Morphological characterization and identification of Phytophthora species causing citrus gummosis in Kenya

Frequent outbreaks of citrus gummosis in Kenyan citrus orchards have been reported, yet the identity and distribution of the Phytophthora species causing the disease are unknown. Work was carried out to (i) characterize and identify Phytophthora species associated with citrus gummosis based on cultural and morphological traits and (ii)determine the distribution of these species associated with gummosis in different agroecological zones (AEZ). Some 59 plant and soil samples obtained from symptomatic trees and the rhizosphere were evaluated by direct isolation and baiting, respectively, using Phytophthora semi-selective media. Phytophthora species were identified on the basis of colony morphology, mycelial characteristics, cardinal growth temperatures, morphology and dimensions of sporangia, oogonia and antheridia. For colony morphology and growth temperature studies, a 5 mm diametermycelial plug of each isolate was transferred to amended cornmeal agar (ACMA) and incubated at 5, 24 and 35°C for 7 days in the dark. Growth rates were evaluated based on daily records of mycelial growth for 7 days. The occurrence and distribution of these species were determined by recording the number of isolates recovered from samples from each AEZ. P. citrophthora was the most prevalent (76.3 %) of all the Phytophthora species identified in all the AEZs, followed by P. nicotianae (22 %). P.syringae was the least (1.7 %) prevalent. P. citrophthora was the only species present in all AEZs sampled whereas P. nicotianae was confined to the coastal lowlands although also present in other zones in a lower scale. P. syringae was present only in low midland zones and was the only species not found in coastal lowland zones. The forty five isolates of P. citrophthora, thirteen isolates of P. nicotianae and one isolateof P. syringae were tested for virulence on fruits of lemon var. rough lemon. The three most virulent isolates of P. citrophthora, two most virulent isolates of P. nicotianae and the only isolate of P. syringae were selected for pathogenicity testing on lemon seedlings. Based on these studies, it may be concluded that P. citrophthora, P. nicotianae (syn. P. parasitica) and P. syringae are the Phytophthora species associated with citrus gummosis in Kenya. Molecular characterization of the pathogens is recommended to confirm true genetic identity of the species

The rolling circle amplification and next generation sequencing approaches reveal genome wide diversity of Kenyan cassava mosaic geminivirus

Rolling circle amplification is a simple approach of enriching populations of single-stranded DNA plant begomovirus genomes (genus, Begomovirus; family,  Geminiviridae). This is an innovative approach that utilizes the robustness of the bacteriophage phi29 DNA polymerase used in circle amplification, together with deep sequencing using Illumina Miseq and bioinformatics to assess population  diversity of begomoviruses in naturally infected cassava. The approach is suitable for detecting rare members in a population in begomoviral populations in situation where mixtures of isolates, strains, and multiple species occur. The main objectives were to increase the sensitivity of detection of next generation sequencing by enriching it using rolling circle amplification then determination of the diversity of  the cassava mosaic geminivirus. This was done by total nucleic acids isolated from symptomatic, field cassava infected plants, then using rolling circle amplification to multiply the less abundant viral  sequences. Enriched and non-enriched virus-libraries were subjected to deep sequencing using Illumina Miseq. Using  bioinformatic CLC Genomics 5.5.1 software programs the quality assessment of reads and contig assembly of viral sequences. This was done through de novo and reference-guided assembly. The identity and diversities of the begomoviral sequences were compared with sequences in Sanger sequencing of viral components deposited in the NCBI Gene Bank. In this study we have demonstrated that RCA increases the chances of detecting the virus by approximately 10 to 1000 fold and wide genome diversity of cassava mosaic geminivirus in various cassava growing zones in Kenya were detected. In conclusion, this approach described herein is simple and will enhance the exploration of begomovirus diversities from cassava infected plants, irrespective of their viral abundance. This will make it possible for routine screening of field samples as the cost of deep sequencing NGS is decreasing and the advances of bioinformatic software development become enhanced. This is the first report of the RCA-Illumina-NGS approach to explore cassava infected with begomoviruses under field conditions and their diversities. Key words: Illumina Miseq sequencing, geminivirus, ssDNA viruses, viral sequence enrichment, de novo genome assembly, rolling cycle amplification (RCA)

Evaluating diversity among Kenyan papaya germplasm using simple sequence repeat markers

Papaya is an important fruit crop, produced in Kenya for local consumption and export. Despite a history of varietal introductions, no attempts concerned on developing varieties suited to Kenyan conditions have been documented. The objective of this study was to provide information on the diversity of germplasm available in Kenya, as a precursor to systematic plant breeding program. Forty two papaya accessions were collected from farmers’ fields located in Coast, Rift Valley, Western, Nyanza, Central and Eastern provinces. Genetic diversity was determined using seven simple sequence repeat (SSR) markers, computing allelic richness and frequency, expected heterozygosity and cluster analysis. Results indicated that themarkers were highly polymorphic among the accessions, with polymorphicinformation content (PIC) varying from 0.75 to 0.852 with an average of 0.81. The genetic similarity among the 42 papaya accessions ranged from 0.764 to 0.932 with an average of 0.844 showing that most papaya accessions used in this study were closely related. About 96.9% of the pair-wise comparisons among papaya accessions exhibited genetic similarity greater than 0.802, while less than 4% (3.1%) showed genetic similarity lower than 0.802. The phylogenetic analysis grouped the 42accessions into two main clusters A and B. Cluster A had four sub-clusters while cluster B had one cluster. Accessions from Coast, and some from Rift Valley Provinces, presented the highest variation, being scattered throughout the tree, with little or no differentiation from most accessions, whereas some accessions from Coast regrouped in clusters A (iv) and B. The genetic differences among the accessions revealed by the formation of distinct clusters suggest significant genetic variability emanation from varying sources of the papaya germplasm in Kenya. Although the level of genetic diversity revealed by SSR markers in this study is sufficient todistinguish between breeding lines for varietal protection, the rather narrow genetic diversity demonstrated indicates the need to introduce new germplasm or use other techniques such as mutation and genetic engineering to provide breeding materials for the future improvement of papaya in Kenya.Key words: Kenya, papaya, genetic diversity

Morphological diversity of Kenyan papaya germplasm

Papaya is one of the major fruit crops of the tropical regions of the world. It shows considerable phenotypic variation in morphological and horticultural traits that can be utilized in its genetic improvement. In Kenya, wide range of papaya germplasm exists and has not been characterized. Therefore, there is difficulty in differentiating the papaya accessions in the different regions of Kenya. Characterization of papaya germplasm is normally accomplished by use of morphological descriptors, hence as a first step, a germplasm collection from within Kenya was gathered and its morphological diversity was assessed. The papaya germplasm was collected from Coast, Nyanza, Western, Rift Valley, Eastern and Central provinces of Kenya and characterized in the field using morphological descriptors based on fruit, flower, stem and leaf characteristics. The morphological characters were recorded andmorphological data from sixty accessions were submitted to principal component and Neighbor- Joining cluster analysis. Accessions from Coastal, Western, Rift Valley and Nyanza provinces showed the widest morphological diversity with those from Eastern and Central provinces showing the least diversity. Fruit shape, fruit diameter, tree habit, leaf size and flower color showed the greatest variation in principal component analysis. The high diversity observed within the accessions points to ample possibilities of obtaining desirable trait combinations in specific cultivars.Keywords: Kenya, papaya, germplasm, morphological characterizatio

Characterization of Kenyan sweet potato genotypes for SPVD resistance and high dry matter content using simple sequence repeat markers

Simple sequence repeat (SSR) markers were used to characterize Kenyan sweet potato genotypes for resistance to the sweet potato virus disease (SPVD) and high dry matter content. Eighty nine (89)genotypes with a mean symptom severity score of between 1 and 1.5 were selected following graft inoculation with SPVD-infected scions and characterized using 6 SSR primers. The 6 SSR primer pairshad an average polymorphic information content (PIC) of 0.47. The average number of alleles within the 89 genotypes across the 6 loci was 13.52. Cluster analyses revealed a 50% variation among the 89genotypes. The dendrogram did not reveal any unique clustering of the genotypes according to dry matter content and reaction to SPVD. The genetic differences among the SPVD resistant genotypes andthose with high dry matter revealed by the distinct groups suggest a significant genetic variability and the presence of different sources of resistance to SPVD and high dry matter. This characterization willgive valuable information for breeders and serve as a baseline for efficient development of new cultivars resistant to SPVD and containing high dry matter

Predictors of maternal and fetal complications in SLE patients: a prospective study

The aim of the study was to evaluate predictors of disease flares during pregnancy and obstetric and fetal complications in a cohort of systemic lupus erythematosus (SLE) patients. One hundred and thirty-two pregnancies in 96 SLE patients were prospectively followed by monthly clinical and laboratory evaluations. Predictors of lupus flares, fetal and obstetric complications during pregnancy were identified performing stepwise logistic regression analysis. Maternal lupus flares occurred in 57 % of pregnancies and were being best predicted by the number of flares before conception. Manifestations during flares were best predicted by the same features occurred before conception: dermatological flares by skin rash, renal flares by nephritis, and hematological flares by hematological abnormalities. There were 110 live births and 22 fetal losses. Among live newborns, 22 % were premature. Fetal loss was best predicted by hypertension at conception; miscarriages by the amount of steroids taken during the last year before conception; stillbirth by the number of flares during the last year before conception; preterm birth by the coexistence of anti-phospholipid antibody syndrome and anti-double-stranded DNA antibody levels before conception; premature rupture of membranes by high ECLAM score during the 6 months before conception, small for gestation age by hypertension at conception; and preeclampsia by positive lupus anticoagulant. Some independent predictors of lupus flares and fetal and obstetric complications were identified, which can help the risk assessment of pregnancy in SLE patients