Kenneth Mburu

Potato (Solanum tuberosum L.) is an important staple and food security crop to many communities in the world. However, potato production and quality is greatly constrained by bacterial wilt, a disease caused by a soil-borne pathogen, Ralstonia solanacearum. Ralstonia solanacearum can be managed through clean seed systems and therefore laboratory testing is a pre-requisite for seed certification to confirm the absence of the pathogen in potato seeds before planting. Molecular diagnostics is the gold standard for detection of R. solanacearum in potato seeds. However, the extraction of genomic DNA from R. solanacearum for molecular diagnostics is complex, tedious, lengthy and/or costly procedure. A simple, rapid and reliable DNA extraction protocol is required for use in routine molecular diagnosis of R. solanacearum, a high-risk quarantine pathogen. In this study, we developed a simple and rapid protocol for extracting genomic DNA from symptomatic and asymptomatic potato tubers infected with R. solanacearum and verified its efficiency for the detection and molecular characterization of the pathogen. The protocol was developed from the evaluation of distilled water, Tris-EDTA (TE) and Tris buffer as a base solution for tissue maceration. The DNA quantity and integrity was determined using the NanoDrop 2000C spectrophotometer and agarose gel electrophoresis, respectively. Both hot and cold solutions produced intact high molecular weight genomic DNA of sufficient yield and purity for molecular-based applications. The detection and determination of phylotypes of R. solanacearum, based on conventional and multiplex polymerase chain reaction (PCR), amplified the expected 280 and 372 bp amplicons, respectively, confirming that the quantity and quality of the extracted pathogen genomic DNA was sufficient for molecular diagnostic applications. The sequencing of the amplified products of the endoglucanase gene produced good quality sequences, which confirmed the R. solanacearum isolates to be members of phylotype II sequevar 1. This protocol is a simple, fast and reliable tool for the extraction of sufficient genomic DNA with high quality, directly from R. solancearum-infected potato tubers for PCR and sequencing applications. Its simplicity and throughput make it valuable for use in routine diagnostics and can be adopted by certification programs to ensure distribution of clean potato seeds to farmers.

Pigeon pea (Cajanus cajan (L.) Millsp.), a potential legume as an economic source of protein, is commonly cultivated in tropical and subtropical regions of the world. It possesses medicinal properties and acts as a cash crop, benefiting low-income farmers economically. The identification of pigeon peas exhibiting drought tolerance has become crucial in addressing water scarcity issues in the agriculture sector. In addition, exploring the genetic diversity among genotypes is important for conservation, management of genetic resources, and breeding programs. The aim of this study was to evaluate the morpho-physiological and biochemical responses of selected pigeon pea genotypes under pot-induced water stress conditions through different field capacities as well as the genetic diversity using start codon targeted (SCoT) markers. A significant variation was observed for the physiological traits studied. The accumulation of fresh weight (FW) and dry weight (DW) was significantly reduced in moderate and severe drought stress conditions. The lowest % DW decrease was found in LM (35.39%), KAT (39.43%), and SM (46.98%) than other genotypes at severe drought stress. Analyses of physiological responses including the photosynthetic efficiency (Phi2), the chlorophyll content (SPAD), and the relative water content (RWC) revealed positive and negative correlations with various parameters, reflecting the impact of drought stress on the chlorophyll content. The results revealed that biochemical traits including the total phenolic content, soluble sugars, proline, total protein, total amino acids, and free amino acids were variably and significantly increased under water stress. Antioxidant enzyme activity levels, specifically ascorbate peroxidase (APX) and catalase, varied among the genotypes and in response to severe water stress, offering further insights into adaptive responses. The eight genotypes analysed by use of 20 SCoT markers revealed 206 alleles and an average of 10.3 alleles per locus. Genetic similarity ranged from 0.336 to 0.676, clustering the pigeon pea genotypes into two major groups by the unweighted pair group method of arithmetic averages (UPGMA) cluster analysis. Principal coordinate analysis (PCoA) explained 43.11% of genetic variation and based on analysis of molecular variance, a high genetic variation (80%) within populations was observed, emphasizing the potential for genetic improvement. Among the eight genotypes studied, LM and KAT were drought tolerant and genetically diverse and therefore could be used as parents for developing drought tolerance in pigeon pea.

Plants of the genus Physalis are of economic interest because of their fleshy edible fruits with high nutritional value. Some species have high medicinal value with a long history of ethno-medicinal use to treat diverse diseases. There is therefore a need to correctly discriminate the different species of Physalis for proper utilization. Although most Physalis species have unique morphologies, their vegetative stages are identical, making it difficult to accurately identify them based on morphological characteristics. DNA barcoding has the potential to discriminate species accurately. In this study, ribulose bisphosphate carboxylase large (rbcL) and internal transcribed spacer 2 (ITS2) regions were used to discriminate Physalis species and to reveal their phylogenetic relationships and genetic diversity. Physalis plant samples were collected from seven counties in Kenya based on the availability of the germplasm. The voucher specimens were identified using the botanical taxonomy method and were deposited in the University of Nairobi herbarium. Genomic DNA was isolated from leaf samples of 64 Physalis accessions and used for PCR amplification and the sequencing of rbcL and ITS2 barcode regions. The discriminatory ability of the barcodes was based on BLASTn comparison, phylogenetic reconstruction and cluster analysis, and the determination of inter- and intra-specific distances. The nucleotide polymorphism, genetic diversity and distance of the identified Physalis species were determined using DnaSP and MEGA 11.0 software. Species discrimination was more robust using ITS2 sequences. The species identified and discriminated by ITS2 sequences were Physalis purpurea, Physalis peruviana and Physalis cordata. The rbcL sequences were only able to identify Physalis to the genus level. There was high interspecific and low intraspecific divergence within the identified Physalis species based on ITS2 sequences. The ITS2 barcode is an ideal DNA barcode for use in the discrimination of species, as well as in genetic diversity studies of Physalis accessions in Kenya.

Physalis species are used as an indigenous food and medicine in Kenya. However, species identification and an analysis of the health-promoting bioactive compounds and antioxidant properties are lacking. In this study, we report the molecular identification and mineral and phytochemical profiling of wild Physalis accessions. Leaf samples of 10 Physalis accessions were collected and used for species identification using nuclear ITS2 and plastid rbcL barcodes. Ripe fruits were collected from the same accessions and analyzed for mineral, total phenolic, tannin, and flavonoid contents, and antioxidant activities. The Physalis species were discriminated based on the ITS2 barcode and identified as Physalis purpurea. The genetic diversity, distance, and polymorphism of the ITS2 region of Physalis accessions were high due to the high rate of singleton and parsimony mutations. No genetic diversity, distance, or polymorphism was observed based on the rbcL barcode. The mineral content was significantly different (p < 0.05) for calcium, zinc, nickel, copper, and lithium among the Physalis accessions. No significant variation (p > 0.05) was found for phenolic acids or flavonoids, but the tannic acid content varied significantly (p < 0.05). DPPH free radical scavenging varied significantly (p < 0.05) among Physalis accessions. In conclusion, nuclear ITS2 was used to successfully identify the Physalis species of all the accessions as Physalis purpurea. The present study confirmed that Physalis purpurea has a significantly high mineral and phytochemical content and antioxidant activity. The findings from this study can be used to facilitate exploitation of Physalis purpurea in genetic breeding, their application in pharmaceutical, cosmetic, and nutritional value as well as conservation and sustainable use.