Charlotte Mienie

Identification and validation of genetic markers associated with Meloidogyne incognita race 2 resistance in soybean, Glycine max (L.) Merr

Host-plant resistance to Meloidogyne incognita race 2 is a useful and cost-effective tool for optimising soybean yield as well as profitability. Locally no nematicide is currently registered on soybean and most crops used in soybean-based rotations are also susceptible to M. incognita race 2. The identification of molecular markers and subsequent application of marker-assisted selection (MAS) is a quick and effective way to expedite a nematode resistance breeding programme. The soybean cultivars LS5995 (resistant) and Prima2000 (susceptible) were therefore used as parents in crosses to obtain a segregating F2 mapping population for the identification of genetic markers linked to the resistance trait in LS5995. Inoculation with ca 10 000 M. incognita race 2 eggs and second-stage juveniles (J2) was performed 16 days after plant emergence. Subsequently, the F2 population was screened with a number of SSR evenly distributed throughout the genome. A major QTL was identified on linkage group (LG) M between the markers Satt201 and Satt590 accounting for 62.4% of gall index values, while a QTL explaining 80% of the variation in eggs and J2 per root system in the segregating F2 population is situated between Satt567 and Satt201. A minor QTL accounting for 37.1% of the variation in gall index values was identified between the markers Satt500 and Satt358 on LG-O and corresponds to a QTL for M. incognita race 3 resistance described in other publications. Presence of the abovementioned markers was confirmed in the progeny of a successive F6 population, as well as in a number of exotic and local soybean genotypes. Meloidogyne incognita race 2 MAS could, therefore, be used in a breeding programme using markers Satt201, Satt358, Satt487 and Satt590, which were identified and validated in this study.

PCR-denaturing gradient gel electrophoresis analysis of microbial community in soy-daddawa, a Nigerian fermented soybean (Glycine max (L.) Merr.) condiment.

Soy-daddawa, a fermented soybean (Glycine max (L.) Merr.) condiment, plays a significant role in the culinary practice of West Africa. It is essential to understand the microbial community of soy-daddawa for a successful starter culture application. This study investigated the microbial community structure of soy-daddawa samples collected from Nigerian markets, by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the V3-V5 region of the 16S rRNA gene of bacteria and internal transcribed spacer 2 (ITS2) region of fungi. Six bacterial and 16 fungal (nine yeasts and seven molds) operational taxonomic units (OTUs)/species were obtained at 97% sequence similarity. Taxonomic assignments revealed that bacterial OTUs belonged to the phyla Firmicutes and Actinobacteria, and included species from the genera Atopostipes, Bacillus, Brevibacterium and Nosocomiicoccus. Densitometric analysis of DGGE image/bands revealed that Bacillus spp. were the dominant OTU/species in terms of population numbers. Fungal OTUs belonged to the phyla Ascomycota and Zygomycota, and included species from the genera, Alternaria, Aspergillus, Candida, Cladosporium, Dokmaia, Issatchenkia, Kodamaea, Lecythophora, Phoma, Pichia, Rhizopus, Saccharomyces and Starmerella. The majority of fungal species have not been previously reported in soy-daddawa. Potential opportunistic human pathogens such as Atopostipes suicloacalis, Candida rugosa, Candida tropicalis, and Kodamaea ohmeri were detected. Variation in soy-daddawa microbial communities amongst samples and presence of potential opportunistic pathogens emphasises the need for starter culture employment and good handling practices in soy-daddawa processing.

Detection of African horse sickness virus in Culicoides imicola pools using RT-qPCR.

ABSTRACT: African horse sickness (AHS) is an infectious, non-contagious arthropod-borne disease of equids, caused by the African horse sickness virus (AHSV), an orbivirus of the Reoviridae family. It is endemic in sub-Saharan Africa and thought to be the most lethal viral disease of horses. This study focused on detection of AHSV in Culicoides imicola (Diptera: Ceratopogonidae) pools by the application of a RT-qPCR. Midges were fed on AHSV-infected blood. A single blood-engorged female was allocated to pools of unfed nulliparous female midges. Pool sizes varied from 1 to 200. RNA was extracted and prepared for RT-qPCR. The virus was successfully detected and the optimal pool size for the limit of detection of the virus was determined at a range between 1 to 25. Results from this investigation highlight the need for a standardized protocol for AHSV investigation in Culicoides midges especially for comparison among different studies and for the determination of infection rate.

Culicoides species composition and environmental factors influencing African horse sickness distribution at three sites in Namibia

African horse sickness (AHS) is one of the most lethal infectious, non-contagious, vector-borne disease of equids. The causative agent, African horse sickness virus (AHSV) is transmitted via Culicoides midges (Diptera: Ceratopogonidae). AHS is endemic to Namibia but detailed studies of Culicoides communities and influencing environmental parameters are limited. This study aims to determine the Culicoides species composition at three different sites and to assess environmental parameters influencing the geographical distribution of AHS in Namibia. Weekly collections of Culicoides were made during the AHS peak season from January to May for 2013 and 2014 using the Onderstepoort 220V UV-light trap. Out of 397 collections made, 124 collections (3287 Culicoides) were analysed for AHSV presence with RT-qPCR. A total of 295 collections were analysed for total Culicoides (all collected Culicoides individuals) and in 75% of these collections the Culicoides were identified to species level. C. imicola was the dominant species with proportional representation of 29.9%. C. subschultzei, C. exspectator and C. ravus each contribute more than 10% to the species composition. The lowest number of Culicoides was collected at Aus 9980, a total of 21819 at Windhoek and the highest number at Okahandja 47343. AHSV was present at all three sites during 2013 but only in Windhoek and Okahandja during 2014. Multivariate analyses of data from the two year survey indicate the environmental parameters in order of importance for the distribution of AHS in Namibia as precipitation>temperature>clay>relative humidity>NDVI. The implication of these findings is that any precipitation event increases Culicoides numbers significantly. Together with these results the high number of species found of which little is known regarding their vector competence, add to the complexity of the distribution of AHS in Namibia.

Identification of Meloidogyne spp. associated with agri- and horticultural crops in South Africa

The four major and globally widespread Meloidogyne spp., M. arenaria (Neal, 1889), M. hapla Chitwood, 1949, M. incognita (Kofoid & White, 1919) and M. javanica (Treub, 1885) (see Jones et al., 2013), are also abundant in South African crop production areas (Kleynhans et al., 1996). Many other species parasitise crops in South Africa, including M. enterolobii Yang & Eisenback, 1983 (see Kleynhans et al., 1996; Marais, 2014). Meloidogyne enterolobii, reported from only a few crop production areas in South Africa (Van den Berg et al., 2017), is very aggressive, has a wide host range and is known to overcome various root-knot nematode resistance genes (Castagnone-Sereno, 2012). As it is morphologically similar to M. incognita and other thermophilic species, it is prone to being misidentified (Adam et al., 2007; Karssen et al., 2013). Various authors have emphasised the limitations in using only morphological characteristics (particularly perineal pattern morphology) for accurate identification of Meloidogyne spp. (Karssen & Van Aelst, 2001; Carneiro et al., 2004; Hunt & Handoo, 2009), and as discriminating Meloidogyne spp. is crucial to optimise control strategies (Adam et al., 2007), the use of accurate isoenzyme and molecular deoxyribonuclease (DNA) methods became popular (Esbenshade & Triantaphyllou, 1985; Karssen et al., 2013). The sequencederived-amplified region (SCAR) polymerase chain reaction (PCR) technique has been proven to be a fast, reliable and accurate method to discriminate Meloidogyne spp. (Zijlstra et al., 2000). This study aims to update distribution knowledge by identifying Meloidogyne spp. from 28 populations isolated from diagnostic samples or experimental sites through the use of the SCAR-PCR technique and perineal-pattern morphology. Meloidogyne spp. eggs and second-stage juveniles (J2) were extracted from infected root samples of various crops cultivated in different crop production areas during the 2013-2014 summer-growing season (Table 1). Root samples (50 g) of each crop were subjected to the adapted NaOCl method (Riekert, 1995) for extraction of eggs and J2. The latter life stages of each of the 28 populations were inoculated on roots of individual rootknot nematode-susceptible tomato seedlings (‘Rodade’) grown in 5000 cm3 capacity pots containing Telone II (a.s. 1-3 dichloropropene; dosage of 150 l ha–1) fumigated sandy-loam soil (5.3% clay, 93.6% sand, 1.1% silt, 0.47% organic matter and pH (H2O) 7.47) in a glasshouse

Ecological guild and enzyme activities of rhizosphere soil microbial communities associated with Bt‐maize cultivation under field conditions in North West Province of South Africa

Insecticidal proteins expressed by genetically modified Bt maize may alter the enzymatic and microbial communities associated with rhizosphere soil. This study investigated the structure and enzymatic activity of rhizosphere soil microbial communities associated with field grown Bt and non‐Bt maize. Rhizosphere soil samples were collected from Bt and non‐Bt fields under dryland and irrigated conditions. Samples were subjected to chemical tests, enzyme analyses, and next generation sequencing. Results showed that nitrate and phosphorus concentrations were significantly higher in non‐Bt maize dryland soils, while organic carbon was significantly higher in non‐Bt maize irrigated field soil. Acid phosphatase and β‐glucosidase activities were significantly reduced in soils under Bt maize cultivation. The species diversity differed between fields and Bt and non‐Bt maize soils. Results revealed that Actinobacteria, Proteobacteria, and Acidobacteria were the dominant phyla present in these soils. Redundancy analyses indicated that some chemical properties and enzyme activities could explain differences in bacterial community structures. Variances existed in microbial community structures between Bt and non‐Bt maize fields. There were also differences between the chemical and biochemical properties of rhizosphere soils under Bt and non‐Bt maize cultivation. These differences could be related to agricultural practices and cultivar type.