A reverse transcription loop-mediated isothermal amplification assay for quick detection of tomato mosaic virus.

Tomato mosaic virus (ToMV), an economically important virus that affects a wide range of crops, is highly contagious, and its transmission is mediated by mechanical means, and through contaminated seeds or planting materials, making its management challenging. To contain its wide distribution, early and accurate detection of infection is required. A survey was conducted between January and May, 2023 in major tomato growing counties in Kenya, namely, Baringo, Kajiado, Kirinyaga and Laikipia, to establish ToMV disease incidence and to collect samples for optimization of the reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) assay. A RT-LAMP assay, utilizing primers targeting the coat protein, was developed and evaluated for its performance. The method was able to detect ToMV in tomato samples within 4:45 minutes, had a 1,000-fold higher sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) method and was specific to ToMV. Furthermore, the practical applicability of the assay was assessed using tomato samples and other solanaecous plants. The assay was able to detect the virus in 14 tomato leaf samples collected from the field, compared to 11 samples detected by RT-PCR, further supporting the greater sensitivity of the assay. To make the assay more amenable for on-site ToMV detection, a quick-extraction method based on alkaline polyethylene glycol buffer was evaluated, which permitted the direct detection of the target virus from crude leaf extracts. Due to its high sensitivity, specificity and rapidity, the RT-LAMP method could be valuable for field surveys and quarantine inspections towards a robust management of ToMV infections.

Loop-Mediated Isothermal Amplification assays for on-site detection of the main sweetpotato infecting viruses.

Globally, Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) occur frequently and in combination cause sweetpotato virus disease (SPVD). Many viral diseases are economically important and negatively impact the production and movement of germplasm across regions. Rapid detection of viruses is critical for effective control. Detection and quantification of viruses directly from sweetpotato remains a challenge. Current diagnostic tests are not sensitive enough to reliably detect viruses directly from the plant or require expensive laboratory equipment and expertise to perform. We developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay for the detection of SPFMV, SPCSV and begomoviruses related to sweet potato leaf curl virus (SPLCV). Laboratory validation recorded 100 % diagnostic sensitivity for all the three viruses. The LAMP assays were customized for field testing using a lyophilized thermostable isothermal master mix in a ready-to-use form that required no cold chain. The average time to positivity (TTP) was: SPFMV 5-30 min, SPCSV 15-43 min s and begomoviruses 28-45 mins. LAMP on-site testing results were comparable to PCR and RT-PCR confirmatory laboratory tests. The LAMP assay is a powerful tool for rapid sweetpotato virus detection at a reasonable cost and thus could serve as quality control systems for planting materials.

Comparative analysis for producing sweetpotato pre-basic seed using sandponics and conventional systems.

In Sub-Saharan Africa, sweetpotato pre-basic seed is multiplied in screenhouses using a sterilized soil substrate. This is expensive and unsustainable. The use of sand substrate with a fertigation system ("sandponics"), is an alternative. The study compared the cost-effectiveness for pre-basic seed production using the sandponics system to the conventional soil substrate for four genotypes. A randomized complete block split plot design was used, and data collected on vine traits over six harvests. Real-time cost data were collected for cost-effectiveness analysis. Results showed a highly significant (p < .0001) 21.8% increase in the vine multiplication rate under the sandponics system. The cost of producing one sweetpotato node in sandponics was significantly lower by 0.009 US$. The cost-effectiveness of producing pre-basic seed in sandponics varied among the genotypes. The future use of sandponics is discussed with respect to the availability of soluble inorganic fertilizers, varietal specific response to nutrients, and labor implications.

Storage Root Yield of Sweetpotato as Influenced by Sweetpotato leaf curl virus and Its Interaction With Sweetpotato feathery mottle virus and Sweetpotato chlorotic stunt virus in Kenya.

In this study, the effect of a Kenyan strain of Sweetpotato leaf curl virus (SPLCV) and its interactions with Sweetpotato feathery mottle virus (SPFMV) and Sweetpotato chlorotic stunt virus (SPCSV) on root yield was determined. Trials were performed during two seasons using varieties Kakamega and Ejumula and contrasting in their resistance to sweetpotato virus disease in a randomized complete block design with 16 treatments replicated three times. The treatments included plants graft inoculated with SPLCV, SPFMV, and SPCSV alone and in possible dual or triple combinations. Yield and yield-related parameters were evaluated at harvest. The results showed marked differences in the effect of SPLCV infection on the two varieties. Ejumula, which is highly susceptible to SPFMV and SPCSV, suffered no significant yield loss from SPLCV infection, whereas Kakamega, which is moderately resistant to SPFMV and SPCSV, suffered an average of 47% yield loss from SPLCV, despite only mild symptoms occurring in both varieties. These results highlight the variability in yield response to SPLCV between sweetpotato cultivars as well as a lack of correlation of SPLCV-related symptoms with yield reduction. In addition, they underline the lack of correlation between resistance to the RNA viruses SPCSV and SPFMV and the DNA virus SPLCV.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

Optimization of nutrient media for sweetpotato ( Ipomoea batatas L.) vine multiplication in sandponics: Unlocking the adoption and utilization of improved varieties.

Background: Sweetpotato, being a vegetatively propagated crop is prone to seed degeneration, and a continuous source for high quality sweetpotato seed is critical for an efficient seed system.  In most Sub-Saharan African countries, the National Agricultural Research Systems use tissue culture to produce limited quantity of pre-basic sweetpotato seed which is then used as starting material to maintain and produce basic seed in mini-screen houses, net tunnels or open field multiplication in low-virus pressure areas by either the private seed companies or vine multipliers. Soil is the predominant media for pre-basic seed multiplication. Multiplying pre-basic sweetpotato seed in sand with fertigation, also known as 'sandponics' is a possible opportunity towards sustainable production of pre-basic sweetpotato seed. It would be beneficial to examine the feasibility and the potential to replace soil system with 'sandponics' for growing pre-basic sweetpotato seed. Methods: Pot experiments were conducted to study how sweetpotato vine propagation is affected by sequentially omitting nitrogen, phosphorus, calcium, sulfur and boron from fertilizer applications on cv. Kabode. The experiment was laid in a randomized complete block design with five levels of the factor fertilizer, replicated four times with two blocks. The effect of fertilization of nitrogen at (0, 100, 150, 200 & 250), phosphorus at (0, 30, 60, 90 & 120), calcium at (0, 100, 200, 300 & 400), sulfur at (0, 30, 60, 90 & 120) and boron at (0, 0.1, 0.2, 0.3 & 0.4) ppm on sweetpotato vegetative growth parameters was measured 45 days after planting. Results: The obtained results showed that application of 200, 60, 200, 120 and 0.3 ppm of N, P, Ca, S and B respectively recorded the highest values in sweetpotato vegetative growth parameters.   Conclusions: These results imply that pre-basic sweetpotato vine yields in sandponics could be increased by using this optimized media.

Genetic diversity of Kenyan native oyster mushroom (Pleurotus).

Members of the genus Pleurotus, also commonly known as oyster mushroom, are well known for their socioeconomic and biotechnological potentials. Despite being one of the most important edible fungi, the scarce information about the genetic diversity of the species in natural populations has limited their sustainable utilization. A total of 71 isolates of Pleurotus species were collected from three natural populations: 25 isolates were obtained from Kakamega forest, 34 isolates from Arabuko Sokoke forest and 12 isolates from Mount Kenya forest. Amplified fragment length polymorphism (AFLP) was applied to thirteen isolates of locally grown Pleurotus species obtained from laboratory samples using five primer pair combinations. AFLP markers and internal transcribed spacer (ITS) sequences of the ribosomal DNA were used to estimate the genetic diversity and evaluate phylogenetic relationships, respectively, among and within populations. The five primer pair combinations generated 293 polymorphic loci across the 84 isolates. The mean genetic diversity among the populations was 0.25 with the population from Arabuko Sokoke having higher (0.27) diversity estimates compared to Mount Kenya population (0.24). Diversity between the isolates from the natural population (0.25) and commercial cultivars (0.24) did not differ significantly. However, diversity was greater within (89%; P > 0.001) populations than among populations. Homology search analysis against the GenBank database using 16 rDNA ITS sequences randomly selected from the two clades of AFLP dendrogram revealed three mushroom species: P. djamor, P. floridanus and P. sapidus; the three mushrooms form part of the diversity of Pleurotus species in Kenya. The broad diversity within the Kenyan Pleurotus species suggests the possibility of obtaining native strains suitable for commercial cultivation.

Genetic diversity in Napier grass (Pennisetum purpureum) cultivars: implications for breeding and conservation.

Napier grass is an important forage crop for dairy production in the tropics; as such, its existing genetic diversity needs to be assessed for conservation. The current study assessed the genetic variation of Napier grass collections from selected regions in Eastern Africa and the International Livestock Research Institute Forage Germplasm-Ethiopia. The diversity of 281 cultivars was investigated using five selective amplified fragment length polymorphism (AFLP) markers and classical population genetic parameters analysed using various software. The number of bands generated was 216 with fragments per primer set ranging from 50 to 115. Mean percentage polymorphic loci was 63.40. Genetic diversity coefficients based on Nei's genetic diversity ranged from 0.0783 to 0.2142 and Shannon's information index ranged from 0.1293 to 0.3445. The Fst value obtained was moderately significant (Fst = 0.1688). Neighbour-joining analysis gave two distinct clusters which did not reflect geographical locations. Analysis of molecular variance showed all variance components to be highly significant (P < 0.001), indicating more variation within (91 %) than between populations (9 %). Results suggested moderate genetic differentiation among Napier grass populations sampled, which could imply a high germplasm exchange within the region. The AFLP markers used in this study efficiently discriminate among cultivars and could be useful in identification and germplasm conservation.