or both PWL1 and PWL2, suggesting the presence of effective R genes against PWL and other effectors. Partial resistance in some Eragrostis curvula accessions to some blast isolates lacking PWL1 and PWL2 also indicated the presence of other interactions between fungal avirulence (AVR) genes and host resistance (R) genes. Related chloridoid species thus harbor resistance genes that could be useful to improve finger millet for blast resistance. Conversely, loss of AVR genes in the fungus could expand its host range, as demonstrated by the susceptibility of Eragrostis curvula to finger millet blast isolates that had lost PWL1 and PWL2. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz).

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava.

Cassava is the world's most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. There is a need for acyanogenic cassava cultivars in order for it to become a consistently safe and acceptable food, and commercial crop. In recent years, the CRISPR/Cas system, has proven to be the most effective and successful genome editing tool for gene function studies and crop improvement. In this study, we performed targeted mutagenesis of the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacterium-mediated transformation. The vector design resulted in knockout in cotyledon-stage somatic embryos regenerated under hygromycin selection. Eight plants were recovered and genotyped. DNA sequencing analysis revealed that the tested putative transgenic plants carried mutations within the MeCYP79D1 locus, with deletions and substitutions being reported upstream and downstream of the PAM sequence, respectively. The levels of linamarin and evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to seven-fold. Nevertheless, the cassava linamarin and cyanide were not completely eliminated by the MeCYP79D1 knockout. Our results indicate that CRISPR/Cas9-mediated mutagenesis is as an alternative approach for development of cassava plants with lowered cyanide content.

A Review of Recent Advances and Future Directions in the Management of Salinity Stress in Finger Millet.

Salinity stress is a major environmental impediment affecting the growth and production of crops. Finger millet is an important cereal grown in many arid and semi-arid areas of the world characterized by erratic rainfall and scarcity of good-quality water. Finger millet salinity stress is caused by the accumulation of soluble salts due to irrigation without a proper drainage system, coupled with the underlying rocks having a high salt content, which leads to the salinization of arable land. This problem is projected to be exacerbated by climate change. The use of new and efficient strategies that provide stable salinity tolerance across a wide range of environments can guarantee sustainable production of finger millet in the future. In this review, we analyze the strategies that have been used for salinity stress management in finger millet production and discuss potential future directions toward the development of salt-tolerant finger millet varieties. This review also describes how advanced biotechnological tools are being used to develop salt-tolerant plants. The biotechnological techniques discussed in this review are simple to implement, have design flexibility, low cost, and highly efficient. This information provides insights into enhancing finger millet salinity tolerance and improving production.

Plasmid mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Treatment of gonorrhea is complicated by the development of antimicrobial resistance in Neisseria gonorrhoeae (GC) to the antibiotics recommended for treatment. Knowledge on types of plasmids and the antibiotic resistance genes they harbor is useful in monitoring the emergence and spread of bacterial antibiotic resistance. In Kenya, studies on gonococcal antimicrobial resistance are few and data on plasmid mediated drug resistance is limited. The present study characterizes plasmid mediated resistance in N. gonorrhoeae isolates recovered from Kenya between 2013 and 2018. METHODS: DNA was extracted from 36 sub-cultured GC isolates exhibiting varying drug resistance profiles. Whole genome sequencing was done on Illumina MiSeq platform and reads assembled de-novo using CLC Genomics Workbench. Genome annotation was performed using Rapid Annotation Subsystem Technology. Comparisons in identified antimicrobial resistance determinants were done using Bioedit sequence alignment editor. RESULTS: Twenty-four (66.7%) isolates had both ß-lactamase (TEM) and TetM encoding plasmids. 8.3% of the isolates lacked both TEM and TetM plasmids and had intermediate to susceptible penicillin and tetracycline MICs. Twenty-six (72%) isolates harbored TEM encoding plasmids. 25 of the TEM plasmids were of African type while one was an Asian type. Of the 36 isolates, 31 (86.1%) had TetM encoding plasmids, 30 of which harbored American TetM, whereas 1 carried a Dutch TetM. All analyzed isolates had non-mosaic penA alleles. All the isolates expressing TetM were tetracycline resistant (MIC> 1 mg/L) and had increased doxycycline MICs (up to 96 mg/L). All the isolates had S10 ribosomal protein V57M amino acid substitution associated with tetracycline resistance. No relation was observed between PenB and MtrR alterations and penicillin and tetracycline MICs. CONCLUSION: High-level gonococcal penicillin and tetracycline resistance in the sampled Kenyan regions was found to be mediated by plasmid borne blaTEM and tetM genes. While the African TEM plasmid, TEM1 and American TetM are the dominant genotypes, Asian TEM plasmid, a new TEM239 and Dutch TetM have emerged in the regions.

Varietal differences in physiological and biochemical responses to salinity stress in six finger millet plants.

Finger millet is an important cereal that is grown in semi-arid and arid regions of East-Africa. Salinity stress is a major environmental impediment for the crop growth and production. This study aimed to understand the physiological and biochemical responses to salinity stress of six Kenyan finger millet varieties (GBK043137, GBK043128, GBK043124, GBK043122, GBK043094, GBK043050) grown across different agroecological zones under NaCl-induced salinity stress (100, 200 and 300 mM NaCl). Seeds were germinated on the sterile soil and treated using various concentrations of NaCl for 2 weeks. Early-seedling stage of germinated plants were irrigated with the same salt concentrations for 60 days. The results indicated depression in germination percentage, shoot and root growth rate, leaf relative water content, chlorophyll content, leaf K+ concentration, and leaf K+/Na+ ratios with increased salt levels and the degree of increment differed among the varieties. On the contrary, the content of proline, malonaldehyde, leaf total proteins, and reduced sugar increased with increasing salinity. At the same time, the leaf Na+ and Cl- amounts of all plants increased substantially with increasing stress levels. Clustering analysis placed GBK043094 and GBK043137 together and these varieties were identified as salt-tolerant based on their performance. Taken together, our findings indicated a significant varietal variability for most of the parameters analysed. The superior varieties identified could be used as promising genetic resources in future breeding programmes directed towards development of salt-tolerant finger millet hybrids. Further analysis at genomic level needs to be undertaken to better understand the genetic factors that promote salinity tolerance in finger millet.

Breeding Strategies and Challenges in the Improvement of Blast Disease Resistance in Finger Millet. A Current Review.

Climate change has significantly altered the biodiversity of crop pests and pathogens, posing a major challenge to sustainable crop production. At the same time, with the increasing global population, there is growing pressure on plant breeders to secure the projected food demand by improving the prevailing yield of major food crops. Finger millet is an important cereal crop in southern Asia and eastern Africa, with excellent nutraceutical properties, long storage period, and a unique ability to grow under arid and semi-arid environmental conditions. Finger millet blast disease caused by the filamentous ascomycetous fungus Magnaporthe oryzae is the most devastating disease affecting the growth and yield of this crop in all its growing regions. The frequent breakdown of blast resistance because of the susceptibility to rapidly evolving virulent genes of the pathogen causes yield instability in all finger millet-growing areas. The deployment of novel and efficient strategies that provide dynamic and durable resistance against many biotypes of the pathogen and across a wide range of agro-ecological zones guarantees future sustainable production of finger millet. Here, we analyze the breeding strategies currently being used for improving resistance to disease and discuss potential future directions toward the development of new blast-resistant finger millet varieties, providing a comprehensive understanding of promising concepts for finger millet breeding. The review also includes empirical examples of how advanced molecular tools have been used in breeding durably blast-resistant cultivars. The techniques highlighted are cost-effective high-throughput methods that strongly reduce the generation cycle and accelerate both breeding and research programs, providing an alternative to conventional breeding methods for rapid introgression of disease resistance genes into favorable, susceptible cultivars. New information and knowledge gathered here will undoubtedly offer new insights into sustainable finger millet disease control and efficient optimization of the crop's productivity.

Induced Expression of Xerophyta viscosa XvSap1 Gene Enhances Drought Tolerance in Transgenic Sweet Potato.

Drought stress often leads to reduced yields and is a perilous delimiter for expanded cultivation and increased productivity of sweet potato. Cell wall stabilization proteins have been identified to play a pivotal role in mechanical stabilization during desiccation stress mitigation in plants. They are involved in numerous cellular processes that modify cell wall properties to tolerate the mechanical stress during dehydration. This provides a plausible approach to engineer crops for enhanced stable yields under adverse climatic conditions. In this study, we genetically engineered sweet potato cv. Jewel with XvSap1 gene encoding a protein related to cell wall stabilization, isolated from the resurrection plant Xerophyta viscosa, under stress-inducible XvPSap1 promoter via Agrobacterium-mediated transformation. Detection of the transgene by PCR, Southern blot, and quantitative real-time PCR (qRT-PCR) analyses revealed the integration of XvSap1 in the three independent events. Phenotypic evaluation of shoot length, number of leaves, and yield revealed that the transgenic plants grew better than the wild-type plants under drought stress. Assessment of biochemical indices during drought stress showed higher levels of chlorophyll, free proline, and relative water content and decreased lipid peroxidation in transgenic plants than in wild types. Our findings demonstrate that XvSap1 enhances drought tolerance in transgenic sweet potato without causing deleterious phenotypic and yield changes. The transgenic drought-tolerant sweet potato lines provide a valuable resource as a drought-tolerant crop on arid lands of the world.

Differential characterization of physiological and biochemical responses during drought stress in finger millet varieties.

Drought is the most perilous abiotic stress that affects finger millet growth and productivity worldwide. For the successful production of finger millet, selection of drought tolerant varieties is necessary and critical stages under drought stress, germination and early seedling growth, ought to be fully understood. This study investigated the physiological and biochemical responses of six finger millet varieties (GBK043137, GBK043128, GBK043124, GBK043122, GBK043094 and GBK043050) under mannitol-induced drought stress. Seeds were germinated in sterile soil and irrigated with various concentrations of mannitol (200, 400 and 600 mM) for 2 weeks. In a comparative analysis relative water content (RWC), chlorophyll, proline and malondialdehyde (MDA) contents were measured to obtain the physiological and biochemical characteristics of drought stress. The results showed that increased levels of drought stress seriously decreased germination and early seedling growth of finger millet varieties. However, root growth was increased. In addition, exposition to drought stress triggered a significant decrease in relative water content and chlorophyll content reduction, and the biochemical parameters assay showed less reduction in RWC. Furthermore, oxidative damage indicating parameters, such as proline concentration and MDA content, increased. Varieties GBK043137 and GBK043094 were less affected by drought than the other varieties as shown by significant changes in their physiological parameters. Our findings reveal the differences between the physiological and biochemical responses of finger millet to drought and are vital for breeding and selecting drought tolerant varieties of finger millet. Further, genomic and molecular investigations need to be undertaken to gain a deeper insight into the detailed mechanisms of drought tolerance in finger millet.

gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Phenotypic fluoroquinolone resistance was first reported in Western Kenya in 2009 and later in Coastal Kenya and Nairobi. Until recently gonococcal fluoroquinolone resistance mechanisms in Kenya had not been elucidated. The aim of this paper is to analyze mutations in both gyrA and parC responsible for elevated fluoroquinolone Minimum Inhibitory Concentrations (MICs) in Neisseria gonorrhoeae (GC) isolated from heterosexual individuals from different locations in Kenya between 2013 and 2017. METHODS: Antimicrobial Susceptibility Tests were done on 84 GC in an ongoing Sexually Transmitted Infections (STI) surveillance program. Of the 84 isolates, 22 resistant to two or more classes of antimicrobials were chosen for analysis. Antimicrobial susceptibility tests were done using E-test (BioMerieux) and the results were interpreted with reference to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The isolates were sub-cultured, and whole genomes were sequenced using Illumina platform. Reads were assembled de novo using Velvet, and mutations in the GC Quinolone Resistant Determining Regions identified using Bioedit sequence alignment editor. Single Nucleotide Polymorphism based phylogeny was inferred using RaxML. RESULTS: Double GyrA amino acid substitutions; S91F and D95G/D95A were identified in 20 isolates. Of these 20 isolates, 14 had an additional E91G ParC substitution and significantly higher ciprofloxacin MICs (p = 0.0044*). On the contrary, norfloxacin MICs of isolates expressing both GyrA and ParC QRDR amino acid changes were not significantly high (p = 0.82) compared to MICs of isolates expressing GyrA substitutions alone. No single GyrA substitution was found in the analyzed isolates, and no isolate contained a ParC substitution without the simultaneous presence of double GyrA substitutions. Maximum likelihood tree clustered the 22 isolates into 6 distinct clades. CONCLUSION: Simultaneous presence of amino acid substitutions in ParC and GyrA has been reported to increase gonococcal fluoroquinolone resistance from different regions in the world. Our findings indicate that GyrA S91F, D95G/D95A and ParC E91G amino acid substitutions mediate high fluoroquinolone resistance in the analyzed Kenyan GC.

Xerophyta viscosa Aldose Reductase, XvAld1, Enhances Drought Tolerance in Transgenic Sweetpotato.

Sweetpotato is a significant crop which is widely cultivated particularly in the developing countries with high and stable yield. However, drought stress is a major limiting factor that antagonistically influences the crop's productivity. Dehydration stress caused by drought causes aggregation of reactive oxygen species (ROS) in plants, and aldose reductases are first-line safeguards against ROS caused by oxidative stress. In the present study, we generated transgenic sweetpotato plants expressing aldose reductase, XvAld1 isolated from Xerophyta viscosa under the control of a stress-inducible promoter via Agrobacterium-mediated transformation. Our results demonstrated that the transgenic sweetpotato lines displayed significant enhanced tolerance to simulated drought stress and enhanced recuperation after rehydration contrasted with wild-type plants. In addition, the transgenic plants exhibited improved photosynthetic efficiency, higher water content and more proline accumulation under dehydration stress conditions compared with wild-type plants. These results demonstrate that exploiting the XvAld1 gene is not only a compelling and attainable way to improve sweetpotato tolerance to drought stresses without causing any phenotypic imperfections but also a promising gene candidate for more extensive crop improvement.